Wai Mun Loke B. Sc (Hons) · men was conducted to compare the acute effects on nitric oxide,...

1

CARDIOVASCULAR PROTECTIVE EFFECTS

OF

DIETARY POLYPHENOLS

Wai Mun Loke

B. Sc (Hons)

This thesis is presented for the degree of Doctor of

Philosophy

School of Biochemical, Biomedical and Chemical Sciences

School of Medicine and Pharmacology

University of Western Australia

2008

2

ACKNOWLEDGEMENT

I will like to express my graditude to the School of Biomedical, Biomolecular and

Chemical Sciences and School of Medicine and Pharmacology at UWA for the

opportunity to undertake a PhD course. I am especially grateful to my supervisors, Prof.

Kevin D. Croft, Dr Allan J. McKinley, Mrs Julie Proudfoot and Dr Scott Stewart for

their relentless support and patient guidance.

I will also like to thank Dr Jason Wu, Dr Jonathan Hodgson, Dr Trevor Mori, Dr Anne

Barden and Dr Henrietta Headlam for their help and kind advices; Karey, Cordellia,

Adeline, I-Jung and all the people working in the laboratory at MRF for the wonderful

times in the laboratory; Ramiz, Samir, Sean and other Chemistry mates for all the fun

we have in Perth.

Most importantly, I thank my wife, Kar Gee and my family for their never-ending

support for me.

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PERSONAL CONTRIBUTION OF AUTHOR

The author participated in the planning and design of these studies through discussions

with his supervisors. All studies were coordinated by the author. The author conducted

all the experiments and sample analyses. The author was responsible for all statistical

analyses under the guidance of Dr Jonathan M. Hodgson. The author was responsible

for summarising data, as well as drafting and subsequent editing of manuscripts where

he is the first author. The author had significant contribution into experiment works and

manuscript preparation into the other listed publication where he is not the first author.

SOURCES OF FUNDING

This work was supported by grants from the National Heart Foundation of Australia,

National Health and Medical Research Council (Australia) and Biotechnology and

Biological Sciences Research Council, UK. The author would like to thank the

University of Western Australia for an International Research Fees Scholarship and

convocation travel award, and Australian Atherosclerosis Society for a travel grant.

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LIST OF PUBLICATIONS, PRESENTATIONS AND AWARDS

Publications arising from works in this thesis:

1. Loke WM, Proudfoot JM, Stewart S, McKinley AJ, Needs PW, Kroon PA,

Hodgson JM, Croft KD. Metabolic transformation has a profound effect on anti-

inflammatory activity of flavonoids such as quercetin: Lack of association

between antioxidant and lipoxygenase inhibitory activity. Biochem. Pharmacol.

2008;75:1045-1053.

2. Loke WM, Proudfoot JM, McKinley AJ, Needs PW, Kroon PA, Hodgson JM,

Croft KD. Quercetin and its in vivo metabolites inhibit neutrophil - mediated

LDL oxidation. J. Agri. Food Chem. 2008;56 3609–3615.

3. Loke WM, Hodgson JM, Proudfoot JM, McKinley AJ, Puddey IB, Croft KD.

Pure dietary flavonoids, quercetin and (-)-epicatechin augment nitric oxide

products and reduce endothelin-1 acutely in healthy human volunteers. Am. J.

Clin. Nutr. 2008;In press.

4. Loke WM, Hodgson JM, Croft KD. The biochemistry behind the potential

cardiovascular protection by dietary flavonoids. In: Fraga CG, ed. Phenolic

Compounds of Plant Origin and Health: The Biochemistry behind their

Nutritional and Pharmacological Value: Wiley & Sons; 2008.

5. Loke WM, Hodgson JM, Proudfoot JM, McKinley AJ, Croft KD. Specific

dietary polyphenols attenuate atherosclerosis in ApoE knockout mice by

alleviating oxidative stress, inflammation and endothelial dysfunction.

Arterioscler. Thromb. Vasc. Biol. 2008;Under review.

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Presentations arising from works in this thesis:

1. UWA Combined Biological Sciences Meeting, 2006, PERTH, AUSTRALIA.

(Poster presentation) Am J Hypertens

2. Society for Free Radical Research (Australasia) Meeting, 2006, PERTH,

AUSTRALIA. (Poster presentation)

3. UWA Combined Biological Sciences Meeting, 2007, PERTH, AUSTRALIA.

(Poster presentation)

4. Australian Atherosclerosis Society Meeting, 2007, FREMANTLE,

AUSTRALIA. (Poster presentation)

5. 4th

Joint Meeting of the Society for Free Radical Research (Australasia &

Japan), 2007, KYOTO, JAPAN. (Oral presentation)

6. Agilent GC/MS User Group Meeting, 2008, PERTH, AUSTRALIA. (Oral

presentation)

7. Institute of Food Research, NORWICH, UNITED KINGDOM.. (Oral

presentation)

8. Society for Free Radical Research (Europe) Meeting 2008, BERLIN,

GERMANY. (Poster presentation)

Awards received:

University of Western Australia Graduate Research School Travel Award 2007

Society for Free Radical Research (Japan) and Procter & Gamble Travel Award

2007 (for best oral presentation)

University of Western Australia Postgraduate Convocation Travel Award 2008

University of Western Australia Grant for Research Student Training 2008

Australian Atherosclerosis Society Travel Grant 2008

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Publications arising from work unrelated to this thesis:

1. Loke WM, Proudfoot JM, McKinley AJ, Croft KD.Augmentation of monocyte

intracellular ascorbate in vitro protects cells from oxidative damage and

inflammatory responses. Biochem. Biophys. Res. Comm. 2006;345:1039-1043.

2. Proudfoot JM, Barden A, Loke WM, Croft KD, Puddey IB, Mori TA. High

Density Lipoprotein is the major carrier of plasma F2-isoprostanes J. Lipid Res.

2008;Under review.

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THESIS ABSTRACT

Polyphenols are naturally-occurring phytochemicals, which form an integral

part of the human diet. Results from epidemiological studies have associated

polyphenol intake with reduced risk of cardiovascular diseases. Previous human

intervention studies suggested that dietary polyphenols exert their cardioprotective

effects through their antioxidant and anti-inflammatory effects. While most in vitro

experiments have not accounted for the bioavailability and metabolism of these

polyphenols, our work has provided direct evidence, using quercetin, that metabolic

transformation, together with bioavailability, exert profound effects on bioactivity. We

examined the effect of quercetin and its major metabolites on the production of pro-

inflammatory eicosanoids by human leukocytes. Studies comparing free radical

scavenging, antioxidant activity and eicosanoid production demonstrate that there are

different structural requirements for antioxidant and anti-inflammatory activity. We also

investigated the effect of metabolic transformation on flavonoid bioactivity by

comparing the activity of quercetin and its major metabolites to inhibit inflammatory

eicosanoid production from human leukocytes. Quercetin was a potent inhibitor of

leukotriene B4 formation in leukocytes (IC50 ~ 2μM), and its activity was dependent on

specific structural features, particularly the 2,3 double bond of the C ring.

Functionalisation of the 3’-OH group with either methyl or sulfate reduced inhibitory

activity up to 50% while a glucuronide substituent at the 3-OH effectively removed the

leukotriene B4 inhibitory activity. The major quercetin metabolite quercetin-3’-O-

sulfate retained considerable lipoxygenase inhibitory activity (IC50 ~ 7 μM) while

quercetin-3-O-glucuronide maintained antioxidant activity but had no lipoxygenase

inhibitory activity at physiologically relevant concentrations. We conclude that

structural modification of quercetin due to metabolic transformation had a profound

effect on bioactivity, and that the structural features required for antioxidant activity of

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quercetin and related flavonoids were unrelated to those required for inhibition of

inflammatory eicosanoids.

Our study also examined the effects of metabolic transformation of the

common dietary flavonoid, quercetin, on its ability to protect LDL from neutrophil-

mediated modification. Quercetin was shown to be effective in protecting LDL against

neutrophil–mediated modification at physiologically relevant concentrations (1 µM),

and appeared to act by inhibiting myeloperoxidase-catalyzed oxidation (IC50 = 1.0 µM).

Quercetin was also shown to protect against radical-induced (AAPH) oxidation (IC50 =

1.5 µM). Studies of structure-activity relationships showed that methylation at the 3'-

position or glucuronidation at the 3- position of quercetin did not significantly affect

inhibition of myeloperoxidase activity, but conjugations at both positions significantly

reduced its activity. Our results suggest that the common dietary flavonoid, quercetin

and some of its major in vivo metabolites are potential inhibitors of myeloperoxidase at

physiologically relevant concentrations. Dietary flavonoids that could modify

myeloperoxidase activity could protect lipoproteins from damage in chronic

inflammatory states such as cardiovascular disease.

Improving endothelium-dependent vasodilation and alleviating oxidative stress

are believed to be possible mechanisms by which dietary polyphenols may reduce

cardiovascular risk. A randomised, placebo controlled, cross-over trial in 12 healthy

men was conducted to compare the acute effects on nitric oxide, endothelin-1 and

oxidative stress after oral administration of 200 mg of quercetin, (-)-epicatechin or

epigallocatechin gallate. Relative to water (control), quercetin and (-)-epicatechin

resulted in a significant increase in plasma S-nitrosothiols, plasma nitrite, and urinary

nitrate concentrations (p < 0.05), but not plasma nitrate or urinary nitrite.

Epigallocatechin gallate did not alter any of the measures of nitric oxide production.

Quercetin and (-)-epicatechin resulted in a significant reduction in plasma endothelin-1

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concentration (p < 0.05), but only quercetin significantly decreased urinary endothelin-1

concentration. All three treatments did not significantly change plasma or urinary F2-

isoprostane concentrations. Significant increases in the circulating concentrations of the

three flavonoids were observed (p < 0.05) after the corresponding treatment. Our results

showed that pure quercetin and (-)-epicatechin, but not epigallocatechin gallate, may

improve endothelial function acutely by augmenting circulating levels of vasoactive

nitric oxide and reducing endothelin-1 production. The increased nitric oxide

bioavailability may occur possibly via the inhibition of NADPH oxidase and activation

of endothelial nitric oxide synthase.

A 26-week dietary intervention study was conducted to investigate whether

pure dietary polyphenols representing different polyphenolic classes, namely quercetin

(flavonol), (-)-epicatechin (flavan-3-ol), theaflavin (dimeric catechin), sesamin (lignan)

and chlorogenic acid (phenolic acid), reduce atherosclerotic lesion formation in the

ApoE knockout mouse. Quercetin and theaflavin (64 mg/ kg body mass daily)

significantly attenuated atherosclerotic lesion formation in the aortic sinus and thoracic

aorta (p < 0.05 vs ApoE control mice). Quercetin significantly reduced urinary and

aortic F2-isoprostane concentrations, vascular superoxide production, vascular

leukotriene B4 production, plasma soluble P-selectin, and augmented vascular

endothelial nitric oxide synthase activity and urinary nitrate concentrations (p < 0.05 vs

control ApoE mice). Theaflavin showed similar though less extensive, significant

effects. While (-)-epicatechin significantly reduced vascular F2-isoprostanes, superoxide

concentrations and endothelin-1 production (p < 0.05 vs control ApoE mice), it had no

significant effect on lesion area. Sesamin and chlorogenic acid treatments exerted no

significant effects. Our data suggest that specific dietary polyphenols, in particular

quercetin and theaflavin, have multiple biological activities that in combination may

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help prevent atherosclerosis and contribute to the cardiovascular protection associated

with diets rich in fruits, vegetables and some beverages.

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ABBREVIATIONS

Abbreviation Full Name

5-HETE 5S-hydroxyeicosatetraenoic acid

5-HPETE 5S-hydro(peroxy)eicosatetraenoic acid

5-LO 5-lipoxygenase

AA arachidonic acid

AAPH 2,2′-Azobis(2-methylpropionamidine) dihydrochloride

ATP adenosine triphosphate

BH4 tetrahydrobiopterin

BHT butylated hydroxytoluene

BSA bovine serum albumin

BSTFA N,O-Bis(trimethylsilyl)trifluoroacetamide

CAD coronary artery disease

COX-1 cyclooxygenase-1

COX-2 cyclooxygenase-2

CPF-1 colony stimulating factor

cPLA2 cytosolic phospholipase A2

CRP C reactive protein

CVD cardiovascular disease

DIPEA N,N-diisopropylethylamine

DMSO dimethyl sulfoxide

eNOS endothelial nitric oxide synthases

ET-1 endothelin-1

FLAP 5-lipoxygenase-activating protein

FMD flow-mediated dilation

GC-MS gas chromatography – mass spectroscopy

GM-CSF granulocyte-macrophage colony stimulating factor

H2O2 hydrogen peroxide

HDL high-density lipoprotein

HIFCS heat-inactivated fetal calf serum

HOCl hypochlorous acid

IFN-γ interferon- γ

IL-1 interleukin-1

IL-10 interleukin-10

IL-13 interleukin-13

IL-4 interleukin-4

IL-6 interleukin-6

LDL low-density lipoproteins

LPS lipopolysaccharide

LTA4 leukotrienes A4

LTB4 leukotrienes B4

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Abbreviation Full Name

LTC4 leukotrienes C4

LTD4 leukotrienes D4

MCP-1 monocyte chemoattractant protein-1

M-CSF macrophage-colony-stimulating factor

mm-LDL minimally oxidised LDL

MMP-9 matrix metalloproteinase-9

mPGES-1 membrane-associated prostaglandin E synthase-1

MPO myeloperoxidase

NADH nicotinamide adenine dinucleotide, reduced

NF-κB nuclear factor-κB

NO• nitric oxide

NOX NADPH oxidase

O2•- superoxide radical anion

ONOO- peroxynitrite

ox-LDL oxidised LDL

PBMC peripheral blood mononuclear cells

PBS phosphate buffered saline

PFBBr pentafluorobenzyl bromide

PGB2 prostaglandin B2

PGE2 prostaglandin E2

PGH2 prostaglandin H2

PMA phorbol 12-myristate 13-acetate

ROS reactive oxygen species

SAA serum amyloid A

SOD superoxide dimutase

TGF-β transforming growth factor

Th1 T-helper-1

TMS tetramethylsilane

TNF tumor necrosis factor

TxA2 thromboxane A2

VCAM-1 vascular cell adhesion molecule-1

XO xanthine oxidase

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LIST OF FIGURES AND TABLES

Chapter Figure Number Page

1 1.1 21

1.2 23

1.3 26

1.4 27

1.5 29

1.6 33

1.7 40

1.8 45

1.9 46

2 2.1 58

2.2 64

2.3 64

2.4 66

2.5 66

2.6 67

2.7 68

2.8 69

2.9 71

2.10 72

2.11 77

3 3.1 84

3.2 85

3.3 87

3.4 89

3.5 90

3.6 93

3.7 95

4 4.1 104

4.2 104

4.3 105

4.4 106

4.5 107

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Chapter Figure Number Page

4.6 108

4.7 110

4.8 111

4.9 112

4.10 113

5 5.1 122

5.2 128

5.3 129

5.4 131

5.5 132

5.6 133

5.7 134

5.8 135

5.9 136

5.10 137

5.11 138

Chapter Table Number Page

4 4.1 108

5 5.1 121

5.2 130

5.3 139

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TABLE OF CONTENTS

Page

Acknowledgement 2

Personal Contribution of Author 3

Sources of Funding 3

List of Publications, Presentations and Awards 4

Thesis Abstract 7

List of Abbreviations 11

List of Figures and Tables 13

Table of Contents 15

CHAPTER 1 LITERATURE REVIEW

1.1. ATHEROSCLEROSIS 20

1.1.1. Atherosclerosis and Inflammation 21

1.1.1.1. Leukocyte Adhesion, Chemotaxis And

Transmigration

1.1.1.2. Differentiation And Proliferation

1.1.1.3. Immune Responses

1.1.2. Atheroslcerosis and Eicosanoids 28

1.1.2.1. 5-Lipoxygenase Pathway In Atherogenesis

1.1.2.2. Cyclooxygenase-2 Pathway In Atherogenesis

1.1.3. Atherosclerosis and Oxidative Stress 34

1.1.3.1. Oxidative Modification Hypothesis Of

Atherosclerosis

1.1.3.2. Reactive Oxygen Species And Their Sources

1.1.4. Atherosclerosis and Endothelial Dysfunction 38

1.1.4.1. Nitric Oxide And Endothelial Nitric Oxide

Synthase

1.1.4.2. Endothelin-1

1.2. DIETARY POLYPHENOLS AND THEIR

CARDIOVASCULAR PROTECTIVE EFFECTS

43

1.2.1. Bioavailability and Metabolism 43

1.2.2. Dietary Polyphenols As In Vivo Antioxidants 46

1.2.3. Dietary Polyphenols And Lipemia 48

1.2.4. Dietary Polyphenols And Inflammation 49

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1.2.5. Dietary Polyphenols And Endothelial Function 50

1.2.6. Dietary Polyphenols And Platelet Reactivity 51

1.3. HYPOTHESES AND AIMS 53

CHAPTER 2 IN VITRO EFFECT OF QUERCETIN AND

METABOLITES ON EICOSANOID BIOSYNTHESIS

2.1. INTRODUCTION 56

2.2. MATERIALS AND METHODS 59

2.2.1. Chemicals And Reagents 59

2.2.2. Isolation Of Peripheral Monocytes And Neutrophils 59

2.2.3. Stimulation And Measurement Of Leukotriene B4

Production

60

2.2.4. Stimulation And Measurement Of Prostaglandin E2

And monocyte chemoattractant protein-1

Production

61

2.2.5. Measurement Of Cellular Quercetin And Its

Metabolites

62

2.2.6. Measurement Of Inhibition Of Lipoprotein

Oxidation

62

2.2.7. Statistical Analysis Of Results 63

2.3. RESULTS 63

2.3.1. Effects Of Quercetin And Its Metabolites On

Leukotriene B4 Production

63

2.3.2. Effects Of Quercetin And Its Metabolites On

Prostaglandin E2 And monocyte chemoattractant

protein -1 Production

65

2.3.3. Effects Of Luteolin, Kaempferol And Taxifolin On

Leukotriene B4 Production

67

2.3.4. Effects Of Quercetin On Leukotriene A4 Hydrolase 68

2.3.5. Cellular Uptake Of Quercetin And Its Metabolites 68

2.3.6. Antioxidant Activity 70

2.4. DISCUSSION 73

CHAPTER 3 IN VITRO EFFECT OF QUERCETIN AND

METABOLITES ON MYELOPEROXIDASE

ACTIVITY



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3.2.2. AAPH – Induced And Neutrophil – Mediated

Modification Of Low-Density Lipoprotein

80


Oxidation

80


Chlorination

81

3.2.5. Measurement Of Inhibition Of Functional

Myeloperoxidase Activity

81

3.2.6. Measurement Of Inhibition Of NADPH Oxidase

Activity

82

3.2.7. Statistical Analysis Of Results 82

3.3. RESULTS 83

3.3.1. Effects On Lipid Peroxidation 83

3.3.2. Effects On Low-Density Lipoprotein Protein

Modification

86

3.3.3. Effects On Functional Myeloperoxidase Activity 88

3.3.4. Effects On O2•- Production 88

3.4. DISCUSSION 90

CHAPTER 4 IN VIVO EFFECT OF PURE FLAVONOIDS ON

NITRIC OXIDE AND ENDOTHELIN-1 STATUS IN

HEALTHY HUMANS




4.2.2. Subjects 98

4.2.3. Experimental Design 98

4.2.4. Measurement Of S-Nitrosothiols 99

4.2.5. Measurement Of Nitrite And Nitrate 100

4.2.6. Measurement Of Endothelin-1 100

4.2.7. Systemic Oxidative Stress 100

4.2.8. Metabolism Of Quercetin, (-)-Epicatechin And

Epigallocatechin Gallate 101

4.2.9. Statistical Analysis 101

4.3. RESULTS 102

4.3.1. S-Nitrosothiols, Nitrite And Nitrate Production 102

4.3.2. Endothelin-1 Production 107

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4.3.3. Systemic Oxidative Stress 109

4.3.4. Quercetin, (-)-Epicatechin And Epigallocatechin

Gallate Absorption 109

4.3.5. Correlations Of Nitric Oxide Products And

Endothelin -1 With Plasma Flavonoid

Concentrations

112

4.4. DISCUSSION 114

CHAPTER 5 SPECIFIC DIETARY POLYPHENOLS ATTENUATE

ATHEROSCLEROSIS IN APOE-/-

MICE VIA

ALLEVIATING OXIDATIVE STRESS,

INFLAMMATION AND ENDOTHELIAL

DYSFUNCTION




5.2.2. C57BL And ApoE-/-

Mice 120

5.2.3. Isolation Of Plasma And Aortic Tissue 122

5.2.4. Histological Analysis Of Mouse Aortas 123

5.2.5. Plasma Cholesterol And Aortic Fatty Acid

Composition 123

5.2.6. Systemic And Vascular Oxidative Stress 124

5.2.7. Ex Vivo Vascular Leukotriene B4 Production 125

5.2.8. Plasma Soluble P-Selectin 125

5.2.9. Vascular endothelial Nitric Oxide Synthase Activity,

Urinary Nitrite, Nitrate And Endothelin-1 125

5.2.10. Statistical analysis 126

5.3. RESULTS 127

5.3.1. Animals And Polyphenol Diets 127

5.3.2. Aortic Lesion Analyses 127

5.3.3. Plasma Cholesterol And Aortic Fatty Acid

Composition 130

5.3.4. Systemic And Vascular Oxidative Stress 131

5.3.5. Ex Vivo Vascular Leukotriene B4 Production And

Plasma sP-Selectin 134

5.3.6. Vascular endothelial Nitric Oxide Synthase Activity,

Urinary Nitrite, Nitrate And Endothelin-1 135

5.4. DISCUSSION 139

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CHAPTER 6 CONCLUSIONS & FUTURE RESEARCH 145

REFERENCES 150

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CHAPTER 1:

LITERATURE REVIEW

1.1. ATHEROSCLEROSIS

Atherosclerosis is the formation of plaque in the inner lining of arteries caused

by the build-up of fatty materials, cholesterol, calcium and matrix proteins (Figure 1.1).

The plaque may grow significantly to reduce the blood flow through the affected artery.

If the plaque becomes too fragile, it ruptures, causing blood clots (thrombosis), which

block off or reduce the blood flow to certain tissues. This event can result in heart

attack, stroke and gangrene of the limbs1. Usually, atherosclerosis does not produce

symptoms until it severely narrows the artery, or until it causes a sudden obstruction.

The risk of developing atherosclerosis increases with high blood pressure, high blood

cholesterol levels, cigarette smoking, diabetes, obesity, a lack of exercise, and

advancing age.

Atherosclerotic lesions are asymmetric focal thickening of the intima of the

artery, consisting of blood-borne inflammatory and immune cells, vascular endothelial

cells, smooth muscle cells, connective-tissue elements and lipids2. The lesion is usually

preceded by a fatty streak, which is an accumulation of lipid-laden macrophages

beneath the endothelium. Fatty streaks do not cause any symptoms and may progress to

atherosclerotic lesions or eventually disappear. Lipid-rich foam cells form the core of

the plaque surrounded by a cap of smooth muscle cells and a collagen-rich matrix.

These foam cells are derived from macrophages and smooth muscle cells which have

accumulated low-density lipoproteins (LDL) by endocytosis. Activated T-cells,

macrophages, and mast cells are abundant in the shoulder region of the growing lesion1.

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Thrombosis occurs when the atherosclerotic lesion prevents blood flow

through the artery via the rupture of the plaque and the resultant formation of an

occluding thrombus on the surface of the plaque1. There are two major causes of

thrombosis: plaque rupture and endothelial erosion. Plaque ruptures may occur where

the fibrous cap is thin and partly destroyed by activated immune cells. They produce

numerous inflammatory molecules and proteolytic enzymes which weaken the cap,

activate cells in the core and transform the stable plaque into a vulnerable, unstable

structure that can rupture, induce a thrombus, and elicit an acute coronary syndrome.

Plaque rupture is dangerous because prothrombotic material from the core of the plaque,

such as phospholipids, tissue factors and platelet-adhesive matrix molecules are exposed

to the blood.

Figure 1.1: The picture on the left shows a normal coronary artery with no

atherosclerosis. The picture on the right shows a coronary artery with severe

atherosclerosis (Picture extracted from http://medweb.bham.ac.uk; 30 July 2005)

1.1.1. ATHEROSCLEROSIS AND INFLAMMATION

The early phase of atherosclerosis may be explained as an inflammatory

response elicited by retention and modification of LDL in the arterial intima3. LDL is

the main carrier of cholesterol in the blood, and is responsible for transporting

cholesterol to peripheral cells4. Studies in animals and humans have shown that

http://medweb.bham.ac.uk/

22

hypercholesterolemia causes activation of endothelium in large and medium-sized

arteries5. When the plasma level of the cholesterol-rich LDL rises, the LDL infiltrates

the artery wall and begins to accumulate in the subendothelial matrix. The normal

arterial endothelium resists prolonged contact with leukocytes including the blood

monocyte. LDL modification, through enzymatic or non-enzymatic oxidation, leads to

the release of bioactive lipids that can activate endothelial cells5.

1.1.1.1. Leukocyte Adhesion, Chemotaxis and Transmigration

The platelet is the first blood cell to accumulate at the site of endothelial

activation where its glycoproteins Ib and IIb/ IIIa engage surface molecules on the

endothelial cells and cause further endothelial activation6. The activated endothelial

cells increase their expression of various leukocyte adhesion molecules, for example, P-

selectin and vascular cell adhesion molecule-1 (VCAM-1) which promotes adhesion of

cells carrying the receptors for VCAM-1 (i.e. monocytes and T-lymphocytes) to the

endothelium7 (Figure 1.2). Atherosclerotic lesions often form at bifurcations of arteries,

regions characterized by disturbed blood flow. This reduces the activity of endothelial

atheroprotective molecules such as nitric oxide (NO•) and favours VCAM-1

expression8. The adhered leukocytes diapedeses between intact endothelial cells and

penetrate into the innermost layer of the arterial wall. The migratory gradient is

facilitated by various chemokines, such as monocyte chemoattractant protein-1 (MCP-

1) which binds to CCR-2 receptors on the surface of monocytes and other leukocytes

bearing similar receptors9. Interferon-γ (IFN-γ)-inducible chemokines bind to CXCR3

receptors on T-lymphocytes10

. Monocytic cells directly interacting with endothelial

cells also increase monocyte matrix metalloproteinase-9 (MMP-9) production, which

causes the breakdown of extracellular matrix between the endothelial cells. This allows

the subsequent infiltration of leukocytes through the endothelial layer and its associated

basement membrane11

.

23

Figure 1.2: Proposed initiation of atherosclerosis showing monocyte adhesion,

chemotaxis and transmigration (Adapted from Libby, 20027).

1.1.1.2. Differentiation and Proliferation

Once resident in the subendothelial matrix, the monocytes, under the influence

of macrophage-colony-stimulating factor (M-CSF), differentiate into macrophages and

express both scavenger receptors and toll-like receptors12, 13

. Scavenger receptors bind

to and internalize a broad range of molecules and particles bearing molecules with

pathogen-like molecular patterns such as bacterial endotoxins, apoptotic cell fragments

and oxidatively modified LDL particles. When the cholesterol derived from the uptake

of the oxidatively modified LDL cannot be mobilized from the macrophages to a

sufficient extent, it accumulates as cytosolic droplets and ultimately, the macrophage is

transformed into a foam cell, the prototypical cell in atherosclerotic lesions3. Early

atherosclerotic lesions (Figure 1.3) are characterized by massive accumulation of foam

cells7. In addition to binding molecules with pathogen-like molecular patterns, toll-like

receptors can initiate a signal cascade that leads to cell activation13

. Similar effects were

observed in dendritic cells, mast cells, and endothelial cells, which also express toll-like

24

receptors. Oxidatively modified LDL particles may activate these receptors stimulating

macrophages and foam cells to secrete MCP-1, VCAM-1, pro-inflammatory cytokines,

vasoactive molecules and a host of other pro-inflammatory mediators which further

amplify the inflammatory responses in the lesion14

. These cells also release reactive

oxygen species (ROS) which may result in LDL oxidation15

. Oxidatively modified LDL

increases the monocyte chemotactic activities of endothelial cells and smooth muscle

cells via the production of MCP-116

. MCP-1 expression is known to be increased in

atherosclerotic lesions and injured arteries17

and recent studies have shown that plasma

MCP-1 levels are associated with risk factors for atherosclerosis 18

. The cytotoxic

oxidized LDL (ox-LDL) may also result in endothelial dysfunction and destruction of

smooth muscle cells16

. In addition, activated macrophages secrete proteolytic enzymes

that may degrade matrix components leading to destabilization of plaques and an

increased risk of plaque rupture and thrombosis19

.

1.1.1.3. Immune Responses

A T-cell infiltrate is always present in atherosclerotic lesions (Figure 1.4).

Such infiltration is predominantly CD4+ T-cells20

. Oxidatively modified LDL activates

T-lymphocytes in the subendothelial tissue and T-cell activation may lead to several

types of effector responses, the T-helper-1 (Th1) response being the most prevalent in

atherosclerotic lesions20

. The activated T-cells differentiate into Th1 effector cells and

begin producing pro-inflammatory cytokines, which amplify the existing inflammatory

responses and promote lesion formation21

. IFN-γ, a major pro-atherogenic Th1

cytokine, improves the efficiency of antigen presentation and augments synthesis of the

inflammatory cytokines tumor necrosis factor (TNF) and interleukin-1 (IL-1)22

. Acting

synergistically, these cytokines promote macrophage and endothelial activation and

production of adhesion molecules, cytokines and chemokines23

. IFN-γ, TNF and IL-1

induce the production of substantial amounts of interleukin-6 (IL-6) in various tissues.

25

IL-6, in turn stimulates the production of large amounts of acute-phase reactants,

including C-reactive protein (CRP), serum amyloid A (SAA) and fibrinogen in the liver.

Powerful anti-inflammatory regulators built into the immune network act as

protective factors in atherosclerosis. T-cell-mediated inflammatory responses are

inhibited by interleukin-10 (IL-10) and transforming growth factor β (TGF-β).

Inhibition of IL-10 aggravates atherosclerosis and exacerbrates coronary thrombosis in

hypercholesterolemic mice24

and abrogation of TGF-β elicits rapid development of

large, unstable atherosclerotic lesions25

. Specific antibodies produced by splenic B-cells

recognize phosphorylcholine, a molecule present in oxidatively modified LDL and

apoptotic cell membranes, and aid in the elimination of oxidatively modified LDL and

dead cells26

.

In summary, atherosclerosis may be regarded as an inflammatory disease.

Immune cells, like monocytes and T-cells infiltrate lesions at all stages of development.

The balance between inflammatory and anti-inflammatory activity controls the

progression of atherosclerosis. Disease development is slowed by inhibition of immune-

cell recruitment and is accelerated by cytokines and antigens27

. Anti-inflammatory and

immunosuppressive mechanisms inhibit atherosclerosis and may be attractive targets for

disease prevention and treatment.

26

Figure 1.3: Schematic diagram showing various stages in the development of

atherosclerosis (Adapted from Libby, 20027).

27

Figure 1.4: Schematic diagram showing the effects of T-cell activation in

atheroslcerosis (Adapted from Hansson et al, 20063).

28

1.1.2. ATHEROSCLEROSIS AND EICOSANOIDS

Arachidonic acid (20:4 n-6) (AA) or all cis-5, 8, 11, 14 eicosatetraenoic acid is

a 20-carbon polyunsaturated fatty acid, found in mammalian tissues. AA can be

converted to a range of oxygenated metabolites, collectively known as eicosanoids,

which are involved in inflammatory and antiinflammatory processes28

. AA may be

oxygenated by either lipoxygenases to generate leukotrienes or by cyclooxygenases to

generate prostaglandins. Alternatively, AA can be oxidised by cytochrome P450

monooxygenases to generate epoxyeicosatrienoic acids and hydroxyeicosatetraenoic

acids.

1.1.2.1. 5-Lipoxygenase Pathway in Atherogenesis

Lipoxygenases belong to a class of enzymes that catalyse the insertion of

oxygen across the cis, cis-nonconjugated diene system in polyunsaturated fatty acids,

such as AA. A common human lipoxygenase, 5-lipoxygenase (5-LO) is expressed by

leukocytes including blood monocytes, tissue macrophages, dendritic cells, neutrophils,

and mast cells and it is so named because it catalyses the insertion of oxygen

specifically at the C-5 of AA29

. In resting leukocytes, 5-LO is a soluble enzyme and is

found in both the nucleus and the cytoplasm. Nuclear 5-LO acts at the nuclear envelope,

whereas cytosolic 5-LO acts at cytoplasmic and perinuclear membranes.

Leukotrienes are powerful lipid mediators derived from the 5-LO cascade of

AA (Figure 1.5)29

. Within leukocytes, AA is hydrolysed from membrane

glycerophospholipids by the action of cytosolic phospholipase A2 (cPLA2). The released

unesterified AA binds to 5-lipoxygenase-activating protein (FLAP) which transfers it to

5-LO. 5-LO catalyses the incorporation of molecular oxygen into AA at position C5 to

form the hydroperoxide, 5S-hydro(peroxy)eicosatetraenoic acid (5-HPETE).

Subsequent conversion of 5-HPETE by 5-LO results in the formation of the epoxide,

29

5-HPETE

5-LO

LTA4

5-HETE

LTB4

LTA4

Hydrolase

20-OH-LTB4

20-COOH-LTB4

LTC4

LTC4 Synthase

LTD4

LTE4

O

OH

O

OH

OOH

AA

O

OH

OH

5-LO

O

OHO

O

OHOHHO

O

OHOHHO

OH

O

OHOHHO

O

OH

O

OHOH

S

Cys GlyGlu

O

OHOH

S

Cys Gly

O

OHOH

S

Cys

CYP450

-hydroxylase

Figure 1.5: 5-lipoxygenase pathway

30

leukotriene A4 (LTA4). Alternatively, 5-HPETE can be reduced via a pseudoperoxidase

activity of 5-LO to the corresponding alcohol, 5S-hydroxyeicosatetraenoic acid (5-

HETE). LTA4 serves as substrate for leukotriene C4 (LTC4) synthase to generate LTC4

and leukotriene D4 (LTD4) or for LTA4 hydrolase to generate leukotriene B4 (LTB4). An

important alternative pathway for leukotriene biosynthesis involves the transcellular

import of leukotrienes. LTA4, exclusively synthesized by leukocytes, is exported to

endothelial cells, where it is converted to LTB4 or LTC4 and LTD4, because these cells

express LTA4 hydrolase and LTC4 synthase30

.

The 5-LO pathway was found to be abundantly expressed in both healthy

arterial walls and atherosclerotic lesions29

. Marked increases in 5-LO expression

localized in macrophages, neutrophils, monocytes, dendritic cells and mast cells were

reported with advancing stages of lesion development31

. The addition of oxidized LDL

to U937 and HL60 myeloid cells increased FLAP and 5-LO transcription levels in vitro

by about 10-fold32

. In contrast, 15-lipoxygenase, which has been implicated to mediate

the oxidative modification of LDL in the arterial wall, was expressed at levels several

orders of magnitude lower than 5-LO in both healthy and diseased arteries31

.

Several lines of evidence suggest that the 5-LO pathway is involved in

atherogenesis. 5-LO pathways may promote leukocyte adhesion by inducing the

expression of adhesion molecules, such as VCAM-133

. LTB4 is a potent chemoattractant

for neutrophils, monocytes and eosinophils leading to their adherence to vessel walls

and the subsequent transmigration into the subendothelial space of arteries29

. The

adhered leukocytes spread across the endothelial monolayer before they diapedese

between intact endothelial cells and penetrate into the innermost layer of the arterial

wall. LTB4 appears to mediate cell spreading after adhesion as its addition was found to

increase the surface area of spread cells34

. The two other 5-LO products, namely 5-

HETE and 5-oxoeicosatetraenoic acid were also shown to induce direction migration

31

and promote cell spreading35

. Blocking the LTB4 receptor in apoE knockout mice

diminished not only the chemostatic activity of LTB4, but also its role in adhesion by

disrupting cell binding to endothelial cells and leukocyte transmigration into the

subendothelial space as well as reducing the extent of atherosclerotic lesions36

. In a

murine model of peritoneal sepsis, LTB4 levels fell by 60% with the injection of an anti-

MCP-1 antibody37

. Conversely, MCP-1 increased LTB4 levels in cultured mouse

macrophages in a dose-dependent manner. Thus, the synergistic interaction between

MCP-1 and LTB4 could modulate the chemotaxis and transmigration of leukocytes into

the intima after their adherence to endothelial cells.

Once in the intima, leukocytes are primed by granulocyte-macrophage colony

stimulating factor (GM-CSF), which also activates 5-LO gene transcription and

expression38

. Initially, GM-CSF increases the availability of AA for the existing 5-LO,

which is followed by a sustained increase in LTB4 synthesis as a consequence of the

augmented 5-LO expression. The result is the generation of an amplification loop

through the accumulation of LTB4 and other 5-LO products in the vascular tissue.

Interactions among endothelial cells, T lymphocytes, macrophages and smooth muscle

cells are probably mediated through leukotrienes because all these cells express distinct

functional leukotriene receptors29

. LTB4 can alter gene expression in monocytes and

macrophages leading to the induction of CD36, MCP-1 and M-CSF39

. CD36 is a

receptor for oxidized LDL involved in the conversion of monocytes to foam cells40

.

Induction of MCP-1 mRNA and protein expression by LTB4 provides a positive loop

for both the recruitment of macrophages and monocytes and further induction of the

components of the 5-LO pathway39

. Similarly, induction of CD36 provides yet another

positive feedback loop through oxidized LDL uptake, which converts macrophages to

foam cells. All these mechanisms, which are mediated directly or indirectly by LTB4

may further enhance disease progression.

32

1.1.2.2. Cyclooxygenase-2 Pathway in Atherogenesis

Cyclooxygenase is the key enzyme catalysing the rate-limiting steps in

prostaglandin biosynthesis41

. It exists in at least two isoforms, designated as

cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2). Although both isoforms

share considerable sequence homology, they differ considerably in tissue distribution,

regulation, and function. COX-1 is constitutively expressed in almost all mammalian

tissues. COX-1 mediates production of platelet thromboxane A2 (TxA2), a potent

vasoconstrictor and platelet agonist41

. In contrast, COX-2 is barely detectable under

normal physiological conditions, but can be induced rapidly and transiently by pro-

inflammatory mediators and mitogenic stimuli including cytokines, endotoxins, growth

factors and oncogenes41

.

COX-2 has been reported to be widely expressed by monocytes, macrophages,

smooth muscle cells and endothelial cells in human atherosclerotic arteries42

and so has

received considerable attention for its potential role in inflammation and development

of atherosclerosis. While COX-1 is expressed abundantly in normal arteries and in

atherosclerotic lesions, COX-2 expression is restricted to atherosclerotic lesions and is

not found in normal arteries42

. The proinflammatory agents, IL-1, TNF and

lipopolysaccharide have all been shown to induce COX-2 expression in monocytes,

whereas COX-2 expression is inhibited by the anitinflammatory cytokines interleukin-4

(IL-4) and interleukin-13 (IL-13)41

. COX-2 catalyses the intial two steps in the

biosynthesis of prostaglandin H2 (PGH2) from the AA substrate and from this stage,

PGH2 is further metabolised by other isomerases to a range of prostaglandins41

(Figure

1.6). Macrophages expressing COX-2 produce pro-inflammatory prostaglandin E2

(PGE2), which has been shown to induce the production of the inflammatory cytokine

IL-643

and promote the release and activation of matrix metalloproteinases, which play

an important role in macrophage migration and plaque stability44

. Unstable plaques

33

O

OH

AA

O

OH

OOH

O

O

PGG2

O

OH

OH

O

O

PGH2

O

OH

OH

PGD2

O

HO

O

OH

OH

PGE2

HO

OO

OH

OH

PGF2

HO

HOOH

PGI2

HO

O

O

OH

O

O

O

OH

OH

TxA2

COX-2

COX-2

PGDS

PGESPGFS

PGIS

TxAS

Figure 1.6: Cyclooxygenase-2 biosynthesis of prostaglandins and thomboxanes.

were found to contain higher levels of COX-2 in association with higher content of the

inducible isoform of PGE2 synthase, membrane-associated prostaglandin E synthase-1

(mPGES-1), compared to stable plaques45

. COX-2-mediated prostaglandin production

by activated macrophages in the artery might promote atherosclerosis through several

mechanisms, including activation of chemotaxis, induction of vascular permeability,

propagation of the inflammatory cytokine cascade and stimulation of smooth muscle

cell migration42

.

Results from various studies examining the impact of selective COX-2

inhibition on the development of atherosclerosis in murine models have been mixed.

Treatment with selective COX-2 inhibitors has been reported to decrease or have no

impact on atherosclerosis46, 47

. COX-2 inhibition seems to exert greater influence over

34

early atherosclerotic inflammatory events and lesion formation than on advanced

lesions46, 47

. Various in vitro studies have shown that oxidised LDL suppresses COX-2

activity and expression in human monocyte-macrophages48

. This is consistent with the

observation that the majority of macrophage-derived foam cells in the lesions of apoE

knockout mice did not express COX-246

. This may explain at least in part, the

diminishing role of COX-2 in the progression of atherosclerosis. Selective COX-2

inhibition may provide a potent anti-inflammatory effect, and could be considered as a

potential therapeutic treatment for early events in atherosclerosis.

1.1.3. ATHEROSCLEROSIS AND OXIDATIVE STRESS

Oxidative stress is defined as the imbalance between oxidants and antioxidants

in favour of oxidants, leading to a disruption of redox signaling and control and/or

molecular damage 49

. It may result from a diminished level of antioxidants or an

increased level of oxidants or both. Growing evidence indicates that oxidative stress is

integral in the development of cardiovascular disease (CVD). Various animal models of

oxidative stress and human investigations support the oxidative stress hypothesis of

atherosclerosis in which reactive oxygen species (ROS) play a causal role. ROS are ions

or very small molecules which are highly reactive due to the presence of unpaired

valence electrons 49

. They are formed as natural byproducts of the normal metabolism of

oxygen and have important roles in cell signaling 49

. The most direct evidence for the

role of oxidative stress in atherosclerosis comes from studies with the apoE knockout

mouse. These mice spontaneously develop atherosclerosis similar to that found in

humans and are an accepted model of atherosclerosis, displaying high cholesterol

concentration, increased lipid peroxidation and low NO• bioavailability

1. F2-

isoprostanes, prostaglandin-like products of the free radical-catalysed peroxidation of

AA, have been found to localize in foam cells in atherosclerotic lesions of humans50

. F2-

isoprostanes are established as biomarkers of in vivo oxidative stress50

.

35

1.1.3.1. Oxidative Modification Hypothesis of Atherosclerosis

According to the oxidative modification hypothesis, LDL in its native state is

not atherogenic and the atherogenic process begins when LDL is oxidatively modified

by the vascular cells within the arterial wall1. In the early phase, LDL is mildly oxidized

to form minimally modified LDL (mm-LDL) in the sub-endothelial space. The mild

oxidation of LDL does not affect its cholesterol component or the apoB, but a

significant proportion of the unsaturated acyl chains of the cholesteryl esters and

phospholipids are oxidized to hydroperoxides, isoprostanes and short-chain aldehydes.

Although mm-LDL is not internalized by macrophages, it promotes monocyte

chemotaxis into the sub-endothelial space and their subsequent differentiation into

macrophages by stimulating the production of MCP-1 and M-CSF 1. Significant

oxidation of LDL is required to promote its uptake by macrophages. At this oxidation

stage, a substantial portion of cholesterol is converted into 7-ketocholesterol and other

oxysterols and the unsaturated fatty acids are oxidized to a complex mixture of

products, including aldehydic products such as malondialdehyde and hydroxynonenal51

.

The apoB protein is also extensively fragmented and modified. Due to the extensive

modification, the ox-LDL particle is not recognized by the LDL receptor, but is taken

up avidly by the scavenger receptor pathway in macrophages leading to cholesterol

accumulation and foam cell formation51

. Ox-LDL augment the atherogenic process by

inhibiting endothelial nitric oxide synthase (eNOS), promoting vasoconstriction and

adhesion, stimulating cytokines such as IL-1 and increasing platelet aggregation52

. Ox-

LDL itself is cytotoxic and can induce apoptosis1. It is also immunogenic and is able to

inhibit macrophage motility so as to retain them in the arterial wall52

. In addition, ox-

LDL stimulates vascular smooth muscle cell proliferation1.

36

1.1.3.2. Reactive Oxygen Species and Their Sources

Different ROS can originate from the cellular and extracellular sources within

the vessel wall. Superoxide radical anion (O2•-), NO

•, hydrogen peroxide (H2O2),

hypochlorous acid (HOCl) and peroxynitrite (ONOO-) are some common ROS found in

the vasculature. Phagocyte NADPH oxidase (NOX) is thought to be a major source of

ROS in the circulatory system53

. During host defense, the activated phagocyte NOX

produces large amounts of O2•- over relatively short periods by using electrons from

NADPH to reduce molecular oxygen to O2•- (Equation 1). While O2

•- is essentially a

reducing agent, it can react with other biomolecules to form powerful secondary

oxidants or may dismutate to form the oxidizing H2O2 (Equation 2)1. Vascular

endothelial cells, smooth muscle cells and fibroblasts also express functional leukocyte-

like NOX and provide further evidence that NOX activity represents a major source of

ROS in the vasculature53

. NOX in phagocytes and vascular cells can be activated by

stimuli such as angiotensin II, thrombin, platelet-derived growth factor, TNF, IL-1,

while NOX in endothelial cells may be activated by shear stress and vascular

endothelial growth factor54

. Xanthine oxidase (XO), an enzyme found in plasma and

endothelial cells, but not in smooth muscle cells, represents another source of vascular

O2•- as it catalyses oxidation of hypoxanthine to xanthine and xanthine to uric acid

1.

Possible roles for NOX and XO in atherosclerosis were suggested in electron spin

resonance studies which showed significant activation of NOX and XO in the coronary

arteries of patients with coronary artery disease55

.

NADPH + 2O2 NADP- + H

+ + 2O2

•- …………………. Equation 1

2O2•- + 2H

+ H2O2 + O2 …………………. Equation 2

H2O2 + Cl- + H

+ HOCl + H2O …………………. Equation 3

37

Myeloperoxidase (MPO), a heme-containing enzyme found in neutrophils,

monocytes and macrophages, catalyses the conversion of H2O2 and chloride ion to

HOCl. (Equation 3)56

. As MPO is the only known human enzyme which produces

HOCl, chlorinated biomolecules are considered oto be oxidation products of reactions

catalysed by this enzyme1. The MPO/H2O2/Cl

- system can give rise to 3-chlorotyrosine

and chlorohydrins of cholesterol and fatty acids56

. The same system can also oxidize

nitrite to nitryl chloride (NO2Cl) and the NO2• radical, both of which promote the

nitration of tyrosine to 3-nitrotyrosine. Finally, the MPO/H2O2/Cl- system and HOCl

can generate a series of secondary oxidation products capable of oxidizing LDL and

thereby can lead to the conversion of macrophages to foam cells during the atherogenic

process56

. MPO has been shown to colocalise with macrophages in the human artery

wall and its characteristic oxidation products have been detected in atherosclerotic

lesions1. It has also been shown that MPO binds to apoA-I protein of high-density

lipoprotein (HDL) within human atherosclerotic lesions, which may explain the loss of

the atheroprotective functional properties of HDL in atherosclerotic plaque57

. MPO may

utilize vascular NOX-derived H2O2 to produce HOCl. The vascular NOX-MPO system

may represent a common pathogenic pathway in vascular disease and a new mechanism

involved in the exacerbation of vascular disease under inflammatory conditions58

. A

case-controlled study involving 158 patients with established coronary artery disease

(CAD) and 175 patients without CAD reported that leukocyte- and blood-MPO levels

were significantly higher in CAD patients than in controls59

, suggesting the association

of MPO levels with the risk of CAD. MPO-mediated endothelial dysfunction in humans

has been reported where serum MPO levels independently predicted endothelial

dysfunction60

. This may be an important mechanistic link between oxidative stress,

inflammation, endothelial dysfunction and atherosclerosis. Other sources of vascular

ROS include the mitochondria1 and eNOS, which will be discussed in a later sections.

38

1.1.4. ATHEROSCLEROSIS AND ENDOTHELIAL DYSFUNCTION

Endothelial dysfunction occurs when the endothelium fails to maintain

vascular homeostasis by failing to sustain the balance between vasodilation and

vasoconstriction, inhibition and stimulation of smooth muscle cells proliferation and

migration, and thrombogenesis and fibrinolysis61

. Endothelial dysfunction initiates a

number of events/ processes that promote or exacerbate atherosclerosis, including

increased endothelium permeability, platelet aggregation, leukocyte adhesion, and

generation of cytokines. Endothelial dysfunction is a critical event in the pathogenesis

of atherosclerosis and its clinical manifestations62

. It accelerates the development of

atherosclerosis and is considered an early marker for this disease, preceding

angiographic or ultrasonic evidence of atherosclerotic plaque62-64

. A number of studies

have shown a correlation between endothelial dysfunction and the presence of coronary

risk factors, such as hypercholesterolemia, hypertension, smoking and diabetes in

human subjects with no clinical evidence of coronary disease65, 66

.

1.1.4.1. Nitric Oxide and Endothelial Nitric Oxide Synthase

Nitric oxide is a major vasodilator released by the endothelium61

. It is formed

in endothelial cells from L-arginine via the enzymatic action of the cell membrane-

bounded eNOS. Under normal conditions, eNOS exists in the coupled state and the

coupled enzyme catalyses the oxidation of L-arginine to L-citrulline and NO•, with

tetrahydrobiopterin (BH4) acting as a cofactor in this process67

. The catalytic

mechanisms of eNOS involve flavin-mediated electron transfer from C-terminal-bound

NADPH to the N-terminal heme centre, where oxygen is reduced and incorporated into

the guanidine group of L -arginine, giving rise to NO• and L-citrulline

61 (Figure 1.7).

Nitric oxide mediates endothelium-dependent vasodilation by opposing the effects of

endothelium-derived vasocontrictors, such as angiotensin II and endothelin-1 (ET-1)61

.

It also inhibits pro-inflammatory and pro-atherosclerotic activities like platelet

39

adherence and aggregation, leukocyte adhesion and infiltration, and proliferation of

vascular smooth muscle cells68

as well as the oxidative modification of LDL69

.

Although the exact mechanisms are still not well defined, chronic treatment with L-

arginine inhibits atherosclerotic lesion formation70

and NOS inhibtors like L-NAME

significantly accelerate atherosclerotic lesion development71

in an animal model of

atherosclerosis. Thus, decreased production or activity of NO•, manifested as impaired

vasodilation, leads to endothelial dysfunction and may be one of the earliest signs of

atherosclerosis.

Oxidative stress can interfere with the production and activity of NO• by a

number of mechanisms which are dependent or independent of LDL. Pro-atherogenic

lipids, such as ox-LDL and lysophosphatidylcholine, inhibit signal transduction from

receptor activation to eNOS activation72

. Ox-LDL increases the synthesis of caveolin-1,

which inactivates eNOS and inhibits production of NO•73

. In vitro studies demonstrated

that eNOS can independently produce O2•- under certain conditions and this eNOS-

mediated O2•- generation is primarily regulated by BH4 availability

74. If the availability

of BH4 or L-arginine decreases, eNOS switches from the coupled state to an uncoupled

state and the uncoupled enzyme reduces molecular oxygen to O2•-. In “uncoupled

eNOS”, the electrons flowing from NADPH to the heme are diverted to molecular

oxygen rather than to L-arginine thereby producing O2•-61

. It is postulated that BH4

donates electrons to the ferrous-dioxygen complex during the formation of NO•, but its

precise role is not completely understood74

. It has been demonstrated that addition of

exogenous BH4 increases NO• production and decreases O2

•- production from

endothelial cells in vitro75

and acute administration of BH4 improves endothelial

dysfunction in chronic smokers76

. Nitric oxide also reacts rapidly with O2•- to generate

ONOO- (Equation 4) and ONOO

- formation is kinetically favoured over both NO

•

autoxidation and O2•- dismutation

1. Increased O2

•- production and subsequent production

40

of ONOO- not only alters endothelium-dependent vascular relaxation and causes

endothelial dysfunction, but the resultant ONOO- can also oxidise BH4

1. This creates a

deficiency in BH4 and the pathogenic uncoupling of eNOS. It is now widely recognized

that eNOS with normal function inhibits atherogenesis by producing the anti-

atherogenic NO• while dysfunctional eNOS is implicated in endothelial dysfunction and

atherosclerosis as a result of diminished NO• and elevated O2

•- production.

NO• + O2

•- ONOO

- …………………. Equation 4

NH2

NHH2N

OO

NH3H

+

+

-

Arginine

NADPH, 1/2O2

PIXP Fe(III)

1/2 NADPH, O2, 1/2 H+

PIXP Fe(III) O OH

NOH

NHH2N

OO

NH3H

+

PIXP Fe(III) O O

NOH

NHH2N

OO

NH3H

+

N

NHH2N

OO

NH3H

ON

NH

OO

NH3H

O

OPIXP Fe(III)

H2N

OH

O

NHH2N

OO

NH3H

N O

PIXP Fe(III) OH

+

Figure 1.7: The endothelial nitric oxide synthase reaction mechanism.

41

1.1.4.2. Endothelin-1

Another important factor in endothelial dysfunction is increased production

and biological activity of the potent vasocontrictor and pro-inflammatory 21-amino acid

peptide ET-1. ET-1 administration in healthy humans impairs endothelium-dependent

dilatation77

. Conversely, administration of endothelin receptor antagonists improves

endothelium-dependent, NO•-mediated relaxation and reduces atherosclerosis in

atherosclerotic patients78

. ET-1 is usually produced in small amounts in endothelial cells

under normal physiological conditions. Under atherogenic conditions, however,

production is stimulated in a large number of different cell types, including endothelial

cells, vascular smooth muscle cells and inflammatory cells such as leukocytes and

macrophages77

.

The biological effects of ET-1 are transduced by two receptor subtypes, ETA

and ETB77

. ETA receptors are mainly expressed by vascular smooth muscle cells and

they mediate potent vasocontriction. ETB receptors are expressed primarily on

endothelial cells, but may also be present on vascular smooth muscle cells. Stimulation

of ETB receptors on vascular smooth muscle cells results in vasocontriction whereas

endothelial ETB receptor stimulation results in NO• release and vasodilation

77. Thus, the

net effect of ET-1 is determined by the location and the balance of ETA and ETB

receptors. Increased expression of ETB receptors on inflammatory cells (leukocytes and

macrophages) and vascular smooth muscle cells in human atherosclerotic arteries has

been demonstrated79

. It was suggested that foam cell macrophages and T-lymphocytes

may modulate the switch from ETA to ETB receptors on vascular smooth muscle cells79

.

Increased expression of ETB receptors compared to ETA receptors has been

demonstrated in hypertensive patients80

.

There are several mechanisms by which ET-1 may influence endothelial

function and atherogenesis. ET-1 may reduce NO• bioavailability by decreasing eNOS

42

activity and downregulating the expression of eNOS in endothelial cells81

. It is also

closely associated with oxidative stress through the induction of ROS, which may result

in decreased NO• bioavailability due to ONOO

- formation. ET-1 increases O2

•-

production by stimulating NOX and by upregulating the expression of NOX in

endothelial cells82

. Apart from its direct vasomotor activity, ET-1 has also shown pro-

inflammatory properties. Sub-nanomolar concentrations of ET-1 stimulated the release

of pro-inflammatory and chemotactic mediators from macrophages, including TNF, IL-

1, IL-6 and IL-883

, resulting in accumulation of macrophages. ET-1 enhances the

expression of adhesion molecules on stimulated endothelial cells which promotes

leukocyte adhesion84

and MPO activity is increased due to stimulation of aggregation

of neutrophils 77

. An important interaction exists between ox-LDL and ET-1, which

may be of significance in atherogenesis. ET-1 augments the uptake of ox-LDL and ox-

LDL in turn stimulates the production of ET-185

. Overall, ET-1 plays an important role

in regulating vascular tone and thereby maintaining endothelial function and its over-

expression has been associated with endothelial dysfunction and several important

atherogenic processes.

43

1.2. DIETARY POLYPHENOLS AND THEIR CARDIOVASCULAR

PROTECTIVE EFFECTS

Polyphenols are natural phytochemicals and are currently the focus of much

nutritional and therapeutic interest. Results of population studies suggest that adopting

polyphenol-rich diets may protect against CVD86-88

. The role of fresh fruits and

vegetables as part of a heart-healthy diet is well recognized. However, the role of

dietary polyphenols and phenolic acids as part of such a diet or as supplements is

unclear. Mechanisms by which these compounds exert their cardiovascular protective

effects are not fully understood. It is widely hypothesized that dietary polyphenols

improve cardiovascular health and may help to prevent CVD by interacting with one or

more of the proposed disease progression mechanisms such as oxidative stress (lipid

and protein peroxidation), inflammation, endothelial dysfunction and platelet

activation89

.

1.2.1. BIOAVAILABILITY AND METABOLISM

Bioavailability is the degree to which a substance is absorbed or becomes

available at the targeted site in the body. The bioavailability of dietary polyphenols has

recently been reviewed in details 90

. Research in this area has increased over the last

decade with improved methods for analysis of specific polyphenols and their

metabolites. While intestinal absorption can be high for some polyphenols, plasma

concentrations of specific polyphenols rarely exceed 1 µM even after oral

supplementation (10 – 100 mg) of that compound 90

. Bioavailabilities of polyphenols

are determined by the processes of biotransformation and absorption, which are in turn

influenced by their molecular structures, but there is yet little understanding of the

relationship between them. Large individual variation in absorption between subjects is

observed. Although a significant amount of absorption occurs before bacteria

44

exposition, the composition of colonic microflora may still play a role in

biotransformation of polyphenols prior to intestinal absorption. A number of simple

phenolic acids have been identified as major metabolites resulting from C ring fission of

parent flavonoids by in vitro incubation of flavonoids with colonic microflora91

.

Phenolic acid absorption was studied using gas chromatography – mass spectroscopy

(GC-MS) following ingestion of red wine92

. Plasma concentrations of the major

phenolic acids, caffeic acid and gallic acid peaked at 2 hours, with all the gallic acid in

the form of 4-O-methylgallic acid (Figure 1.9). The same study also showed that the

antioxidant activity of methylated gallic acid was significantly less than the

unmethylated form. Methylation, sulfation and glucuronidation (i.e. conjugation with

methyl, sulfate or glucuronide groups) are three major biotransformations of

flavonoids93

. When the absorption and metabolism of catechin, a major flavonoid in red

wine, was studied using GC-MS94

, circulating catechin was found to be present almost

exclusively as 3′-O-methylcatechin and other conjugated forms (Figure 1.8). Plasma

total catechin concentration peaked at 0.09 µM, 1 hour after ingestion of 120 mL of red

wine containing 35 mg of catechin, regardless of whether the wine was dealcoholised or

not. Quercetin and (-)-epicatechin are major flavonoids present in our diet and their

metabolites (3′-O-methylquercetin93

, quercetin-3′-O-sulfate93

, quercetin-3-O-

glucuronide93

, 3′-O-methylquercetin 3-O-glucuronide93

, (-)-epicatechin-3′-O-

glucuronide94

, 3′-O-methyl-(-)-epicatechin95

and 4′-O-methyl-(-)-epicatechin-3′-O-

glucuronide96

) are identified in the circulation 1 – 2 hours after ingestion of the parent

compounds (Figure 1.8). Previous studies have also identified a number of simple

phenolic acids which are specifically increased in the circulation and excreted in urine

following polyphenol supplementation (Figure 1.9). These compounds include, but are

not limited to, 3-O-methyl gallic acid97

, 4-O-methyl gallic acid97

, 3-

hydroxyphenylacetic acid98

, 4-hydroxyphenylacetic acid98

, 4-hydroxy-3-

45

methoxyphenylacetic acid98

, 3-(3-hydroxyphenyl) propionic acid98

, 3-(4-

hydroxyphenyl) propionic acid98

, 3-(4-hydroxy-3-methoxyphenyl) propionic acid98

and

3,4-dihydroxyphenylacetic acid98

. Pure authentic standards of these compounds are

either commercially available or readily synthesized94, 98, 99

.

OHO

OH O

OH

OH

OH

OHO

OH O

OH

O

OH

CH3

OHO

OH O

OH

OH

O

O

COOHHO

HO

OH

Quercetin 3′-O-methyl-quercetin Quercetin-3-O-

glucuronide

OHO

OH O

OH

O

O

O

COOHHO

HO

OH

CH3

OHO

OH O

OH

O

OH

S OH

O

O

HO

OH

O

OH

OH

OH

3′-O-methylquercetin-3-

O-glucuronide

Quercetin-3′-O-sulfate (-)-Epicatechin

HO

OH

O

OH

OH

O CH3

HO

OH

O

OH

OH

O

O

COOH

OHHO

OH

HO

OH

O

OH

O

O

O

COOH

OHHO

OH

CH3

3′-O-methyl-(-)-

epicatechin

(-)-epicatechin-3′-

glucuronide

4′-O-methyl-(-)-

epicatechin-3′-O-

glucuronide

Figure 1.8: Structures of quercetin, (-)-epicatechin and their metabolites present in

human circulation.

46

OHO

HO COOH

CH3

OHO

HO COOH

H3C

HO OOH

3-O-methyl gallic acid 4-O-methyl gallic acid 3-hydroxyphenylacetic

acid

HO

OOH

HO

HO OOH

HO

OH3C OOH

4-hydroxyphenylacetic

acid

3,4-dihydroxyphenylacetic

acid

4-hydroxy-3-

methoxyphenylacetic acid

O

OH

HO

O

OHHO

O

OH

OH3C

HO

3-(3-hydroxyphenyl)

propionic acid

3-(4-hydroxyphenyl)

propionic acid

3-(4-hydroxy-3-

methoxyphenyl) propionic

acid

Figure 1.9: Structures of some simple phenolic acids present in human circulation.

1.2.2. DIETARY POLYPHENOLS AS IN VIVO ANTIOXIDANTS

The concept that lipid peroxidative damage may be a critical step in the

pathogenesis of atherosclerosis1 and the well recognized in vitro antioxidant activity of

many polyphenols, has led to the proposal that the mechanism for the apparent

beneficial properties of polyphenols may involve antioxidant effects100, 101

. Polyphenols

can potentially prevent free radical-related injury as they exhibit powerful antioxidant

activities in vitro, being able to scavenge a wide range of reactive oxygen, nitrogen and

chlorine species100, 102, 103

as well as being able to inhibit the production of such reactive

species104, 105

. Polyphenols can also chelate metal ions and often decrease metal ion

prooxidant activity106

. Ishige et al, 2001 proposed that polyphenols may protect against

47

oxidative stress by increasing intracellular glutathione, directly reducing reactive

oxygen species and preventing the influx of Ca2+107

. However, the studies carried out in

this area should be interpreted with caution as the native unmodified forms of

polyphenols found in the diet were utilised in in vitro experiments instead of the

metabolites found in vivo.

Recent evidence on the bioavailability and metabolism of these compounds in

vivo suggests that dietary polyphenols are less likely to act as antioxidants. Most

polyphenols may not have significant antioxidant activity in vivo for two reasons: 1)

bioavailability of dietary polyphenols is very poor (their concentration in vivo is likely

to be much lower than vitamin C or vitamin E, although they may act as co-

antioxidants)90

and 2) biotransformation may lead to diminished antioxidant activity92

.

Methylation, sulfation and glucuronidation block the radical scavenging phenolic

hydroxyl groups and may decrease antioxidant activity. Thus, the antioxidant activity of

the metabolites should be examined instead of the parent compounds. Studies

examining whether dietary polyphenols exert antioxidant effects in vivo have produced

confusing and contradictory data. O’Reilly et al (2001) reported that F2-isoprostanes

concentrations and plasma levels of oxidised LDL were the same whether the healthy

volunteers were on a flavonoid-rich diet or flavonoid-poor diet108

. A similar result was

observed when rutin supplementation had no effect on urinary concentrations of 8-

hydroxy-2′-deoxyguanosine, F2-isoprostanes or malondialdehyde in human

volunteers109

. While Thompson et al (1999) reported decreased lipid peroxidation

markers (F2-isoprostanes and malondialdehyde) in subjects who consumed more fruits

and vegetables110

, the consumption of fruits and vegetables failed to decrease markers

of oxidative damage, including F2-isoprostanes and DNA damage markers, in a chronic

intervention study111

. Dietary intervention studies involving green tea112, 113

, black tea114,

115 and red wine

92, 116, which represent rich sources of polyphenols in human diets,

48

produced similar contradictory results. These inconsistent data may result from the

choice of biomarkers of lipid and protein peroxidation used in these studies, as some do

not fulfil the criteria for ideal biomarkers117

. Much more work must be done in order to

draw conclusions on the antioxidant effects of dietary polyphenols.

1.2.3. DIETARY POLYPHENOLS AND LIPEMIA

Hypercholesterolemia is well established as a risk factor in atherosclerosis and

dietary polyphenols are thought to protect against the disease by exerting

hypocholesterolemic effects. However, clinical studies do not conclusively support this

hypothesis. Clinical studies using both normo- and hypercholesterolemic subjects taking

different polyphenol sources for 1 – 13 weeks showed improvement or no change in

lipid profiles. Consumption of black tea (5 servings/ day) by hypercholesterolemic

subjects over a period of 3 weeks resulted in significant reductions in total cholesterol,

LDL cholesterol and apolipoprotein B levels118

. HDL cholesterol levels in healthy

volunteers were elevated, but LDL/HDL ratio remained unaffected after 4 weeks of

intervention with cocoa powder and dark chocolate119

. Two independent studies on

healthy subjects and smokers showed no change in the levels of triglyceride, LDL, HDL

and total cholesterol after red wine intervention120, 121

. Lyophilised grape powder

significantly decreased triglyceride and LDL cholesterol concentrations in both pre- and

postmenopausal women122

.

Mechanisms by which dietary polyphenols may influence plasma lipids are yet

to be defined. Polyphenols may reduce cholesterol absorption via interaction with

cholesterol carriers and transporters across the brush border membrane123

. This

reduction in cholesterol absorption decreases the delivery of cholesterol to the liver,

which in turn upregulates the expression of the LDL receptor to compensate for less

substrate availability and induces reductions in plasma cholesterol124

. Dietary

polyphenols were also shown to affect hepatic production of lipoproteins and inhibit

49

cholesterol esterification through their binding with the plasma membrane transporter P-

glycoprotein125

. Reduction in plasma triglyceride by polyphenols may occur as a result

of lower microsomal transfer protein activity and increased lipoprotein lipase activity,

which may further alter the delipidation cascade, yielding less LDL in circulation124

.

1.2.4. DIETARY POLYPHENOLS AND INFLAMMATION

As inflammation is now recognised as a key process in atherogenesis7, the

potential for dietary polyphenols to inhibit inflammatory activities is of particular

interest. A potential anti-inflammatory feature of polyphenols is the ability to inhibit the

biosynthesis of eicosanoids. Selected phenolic acids and some flavonoids have been

shown to inhibit both cyclooxygenase and 5-LO pathways89, 126

. (-)-Epicatechin and

related flavonoids have been shown to inhibit the synthesis of pro-inflammatory

cytokines in vitro127

and plasma metabolites of catechin and quercetin inhibit the

adhesion of monocytes to cultured endothelial cells128

. Silymarin has been shown to

inhibit the production of inflammatory cytokines, such as IL-1, IFN-γ, and TNF, from

macrophages and T-cells129

. Some flavonoids can inhibit neutrophil degranulation,

diminishing the release of free AA130

. These activities may be important because the

COX-2 and leukotriene pathways may have a role in atherosclerosis131

.

Even though in vitro studies have provided extensive evidence for anti-

inflammatory effects of various dietary polyphenols, human studies have provided only

a few supporting results. Polyphenols may exert anti-inflammatory effects through

modulation of immune processes involving cytokines, inflammatory mediators as well

as circulating adhesion molecules. TNF-induced adhesion of monocytes to endothelial

cells was virtually abolished after red wine consumption in human volunteers and was

only partially reduced after gin consumption132

. Similar observations were made in an

11-week study comparing red wine and gin consumption in healthy men where

adhesion molecules and monocyte adhesion to endothelial cells were significantly

50

altered due to red wine133

. Lyophilized grape powder treatment was shown to

significantly decrease TNF and IL-6 concentrations in both pre- and postmenopausal

women122

. Circulating soluble P-selectin in healthy human volunteers was significantly

reduced after black tea consumption134

. However, supplementation with cocoa did not

affect circulating concentrations of cytokines135

, nor urinary thromboxane B2 or 6-keto-

prostaglandin F1α119

in healthy human volunteers. Circulating VCAM-1 concentrations

were significantly reduced after 6 weeks administration of formononetin-enriched

isoflavones136

. This effect was totally absent in another human intervention study

involving 6 weeks intake of soy isoflavones137

. As the transcription factor, nuclear

factor-κB (NF-κB) is responsible for activating cytokines, adhesion molecules and other

pro-inflammatory mediators, polyphenols may act by inhibiting NF-κB138

. The limited

anti-inflammatory effects of polyphenols in these human studies may be due to the lack

of inflammatory immune responses in healthy subjects.

1.2.5. DIETARY POLYPHENOLS AND ENDOTHELIAL FUNCTIONS

The endothelium regulates vascular tone by balancing the production of

vasodilators, most importantly NO•139

and vasoconstrictors, such as ET-1140

. It

maintains vascular homeostasis through multiple complex interactions with cells in the

vessel wall. Therefore, endothelial function may serve as an indication for

cardiovascular health and may be used for evaluation of new therapeutic strategies61

.

Improving endothelial function is believed to be one possible mechanism by which

polyphenols may reduce cardiovascular risk141

.

The dysfunction of vascular endothelial cells can be measured by flow-

mediated dilatation (FMD) of the brachial artery. The majority of clinical studies

involving polyphenol-rich food have shown a net beneficial effect on FMD.

Dealcoholised red wine and purple grape juice were reported to significantly improve

FMD in healthy volunteers and patients with coronary heart disease142, 143

. Acute or

51

long-term black tea intake increased FMD in patients with coronary heart disease to

values comparable to healthy volunteers144

and similar effects were observed in healthy

subjects145

. (-)-Epicatechin present in cocoa has also been shown to improve endothelial

function in healthy volunteers146

. Dietary polyphenols in these foods are thought to be

the bioactive constituents that improve endothelial function, but more clinical studies

involving pure polyphenol compounds should be conducted to determine their vascular

effects.

Dietary polyphenols may operate by increasing the bioavailability of the

vasodilating NO•. Grape juice and red wine polyphenols have been reported to induce

endothelium-dependent vasorelaxation in rat aorta, which was inhibited by NOS

inhibitors147

. Due to their antioxidant properties, polyphenols may scavenge O2•- and

therefore protect NO• from O2

•--driven inactivation. They may also prevent the

oxidation of BH4 and the subsequent eNOS uncoupling. On the other hand, polyphenols

may scavenge NO•, although the rate of NO

• scavenging by polyphenols is much lower

than that of the physiological NO• scavenger haemoglobin

148. Red wine polyphenols

were shown to enhance eNOS expression and subsequent NO• release from endothelial

cells149

. ET-1 may also be a target for polyphenols. Quercetin (0.5 – 50 µM) was

reported to inhibit ET-1 release in cultured human umbilical vein endothelial cells150

.

No datum is yet available on the in vivo effect of polyphenols on ET-1 production.

1.2.6. DIETARY POLYPHENOLS AND PLATELET REACTIVITY

Platelet aggregation plays a critical role in the pathogenesis of acute coronary

syndromes with increasing evidence that antiplatelet therapy reduces CVD risk151

.

Dietary polyphenol reduction of platelet activity may provide one important

mechanistic explanation for the available epidemiologic data regarding polyphenols and

CVD.

52

Demrow et al (1995) used the Folts model of unstable coronary stenosis,

which closely mimics ruptured atherosclerotic plaque causing unstable angina, to

examine the effects of grape juice on platelet function in vivo152

. In this model, transient

platelet aggregation and release are reflected in cyclic variations in coronary blood flow.

Acute intragastric administration of red wine or grape juice was associated with marked

reductions in cyclic flow variations, which was indicative of an anti-platelet effect. Tea

consumption reduced plasma concentrations of P-selectin (a marker of in vivo platelet

aggregation)134

, but another study which examined the effects of short-term and long-

term tea consumption on ex vivo platelet aggregation in patients with coronary artery

disease did not demonstrate any effect on platelet function144

. More studies are required

to define the effects of tea consumption on platelet function. Cocoa decreased

epinephrine-stimulated or adenosine diphosphate-stimulated glycoprotein IIb/ IIIa and

P-selectin expression in human subjects 2 and 6 hours after consumption153

. Platelet-

related primary homeostasis, measured using a platelet function analyser, was inhibitied

6 hours after polyphenol-rich cocoa ingestion153

. Quercetin has been implicated as a

dietary inhibitor of platelet cell signalling in a human acute intervention study154

.

Platelet aggregation was inhibited 30 and 120 minutes after ingestion of 150 mg and

300 mg of quercetin-4′-O-β-glucoside, accompanied by reduced tyrosine

phosphorylation of the tyrosine kinase Syk and phospholipase Cγ2 components of the

platelet glycoprotein VI collagen receptor signalling pathway154

.

The exact mechanisms by which polyphenols inhibit platelet activity are not

yet fully understood, but it is possible that polyphenols effect changes in membrane

fluidity, ligand-receptor affinity and intracellular signalling pathways. Polyphenols may

mediate their effects through antioxidant and NO•-related pathways. Catechin and

quercetin were shown to inhibit platelet function in vitro by inhibiting protein kinase C-

dependent NOX activation155

. Catechin was also shown to attenuate oxidant induced

53

platelet activation in vivo156

. The addition of grape juice to platelets ex vivo reduced

platelet aggregation, decreased platelet production of O2•- and increased platelet

production of NO•157

. Polyphenols, or polyphenol-rich foods were shown to inhibit

platelet 12-lipoxygenase and 5-LO in vitro158

. Polyphenols can also modulate

membrane fluidity by interacting with the lipid bilayer159

, which in turn can result in

changes in membrane receptor function and enzymatic activity160

. Lipid rafts are

described as important membrane components involved in cell signalling and platelet

activation 161

. Recently, epigallocatechin-3-O-gallate was found to associate strongly

with the cholesterol component of the lipid raft in human basophilic KU812 cells162

,

suggesting that certain polyphenols may modulate cellular activation through interaction

with lipid rafts.

1.3. HYPOTHESES AND AIMS

If dietary polyphenols are cardiovascular protective nutrients, their bioactivity

must reside in their metabolites rather than in the native forms present in our food and

beverages, since metabolites are the form found in the circulation. These metabolites

may be present in the circulation at sufficient concentrations to reduce oxidative stress,

inflammation, endothelial dysfunction and platelet activation, which may ultimately

influence the disease process. A major weakness of many earlier in vitro studies on the

bioactivity of polyphenols was that the compounds were studied in the native form

found in the diet rather than the metabolised forms found in the circulation. To

understand the mechanism of action of dietary polyphenols either as antioxidants or as

modulators of cell signalling and inflammatory pathways, it is important to identify

their metabolites in vivo as well as to study the consequences of interaction of these

circulating metabolites with cells.

54

The effects of polyphenols on biomarkers of oxidative stress, lipemia and

inflammation are inconclusive, while more consistent effects have been observed on

endothelial function and platelet activity. Previous clinical studies using foods or

beverages containing a mixture of different polyphenols make it difficult to determine

the exact nature of the active polyphenols. The lack of effect of polyphenols in some

studies may be explained by low bioavailability or rapid elimination after absorption.

Until the activity of pure polyphenols in humans is determined and their targets of

action are identified, it remains difficult to predict their protection against CVD. Animal

and human studies need to be conducted to identify the in vivo mechanism of action of

pure dietary polyphenols. Results from these studies are crucial in the evaluation of the

potential of dietary polyphenols as cardiovascular protective agents and may influence

specific advice regarding intake of foods and beverages rich in polyphenols and

attempts to enhance polyphenol content of foods.

The studies described in this thesis were designed to test the following hypotheses:

Metabolic transformation has a profound effect on the bioactivity of dietary

polyphenols in vitro;

Pure flavonoids, such as quercetin, (-)-epicatechin and epigallocatechin gallate

improve markers of human endothelial function;

Dietary polyphenols protect against atherosclerosis in the apoE knockout mouse

through inhibition of pro-inflammatory pathways, endothelial dysfunction and

oxidative stress.

These hypotheses were examined by the following aims:

To examine the effects of specific dietary flavonoids, such as quercetin and its in-

vivo metabolites on inflammatory eicosanoid pathways and MPO activity in human

monocytes and neutrophils;

55

To examine the effect of pure dietary flavonoids such as quercetin, (-)-epicatechin

and epigallocatechin gallate on NO• and ET-1 release, as well as oxidative stress

biomarker (F2-isoprostanes) in human volunteers;

To examine the effect of pure dietary polyphenols on lesion formation in the apoE-/-

mouse and to determine effects on markers of oxidative stress (F2-isoprostanes),

NO• production, proinflammatory chemokines and adhesion molecules.

56

CHAPTER 2:

IN VITRO EFFECT OF QUERCETIN AND

METABOLITES ON EICOSANOID BIOSYNTHESIS

(The results from this chapter have been published in Biochem. Pharmacol.

2008;75:1045-1053.)

2.1. INTRODUCTION

There is considerable research interest in the potential health benefits of

flavonoids. Results of population studies suggest that dietary flavonoids provide

protection against CVD163-165

. There is also a growing body of evidence from controlled

trials that dietary flavonoids can improve endothelial and platelet function and reduce

blood pressure in humans166

, and that they may inhibit the development of

atherosclerosis in animal models167

. Since oxidative stress has been implicated in

atherosclerosis and CVD, one of the main properties of flavonoids thought to explain

their effect is the antioxidant activity of this group of polyphenols168

. However, there is

some doubt as to whether dietary flavonoids can act as antioxidants in vivo and the

results of intervention studies have yielded conflicting results169, 170

. These results may

be due to several factors including variations in the absorption and metabolism of

flavonoids which may alter antioxidant activity as well as other biological activities171

.

In particular there is doubt about the interpretation of in vitro studies of antioxidant

activity where issues of bioavailability and metabolic transformation have not been

considered172

.

Inflammation and leukocyte recruitment are play key roles in atherogenesis7.

Inflammatory processes in the vascular wall may be mediated by a range of factors,

such as cytokines, eicosanoids (such as LTB4), ROS (generated by NOX173

and MPO

activities174

) and NO•, which in turn modulate cellular signaling, cell growth and

57

differentiation and a variety of other cellular processes. Arterial leukocyte recruitment is

an important initiating step in atherogenesis7. Leukocyte-endothelial interactions and

leukocyte migration to the sub-endothelium occur in response to cytokines and

chemokines such as MCP-1. There is evidence that potent chemotactic molecules such

as MCP-1 and LTB4 are involved in inflammatory diseases such as rheumatoid arthritis7

and atherosclerosis175

. Stimulated neutrophil LTB4 synthesis has recently been

suggested as a useful marker for assessing the leukotriene pathway in humans131

.

Human atherosclerotic lesions produce LTB4 and the enzymes responsible for its

production (5-LO and LTA4 hydrolase) are associated with symptoms of plaque

instability176

.

We were particularly interested in examining the effects of dietary flavonoids

on the production of proinflammatory eicosanoids such as LTB4 and PGE2 by human

leukocytes. Quercetin is a common dietary flavonoid which has been shown to inhibit

proinflammatory cytokines in mononuclear cells177

and block airway epithelial

chemokine expression178

. A recent study has demonstrated that quercetin and related

flavonoids can attenuate TNF stimulated adhesion molecule expression in human aortic

endothelial cells, however, exposure to cultured hepatocytes (mimicking first pass

metabolism) greatly diminished this activity169

.

To address the issue of the effect of metabolic transformation on flavonoid

bioactivity we have compared the ability of quercetin and its major human metabolites

to inhibit inflammatory eicosanoid (LTB4 and PGE2) and MCP-1 production from

human leukocytes. We have examined quercetin, structural analogues of quercetin, and

a series of quercetin phase-2 conjugates of known structure to determine structural

features important for antioxidant and anti-inflammatory activity (see Fig. 1.7 for

structures and Fig. 2.1 for flow diagram of experiments). We found that structural

modification of quercetin due to metabolic transformation had a profound effect on

58

bioactivity. The structural features required for antioxidant activity of quercetin and

related flavonoids were unrelated to that required for inhibition of inflammatory

eicosanoids.

Figure 2.1: Flow-diagram of the experiments.

Anti-inflammatory

activity

Antioxidant

activity

Monocytes Neutrophils

LTB4 Lipid

hydroperoxides

LTB4

LTA4 hydrolase

activity

F2-isoprostanes PGE2

Inhibition of

AAPH-induced

LDL oxidation

Quercetin and metabolites

59

2.2. MATERIALS AND METHODS

2.2.1. Chemicals and reagents

Bovine serum albumin (BSA), calcium chloride, calcium ionophore A23187,

Hepes, lipopolysaccharide (LPS), MK886, quercetin, sodium phosphate dibasic, sodium

bromide, sodium chloride, sodium hydrogencarbonate, trifluoroacetic acid, luteolin,

kaempferol, taxifolin, xylenol orange, ammonium ferrous sulphate, butylated

hydroxytoluene (BHT), hydrogen peroxide (50% by volume) and 2,2′-Azobis(2-

methylpropionamidine) dihydrochloride (AAPH) were purchased from Sigma Aldrich

(St Louis, MO, USA); acetonitrile, magnesium sulphate and sulfuric acid from Univar

(WA, Australia); ficoll-paque from GE Healthcare (Uppsala, Sweden); phosphate

buffered saline (PBS), heat-inactivated fetal calf serum (HIFCS) and RPMI 1640 from

Gibco™ Invitrogen (Calsbad, CA, USA); dextran 500 from Amersham Biosciences

(Uppsala, Sweden); glucose and potassium phosphate monobasic from Merck (VIC,

Australia); methanol and ethanol from Mallinckrodt (NJ, USA); and prostaglandin B2

(PGB2) and LTA4 methyl ester from Cayman Chemical (Michigan, USA). 3′-O-

methylquercetin was purchased from Advanced Technology & Industrial Co., Ltd,

Hong Kong , while quercetin-3′-O-sulfate, quercetin-3-O-glucuronide and 3′-O-

methylquercetin-3-O-glucuronide were synthesized as previously described99

.

2.2.2. Isolation of peripheral monocytes and neutrophils

Peripheral blood mononuclear cells (PBMC) were isolated from human whole

blood (containing 1 mg/mL EDTA) by centrifugation on Ficoll-Paque at 500xg for 30

minutes at 20 ºC. The collected PBMC layer was further purified by washing with

MACS buffer (0.5% bovine serum albumin, 2 mM EDTA in PBS; pH 7.2) and

centrifuging at 100xg for 10 minutes at 4 ºC to remove platelets. MACS®

human CD14

Micro-beads (Miltenyl Biotec, CA, USA) (20L / 107 cells final concentration) were

60

incubated with PBMC for 15 min at 4 ºC. The resulting mixture was passed through

MACS® separation column (Miltenyl Biotec, CA, USA), which separated peripheral

blood monocytes from other mononuclear cells. The neutrophils were isolated from the

neutrophil/erythrocyte pellet from the Ficoll-Paque gradient by dextran sedimentation of

red cells as previously described. Cell viability was assessed using trypan blue

exclusion and was typically >98%.

2.2.3. Stimulation and measurement of LTB4 production

The effects of quercetin and its metabolites on the 5-LO pathway were

examined using freshly isolated human peripheral monocytes and neutrophils. The

freshly isolated monocytes and neutrophils were resuspended in HBHS [CaCl2.2H2O

(0.09 g), glucose (0.50g), Hepes (0.06 g), KCl (0.20 g), KH2PO4 (0.03 g), MgSO4.7H2O

(0.10 g), NaHCO3 (0.18 g), NaCl (4.00 g), Na2HPO4 (0.02 g) and BSA (0.50 g) in pure

water (500 mL); pH 7.4] at a concentration of 5 x 106 cells/ mL. The cell suspension (1

mL) was incubated with either quercetin, 3′-O-methylquercetin, quercetin-3′-O-sulfate,

quercetin-3-O-glucuronide, 3′-O-methylquercetin-3-O-glucuronide, luteolin, kaempferol

or taxifolin (2-10 µM final concentration) at 37 ºC for 5 minutes prior to 5-LO

stimulation. Quercetin and 3′-O-methylquercetin were added using ethanol as vehicle,

while the other metabolites used water as their vehicles. The cells were stimulated with

calcium ionophore A23187 (2.5 µg/mL final concentration) at 37 ºC for 15 minutes.

The supernatant from the cell suspension was collected and stored at -80 ºC before

LTB4 extraction and analysis. Untreated cells with ethanol and water vehicles were used

as positive controls, while untreated cells incubated with the leukotriene biosynthesis

inhibitor MK886 (300 nM) served as negative controls179

. In another set of experiments

designed to examine specific inhibition of LTA4 hydrolase, cells incubated with LTA4

(final concentration 15 µM) only or LTA4 (final concentration 15 µM) and quercetin

(final concentration 10 µM) were stimulated with calcium ionophore as above. The

61

release of LTB4 from stimulated neutrophils was measured by HPLC. Briefly, the

released eicosanoids were extracted from cell supernatant (after acidification with

formic acid, and addition of a PGB2 internal standard) with ethyl acetate, dried under

nitrogen and re-suspended in mobile phase [methanol : acetonitrile : water (1:1:2)].

Components were separated by reverse phase chromatography on a C18 column

(Agilent Technologies LiChrospher 100 RP-18, 5 micron) using methanol, acetonitrile,

water, trifluoroacetic acid (40:40:80:0.1 v/v; pH 3) mobile phase (solvent A) with

increasing gradient of methanol:acetonitrile (1:1) (solvent B) at a flow rate of 1 mL /

min over 30 minutes (50% A and 50% B) using a Hewlett Packard Series 1050 HPLC.

Wavelength detection at 270 nm was used to detect conjugated trienes. Peak area was

determined using Agilent Technologies Chemstation software package. The production

of LTB4 from the monocytes was measured using a specific LTB4 enzyme

immunoassay kit (Cayman Chemical).

2.2.4. Stimulation and measurement of PGE2 and MCP-1 productions

Freshly isolated peripheral blood monocytes were used to evaluate the effects

of quercetin and its metabolites on the COX-2 pathway and MCP-1 production. The

cells were resuspended in 10 % HIFCS in RPMI (3 x 106 cells/ mL final concentration).

The cell suspension was then incubated with quercetin or its metabolites (2-10 µM final

concentrations) at 37 ºC for 5 minutes. COX-2 stimulation was carried out by

incubating cells with LPS (1 µg/mL final concentration) for 20 hours at 37 ºC. Positive

controls (LPS treated without polyphenol treatment) and negative controls (without both

polyphenols and LPS treatment) were also studied. Cell supernatants were collected at

the end of the incubation. The production of PGE2 was measured by specific PGE2

enzyme immunoassay kit (Cayman Chemical), while MCP-1 production was measured

by using BD OptEIA™ Human MCP-1 ELISA Set.

62

2.2.5. Measurement of cellular quercetin and its metabolites

Freshly isolated neutrophils suspended in HBHS were incubated with

quercetin or its metabolites at concentrations ranging from 0 to 10 µM at 37 ºC as

described above, for a period of 20 minutes. The cell pellet was obtained after

centrifugation at 2000xg for 5 minutes at 4 ºC, and washed once with HBBS. The cells

were then lysed in buffer (30 mM NaH2PO4, adjusted to pH 3.0 with H3PO4) by

sonication. The supernatant was collected and stored at – 80 ºC prior to HPLC analysis.

An HPLC assay has previously been described to simultaneously measure the cellular

amount of quercetin and its metabolites180, 181

. Components were separated by reverse

phase chromatography using a LiChrospher 100 RP-18, 5 micron column (Agilent

Technologies) with sodium orthophosphate (30 mM; adjusted to pH 3 with phosphoric

acid) : acetonitrile (15:85 v/v) mobile phase (solvent A) with increasing gradient of

acetonitrile (solvent B) at a flow rate of 0.8 mL/ min over 20 minutes (50% A and 50%

B). Dual wavelength detection at 370 and 270 nm was used to detect the B ring and C

ring contained within the 2-phenyl-γ-benzopyrone structure of quercetin respectively.

2.2.6. Measurement of inhibition of lipoprotein oxidation

LDL was isolated from blood plasma by density gradient ultracentrifugation as

previously described182

. The anti-oxidant activity of each compound (quercetin, 3′-O-

methylquercetin, quercetin-3′-O-sulfate, quercetin-3-O-glucuronide, 3′-O-

methylquercetin-3-O-glucuronide, luteolin, kaempferol and taxifolin) to inhibit AAPH-

induced LDL oxidation was analysed by measuring the formation of lipid

hydroperoxides using the FOX assay183

, or F2-isoprostanes measured by GC-MS184

.

Briefly, the test compound (final concentration 10 µM) was added to LDL (final protein

concentration 0.1 mg/ mL) and AAPH (final concentration 5 mM) at 37 ºC. Aliquots of

the mixture were analysed for lipid peroxidation products at specific time points up to 3

hours and compared to control incubations without the addition of test compounds.

63

2.2.7. Statistical analysis of results

Statistical analysis of results (n = 3 or 5 independent experiments,) was

performed using SPSS version 11.5. One-way ANOVA185

and Bonferroni post hoc

analyses were performed on specific concentration points as well as the areas under the

curves186

in concentration-response results. The results analysed were considered

significantly different if p value ≤ 0.05 based on 95% confidence.

2.3. RESULTS

2.3.1. Effects of quercetin and its metabolites on LTB4 production

The LTB4 inhibiting actions of quercetin and its major circulating metabolites

(Fig. 1.7) in human neutrophils and monocytes are presented in Figures 2.2 and 2.3

respectively. Inhibitory activity is expressed as the percentage reduction in LTB4

production compared to the untreated positive control (producing 7.0 ng/106 cells).

None of the negative controls (MK 886 treated) showed measurable LTB4. Quercetin

exhibited a dose-dependent inhibitory effect on LTB4 production with an IC50 value of 2

µM, while its metabolites showed reduced inhibitory activity at this concentration. At

this low concentration (2 µM) only quercetin and 3′-O-methylquercetin showed any

LTB4 inhibitory activity in peripheral neutrophils (Fig. 2.2). Over the concentration

range tested, all quercetin metabolites showed less activity than the parent compound

(quercetin). Among the metabolites, 3′-O-methylquercetin and quercetin-3′-O-sulfate

exhibited significant dose response effects while quercetin-3-O-glucuronide and 3′-O-

methylquercetin-3-O-glucuronide showed virtually no activity up to a concentration

of10 μM. In peripheral monocytes, quercetin, 3′-O-methylquercetin and quercetin-3′-O-

sulfate all inhibited LTB4 production at 2 µM (approx 50% inhibition compared to

controls, on average producing 2.2 ng/106 cells) while quercetin-3-O-glucuronide and3′-

O-methylquercetin-3-O-glucuronide showed minimal activity (Fig. 2.3). Quercetin and

64

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 (

%)

Figure 2.2: Leukotriene B4 inhibition in peripheral neutrophils by quercetin (Q), 3′-O-

methylquercetin (MQ), quercetin-3′-O-sulfate (QS), quercetin-3-O-glucuronide (QG)

and 3′-O-methylquercetin-3-O-glucuronide (MQG) at concentrations up to 10 µM (n =

5). * p < 0.05 vs all quercetin metabolites using comparison of area under the curve

(AUC) (ANOVA). # p < 0.05 vs QG and MQG using AUC (ANOVA). + p < 0.05 for

Q compared to QS, QG and MQG at 2 μM (ANOVA). ++ p < 0.05 for MQ compared to

QS, QG and MQG at 2 μM (ANOVA).

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 (

%)

Figure 2.3: Leukotriene B4 inhibition in peripheral monocytes by quercetin (Q), 3′-O-


and 3′-O-methylquercetin-3-O-glucuronide (MQG) at concentrations up to 10 µM (n =

3). * p < 0.001 for Q and MQ compared to QS, QG and MQG using AUC (ANOVA). #

p < 0.001 for QS compared to Q, MQ, QG and MQG using AUC (ANOVA). + p < 0.05

for Q, MQ and QS compared to QG and MQG at 2 μM (ANOVA).

65

3′-O-methylquercetin were significantly more effective in reducing LTB4 production in

monocytes (p < 0.001) compared to quercetin-3′-O-sulfate, quercetin-3-O-glucuronide

and 3′-O-methylquercetin-3-O-glucuronide (Fig. 2.3). In neutrophils the LTB4

inhibiting activity observed follows the same declining order: quercetin > 3′-O-

methylquercetin > quercetin-3′-O-sulfate >> quercetin-3-O-glucuronide > 3′-O-

methylquercetin-3-O-glucuronide. LTB4 inhibiting activity was similar in monocytes,

except that quercetin and 3′-O-methylquercetin were equipotent in monocytes.

2.3.2. Effects of quercetin and its metabolites on PGE2 and MCP-1 production

The effects of quercetin and its metabolites on PGE2 and MCP-1 production were

examined in LPS treated peripheral monocytes. Without LPS stimulation (negative

controls) no PGE2 or MCP-1 was measurable. Quercetin and 3′-O-methylquercetin

exhibited similar dose-dependent reduction of PGE2 production in peripheral monocytes

(IC50 = 4 µM) compared to the untreated positive controls (producing 3.2 ng/ 106 cells),

while quercetin-3′-O-sulfate, quercetin-3-O-glucuronide and 3′-O-methylquercetin-3-O-

glucuronide showed minimal activity (Fig. 2.4). At 2 µM, both quercetin and 3′-O-

methylquercetin showed significantly greater PGE2 inhibition (25%) than the other

three metabolites (p < 0.001). While quercetin reduced MCP-1 production by stimulated

monocytes in a dose-dependent manner (IC50 = 5.5 μM), its metabolites (3′-O-

methylquercetin, quercetin-3′-O-sulfate, quercetin-3-O-glucuronide and 3′-O-

methylquercetin-3-O-glucuronide) had no effect on MCP-1 production up to 10 μM

treatment concentration (Fig. 2.5).

66

0 1 2 3 4 5 6 7 8 9 100

25

50

75

100

Q

MQ

QGMQG

#

QS*

[Polyphenol] (M)

PG

E2 I

nhib

itio

n (

%)

Figure 2.4: Prostaglandin E2 inhibition in LPS stimulated peripheral monocytes by

quercetin (Q), 3′-O-methylquercetin (MQ), quercetin-3′-O-sulfate (QS), quercetin-3-O-

glucuronide (QG) and 3′-O-methylquercetin-3-O-glucuronide (MQG) at concentrations

up to 10 µM (n = 3). * p < 0.001 for Q and MQ compared to QS, QG and MQG at 2 µM

(ANOVA). # p < 0.001 for Q and MQ compared to QS, QG and MQG using AUC

(ANOVA).

0.0 2.5 5.0 7.5 10.00

25

50

75

100

Q

MQ

QS

QG

MQG

*

#

[Polyphenols] (M)

MC

P-1

Inhib

itio

n (

%)

Figure 2.5: Monoctye chemoattractant protein-1 inhibition in LPS stimulated peripheral

monocytes by quercetin (Q), 3′-O-methylquercetin (MQ), quercetin-3′-O-sulfate (QS),

quercetin-3-O-glucuronide (QG) and 3′-O-methylquercetin-3-O-glucuronide (MQG) at

concentrations up to 10 µM (n = 3). * p < 0.001 for Q compared to MQ, QS, QG and

MQG at 2 µM (ANOVA). # p < 0.001 for Q compared to MQ, QS, QG and MQG using

AUC (ANOVA).

67

2.3.3. Effects of luteolin, kaempferol and taxifolin on LTB4 production

The LTB4 inhibiting activity of quercetin was compared with luteolin, kaempferol and

taxifolin (see structures in Fig. 2.11). These compounds bear close structural

resemblance to quercetin but have specific OH functional group substitutions that

enable a structure activity relationship (SAR) to be made. Luteolin showed similar

LTB4 inhibiting activity as quercetin, while kaempferol exhibited a much reduced

activity level compared to quercetin (p < 0.001, Fig. 2.6). Taxifolin showed minimal

activity compared to all the compounds tested (p < 0.001). At 2 µM concentration, there

were no significant differences in activity between quercetin and luteolin and between

kaempferol and taxifolin, but significant differences were observed between these two

sets of flavonoids (p < 0.001).

0.0 2.5 5.0 7.5 10.00

25

50

75

100

L

K

Q

T

*

#

+

[Polyphenol] (M)

LT

B4 In

hib

itio

n (

%)

Figure 2.6: Leukotriene B4 inhibition in peripheral neutrophils by quercetin (Q),

luteolin (L), kaempferol (K) and taxifolin (T) at concentrations up to 10 µM (n = 5). * p

< 0.001 for Q and L compared to K and T using AUC (ANOVA). # p < 0.001 T

compared to Q, L and K by AUC. + p < 0.001 for Q and L compared to K and T at 2 µM

(ANOVA).

68

2.3.4. Effects of quercetin on LTA4 hydrolase

To examine the effect of quercetin on LTA4 hydrolase we measured the amount of

LTB4 produced by peripheral neutrophils in the presence or absence of added LTA4 (15

µM) with or without quercetin treatment (Fig. 2.7). Peripheral neutrophils produced

significantly higher amounts of LTB4 after LTA4 supplementation and this was not

affected by the presence or absence of quercetin (p < 0.001) compared to the control

(without LTA4 and quercetin). This result shows that quercetin does not exert its

inhibitory effect on the enzymatic hydrolysis of LTA4 to LTB4.

2.3.5. Cellular uptake of quercetin and its metabolites

Since many flavonoids do adsorb on the cell surface, it is very difficult to know how

much is inside and how much is adsorbed on the outside surface of the cells. The

0

20

40

60*

*

LT

B4 (

ng/

10

6ce

lls)

quercetin - + - +

LTA4 - - + +

Figure 2.7: The effect of quercetin (10 µM) on Leukotriene A4 hydrolase activity in

peripheral neutrophils (n = 3). LTA4 (15 µM) was added to calcium ionophore A23187

stimulated cells in the presence or absence of quercetin (filled bars) and compared to

controls (without added LTA4 ± quercetin, open bars). * p < 0.001 compared to control

(ANOVA).

69

cellular amounts of quercetin and its metabolites, which represent the total amounts

present inside and adsorbed on the outer cell surfaces, are expressed as their

concentrations based on the published average cell volume of human peripheral

neutrophils (~330 fL/ cell)187

. Neutrophils were able to accumulate quercetin and its

metabolites within their cellular matrices up to a concentration of 25 µM when they

were incubated with a 2 µM concentration and there was no significant difference in

cellular uptake between any of the compounds. However, when the treatment

concentrations were increased to 10 µM, significantly lower uptakes of quercetin-3′-O-

sulfate, quercetin-3-O-glucuronide and 3′-O-methylquercetin-3-O-glucuronide were

observed (p < 0.05) compared to that of quercetin, while cellular uptake of quercetin

and 3′-O-methylquercetin did not differ significantly (Fig. 2.8).

Q MQ MQG QG QS0

50

100

150

200

250 * *

Cel

lula

r co

nc.

(

M)

Figure 2.8: Cellular concentrations of quercetin and its metabolites in peripheral

neutrophils after incubation with each compound at a concentration of 10 µM.

Concentrations are based on the cellular volume of neutrophils. n = 3. * p < 0.05 for Q,

MQ and QS compared to QG and MQG (ANOVA).

70

2.3.6. Antioxidant activity

To determine the antioxidant activity of these compounds in a physiological setting, we

investigated the formation of lipid hydroperoxides and F2-isoprostanes in AAPH-

induced LDL oxidation. Figure 2.9A shows the time course for LDL lipid

hydroperoxide formation in the presence of quercetin and its metabolites, while figure

2.9B shows the comparison of the effect of quercetin with luteolin, kaempferol and

taxifolin. Figure 2.10 shows the formation of F2-isoprostanes (a stable biomarker of

lipid peroxidation) at 120 min after exposure of LDL to AAPH in the presence or

absence of quercetin or its metabolites or structural analogues. These data show that

quercetin, and to a slightly lesser extent 3′-O-methylquercetin and quercetin-3-O-

glucuronide, are very effective at inhibiting LDL oxidation at 10 μM (p < 0.001

compared to LDL control). Quercetin-3′-O-sulfate and 3′-O-methylquercetin-3-O-

glucuronide were only partially capable of inhibiting LDL oxidation at this

concentration. The quantitation of F2-isoprostanes at 120 min (the time at which F2-

isoprostanes reach maximal concentration in AAPH-induced LDL oxidation) confirms

the same order of antioxidant activity for quercetin and its metabolites as seen for lipid

hydroperoxide formation. Similarly the inhibition of LDL oxidation by luteolin,

kaempferol and taxifolin (Fig. 2.9B) is mirrored by the quantitation of F2-isoprostanes

at 120 min (Fig. 2.10). Using these data we can construct an order of antioxidant

potency; quercetin > 3′-O-methylquercetin = taxifolin > quercetin-3-O-glucuronide >

kaempferol > luteolin > quercetin-3′-O-sulfate > 3′-O-methylquercetin-3-O-

glucuronide.

71

0 30 60 90 120 150 1800

25

50

75 LDLQ + LDLMQ + LDL

QS + LDL

QG + LDL

MQG + LDL

*

#

(A)

Time (min)

[Hydro

per

oxid

es]

( M

)

0 30 60 90 120 150 1800

25

50

75

LDL

L + LDL

K + LDLT + LDL

*Q + LDL

#

(B)

Time (min)

[Hydro

per

oxid

es]

( M

)

Figure 2.9: Time course for the production of lipid hydroperoxides in LDL after

exposure to peroxyl radicals produced by AAPH. A) quercetin and its metabolites were

incubated with LDL (0.1 mg/ mL) at a concentration of 10 µM and lipid hydroperoxides

determined by FOX assay at time points up to 180 min (n = 3). * p < 0.001 for control

compared to all other treatments, # p < 0.05 for MQG and QS compared to Q (using

AUC). B) quercetin is compared to luteolin (L), kaempferol (K) and taxifolin (T) at a

concentration of 10 µM (n = 3).* p < 0.001 for control compared to all other treatments,

# p < 0.05 for L compared to Q (using AUC).

72

LDL Q MQ QS QG MQG L K T0

2

4

6

8

10

12

14 *

##

#

#

F2-i

sopro

stan

es (

ng/

mL

)

Figure 2.10: The effect of flavonoid compounds on production of F2-isoprostanes

during AAPH-induced LDL oxidation. The compounds (10 µM) were added to LDL

(0.1 mg/ mL) prior to incubation with AAPH and samples removed for F2-isoprostane

analysis at 120 min (n = 3). Compounds tested were Quercetin (Q), 3′-O-


and 3′-O-methylquercetin-3-O-glucuronide (MQG), luteolin (L), kaempferol (K) and

taxifolin (T). * p < 0.001 for control compared to all other treatments, # p < 0.05 for

MQG, QS, L and K compared to Q.

73

2.4. DISCUSSION

In this study we have shown that quercetin is a very potent inhibitor of LTB4

production in human peripheral monocytes and neutrophils at a realistic physiologically

relevant concentration (~ 2 µM), but is less effective in reducing PGE2 (IC50 = 4 µM)

and MCP-1 (IC50 = 5.5 µM) production from human peripheral monocytes. However,

some of its major metabolites show significantly diminished activity. Conjugation at 3′-

OH of quercetin’s phenylbenzopyran-4-one structure (3′-O-methylquercetin and

quercetin-3′-O-sulfate, Fig. 1.3) decreased LTB4 inhibitory activity by up to 50% while

metabolism at the 3-OH (quercetin-3-O-glucuronide and 3′-O-methylquercetin-3-O-

glucuronide, Fig. 1.3) greatly diminished LTB4 inhibiting activity within the

physiologically relevant concentration range tested. When quercetin was compared with

structural analogues (luteolin, kaempferol and taxifolin, Fig. 2.11), it became apparent

that the 3′-OH of the B-ring played a more critical LTB4 inhibiting role than the 3-OH

of the C-ring. The absence of the 3-hydroxyl group of the C-ring in luteolin had

minimal effect on its action, while the absence of 3′-hydroxyl group of the B-ring in

kaempferol reduced its effect by up to 60%. These results also highlighted that the 2,3–

double bond within the C-ring in quercetin is an essential structural requirement for

inhibition of LTB4 production in neutrophils, as its absence (as in taxifolin) totally

diminished the inhibiting action (Fig. 2.11). In addition, we have observed a remarkable

dissociation between structural features that determine anti-inflammatory activity and

antioxidant activity, which are illustrated in Figure 2.11. The profound effect that

structural modification can have on bioactivity as reported in this work further

highlights the need for in vitro studies to use actual metabolic forms of flavonoids rather

than the free aglycone or glycosides occurring in the diet.

Daily intake of flavonols such as quercetin has been estimated at between 20-

35 mg/day, in the form of various glycosides, although intact glycosides are not found

74

in plasma. Following supplementation with flavonol rich foods (such as onions or

apples), or various quercetin-glycosides, at doses of 50-200 mg equivalents, the plasma

concentration of quercetin can reach between 2-7 μM90

. In plasma, quercetin is not

present as the aglycone but only in conjugated forms, with 20-40% methylated at the 3′

position and other identified metabolites being the 3-O-glucuronide, 3′-O-sulphate or

the 3′-O-methylquercetin-3-O-glucuronide90

. The synthesis of each of these metabolites

has enabled us to study the activity of each individual metabolite and evaluate the likely

consequences of metabolic conversion of flavonoids such as quercetin. It has been

previously demonstrated that exposure of flavonols such as kaempferol to cultured

hepatocytes (mimicking first pass metabolism) greatly diminished inhibitory activity

towards endothelial cell adhesion molecule expression, although the nature of the

metabolic conversion was not identified169

.

The production of LTB4 by leukocytes is dependent on the translocation of 5-

LO from the cytosol to the nuclear membrane in response to increased intracellular

calcium188

. It requires substrate, AA, which must be generated from membrane

phospholipids via PLA2. FLAP acts as a docking protein on the nuclear membrane and

together with LTA4 hydrolase and PLA2 completes the complex that is required for

LTB4 synthesis188

. Certain indole derivatives such as MK-886 selectively bind to FLAP

and prevent the activation of 5-LO and subsequent synthesis of leukotrienes188

. Other

compounds such as certain redox active flavonoids and phenols can directly inhibit 5-

LO, presumably by reducing the iron at the active site. We have been able to establish

that quercetin does not suppress the hydrolysis of LTA4 to LTB4 (Fig. 2.7) indicating

that quercetin acts directly on 5-LO or possibly FLAP. There is evidence that quercetin

and other flavonols such as epicatechin can directly inhibit human 5-LO. Using a

recombinant enzyme, Schewe et al (2002) showed direct inhibition in a cell free system

with quercetin having IC50 = 0.6 μM for 5-LO and 4 μM for inhibition of 15-

75

lipoxygenase189

. Since these were cell membrane-free systems the involvement of

FLAP appears to be ruled out. Moreover the flavonols appear to be non-specific

lipoxygenase inhibitors189

.

Whatever the mechanism of action for the inhibition of 5-LO by flavonoids, it

appears to be distinct from the antioxidant properties of these studied compounds. Our

results comparing antioxidant activity with leukotriene inhibitory activity clearly

demonstrate this distinction. For example, the 2,3–double bond is critical for leukotriene

inhibitory activity but has little effect on antioxidant activity as observed with taxifolin.

The C3-hydroxyl group is not critical for leukotriene inhibitory activity but its absence

significantly reduces antioxidant properties as seen with luteolin. The results are

consistent with previous reports that the 2, 3–double bond of the C-ring in flavonols is

essential for inhibition of adhesion molecule expression in endothelial cells169

and

inflammatory cytokine production in mouse macrophages190

.

Similarly metabolism of quercetin which introduces a glucuronide at the C3-

oxygen position (quercetin-3-O-glucuronide and 3′-O-methylquercetin-3-O-

glucuronide) almost completely abolishes leukotriene inhibitory activity. This result is

in agreement with a previous observation that formation of the 3-O-glucuronide by

incubation of quercetin with liver cell-free extracts substantially reduces lipoxygenase

inhibitory activity191

. Interestingly, glucuronide formation at other sites (3′, 4′ and 7)

had little effect on soybean lipoxygenase inhibition in a cell free preparation191

.

Metabolites with groups attached to the 3′-O position of the B ring (3′-O-

methylquercetin and quercetin-3′-O-sulfate) maintain some inhibitory activity towards

both leukotriene production and PGE2 formation. Another factor which may influence

the activity of quercetin metabolites is change in polarity and reduced cellular uptake.

This is particularly noticeable with the polar moieties such as glucuronide and sulphate,

76

while methylation has little effect on cellular uptake (Fig. 2.8). However, the actual

mechanisms by which neutrophils sequester quercetin remain unknown at this time.

Overall our study suggests that at least two of the major in vivo metabolites of

quercetin retain significant activity for the inhibition of pro-inflammatory eicosanoids

such as LTB4 and PGE2. While in vivo studies of the anti-inflammatory action of

flavonols is limited, flavonols have been shown to reduce leukotriene production in

humans192

and direct injection of quercetin into joints of the rat significantly reduced

inflammation193

. The availability of synthetic standards of specific flavonoid

metabolites will enable their bioactivity to be more clearly defined. It is now becoming

clear that structural modification of flavonoids by metabolic transformation is likely to

have a profound effect on biological activity.

77

O

OOH

HO

OH

OH

OH

O

OOH

HO

OH

OH

OH

O

OOH

HO

OH

OH

OH

Luteolin (L) Kaempferol (K) Taxifolin (T)

Anti-inflammatory

activity

Strong activity, similar to

Q

Some activity

No activity

Antioxidant activity

Much less activity than Q Modest activity Strong activity

Figure 2.11: Structures of luteolin (L), kaempferol (K) and taxifolin (T). The structural features shown in grey

represent the difference in structure between these compounds and quercetin (Q). A comparison of the

antioxidant and anti-inflammatory (inhibition of LTB4) activity with that of quercetin (Q) is summarized.

78

CHAPTER 3:

IN VITRO EFFECT OF QUERCETIN AND

METABOLITES ON MYELOPEROXIDASE ACTIVITY

(The results from this chapter have been published in J. Agri. Food Chem. 2008;56

3609–3615.

3.1. INTRODUCTION

Oxidative damage to LDL has been proposed as a critical step in the

development of atherosclerosis1. Studies with human populations have demonstrated

that circulating ox-LDL is associated with preclinical atherosclerosis, coronary arterial

atherosclerosis, acute coronary syndrome and vulnerable plaques194

. Circulating ox-

LDL is believed to be a useful marker for identifying patients with coronary heart

disease194

. Nitrotyrosine, a marker of LDL protein damage, is nearly 100-fold higher in

LDL recovered from human atherosclerotic aorta than that in circulating LDL from

healthy donors195

.

One pathway for LDL modification in vivo is through reactive species

generated by MPO196

. MPO has been suggested as a physiological catalyst for in vivo

LDL modification in studies using monocytes and neutrophils isolated from humans197

.

Previous reports of elevated plasma MPO in early adverse cardiac events198

, in acute

coronary syndrome 199

, and after acute myocardial infarction200

, provide further

evidence that MPO may contribute to CVD. More recently MPO has been implicated in

the production of cyanate which can cause carbamylation of proteins196

.

Population studies suggest that dietary flavonoids may provide protection

against CVD164

. Recent controlled intervention trials report that flavonoids – rich food

can improve endothelial and platelet function and reduce blood pressure in humans166,

201, and may inhibit the development of atherosclerosis in animal models

167. Since

79

oxidative stress has been implicated in atherosclerosis and CVD, one of the main

properties of flavonoids thought to explain their health benefit is their antioxidant

activity168

. However, the results of intervention studies have yielded conflicting

results170

. This may be due to variations in the absorption and metabolism of flavonoids

which may alter antioxidant activity as well as other biological activities171

. In particular

there is doubt about the interpretation of in vitro studies of antioxidant activity where

issues of bioavailability and metabolic transformation have not been considered172

.

To address the issue of the effect of metabolic transformation on flavonoid

bioactivity we have compared the ability of quercetin and its major in vivo metabolites

to inhibit LDL modification by human neutrophils, a rich source of MPO. We have also

examined the structural features important for antioxidant activity by comparing

quercetin and its major metabolites (Figure 1.8) as well as other structurally related

flavonoids.



BSA, calcium chloride, guaiacol, sodium acetate, quercetin, sodium phosphate

dibasic, sodium phosphate monobasic, sodium bromide, sodium chloride, sodium

hydrogencarbonate, luteolin, kaempferol, taxifolin, butylated hydroxytoluene, hydrogen

peroxide (50% by volume), phorbol 12-myristate 13-acetate (PMA), methane sulfonic

acid, phenol, benzoic acid, superoxide dimutase (SOD), ferricytochrome c, AAPH and

diethylenetriamine penta-acetic acid were purchased from Sigma Aldrich (St Louis,

MO, USA); acetonitrile, magnesium sulfate and sulfuric acid from Univar (WA,

Australia); ficoll-paque from GE Healthcare (Uppsala, Sweden); HBSS, PBS, HIFCS

and RPMI 1640 from Gibco™ Invitrogen (Calsbad, CA, USA); dextran 500 from

Amersham Biosciences (Uppsala, Sweden); glucose and potassium phosphate

80

monobasic from Merck (VIC, Australia) and methanol and ethanol from Mallinckrodt

(NJ, USA). 3′-O-methylquercetin, quercetin-3′-O-sulfate, quercetin-3-O-glucuronide

and 3′-O-methylquercetin-3-O-glucuronide were synthesized as previously described99

.

3.2.2. AAPH – induced and neutrophil – mediated modification of LDL

Neutrophils were isolated from the neutrophil/erythrocyte pellet after Ficoll-

Paque gradient centrifugation and dextran sedimentation of red cells as previously

described202

. Cell viability was assessed using trypan blue exclusion and was typically

>98%. LDL was isolated immediately from fresh

plasma by density gradient

ultracentrifugation and its protein concentration was determined as previously

described203

. LDL (final protein concentration 0.1 mg/ mL) was incubated at 37 ºC in

the presence of either PMA – activated peripheral neutrophils (5 x 106 cells/ mL HBSS;

final PMA concentration 200 nM) or AAPH (final concentration 5 mM). The

supernatant from the cell suspension was collected at regular time intervals and stored at

-80 ºC before F2-isoprostane and 3-chlorotyrosine analyses. LDL incubated with PMA –

activated cells were used as positive controls while LDL incubated with untreated cells

without PMA activation served as negative controls.

3.2.3. Measurement of inhibition of lipoprotein oxidation

The anti-oxidant ability of each compound (quercetin, 3′-O-methylquercetin,

quercetin-3′-O-sulfate, quercetin-3-O-glucuronide, 3′-O-methylquercetin-3-O-

glucuronide, luteolin, kaempferol and taxifolin) to inhibit AAPH-induced or

neutrophils-mediated LDL oxidation was measured by the formation of F2-isoprostanes

(stable marker of lipid peroxidation) quantitated by GC-MS184

. Briefly, the test

compound (final concentrations 1, 2 and 10 µM) was added to the LDL and neutrophils

(prior to PMA stimulation) or AAPH addition. Aliquots of the mixture were analysed

for lipid peroxidation products at specific time points up to 3 h and compared to control

81

incubations without the addition of test compounds. LDL oxidation by neutrophils was

quantified by measuring the amount of 15-F2t-isoprostanes produced after 120 min,

calculated relative to an internal standard (15-F2t-isoprostanes-d4). The intra - and inter

– assay coefficient of variation (CV) for this assay were 8% (n = 8) and 5.6% (n = 3).

3.2.4. Measurement of inhibition of lipoprotein chlorination

Inhibition of lipoprotein chlorination by quercetin, its metabolites, and its

structural analogues, luteolin, kaempferol and taxifolin was determined by measuring 3-

chlorotyrosine production204

. Briefly, the sample was extracted with acetone and the

residue containing total proteins was hydrolysed with methane sulfonic acid (6 M with

1% phenol and 1% benzoic acid) at 50 ºC for 12 h. The hydrolysate was dissolved in the

HPLC mobile phase (20 mM sodium phosphate buffer, pH 3.0 with 5% (v/v) methanol)

and an aliquot (20 µL) of the supernatant was injected into the HPLC-ECD system.

Chromatography was performed on a 15 mm x 5 mm i.d., 5 µm, LiChrospher 100 RP-

18 reverse phase column (Agilent Technologies) at a flow rate of 1 mL/ min using a

Hewlett Packard Series 1100 HPLC. The voltage of the analytical cell was set at 550

mV. Results were obtained with within and between assay reproducibility of 7% (n =

10) and 3.5% (n = 3) respectively.

3.2.5. Measurement of inhibition of functional Myeloperoxidase activity

To test the effect of quercetin, its metabolites and its structural analogues,

luteolin, kaempferol and taxifolin on MPO activity in peripheral neutrophils, freshly

isolated cells (1 x 106 cells/ mL in HBSS) were incubated with these compounds (2 µM)

for 5 min at 37 ºC before the incubate was removed. The cells were resuspended on

fresh HBSS and lysed by sonication. Functional MPO activity was determined by

measuring its catalytic action on the oxidation of guaiacol in the presence of hydrogen

82

peroxide as described previously205

. The intra - and inter – assay CV of the results

obtained were 1.3% (n = 6) and 2.9% (n = 3) respectively.

3.2.6. Measurement of inhibition of NOX activity

The effects of quercetin, its metabolites and its structural analogues, luteolin,

kaempferol and taxifolin on NOX activity in peripheral neutrophils was investigated by

measuring O2•- as described by Johnston and Richard (1984)

206. Freshly isolated

neutrophils (1 x 106 cells/ mL) in HBSS were incubated with the compounds (1, 2 and

10 µM) for 5 min at 37 ºC before PMA was added (final concentration 200 nM). O2•-

production was determined by measuring the reduction of cytochrome c (final

concentration 100 M) in the presence and absence of SOD (final concentration 150

units/ mL). The intra - and inter – assay CV of the results obtained were 3% (n = 8) and

2.4% (n = 3).

3.2.7. Statistical analysis of results

Statistical analysis of results was performed using SPSS version 11.5. One-

way ANOVA185

and Bonferroni post hoc analyses were performed on specific

concentration points. The results analysed were considered significantly different if p

value ≤ 0.05 based on 95% confidence interval. Error bars in all the figures were

presented as standard error of means (SEM).

83

3.3. RESULTS

3.3.1. Effects on lipid peroxidation

Neutrophils did not produce F2-isoprostanes from LDL over 120 min in the

absence of PMA. LDL incubated with PMA – activated neutrophils produced

significant amounts of F2-isoprostanes up to 10-fold the initial level over the 120-min

period (mean ± S.E.M.: 145.5 ± 2.5 pg/ mL vs 1385.7 ± 3.2 pg/ mL, p < 0.001).

Quercetin and its metabolites exerted dose – dependent inhibition of F2-isoprostane

formation (Figure 3.1A). Quercetin protected LDL against neutrophil-mediated lipid

peroxidation with an IC50 of approximately 1 µM while the IC50 for its metabolites

ranged from 2 to 4 µM. All the metabolites had significantly lower inhibitory activities

(p < 0.05 by ANOVA analysis of the area under dose – response curve) compared to the

parent molecule. Quercetin and its metabolites were effective in inhibiting lipid

peroxidation at concentrations ranging from 1 – 10 µM (Figure 3.1A). At the realistic

physiological concentration (1 µM), quercetin and all its major in vivo metabolites

showed significant protection against lipid peroxidation (p < 0.05 vs positive control)

(Figure 3.1A). Quercetin and quercetin-3-O-glucuronide were significantly more

effective in reducing lipid peroxidation (p < 0.05) compared to the other metabolites

(Figure 3.1A). There was no significant difference in activity between quercetin and its

metabolites at 2 µM concentration, even though the metabolites appeared to have

diminished effects at both concentrations (Figure 3.1A). Quercetin and its metabolites

exhibited dose – dependent inhibition of AAPH – initiated LDL peroxidation (Figure

3.2A), with IC50 of approximately 1.5 µM for quercetin and IC50 ranging from 2 to 10

µM for its metabolites. At 1 µM concentration, quercetin, 3′-O-methylquercetin,

quercetin-3′-O-sulfate and quercetin-3-O-glucuronide offered significant protection

against AAPH-initiated LDL oxidation (p < 0.05 vs positive control) (Figure 3.2A).

84

1 2 100

25

50

75

100

*

#(A)

Concentration (M)

F2-i

sopro

stan

es (

%)

1 2 100

25

50

75

100

#

*

**

Concentration (M)

F2-i

sopro

stan

es (

%)

(B)

Figure 3.1: (A) Dose – dependent effects of quercetin (Q ), 3′-O-methylquercetin

(MQ ), quercetin-3′-O-sulfate (QS ), quercetin-3-O-glucuronide (QG )

and 3′-O-methylquercetin-3-O-glucuronide (MQG ) on the production of F2-

isoprostanes by PMA-activated neutrophils (n = 3). * p < 0.05 vs quercetin-3′-O-sulfate

and 3′-O-methylquercetin-3-O-glucuronide at 1 µM concentration. # p < 0.05 vs the

positive control. (B) Dose – dependent effects of quercetin (Q ), luteolin (L ),

kaempferol (K ) and taxifolin (T ) on the production of F2-isoprostanes by

PMA-activated neutrophils (n = 3). * p < 0.05 vs quercetin at 2 µM concentration. ** p

< 0.05 vs quercetin at 10 µM concentration. # p < 0.05 vs the positive control.

85

1 2 100

25

50

75

100

#

*

## ###

**

***

Concentration (M)

F2-i

sopro

stan

es (

%)

(A)

1 2 100

25

50

75

100

***

**

#

*

Concentration (M)

F2-i

sopro

stan

es (

%)

(B)



and 3′-O-methylquercetin-3-O-glucuronide (MQG ) on the production of F2-

isoprostanes after AAPH treatment of LDL (n = 3). * p < 0.05 vs all metabolites at 1

µM concentration. ** p < 0.05 vs quercetin-3′-O-sulfate and 3′-O-methylquercetin-3-O-

glucuronide at 2 µM concentration. *** p < 0.05 vs quercetin-3′-O-sulfate and 3′-O-

methylquercetin-3-O-glucuronide at 10 µM concentration. # p < 0.05 vs the positive

control at 1 µM concentration. ## p < 0.05 vs the positive control at 2 µM

concentration. ### p < 0.05 vs the positive control at 10 µM concentration. (B) Dose –

dependent effects of quercetin (Q ), luteolin (L ), kaempferol (K ) and

taxifolin (T ) on the production of F2-isoprostanes after AAPH treatment of LDL

(n = 3). * p < 0.05 vs luteolin and kaempferol at 1 µM concentration. ** p < 0.05 vs

luteolin and kaempferol at 2 µM concentration. *** p < 0.05 vs luteolin and

kaempferol at 10 µM concentration. # p < 0.05 vs the positive control.

86

Quercetin, 3′-O-methylquercetin and quercetin-3-O-glucuronide were significantly

more effective (p < 0.05) compared to quercetin-3′-O-sulfate and 3′-O-methylquercetin-

3-O-glucuronide (Figure 3.2A). When the structural analogues of quercetin, luteolin,

kaempferol and taxifolin were tested, all three significantly inhibited LDL oxidation (p

< 0.05) (Figure 3.1B). Luteolin and kaempferol showed significantly less inhibition of

neutrophils-mediated oxidation of LDL than quercetin (p < 0.05) at 2 µM and 10 µM

concentrations, whereas only kaempferol was significantly less active than quercetin at

1 µM concentration. The activity of taxifolin was not significantly different to quercetin

(Figure 3.1B). Similar trends in activity were observed during the AAPH oxidation of

LDL, with all, except taxifolin, showing significantly diminished activity (p < 0.05)

compared to quercetin (Figure 3.2B).

3.3.2. Effects on LDL protein modification

Myeloperoxide is an enzyme in neutrophils producing hypochlorous acid

which reacts with tyrosine residues in proteins to form 3-chlorotyrosine. Activated

neutrophils produced at least 10 fold more LDL 3-chlorotyrosine compared to

unstimulated cells (mean ± S.E.M.: 9.80 ± 0.55 µM vs 0.87 ± 0.05 µM, p < 0.001).

Quercetin and its metabolites exhibited dose – dependent inhibition of 3-chlorotyrosine

formation (Figure 3.3A). Quercetin suppressed tyrosine chlorination, with an IC50 of

approximately 1 µM, while its metabolites showed diminished activity with IC50 values

ranging from 2 to 10 µM. However, the reduction in activity reached significance only

when quercetin-3′-O-sulfate and 3′-O-methylquercetin-3-O-glucuronide were compared

to the parent molecule (p < 0.05 by ANOVA analysis) (Figure 3.3A and 3.3B). Only

quercetin, 3′-O-methylquercetin and quercetin-3-O-glucuronide offered significant

protection against tyrosine chlorination (p < 0.05) (Figure 3.3A). Taxifolin exhibited

similar activity to quercetin while luteolin and kaempferol showed lower activity

(Figure 3.3B).

87

1 2 100

20

40

60

80

100

Concentrations (M)

* *** **

**

***

*

3-c

hlo

roty

rosi

ne

(%)

(A)

C Q L K T MQ QS QG MQG0

20

40

60

80

100

** *

*

**

3-c

hlo

roty

rosi

ne

(%)

(B)



and 3′-O-methylquercetin-3-O-glucuronide (MQG ) on the production of 3-

chlorotyrosine by PMA-activated neutrophils (n = 3). * p < 0.05 vs the positive control

at 1 µM concentration. ** p < 0.05 vs the positive control at 2 µM concentration. *** p

< 0.05 vs the positive control at 10 µM concentration. (B) 3-chlorotyrosine production

by PMA – activated neutrophils in the absence (C) or presence of quercetin (Q), luteolin

(L), kaempferol (K), taxifolin (T), 3′-O-methylquercetin (MQ), quercetin-3′-O-sulfate

(QS), quercetin-3-O-glucuronide (QG) and 3′-O-methylquercetin-3-O-glucuronide

(MQG) (1 µM) (n = 3). * p < 0.001 vs control.

88

3.3.3. Effects on functional MPO activity

The effects of quercetin, its metabolites, and structural analogues luteolin,

kaempferol and taxifolin on functional MPO enzyme activity were compared to the

MPO enzyme activity of untreated control neutrophil lysate. The mean rate of guaiacol

oxidation by the untreated control neutrophil lysate was 1.36 ± 0.31 units/ mL (n = 3).

Dose – dependent effects on MPO activity were observed for quercetin (IC50 ≈ 1.5 µM)

and its metabolites (IC50 ranging from 2 to > 10 µM) (Figure 3.4A). The four quercetin

metabolites as well as the analogues luteolin and kaempferol were less effective at

suppressing MPO enzyme activity than quercetin while the analogue taxifolin had

similar activity to quercetin (Figure 3.4B). Quercetin-3′-O-sulfate and 3′-O-

methylquercetin -3-O-glucuronide were the only molecules where the decrease in

activity compared to the parent molecule, reached significance (p < 0.05) (Figure 3.4A).

3.3.4. Effects on O2•- production

O2•- is a reactive oxygen species produced by the enzyme, NOX in phagocytic

cells. Stimulated neutrophils produced approximately 6 fold more O2•- than

unstimulated neutrophils (data not shown). Quercetin (IC50 ≈ 4.5 µM) exhibited dose –

dependent inhibition of O2•- formation (Figure 3.5). At 1 µM, quercetin significantly

reduced O2•- formation by about 20% (Figure 3.5). Quercetin metabolites showed

diminished inhibition, but only quercetin-3′-O-sulfate and 3′-O-methylquercetin-3-O-

glucuronide were significantly less active (p < 0.05 using ANOVA analysis of the area

under dose – response curve) compared to the parent molecule (Figure 3.5).

89

1 2 100

20

40

60

80

100

Concentration (M)

*

**

**

**

**

***

MP

O E

nzy

me

Act

ivit

y (

%)

(A)


20

40

60

80

100

*

*

**

**

MP

O A

ctiv

ity (

%)

(B)

Figure 3.4: (A) Dose – dependent inhibition of functional MPO enzyme activity in

lysed PMA-activated neutrophils by quercetin (Q ), 3′-O-methylquercetin (MQ

), quercetin-3′-O-sulfate (QS ), quercetin-3-O-glucuronide (QG ) and

3′-O-methylquercetin-3-O-glucuronide (MQG ) (n = 3). * p < 0.05 vs the positive

control at 1 µM concentration. ** p < 0.05 vs the positive control at 2 µM

concentration. *** p < 0.05 vs the positive control at 10 µM concentration. (B)

Functional MPO enzyme activity in lysed PMA – activated neutrophils in the absence

(C) or presence of quercetin (Q), luteolin (L), kaempferol (K), taxifolin (T), 3′-O-


and 3′-O-methylquercetin-3-O-glucuronide (MQG) (1 µM) (n = 3). * p < 0.05 vs all

except quercetin-3′-O-sulfate and 3′-O-methylquercetin-3-O-glucuronide.

90

3.4. DISCUSSION

Our results showed that quercetin and two of its metabolites, 3′-O-

methylquercetin and quercetin-3-O-glucuronide inhibited neutrophil–mediated

modification of LDL as measured by F2-isoprostanes and 3-chlorotyrosine formation

(Figure 3.1A and 3.3A) at concentrations which may be achieved in vivo after

consumption of a quercetin – rich diet or supplementation. However, the metabolites,

quercetin-3′-O-sulfate and 3′-O-methylquercetin-3-O-glucuronide consistently showed

reduced activity compared to quercetin. The activities of 3′-O-methylquercetin and

quercetin-3-O-glucuronide were compared with those of luteolin and kaempferol as the

absence of 3-OH group in luteolin and 3′-OH group in kaempferol are similar to the 3-

O- and 3′-O- modifications in 3′-O-methylquercetin and quercetin-3-O-glucuronide.


20

40

60

80

100

* *S

uper

oxid

e pro

duct

ion (

%) ** **

Figure 3.5: Superoxide anion production by PMA – activated neutrophils (5 x 106 cells/

mL) after incubation for 120 min in the absence (C) or presence of quercetin (Q),

luteolin (L), kaempherol (K), taxifolin (T), 3′-O-methylquercetin (MQ), quercetin-3′-O-

sulfate (QS), quercetin-3-O-glucuronide (QG) and 3′-O-methylquercetin-3-O-

glucuronide (MQG) (1 µM) (n = 3). * p < 0.05 vs control by ANOVA analysis. ** p <

0.05 vs quercetin and taxifolin by ANOVA analysis.

91

Taxifolin showed similar activity to quercetin, suggesting that removal of the double

bond in the C-ring had little effect. Taxifolin also showed inhibition of AAPH oxidation

of LDL similar to quercetin in contrast to luteolin, kaempferol and the quercetin

metabolites which were less effective. MPO-catalyzed LDL oxidation was more

sensitive to quercetin and its metabolites than the AAPH-initiated LDL oxidation

(Figure 3.1A and 3.2A). Levels of 3-chlorotyrosine, a product of MPO activity, were

reduced in LDL after treatment with all compounds except for quercetin-3′-O-sulfate

and 3′-O-methylquercetin-3-O-glucuronide showing again that these particular

metabolic transformations render quercetin less active (Figure 3.3A and 3.3B). Similar

trends in activity were seen when MPO activity was measured in lysed neutrophils

(Figure 3.4A and 3.4B). Metabolic modifications resulting in 3′-O-methylquercetin and

quercetin-3-O-glucuronide did not have a significant effect on MPO inhibitory activity

of quercetin. Production of O2•- by neutrophils was only slightly decreased in the

presence of quercetin, 3′-O-methylquercetin and quercetin-3-O-glucuronide. These

results suggest that inhibition of neutrophil-mediated LDL modification by quercetin

and related compounds is predominantly due to inhibition of MPO as well as direct

free-radical scavenging action as evidenced by the compounds′ ability to inhibit AAPH-

initiated oxidation.

Quercetin metabolites had diminished ability (IC50 of 2 – 4 µM) to protect

LDL from oxidative damage and to inhibit MPO activity when compared to the parent

molecule (IC50 of 1 µM) (Figure 3.1A, 3.3A and 3.4A). By comparing the activity of

quercetin with its structural analogues (luteolin, kaempferol and taxifolin), we have

proposed a structural activity relationship of quercetin in relation to its free radical

scavenging and MPO inhibiting activities (Figure 3.6). Our experiments, which were

carried out at physiologically relevant concentrations90

, showed that the 3′-OH (luteolin)

and 3-OH (kaempferol) groups on the B and C-rings respectively played similar roles in

92

both radical scavenging and MPO inhibiting activities. The 2,3 double bond on the B-

ring (absent in taxifolin) played a negligible part in protecting LDL against oxidative

damage (Figure 3.1B, 3.2B, 3.3B and 3.4B), because taxifolin retained similar activity

to quercetin. Similar observations were reported for cell – free Cu2+

and AAPH-

mediated LDL oxidation under non – physiologically relevant concentrations207, 208

. Our

results support the equivalence of the 3′-OH and 3-OH modifications with the observed

similarity in activities of 3′-O-methylquercetin and quercetin-3-O-glucuronide which

were in turn similar to luteolin and kaempferol (Figure 3.1A, 3.1B, 3.2B, 3.3B and

3.4B). Interestingly, conjugation at either 3- or 3′- positions did not significantly reduce

quercetin activity against MPO-catalyzed events (Figure 3.1B, 3.3B and 3.4B) unlike

that against radical scavenging activities (Figure 3.2B). Our results are consistent with

previous reports that the catechol structure at the B ring and the 3-OH group in the C

ring account for the radical scavenging effects of flavonoids207, 208

. They also agree with

a recent report that the presence of a resorcinol group in the A ring is the main

contributors to the inhibitory effects of flavonoids on MPO209

. The significant lack of

activity observed for quercetin-3′-O-sulfate may be explained by the possible hydrogen

bond between the sulphate group and 3-OH group (on the C ring) or 4′-OH group (on

the B ring), which would limit the availability of the 3-OH group.

We have shown that quercetin protects LDL from neutrophil-mediated

modification at physiologically relevant concentrations (1 µM). These results were

particularly relevant as the neutrophil concentration used reflects their possible numbers

at the site of inflammation210

. It has been well established that quercetin with its

phenolic OH groups protects against free radical damage via radical scavenging

activity102

. However, low bioavailability (~ 1 µM) and metabolic transformation reduce

the likely in vivo scavenging action of quercetin. The effect of metabolic transformation

93

O

OOH

HO

OH

OH

OH

O

OOH

HO

OH

OH

OH

O

OOH

HO

OH

OH

OH

Luteolin (L) Kaempferol (K) Taxifolin (T)

MPO inhibiting

activity

Diminished activity

compared to quercetin,

similar to kaempferol.

Diminished activity


similar to luteolin.


quercetin.

Radical scavenging

activity

Diminished activity


similar to kaempferol.

Diminished activity


similar to luteolin


quercetin.

Figure 3.6: Structural activity relationship for quercetin, luteolin, kaempherol and taxifolin.

94

on bioactivity has been reported previously169, 211

. The importance of quercetin as an

antioxidant is brought into question given the higher bioavailabilities of other in vivo

anti-oxidants, like ascorbic acid and α-tocopherol212

, although quercetin may exert

synergistic and additive effects with other in vivo antioxidants. Our results suggest that

quercetin is not effective at suppressing/ scavenging O2•- ion radical production (IC50 =

4.5 µM) in activated neutrophils at physiologically relevant concentrations. It had been

previously reported that quercetin exhibited a dose dependent inhibition of the

generation of O2•- anion radical (IC50 = 5.0 µM) from xanthine/XO

213. Pincemail et al

reported that human MPO activity was inhibited by quercetin (IC50 = 3.5 µM) in

vitro105

. We have shown similar inhibition by quercetin on MPO enzyme activity, as

well as 3-chlorotyrosine production (an established biomarker of MPO activity) by

peripheral neutrophils with an IC50 value of 1 - 1.5 µM (Figure 3.3A and 3.4A).

Quercetin was also shown to be more effective at protecting against MPO-mediated

events (IC50 = 1 µM) (Figure 3.3A and 3.4A) than O2•--initiated damage (IC50 > 10 µM)

in neutrophils. Possible routes by which quercetin may inhibit MPO – catalyzed

activities are summarised in Figure 3.7. Flavonoids were reported to be substrates for

MPO as they were oxidised by MPO in the absence of LDL to produce reactive free

radicals and quinones214

. Quercetin was found to be a powerful scavenger of

hypochlorous acid104

, thus limiting the MPO – catalyzed chlorination of LDL proteins.

It was proposed that quercetin also scavenged the ∙NO2 radical215

, which is a powerful

oxidant produced by the MPO-catalyzed reaction between hydrogen peroxide and nitrite

ions216

. Quercetin and other flavonoids have also been shown to act as substrate for

chlorination217

, thereby protecting the proteins from chlorination damage.

In conclusion, our study has shown that quercetin and at least one of its in vivo

major metabolites (quercetin-3-O-glucuronide) are potent inhibitors of cell-mediated

LDL modification at physiologically relevant concentrations, mainly through the

95

inhibition of MPO. Metabolic transformation of quercetin, as well as its low

bioavailability, did not render the molecule significantly less potent as a MPO inhibitor.

The recently reported presence of quercetin in human atherosclerotic lesions218

suggests

that quercetin may be available to prevent LDL oxidation. These results are particularly

interesting because of the potential health benefits of diet derived flavonoids such as

quercetin.

Figure 3.7: Flow diagram showing possible routes by which MPO can catalyze the

modification of LDL. The arrows indicate the points at which quercetin (Q) may inhibit

the process.

Q Q

Q

H2O

MPO MPO

Neutrophils +

other sources

∙O2-

H2O2

∙NO2 HOCl

∙NO

eNOS

Cl-

Lipid

peroxidation

(major product)

3-nitrotyrosine

formation

(minor

product)

3-chlorotyrosine

formation (major

product)

96

CHAPTER 4:

IN VIVO EFFECT OF PURE FLAVONOIDS ON NITRIC

OXIDE AND ENDOTHELIN-1 STATUS IN HEALTHY

HUMANS

(The results from this chapter have been accepted for publication in Am. J. Clin. Nutr.

2008.)

4.1 INTRODUCTION

Endothelial dysfunction is a critical event in the pathogenesis of

atherosclerosis and its clinical manifestations219, 220

. It accelerates the development of

atherosclerosis and may be one of the earliest manifestations of this disease63, 64

.

Therefore, endothelial function may serve as an indication for cardiovascular health and

be used for evaluation of new therapeutic strategies221

. The endothelium maintains

vascular homeostasis and regulates vascular tone by balancing the production of

vasodilators, most importantly NO•139

and vasoconstrictors, such as ET-1140

. Because of

its low concentration and short half-life222

, it is difficult to measure free NO• in

biological systems. Recent studies have reported that S-nitrosothiols, nitrite and nitrate,

all metabolites of NO•, can be used as reliable measures of endogenous NO

•

production223, 224

. ET-1 is a 21-amino acid vasoconstrictor peptide produced by

endothelial cells that has been identified as one of the strongest vasoconstrictors in

human vasculature79

.

Flavonoids are ubiquitous in plant foods. Important dietary sources can

include tea, red wine, apples and cocoa225, 226

. Many flavonoids are potent antioxidants

in in vitro systems227

. Epidemiologic studies have reported a reduced risk for CVD in

subjects with high flavonoid intake86

. Flavonoid rich tea145

, purple grape juice143

, and

cocoa228

have all been found to improve endothelial function in acute and short-term

97

intervention trials in humans170

. Inclusion of small amounts of polyphenol-rich dark

chocolate as part of a usual diet efficiently reduced blood pressure and improved

formation of vasodilative nitric oxide201

. Improving endothelium-dependent

vasodilation is believed to be one possible mechanism by which flavonoids may reduce

cardiovascular risk229

. There are many hundreds of flavonoids in the human diet.

However, it is likely that bioactivity relevant to endothelial function is limited to fewer

compounds. We have studied three compounds: quercetin, (-)-epicatechin and

epigallocatechin gallate (EGCG). There is consistent evidence from population studies

that flavonols such as quercetin can reduce the risk of CVD. The population data for

flavan-3-ols, such as (-)-epicatechin and EGCG is less consistent. However, in

intervention studies flavan-3-ol rich foods and beverages, such as tea and cocoa,

consistently improve endothelial function170

. In addition, isolated (-)-epicatechin146

and

EGCG230

have been found to acutely improve endothelial function in humans. Isolated

(-)-epicatechin has also been shown to augment NO• status

146.

Our study investigates the acute effects of quercetin, (-)-epicatechin and

EGCG on NO• status and ET-1 production effects which have ultimate implications for

endothelial function. We also investigated whether these compounds induce changes in

oxidative stress which might also be a determinant of any effects on endothelial

function.



N-ethylmaleimide (NEM), sulphanilamide (SFA), potassium iodide, copper(II)

sulfate, sodium nitrite-15

N, sodium nitrate-15

N, 2,3,4,5,6-pentafluorophenylbromide

(PFPBr), 1-hydroxy-2-naphthoic acid, β-glucuronidase, N,O-bis(trimethylsilyl)trifluoro

acetamide (BSTFA), pyridine, toluene, quercetin and (-)-epicatechin were purchased

98

from Sigma Aldrich (St Louis, MO, USA); acetone, acetonitrile, hydrochloric acid,

glacial acetic acid and sulfuric acid from Univar (WA, Australia); methanol and ethanol

from Mallinckrodt (NJ, USA); NOA antifoam agent from GE Sievers (CO, USA); 3′-O-

methyl-(-)-epicatechin from Nacalai Tesque Inc (Kyoto, JAPAN); and 3′-O-

methylquercetin from Advanced Technology & Industrial Co., Ltd (Kln, Hong Kong).

4.2.2. Subjects

Twelve healthy male subjects participated in the study. The study was

approved by and performed under the guidelines of the Human Ethics Committee of

University of Western Australia, and informed consent was obtained from each of the

subjects before commencement of the study. All subjects were healthy with no evidence

of chronic disease. None of the subjects consumed > 20 g alcohol/ day or were taking

other medications, antioxidants, or vitamin supplements. The study group had a mean (±

SEM) age of 43.2 ± 4.3 years, a mean (± SEM) body weight of 76.8 ± 2.3 kg, and a

mean body mass index (BMI) of 25.1 ± 0.8 kg/m2. All subjects had normal blood

pressure (mean systolic blood pressure of 122.8 ± 2.1 mmHg and mean diastolic blood

pressure of 78.0 ± 2 mmHg). The mean concentrations of serum total, LDL and HDL

cholesterol, and triglycerides in the subjects were 4.92 ± 0.28, 2.74 ± 0.31, 1.48 ± 0.24

and 1.65 ± 0.45 mmol/L respectively.

4.2.3. Experimental Design

The acute effects of 3 common dietary flavonoid aglycones; quercetin, (-)-

epicatechin and EGCG on plasma and urinary NO• metabolites were assessed and

compared with a placebo treatment (water only). A total of 4 clinic visits were

conducted in the morning 1 week apart on the same day of the week and at the same

time of the day. Subjects received each of the following four treatments in random

order: water (control) 300 mL, quercetin (200 mg) dissolved in 300 mL of water, (-)-

99

epicatechin (200 mg) dissolved in 300 mL of water and EGCG (200 mg) dissolved in

300 mL of water. These amounts were chosen as they represent a reasonable dose which

could be achieved by eating flavonoids – rich foods such as chocolate, onions and tea.

The order of the treatments was randomly assigned using computer – generated random

numbers. The subjects had not fasted, but they were asked to follow a moderately

restricted flavonoid diet: no tea, red wine, chocolate, cocoa or fruit juice for 48 hours

prior to each treatment. In addition, subjects consumed a meal of the same composition

for breakfast prior to each treatment. They also did not consume alcohol or engage in

vigorous physical activity for 24 hours before each visit. On the treatment day, a blood

and spot urine sample were collected from the subjects prior to the prescribed treatment.

A second blood sample was taken two hours after oral administration of the treatment.

A 5-hour total urine collection was also performed. Subjects consumed each of the four

drinks over 2-3 minutes, one at each visit, in random order.

4.2.4. Measurement of S-nitrosothiols

Plasma levels of circulating NO• pool (S-nitrosothiols, N-nitrosothiols and

iron-nitrosyl complexes) were measured using a previously described gas phase

chemiluminescence method231

. Analyses were performed immediately after blood

collection. Briefly, a mixture of fresh plasma (2.5 mL), NEM (5 mM), SFA (0.5% in 0.1

M HCl) and antifoam (200 µL) was injected into the radical purger containing

potassium iodide (75 mM) and copper(II) sulphate (10 mM) in glacial acetic acid (10

mL) at 70 ºC. NO• liberated by the redox reactions was quantified by its

chemiluminescence reaction with ozone using the Nitric Oxide Analyzer (Sievers NOA

280i).

100

4.2.5. Measurement of nitrite and nitrate

Nitrite and nitrate concentrations in plasma and urine were determined

simultaneously using a previously published GC-MS method232

. Briefly, the sample

fluid was spiked with internal standards, sodium nitrite-15

N (6 ng) and sodium nitrate-

15N (40 ng). The spiked sample was derivatised with acetone and PFPBr at 50 ºC for 30

minutes. After the removal of acetone, the remaining aqueous phase was extracted with

toluene and the organic extract (0.5 µL) was analyzed using an Agilent 6890 gas

chromatograph coupled to a 5973 mass spectrometer fitted with a cross-linked methyl

silicone column (25m x 0.20 mm, 0.33 mm film thickness, HP5-MS) using negative-ion

chemical ionisation. Samples (1.0 μL) were injected in the splitless mode and the oven

temperature was held at 70 °C for 1 min, then increased to 160 °C at a rate of 20 °C/

min and finally to 280 °C at a rate of 30 °C/min. Helium (92.5 kPa and flow rate 0.7

mL/ min) was used as the carrier and methane as the reagent gas for negative-ion

chemical ionization. Peak identification was based on retention time and mass spectra

compared with N-15-labeled authentic standards (sodium [15

N]nitrite and sodium

[15

N]nitrate). Quantification was performed using calibration curves obtained from

authentic standards and labeled standards.

4.2.6. Measurement of endothelin-1

The acute effects of quercetin, (-)-epicatechin and EGCG on systemic ET-1

production were investigated by measuring its concentration in the initial and 5-hour

urine samples using a commercially available ET-1 (human) EIA kit (Assay Design,

GA). ET-1 concentrations were adjusted for creatinine levels.

4.2.7. Systemic oxidative stress

Plasma and urinary F2-isoprostanes, a well established marker of systemic

oxidative stress, were determined by GC-MS using a previously described method184

.

101

4.2.8. Metabolism of quercetin, (-)-epicatechin and epigallocatechin gallate

Quercetin and (-)-epicatechin are present in plasma and urine as glucuronides,

sulfates and in their methylated forms with very small amounts present in their free

forms93, 233

. Absorption of quercetin, (-)-epicatechin and EGCG were determined by

measuring the amounts of free quercetin, 3′-O-methylquercetin, (-)-epicatechin, 3′-O-

methyl-(-)-epicatechin and EGCG after the hydrolysis of conjugates in the baseline

plasma and urine, 2-hour plasma and 5-hour urine samples using a previously reported

GC-MS method234

. Briefly, 1-hydroxy-2-naphthoic acid (50 ng, internal standard) was

added to plasma or urine (750 μL) and acidified to pH 4.8 with dilute hydrochloric acid.

Thirty microliters of β-glucuronidase with sulfatase activity (3000 units of

glucuronidase activity and 1500 units of sulfatase activity) was added, mixed, and

incubated at 37 °C for 24 hours with occasional mixing. Samples were then extracted

twice with ethyl acetate (1 mL), dried under nitrogen, and derivatized with BSTFA (100

μL) and pyridine (50 μL) at 40 °C for 60 min. The trimethylsilyl (TMS) derivatives

were analyzed on an Agilent 6890 gas chromatograph coupled to a 5973 mass

spectrometer using a cross-linked methyl silicone column (25m x 0.20 mm, 0.33 mm

film thickness, HP5-MS). Aliquots (1.0 μL) were injected in the splitless mode, the

column temperature was held at 150 °C for 1 min, and then increased to 300 °C at a rate

of 20 °C/ min and to 320 °C at a rate of 30 °C/min. Helium (0.7 ml/min) was used as the

carrier gas. Peak identification was based on retention time and mass spectra compared

with authentic standards (quercetin, 3′-O-methylquercetin, (-)-epicatechin, 3′-O-methyl-

(-)-epicatechin and EGCG). Quantification was performed using calibration curves

obtained from authentic standards and internal standard.

4.2.9. Statistical analysis

Statistical analyses were performed using SAS version 9.0 or SPSS version

11.5. Data is presented as mean ± SEM. The baseline-adjusted between group

102

differences were analysed with random effects models using PROC MIXED (SAS) with

Tukey adjustment for multiple comparisons. In these models, subject was treated as the

random effect, and treatment, period and treatment order as the fixed effects. The results

analysed were considered significantly different if p value < 0.05.

4.3. RESULTS

4.3.1. S-Nitrosothiols, nitrite and nitrate production

The acute effects of quercetin, (-)-epicatechin and EGCG on NO• status were

investigated by measuring the amounts of well-established in-vivo products of NO•. No

significant difference in the baseline concentrations of plasma S-nitrosothiols, plasma

nitrite and nitrate, and urinary nitrite and nitrate was observed among the four groups.

Acute treatment with quercetin (200 mg) significantly increased plasma S-nitrosothiols,

plasma nitrite and urinary nitrate concentrations (p < 0.0001 vs water control), as well

as plasma nitrite and urinary nitrate concentrations (Figure 4.1, 4.2, 4.3 and 4.4).

Similar significant increases in S-nitrosothiols (p < 0.05 vs water control), plasma nitrite

and urinary nitrate concentrations were observed with (-)-epicatechin (200 mg)

treatment (p < 0.05 vs water control) (Figure 4.1, 4.2, 4.3 and 4.4). No significant

changes in plasma nitrate and urinary nitrite levels were observed for any treatments.

EGCG did not significantly alter any measure of NO• products (Figure 4.1, 4.2, 4.3 and

4.4).

103

Baseline 2 h0

10

20

30708090

Pla

sma

[SN

O]

(nM

equiv

. of

NO

)

(A)

Baseline 2 h0

10

20

30

Pla

sma

[SN

O]

(nM

equiv

. of

NO

)

(B)

Baseline 2 h0

5

10

15

Pla

sma

[SN

O]

(nM

equiv

. of

NO

)

(C)

104

Baseline 2 h0

5

10

15

Pla

sma

[SN

O]

(nM

equiv

. of

NO

)

(D)

Figure 4.1: Plasma S-nitrosothiols concentrations before and 2 hours after ingestion of

(A) quercetin, (B) (-)-epicatechin, (C) epigallocatechin gallate (200 mg each in 300 mL

water) and (D) water (300 mL) for 12 healthy male volunteers.

Q EC EGCG W0

5

10

15

20

25

30

35 *

**

Treatment

Pla

sma

[SN

O]

(nM

eq

uiv

. of

NO

)

Figure 4.2: Plasma S-nitrosothiols concentrations (nM equiv. of NO•) before (white bar)

and 2 hours after (black bar) ingestion of quercetin (Q), (-)-epicatechin (EC),

epigallocatechin gallate (EGCG) (200 mg each) compared with water (W) (placebo)

(Mean ± SEM, n = 12). No significant difference among groups was observed at

baseline. * p < 0.0005 vs water (mixed model analysis with Tukey test) after baseline

adjustment. ** p < 0.05 vs water (mixed model analysis with Tukey test) after baseline

adjustment.

105

Q EC EGCG W0

1

2

3

4

5

6*

*(A)

Treatment

Pla

sma [

Nit

rite

]

( M

)

Q EC EGCG W0

5

10

15

20

25(B)

Treatment

Pla

sma

[Nit

rate

]

( M

)

Figure 4.3: Plasma (A) nitrite and (B) nitrate concentrations (μM) before (white bar)



(Mean (± SEM), n = 12). No significant difference among groups was observed at


adjustment.

106

Q EC EGCG W0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75(A)

Treatment

Uri

nar

y [

Nit

rite

]

( M

)

Q EC EGCG W0123456789

101112

*

**(B)

Treatment

Uri

nar

y [

Nit

rate

]

( M

)

Figure 4.4: Urinary (A) nitrite and (B) nitrate concentrations (μM) before (white bar)



(Mean (± SEM), n = 12). No significant difference among groups was observed at


adjustment.

107

4.3.2. Endothelin-1 production

There was no significant difference in the baseline concentrations of plasma

and urinary ET-1 among the four treatments. Acute treatment with quercetin and (-)-

epicatechin significantly reduced plasma concentrations of ET-1 (p < 0.001 and p < 0.05

for quercetin and (-)-epicatechin respectively vs water control) 2 hours after ingestion

(Figure 4.5). Only quercetin (200 mg) produced a significant acute reduction in urinary

ET-1 concentrations (p < 0.001 vs baseline and water control) over the 5 hours after oral

ingestion (Figure 4.6). The reduction of ET-1 levels observed with (-)-epicatechin

treatment was not significant when compared with either the water control or baseline.

EGCG did not show any effect on ET-1 production (Figure 4.6).

Q EC EGCG W0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75* #

Treatment

Pla

sma

[ET

-1]

(pg/

mL

)

Figure 4.5: Plasma endothelin-1 concentrations (pg/ mL) before (white bar) and 2 hours

after (black bar) ingestion of quercetin (Q), (-)-epicatechin (EC), epigallocatechin

gallate (EGCG) (200 mg each) compared with water (placebo) (Mean (± SEM), n = 12).

No significant difference among groups was observed at baseline. * p < 0.001 vs water

after baseline adjustment. # p < 0.05 vs water after baseline adjustment (mixed model

analysis with Tukey test).

108

Q EC EGCG W0

102030405060708090

100110120

*

Treatment

Uri

nary

[E

T-1

]

(pg/

mm

ol

crea

tinin

e)

Figure 4.6: Urinary endothelin -1 concentrations before (white bar) and 5 hours (black

bar) after ingestion of quercetin (Q), (-)-epicatechin (EC), epigallocatechin gallate

(EGCG) (200 mg each) compared with water (placebo) (n = 12). *p < 0.0005 vs

baseline (paired t-test) and water (mixed model analysis).

Plasma F2-isoprostanes

(pg/ mL)

Urinary F2-isoprostanes

(pg/ mmol creatinine)

Baseline 2 hours Baseline 5 hours

Water (Placebo)

509.3±33.1

544.0±40.1

205.4±40.4

226.4±39.9

Quercetin (200 mg)

501.9±36.7

530.6±38.1

248.0±61.6

235.1±51.4

(-)-Epicatechin (200 mg)

493.9±19.5

482.5±27.0

226.7±68.7

216.3±56.5

Epigallocatechin

Gallate (200 mg)

524.7±52.4

487.8±26.4

244.0±71.3

242.7±65.7

Table 4.1: Plasma F2-isoprostanes concentrations and urinary F2-isoprostanes

concentrations before and after ingestion of quercetin (Q), (-)-epicatechin (EC),

epigallocatechin gallate (EGCG) (200 mg each) compared with water (placebo) (n =

12).

109

4.3.3. Systemic oxidative stress

The acute effects of the three flavonoids on systemic oxidative stress were

determined by measuring the plasma and urinary F2-isoprostanes concentration before

and after the treatments. None of the treatments significantly affected acute plasma or

urinary F2-isoprostanes concentrations (Table 4.1).

4.3.4. Quercetin, (-)-Epicatechin and EGCG absorption

The absorption of quercetin and (-)-epicatechin was investigated by measuring

the total quercetin and (-)-epicatechin concentrations present in the circulation 2 hours

after ingestion and the amounts excreted 5 hours after ingestion. The total flavonoid

concentration was calculated as the sum of the flavonoid and its 3′-O-methyl-derivatives

after enzymatic hydrolysis with glucuronidase and sulfatase. The mean (± SEM)

baseline circulating concentrations of total quercetin and total (-)-epicatechin were 0.84

± 0.39 µM and 0.70 ± 0.34 µM respectively. Acute treatment with quercetin and (-)-

epicatechin significantly increased the total circulating concentration of each flavonoid

(3.54 ± 1.57 µM for quercetin and 3.57 ± 1.21 µM for (-)-epicatechin) (p < 0.001 vs

baseline) (Figure 4.7A and 4.7B). There were significant increases in the circulating

concentrations of quercetin (p < 0.001), 3′-O-methylquercetin (p < 0.05), (-)-epicatechin

(p < 0.001) and 3′-O-methyl-(-)-epicatechin (p < 0.005) 2 hours after the flavonoid

ingestion when compared to their baseline levels.

The baseline concentration of total quercetin and total (-)-epicatechin present

in the urine were 0.61 ± 0.15 µmol/ mmol creatinine and 0.50 ± 0.28 µmol/ mmol

creatinine respectively. Acute treatment with quercetin and (-)-epicatechin significantly

increased the total amount of each flavonoid excreted over the 5-hour period (p < 0.001

vs baseline) to 2.51 ± 0.65 µmol/ mmol creatinine for quercetin and 2.62 ± 0.98 µmol/

mmol creatinine for (-)-epicatechin respectively (Figure 4.8A and 4.8B).

110

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

*

*

#

(B)

Treatment

Pla

sma

[Fla

vonoid

s](

M)

Figure 4.7: (A) Plasma quercetin (Q) and 3′-O-methylquercetin (3′-MQ) concentrations

(μM) (after hydrolysis of glucuronide and sulfate conjugates) and total Q concentration

before (white bar) and 2 hours after (black bar) ingestion of Q (200 mg each) (Mean (±

SEM), n = 12). (B) Plasma (-)-epicatechin (EC) and 3′-O-methyl-(-)-epicatechin (3′-

MEC) concentrations (μM) (after hydrolysis of glucuronide and sulfate conjugates) and

total EC concentration before (white bar) and 2 hours after (black bar) ingestion of EC

(200 mg each) (Mean (± SEM), n = 12). * p < 0.001 vs baseline levels (paired t-test). #

p < 0.005 vs baseline level (paired t-test).

111

Q 3'-MQ Total Q0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5* *

*

(A)

Treatment

Uri

nar

y [

Fla

vonoid

s]

( m

ol/

mm

ol

crea

tinin

e)

EC 3'-MEC Total EC0

1

2

3

4

*

*(B)

Treatment

Uri

nar

y [

Fla

vonoid

s]

( m

ol/

mm

ol

crea

tinin

e)

Figure 4.8: (A) Urinary quercetin (Q) and 3′-O-methyl quercetin (3′-MQ)

concentrations (μmol/ mmol creatinine) (after hydrolysis of glucuronide and sulfate

conjugates) and total Q concentration before (white bar) and 5 hours after (black bar)

ingestion of Q (200 mg each) (Mean (± SEM), n = 12). * p < 0.05 vs baseline levels

(paired t-test). (B) Urinary (-)-epicatechin (EC) and 3′-O-methyl-(-)-epicatechin (3′-

MEC) concentrations (μmol/ mmol creatinine) (after hydrolysis of glucuronide and

sulfate conjugates) and total (-)-epicatechin concentration before (white bar) and 5

hours after (black bar) ingestion of EC (200 mg each) (Mean (± SEM), n = 12). *p <

0.05 vs baseline levels (paired t-test).

112

Pre Post0.000.010.020.030.040.050.060.070.080.090.100.110.12 *

Pla

sma

[EG

CG

]

( M

)

Figure 4.9: Plasma epigallocatechin gallate (EGCG) concentrations (µM) (after

hydrolysis of glucuronide and sulfate conjugates) before (white bar) and 2 hours (black

bar) after ingestion of EGCG (200 mg ) (n = 12). * p < 0.05 vs baseline.(paired t-test).

Treatment with EGCG significantly augmented the amount of circulating

EGCG (p < 0.05 vs baseline) with its mean circulating concentration increasing from

0.06 ± 0.01 µM to 0.10 ± 0.01 µM (Figure 4.9). We were unable to reliably detect

EGCG excretion in urine using GCMS methods.

4.3.5. Correlations of NO• products and ET-1 with plasma flavonoid concentrations

Only plasma S-nitrosothiols levels showed a significant positive correlation

with plasma quercetin (r = 0.815, p < 0.01) and (-)-epicatechin (r = 0.840, p < 0.01)

concentrations (Figure 4.10). Plasma nitrite, urinary nitrate and urinary ET-1 levels

were not found to be significantly correlated to the plasma concentrations of the

flavonoids.

113

(A)

0

10

20

30

40

50

60

70

80

0 1 2 3 4 5 6

Change in plasma quercetin concentration

Chan

ge

in p

lasm

a S

-nitro

soth

iols

conce

ntr

atio

nr = 0.815

p < 0.01

(B)

0

5

10

15

20

25

0 1 2 3 4 5 6

Change in plasma (-)-epicatechin concentration

Chan

ge

in p

lasm

a S

-nitro

soth

iols

conce

ntr

atio

n

r = 0.840

p < 0.001

Figure 4.10: Linear correlation between plasma concentrations of (A) quercetin, (B) (-)-

epicatechin and plasma S-nitrosothiols.

114

4.4. DISCUSSION

We showed that acute treatment with quercetin and (-)-epicatechin, but not

EGCG, augmented endogenous NO• products (S-nitrosothiols, nitrite and nitrate).

Quercetin in addition reduced ET-1 production. These molecules may therefore improve

endothelial function in healthy human subjects. NO• reacts with the free thiol groups in

proteins under physiological conditions to form S-nitrosothiols223

. These S-nitrosothiols

possess the same effects as NO• upon vasodilatation and platelet inhibition

235, but with

half-lives of the order of hours223

. In addition, they exhibit the physiological benefit of

being resistant to, unlike free NO•236

. The comparably higher concentrations of S-

nitrosothiols in the human circulation (~7 µM S-nitrosothiols vs ~3 nM free NO• in

human plasma) suggests that plasma S-nitrosothiols may serve as a reservoir for NO•,

effectively buffering its concentration and thereby maintaining NO• homeostasis

237.

Free NO• can also undergo oxidation in human plasma to form mainly nitrite but also

nitrate238

. However, recent in vivo studies have reported that circulating nitrite, rather

then nitrate reflects endothelial-dependent NO• synthesis in humans and mammals

239,

240.

Numerous studies have shown that acute and repetitive consumption of

flavonoid – rich foods for up to four weeks can improve endothelial function in both

subjects with coronary artery disease and healthy volunteers143, 241

. Flavonoids are

presumed to be the active constituents. However, to date there is little direct evidence

that flavonoids are the bioactive molecules responsible. We have demonstrated that oral

administration of pure dietary flavonoids, quercetin and (-)-epicatechin augment NO•

status in healthy male volunteers. This is shown by the significant elevation of

circulating S-nitrosothiols and nitrite concentrations (Figure 4.2 and 4.3). The changes

in plasma S-nitrosothiols concentrations were also shown to be significantly correlated

to the changes in plasma quercetin and (-)-epicatechin concentrations (Figure 4.10). Our

115

results confirm that flavonoids, such as quercetin and (-)-epicatechin do indeed

influence NO• status in humans as reported in recent in vitro experiments

242 and animal

studies243

. Quercetin may have increased NO• production by increasing eNOS activity

243, 244 or enhancing the bioavailability of endothelium-derived NO

• 245

. (-)-Epicatechin

has been shown to elevate NO• in endothelial cells in-vitro via the inhibition of NOX

242.

Schroeter H. et al146

reported that oral administration of pure (-)-epicatechin to humans

closely emulated the acute vascular effects of the flavonol-rich cocoa. Quercetin and (-)-

epicatechin may also act as antioxidants reducing nitrites and nitrates to free NO• 246

.

Quercetin treatment showed a significant reduction in systemic ET-1

production in this study while (-)-epicatechin may have shown a decrease but not at the

95% confidence level (Figure 4.6). ET-1 has been demonstrated to be associated with

increased oxidative stress and endothelial dysfunction in humans. ET-1 stimulates O2•-

production and vasoconstriction through activation of NOX and uncoupled NOS in the

rat aorta82

. It also reduces NO• bioavailability via interference with the expression and

activity of eNOS81

, indicating that diminished ET-1 level may be accompanied by

elevated NO• bioavailability. It was reported that NO

• inhibits ET-1 production through

the suppression of nuclear factor κB247

. There seems to be an inverse relationship

between NO• and ET-1, which may serve to modulate endothelial function in the

vasculature. Quercetin was shown to decrease ET-1 production in thrombin-stimulated

cultured human umbilical vein endothelial cells in a dose-dependent manner with an

IC50 of 1.54 µM150

. Red wine polyphenols were recently shown to prevent vascular

oxidative stress by inhibiting NOX activity and/or by reducing ET-1 release248

.

Surprisingly, EGCG did not show the same augmentation of NO• products as

quercetin and (-)-epicatechin (Figure 4.2, 4.3 and 4.4). EGCG has been widely assumed

to be the vaso-active flavonoid present in green tea which offers vascular protection

against CVD249

. EGCG was shown to mediate NO• -dependent vasodilation in rat aortic

116

rings250

and was found to work primarily by the rapid activation of eNOS and an

increase in eNOS activity, independent of an altered eNOS protein content251

. However,

it must be noted that these studies reported the effects of EGCG at non-physiologically

relevant concentrations. We carried out experiments to ascertain if EGCG had degraded

during the process of dissolution and found that EGCG does degrade with time (up to

45% in 30 minutes) in the aqueous mixture prepared for this study (200 mg/ 300 mL

water). However, at least 95% of the prescribed 200 mg dose was present in the aqueous

mixture at the time of consumption (1-2 minutes after dissolution) (data not shown).

EGCG was present at much lower concentrations (0.1 ± 0.01 µM) in the circulation than

quercetin (3.54 ± 1.57 µM) and (-)-epicatechin (3.57 ± 1.21 µM) after acute treatment

(Figure 4.7 and 4.9). Similar circulating concentrations of EGCG were reported in a

recent study in which a 300 mg dose of EGCG acutely improved brachial artery flow-

mediated dilation measured by vascular ultrasound in humans with coronary artery

disease230

. If improved endothelial function is brought about by EGCG, the compound

is likely to have exerted its effects through mechanisms other than those mediated by

NO• or ET-1.

Oral administration of quercetin, (-)-epicatechin and EGCG (200 mg) did not

acutely affect plasma and urinary F2-isoprostanes (Table 4.1). There is a growing body

of evidence from controlled human trials casting doubt as to whether flavonoids can act

as antioxidants in vivo. While plasma (-)-epicatechin and EGCG concentrations were

increased after consumption of dark chocolate and black tea, they neither improved

plasma antioxidant capacity nor reduced urinary 8-isoprostane levels114, 135, 252

. In

another trial where subjects consumed onions, significantly elevated plasma quercetin

levels did not result in any significant effects on plasma F2-isoprostanes

concentrations108

. As oxidative stress is implicated in the development of CVD, one of

the main properties thought to explain flavonoids effects is their antioxidant property100

.

117

However, recent studies carried out in this area should be interpreted with caution as the

native unmodified forms of flavonoids found in the diet were utilised in in vitro

experiments instead of the metabolites found in vivo. We have recently shown that

structural modification of flavonoids, such as quercetin, by metabolic transformation, is

likely to have a profound effect on biological activity253, 254

. There is also the issue of

bioavailability, where plasma concentration of flavonoids may reach between 2-5 μM

following supplementation with flavonol rich foods (such as onions or apples), or

various flavonoid-glycosides, at doses of 50-200 mg equivalents90

. Since metabolism of

flavonoids is likely to influence bioactivity it is interesting to note that metabolites

possessing the 3′-O methyl function have increased activity as inhibitors of NOX255

.

Overall our study suggests that pure dietary flavonoids, such as quercetin and

(-)-epicatechin may improve endothelial function acutely by modulating the circulating

levels of vasoactive NO• products and ET-1. These effects may be exerted possibly via

the inhibition of NOX and activation of eNOS. Similar studies, using pure dietary

flavonoids, should be performed over a longer trial period to investigate their chronic

effects.

118

CHAPTER 5:

SPECIFIC DIETARY POLYPHENOLS ATTENUATE

ATHEROSCLEROSIS IN APOE KNOCKOUT MICE VIA

ALLEVIATING OXIDATIVE STRESS, INFLAMMATION

AND ENDOTHELIAL DYSFUNCTION

5.1 INTRODUCTION

Atherosclerosis is a multi-factorial disease developing over many years with

symptoms becoming apparent in the late stages of the disease. Inflammation7, oxidative

stress1 and endothelial dysfunction

62 are associated with the pathogenesis of

atherosclerosis. Polyphenols are naturally-occurring compounds found in fruits and

vegetables and are currently the focus of much nutritional and therapeutic interest.

Results of population studies suggest that adopting polyphenol-rich diets may protect

against CVD86-88

, while animal and human intervention studies indicate cardiovascular

protective effects of polyphenol-rich food122, 144, 146, 252, 256

. Mechanisms by which these

compounds exert their cardiovascular protective effects are inconclusive. It is widely

hypothesized that dietary polyphenols protect against atherosclerosis by preventing one

or more of the processes involved in disease progression such as oxidative stress (lipid

and protein peroxidation), inflammation and endothelial dysfunction89

. However, there

are many hundreds of polyphenols in the human diet and it is not yet known if some

compounds offer more cardiovascular protection than others.

In this study, we selected a few common dietary polyphenols (structures in Fig.

5.1), such as quercetin (a flavonol found in the diet from fruits, vegetables and tea), (-)-

epicatechin (a flavan-3-ol from cocoa and tea), theaflavin (a dimeric catechin from

black tea), sesamin (a lignan from sesame seeds) and chlorogenic acid (a phenolic acid

from coffee and some fruits) for this study. Quercetin from grapes has been shown to

119

improve the lipoprotein profile and reduce plasma inflammatory biomarkers and

oxidized low density lipoprotein in healthy human subjects, which may decrease

cardiovascular disease risk122, 256

. Quercetin reduces blood pressure and improves

endothelial function in several rat models of hypertension257

. Previous human

intervention studies have indicated that (-)-epicatechin from cocoa improves endothelial

function146

and reduces inflammation252

. We have demonstrated in an acute human

intervention study that quercetin and to a lesser extent (-)-epicatechin are able to

augment NO• production and reduce ET-1, whereas the larger molecular weight

catechin, epigallocatechin gallate, had no effect (Chapter 4)258

. In vitro studies with

leukocytes indicate that anti-inflammatory activity of flavonoids may be dissociated

from their antioxidant activity (Chapter 2)253

. Theaflavin was included in our study

because it is a major constituent of black tea which is widely consumed in many

countries and may offer similar antioxidant potency as green tea catechins259

. Black tea

consumption has been shown to improve endothelial function in human patients with

coronary artery disease144

. Sesamin is a bioactive lignan in sesame seeds which was

shown to reduce LDL cholesterol and interfere with the metabolism of the γ-

tocopherol260

. Chlorogenic acid, a major constituent of coffee, acts as an anti-oxidant in

vitro261

. Given concerns about the bioavailability of polyphenols in vivo90

, we examined

their effects when incorporated into the diet of a well established mouse model of

atherosclerosis. We determined if these pure polyphenols prevent or reduce the

formation of atherosclerotic lesions in ApoE-/-

mice and investigated the mechanisms by

which these compounds may exert their anti-atherosclerotic effects. This study may

provide insight into the beneficial effects of consuming polyphenol-rich diets.

120



(-)-Epicatechin, PFPBr, adenosine triphosphate (ATP), arabic gum, BSA,

BHT, calcium ionophore, calcium chloride, chlorogenic acid, formaldehyde, glucose,

Hepes, heptadecanoic acid, Kaiser's glycerine glycol, lucigenin, magnesium sulphate,

BSTFA, nicotinamide adenine dinucleotide, reduced (NADH), potassium chloride,

potassium phosphate monobasic, pyridine, quercetin, sodium chloride, sodium

hydrogencarbonate, sodium nitrate-15

N, sodium nitrite-15

N, sodium phosphate dibasic,

Sudan (IV) and toluene were purchased from Sigma Aldrich (St Louis, MO, USA);

arachidonic acid from Cayman Chemical (Michigan, USA); PBS from Gibco™

Invitrogen (Calsbad, CA, USA); acetone, chloroform, ethanol, hexane, methanol, n-

heptane, sulfuric acid from Univar (WA, Australia). Sesamin and theaflavin were kindly

provided by Suntory (Japan) and Unilever (Netherlands) respectively.

5.2.2. C57BL and ApoE-/-

Mice

The study was approved by and performed under the guidelines of the Animal

Ethics Committees of the University of Western Australia and Royal Perth Hospital.

One hundred and fifty, 4 week-old male, ApoE-/-

mice and twenty five C57BL mice

were obtained from the Animal Resource Centre (Canningvale, WA, Australia). The

mice were housed in groups of five and were immediately placed on a non-purified

stock diet AIN93M (Glenforrest Stockfeeds, WA, Australia) during the entire course of

the study (calculated nutritional parameters in Table 5.1). One hundred and twenty five

ApoE-/-

mice were randomised to receive either quercetin (1.3 mg daily; 64 mg/ kg body

mass daily), (-)-epicatechin (1.3 mg daily; 64 mg/ kg body mass daily), theaflavin (1.3

mg daily; 64 mg/ kg body mass daily), sesamin (1.3 mg daily; 64 mg/ kg body mass

daily) or chlorogenic acid (1.3 mg daily; 64 mg/ kg body mass daily) (n=25 for each

treatment group) (See Figure 1 for structures). The prescribed dosage is approximately

121

Table 5.1.: Calculated nutritional parameters of AIN93M mouse diet

Protein 13.6%

Total fat 4%

Crude fibre 4.7%

Acid detergent fibre 4.7%

Digestive energy 15.1 MJ/ kg

equivalent to 350 mg/ day dosage in humans262

. The treatment compounds were blended

with the mouse feed and stored at 0 °C until used. The control groups of 25 ApoE-/-

mice and 25 C57BL wild-type mice received the untreated mouse feed. The mice were

started on the prescribed treatment from the age of 6 weeks till the end of the study. All

fluids were changed at least three times a week. Food, fluid intake and body weight

were monitored on a regular basis throughout the study. Urine was collected from each

group in metabolic cages at 16 and 26 weeks of age. After 10 weeks of treatment when

the mice were 16 weeks of age, 5 mice from each group were killed for analysis of early

lesion development as well as plasma and aortic biochemistry. The remaining mice

were killed for the same analyses at 26 weeks of age (i.e. following 20 weeks of

treatment). Animal numbers were based on the power analysis performed on the desired

end-points (p < 0.0083 for multiple comparisons between the treatment groups and

control groups) as well as on a previous study which showed significant differences in

lesion area by 26 weeks of age167

.

122

OHO

OH O

OH

OH

OH

Quercetin

HO

OH

O

OH

OH

OH

(-)-Epicatechin

O

OH

HO

OH

HO

OHOH

O

O

OH

OH

RR

Theaflavin

RR

HO

O

O

O O

H

HO

O

S RSR

Sesamin

HO

HO

OH

COOH

O

O

OH

OH

R

RR

R

Chlorogenic Acid

Figure 5.1: Structures of polyphenols used in the study.

5.2.3. Isolation of plasma and aortic tissue

Non-fasting mice from each group were studied at 16 weeks (n = 5) and 26

weeks (n = 20) of age. After the mice were anesthetized with the use of an intra-

peritoneal injection of Nembutal (Boehringer Mannheim, Mannheim, Germany), the

abdominal and thoracic cavities were opened by ventral incision. A blood sample was

obtained via vena cava puncture and collected into microfuge tubes containing 50 µL

EDTA (1 g/ 10 mL in 0.9% saline). The blood plasma was collected by centrifugation at

5000xg for 10 minutes at 4 ºC, and was stored after addition of BHT (8 μg/ mL) at -80

°C. The aortic sinus, thoracic and abdominal aorta were removed and stripped of any

123

external fatty deposits. Aortas for histopathologic analysis were fixed in phosphate-

buffered formaldehyde (4% by volume, pH 7.0 – 7.3) and stored at 4 ºC167

. After

removal and cleansing of extraneous fat, aortas for biochemical analysis were washed in

PBS containing EDTA (0.38 mg/mL) and butylated hydroxytoluene (20µM), blotted

dry, and immediately stored in fresh PBS at -80 °C263

.

5.2.4. Histological analysis of mouse aortas

The amount of atherosclerotic lesion in the mouse aorta was determined by

measuring the cross-sectional lesion area using procedures described previously263

. The

aorta was rinsed in PBS after removal from the phosphate-buffered formaldehyde (4%

by volume, pH 7.0 – 7.3). It was then placed in 0.5 mL of a gum sucrose solution (15%

sucrose and 1% arabic gum in water, w/w) and left overnight at 4 ºC. The next day, the

aortic tissue was rinsed in PBS and blotted dry before it was completely frozen in OCT

compound (Tissue-Tek®) and then cryostat-sectioned (20 µm thickness) using a cryostat

(Leica CM3050S). The section was stained with Sudan (IV) solution (0.5 g Sudan (IV),

35 mL ethanol, 50 mL acetone, and 15 mL water), rinsed with 80% ethanol, blotted dry

and covered with a coverslip using Kaiser's glycerine glycol. The specimen was

examined using light microscopy with a built-in camera under 10 x magnification

(Nikon Eclipse TS100). The total aortic tissue and lesion areas were analysed on the

image collected using Nikon NIS Elements Imaging Software BR 2.30, SP2 (Build

361). Lesions were stained red. Cross-sectional lesion areas were measured at both the

aortic sinus and the region immediately below the thoracic arch. The observer was

blinded to treatment groups.

5.2.5. Plasma cholesterol and aortic fatty acid composition

The total cholesterol content of the mouse plasma samples was measured

using a commercially available cholesterol assay kit (Boehringer Mannheim). To

124

measure the aortic fatty acid composition and F2-isoprostanes, frozen aortic tissue was

thawed, weighed, and homogenised in 2 mL PBS. Aortic F2-isoprotanes and fatty acids

were extracted using ice-cold Folch solution (chloroform:methanol, 2:1 by volume

containing 0.1 mM BHT). The chloroform layer (containing the F2-isoprostanes and

fatty acids) was collected and divided into two equal volumes before drying under

nitrogen. For fatty acid analysis, one of the dried lipid extracts from the mouse aortas

was heated in boiling water for 10 minutes with 2 mL 4% H2SO4 in methanol and 50 µL

heptadecanoic acid (internal standard, stock 1 mg/ mL). The methyl esters of fatty acids

were analysed by gas chromatography as previously described167

.

5.2.6. Systemic and vascular oxidative stress

Systemic and vascular oxidative stress were assessed by measuring F2-

isoprostanes in the urine and aortic tissue respectively by GC-MS using a previously

described method184

. Aortic F2-isoprostanes were measured in the second lipid extract

from the mouse aortas (above) and corrected for the arachidonic acid content of the

aortic tissue. Urinary F2-isoprostanes concentrations were corrected for urinary

creatinine levels.

Superoxide analysis by lucigenin-derived chemiluminescence264

was

performed to assess vascular oxidative stress. Briefly, the fresh aortic tissue was

weighed before placement in an opaque 96-well microtiter plate in PBS at pH 7.5 with

100 µM NADH followed by incubation at 37 °C under 95% O2, 5% CO2 for 30 minutes

and luminescence measurement with a Wallac Victor-II (PerkinElmer Life Sciences) in

the luminometry mode. Lucigenin at a final concentration of 5 µM was added and

luminescence count recorded at 1-minute intervals for 30 minutes. The concentration of

lucigenin was kept at 5 µM to prevent redox-recycling which can lead to increased O2•-

production265

. The residual aortic tissue was then homogenised in 1 mL PBS and the

protein content in the homogenate was determined as previously described203

. The

125

amount of O2•- radical ion produced was corrected for the protein content of the aortic

tissues.

5.2.7. Ex vivo vascular Leukotriene B4 production

The effects of the polyphenol treatments on 5-lipoxygenase enzyme activity

were determined by measuring vascular leukotriene B4 (LTB4) production ex vivo. Fresh

aortic tissue was weighed before immersing in HBHS [CaCl2.2H2O (0.09 g), glucose

(0.50g), Hepes (0.06 g), KCl (0.20 g), KH2PO4 (0.03 g), MgSO4.7H2O (0.10 g),

NaHCO3 (0.18 g), NaCl (4.00 g), Na2HPO4 (0.02 g) and BSA (0.50 g) in pure water

(500 mL); pH 7.4]. The tissue was then incubated with ATP (final concentration 2 mM),

Ca ionophore (final concentration 2.5 µg/ mL) and arachidonic acid (final concentration

10 µM) at 37 ºC for 30 minutes. The supernatant was analysed for LTB4 using a specific

LTB4 enzyme immunoassay (EIA) kit (Cayman Chemical, Michigan, USA). The

residual aortic tissue was then homogenised in 1 mL PBS and the protein content in the

homogenate was determined as previously described203

.

5.2.8. Plasma soluble P-Selectin

The chronic effects of quercetin, (-)-epicatechin, theaflavin, sesamin and

chlorogenic acid on platelet reactivity were investigated by measuring the plasma

concentrations of soluble P-selecitn (sP-selectin) using a commercially available mouse

sP-selectin EIA kit (R&D Systems, MN, USA).

5.2.9. Vascular eNOS activity, urinary nitrite, nitrate and endothelin-1

The effects of the prescribed polyphenols on vascular NO• production were

investigated by measuring vascular eNOS activity as well as urinary nitrite and nitrate.

NOS activity in aortic homogenates was determined by monitoring the conversion of L-

[3H]arginine to L-[

3H]citrulline using NOS activity assay kit (Cayman Chemical,

126

Michigan, USA). Results were expressed as picomoles of L-citruline per milligram of

protein per 60 min. Nitrite and nitrate concentrations in urine were determined

simultaneously using a previously published GC-MS method232

. Briefly, the urine was

spiked with internal standards, sodium nitrite-15

N (6 ng) and sodium nitrate-15

N (40 ng).

The spiked sample was derivatised with acetone and PFPBr at 50 ºC for 30 minutes.

After the removal of acetone, the remaining aqueous phase was extracted with toluene

and the organic extract (0.5 µL) was analyzed using an Agilent 6890 gas chromatograph

coupled to a 5973 mass spectrometer fitted with an cross-linked methyl silicone column

(25m x 0.20 mm, 0.33 mm film thickness, HP5-MS) using negative-ion chemical

ionisation. Samples (1.0 μL) were injected in the splitless mode and the oven

temperature was held at 70 °C for 1 min, then increased to 160 °C at a rate of 20 °C/

min and finally to 280 °C at a rate of 30 °C/min. Helium (92.5 kPa and flow rate 0.7

mL/ min) were used as the carrier and methane as the reagent gas for negative-ion

chemical ionization. Peak identification was based on retention time and mass spectra

compared with 15-N-labeled authentic standards (sodium [15

N]nitrite and sodium

[15

N]nitrate). Quantification was performed using calibration curves obtained from

authentic standards and labeled standards. Results were adjusted for creatinine levels.

The effects of quercetin, (-)-epicatechin, theaflavin, sesamin and chlorogenic

acid on systemic ET-1 production were investigated by measuring its concentration in

the urine samples using a commercially available ET-1 (human) EIA kit (Assay Design,

GA). ET-1 concentrations were adjusted for creatinine levels.

5.2.10. Statistical analysis

Statistical analyses were performed using SPSS version 15. Data are presented

as mean ± SEM. One-way ANOVA185

with post hoc analyses using Tukey’s honestly

significant difference (Tukey’s HSD) were used to compare treatments. Initially, the

ApoE-/-

control mice were compared to the C57BL mice. Polyphenol treatments in the

127

ApoE-/-

mice were then compared to the ApoE-/-

control mice. The results analysed were

considered significantly different if p value < 0.05.

5.3. RESULTS

5.3.1. Animals and Polyphenol Diets

The C57BL and ApoE-/-

mice grew steadily over 26 weeks (from mean body

mass of 9.5±0.5 g at week 4 to 28.0±0.3 g at week 26) with no significant difference in

body mass observed at week 26. The daily intake of the polyphenols was calculated for

each diet group based on the daily consumption of the polyphenols incorporated into the

mouse feed (mean daily intakes in mg/ kg body mass: quercetin, 63.8±0.5; (-)-

epicatechin, 64.0±0.3; theaflavin, 63.5±0.8; sesamin, 63.7±0.4 and chlorogenic acid,

63.8±0.6). No significant difference between the groups was observed.

5.3.2. Aortic lesion analyses

The amount of lesion in the transverse section of the aorta (Figure 5.2) was

expressed as the percentage of the area of the lesion to the total area of the aortic tissue.

No significant atherosclerotic lesion was observed in C57BL and ApoE-/-

mice at 16

weeks of age (Data not shown). At 26 weeks of age, ApoE-/-

mice exhibited

significantly higher amounts of lesions at the aortic sinus and the thoracic aortic region

just below the aortic arch compared to the C57BL control mice (Figure 5.3A and Figure

5.3B; p < 0.05 vs C57BL mice). Lesion formation at both locations was significantly

reduced in ApoE-/-

mice fed a diet containing quercetin or theaflavin (Figure 5.3A and

Figure 5.3B; p < 0.05 vs ApoE-/-

control mice). Quercetin and theaflavin significantly

attentuated lesion formation (quercetin, 60% to 80% vs ApoE-/-

control mice; theaflavin,

45% to 55% vs ApoE-/-

control mice). Although treatments with (-)-epicatechin,

sesamin and chlorogenic acid appeared to diminish lesion formation (14%, 40% and

128

29% respectively vs ApoE-/-

mice), but those differences were not statistically

significant.

(A)

(B)

(C)

(D)

Figure 5.2: Thoracic aorta transverse sections from (A) C57BL, (B) ApoE/ control, (C) ApoE/

quercetin and (D) ApoE/ theaflavin at 26 weeks of age under 10x magnification after Sudan(IV)

staining.

129

C57

BL/ c

ontro

l

Apo

E/ con

trol

Apo

E/ Que

rcet

in

Apo

E/ (-)-e

pica

tech

in

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)

C57

BL/ c

ontro

l

Apo

E/ con

trol

Apo

E/ Que

rcet

in

Apo

E/ (-)-e

pica

tech

in

Apo

E/ the

afla

vin

Apo

E/ ses

amin

Apo

E/ chl

orog

enic

aci

d

0

5

10

15

20

25

30

35

40

45

* **

(B)

Diets

Thora

cic

aort

ic l

esio

n a

rea

(% o

f to

tal

aort

ic

cross

-sec

tional

are

a)

Figure 5.3: Cross-sectional lesion area (% of total cross-sectional area) in the (A) aortic

sinus and (B) thoracic region just below the aortic arch from C57BL and ApoE-/- mice

after 20 weeks of different dietary treatments: C57BL/ control (n = 20), ApoE/ control

(n = 20), ApoE/ quercetin (n = 18), ApoE/ (-)-epicatechin (n = 19), ApoE/ theaflavin (n

= 20), ApoE/ sesamin (n = 18) and ApoE/ chlorogenic acid (n = 19). * p < 0.05 vs

ApoE/ control using one-way ANOVA analysis with Tukey’s HSD post hoc analysis.

130

5.3.3. Plasma cholesterol and aortic fatty acid composition

ApoE-/-

mice had significantly elevated plasma concentrations of cholesterol

compared to the C57BL mice (Figure 5.4; p < 0.05). The polyphenols did not

significantly affect plasma cholesterol concentrations in the ApoE-/-

mice after 20 weeks

of treatment. No polyphenol treatment exerted any significant effect on the

concentrations of any of the fatty acids in the aortas compared to the control ApoE-/-

mice (Table 5.2).

Table 5.2: Aortic tissue fatty acid content after 20 weeks of different dietary treatments

Fatty acids (µg/ mg of aortic tissue)

Group (n) Palmitic Stearic Oleic Linoleic Arachidonic

C57BL/ control (20) 12.06±2.29 10.28±2.12 4.55±0.79* 0.66±0.10 0.06±0.01

ApoE/ control (19) 12.60±1.53 12.06±1.49 2.96±0.47 0.72±0.12 0.08±0.01

ApoE/ quercetin (18) 11.25±2.00 10.37±1.95 2.63±0.50 0.49±0.08 0.07±0.01

ApoE/ (-)-epicatechin

(18)

10.96±2.14 9.88±1.93 2.52±0.68 0.68±0.10 0.10±0.01

ApoE/ theaflavin (19) 12.87±2.03 12.52±2.00 2.16±0.36 0.50±0.07 0.09±0.02

ApoE/ sesamin (18) 8.91±1.41 8.30±1.41 1.83±0.24 0.35±0.03 0.06±0.01

ApoE/ chlorogenic

acid (18)

7.86±1.36 7.52±1.49 2.00±0.47 0.44±0.11 0.05±0.01

Values are mean±SEM.

* p < 0.05 vs ApoE/ control using one-way ANOVA analysis.

131

C57

BL/ c

ontro

l

Apo

E/ con

trol

Apo

E/ Que

rcet

in

Apo

E/ (-)-e

pica

tech

in

Apo

E/ the

afla

vin

Apo

E/ ses

amin

Apo

E/ chl

orog

enic acid

C57

BL/ c

ontro

l

Apo

E/ con

trol

Apo

E/ Que

rcet

in

Apo

E/ (-)-e

pica

tech

in

Apo

E/ the

afla

vin

Apo

E/ ses

amin

Apo

E/ chl

orog

enic acid

0.0

2.5

5.0

7.5

10.0

12.5

16 week 26 week

* *

Diets

Chole

ster

ol

(mm

ol/

L)

Figure 5.4: Plasma total cholesterols concentrations of C57BL and ApoE-/- mice after

10 weeks (16 week of age) and 20 weeks (26 week of age) of different dietary

treatments: C57BL/ control (n = 5 for 16th

week, n = 20 for 26th

week), ApoE/ control

(n = 5 for 16th


week), ApoE/ quercetin (n = 5 for 16th

week, n = 19

for 26th

week), ApoE/ (-)-epicatechin (n = 5 for 16th


week), ApoE/

theaflavin (n = 5 for 16th


week), ApoE/ sesamin (n = 5 for 16th


week) and ApoE/ chlorogenic acid (n = 5 for 16th

week, n = 18 for

26th

week),. * p < 0.05 vs all other groups at the two time points using one-way

ANOVA analysis with Tukey’s HSD post hoc analysis.

5.3.4. Systemic and vascular oxidative stress

At 26 weeks of age, ApoE-/-

mice had significantly higher concentrations of

aortic F2-isoprostanes than C57BL mice (Figure 5.5A; p < 0.05). Diets incorporating

quercetin and (-)-epicatechin significantly reduced aortic F2-isoprostanes concentrations

in ApoE-/-

mice (Figure 5.5A; p < 0.05 vs ApoE-/-

control mice). Theaflavin, sesamin

and chlorogenic acid treatments did not show any significant effect on aortic F2-

isoprostanes concentrations. Significant increases in urinary F2-isoprostanes

concentrations were also observed in the ApoE-/-

mice compared to the C57BL mice

132

C57

BL/ c

ontro

l

Apo

E/ con

trol

Apo

E/ que

rcetin

Apo

E/ (-)-e

pica

tech

in

Apo

E/ the

afla

vin

Apo

E/ ses

amin

Apo

E/ chl

orog

enic

aci

d0

10

20

30

*

*

*

(A)

*

Diets

Aort

ic t

issu

e

F2-i

sopro

stan

es

(ng/

g a

ort

ic a

rach

idonic

acid

)

C57

BL/ c

ontro

l

Apo

E/ con

trol

Apo

E/ que

rcet

in

Apo

E/ (-)-e

pica

tech

in

Apo

E/ the

afla

vin

Apo

E/ ses

amin

Apo

E/ chl

orog

enic

aci

d

C57

BL/ c

ontro

l

Apo

E/ con

trol

Apo

E/ que

rcet

in

Apo

E/ (-)-e

pica

tech

in

Apo

E/ the

afla

vin

Apo

E/ ses

amin

Apo

E/ chl

orog

enic

aci

d

0

1000

2000

3000

4000

**

*

16 week 26 week(B)

Diets

Uri

nar

y F

2-i

sopro

stan

es

(ng/

mm

ol

crea

tinin

e)

Figure 5.5: (A) Aortic F2-isoprostanes concentrations of C57BL and ApoE-/-

mice,

expressed per unit mass of aortic arachidonic acid (ng/ mg aortic arachidonic acid), after

20 weeks of different dietary treatments: C57BL/ control (n = 20), ApoE/ control (n =

19), ApoE/ quercetin (n = 18), ApoE/ (-)-epicatechin (n = 18), ApoE/ theaflavin (n =

19), ApoE/ sesamin (n = 18) and ApoE/ chlorogenic acid (n = 18). * p < 0.05 vs ApoE/

control using one-way ANOVA analysis with Tukey’s HSD post hoc analysis.

(B) Urinary F2-isoprostanes concentrations of C57BL and ApoE-/-

mice (ng/ mmol

creatinine) after 20 weeks (26 weeks of age) of different dietary treatments: C57BL/

control (n = 5), ApoE/ control (n = 5), ApoE/ quercetin (n = 5), ApoE/ (-)-epicatechin (n

= 5), ApoE/ theaflavin (n = 5), ApoE/ sesamin (n = 5) and ApoE/ chlorogenic acid (n =

5). * p < 0.05 vs ApoE/ control at 26 week of age using one-way ANOVA analysis with

Tukey’s HSD post hoc analysis.

133

(Figure 5.5B; p < 0.05). Only quercetin and theaflavin treatments significantly reduced

urinary F2-isoprostanes after 20 weeks of treatment (Figure 5.5B; p < 0.05).

Aortic tissues from the ApoE-/-

mice produced similar amounts of O2•- anion radical as

the C57BL mice at 26 weeks of age (Figure 5.6), indicating similar levels of NOX

activity in both mice strains. Quercetin and (-)-epicatechin treatments significantly

attenuated the production of vascular O2•- anion radicals (Figure 5.6; p < 0.05 vs ApoE

-/-

control mice), while no significant effect was observed for the other treatments (Figure

5.6).

C57

BL/ c

ontro

l

Apo

E/ con

trol

Apo

E/ que

rcet

in

Apo

E/ (-)-e

pica

tech

in

Apo

E/ the

afla

vin

Apo

E/ ses

amin

Apo

E/ chl

orog

enic

aci

d

0.0

0.5

1.0

1.5

2.0

* *

Diets

Vas

cula

r S

uper

oxid

e

(RU

/ m

in/

g p

rote

ins)

Figure 5.6: Superoxide anion radical production in abdominal aortic tissues from

C57BL and ApoE-/-

mice after 20 weeks of different dietary treatments: C57BL/ control

(n = 20), ApoE/ control (n = 19), ApoE/ quercetin (n = 19), ApoE/ (-)-epicatechin (n =

18), ApoE/ theaflavin (n = 20), ApoE/ sesamin (n = 18) and ApoE/ chlorogenic acid (n

= 19). * p < 0.05 vs ApoE/ control using one-way ANOVA analysis with Tukey’s HSD

post hoc analysis.

134

5.3.5. Ex vivo vascular LTB4 production and Plasma sP-Selectin

Aortic tissues from ApoE-/-

mice produced significantly higher amounts of

LTB4 compared to the C57BL mice (Figure 5.7; p < 0.05). Only quercetin and

theaflavin significantly reduced the production of LTB4 in the aortic tissues (Figure 5.7;

p < 0.05).

ApoE-/-

mice expressed significantly higher plasma concentrations of sP-

selectin than C57BL mice at 26 weeks of age (p < 0.005) (Figure 5.8). Treatment with

quercetin, (-)-epicatechin and theaflavin significantly lowered the plasma sP-selectin

concentrations (p < 0.005 vs ApoE-/-

control mice). Sesamin and chlorogenic acid did

not significantly affect plasma sP-selectin concentrations when compared to the ApoE-/-

control mice (Figure 5.8).

C57

BL/ c

ontro

l

Apo

E/ con

trol

Apo

E/ que

rcet

in

Apo

E/ (-)-e

pica

tech

in

Apo

E/ the

afla

vin

Apo

E/ ses

amin

Apo

E/ chl

orog

enic

aci

d

0

5

10

15

20

25

30

35

* **

Diets

Ex

vivo

vas

cula

r L

TB

4

pro

duct

ion

(ng/

mg p

rote

in)

Figure 5.7: Ex vivo LTB4 production in abdominal aortic tissues from C57BL and

ApoE-/-

mice after 20 weeks of different dietary treatments: C57BL/ control (n = 20),

ApoE/ control (n = 20), ApoE/ quercetin (n = 19), ApoE/ (-)-epicatechin (n = 19),

ApoE/ theaflavin (n = 20), ApoE/ sesamin (n = 19) and ApoE/ chlorogenic acid (n =

18). * p < 0.05 vs ApoE/ control using one-way ANOVA analysis with Tukey’s HSD

post hoc analysis.

135

C57

BL/ c

ontro

l

Apo

E/ con

trol

Apo

E/ Que

rcet

in

Apo

E/ (-)-e

pica

tech

in

Apo

E/ the

afla

vin

Apo

E/ ses

amin

Apo

E/ chl

orog

enic

aci

d

0102030405060708090

100110120130

*** *

Diets

Pla

sma

[sP

-Sel

ecti

n]

( M

)

Figure 5.8: Plasma sP-Selectin concentrations of C57BL and ApoE-/-

mice (pg/ mmol

creatinine) after 10 weeks (16 weeks of age) and 20 weeks (26 weeks of age) of

different dietary treatments: C57BL/ control (n = 5), ApoE/ control (n = 5), ApoE/

quercetin (n = 5), ApoE/ (-)-epicatechin (n = 5), ApoE/ theaflavin (n = 5), ApoE/

sesamin (n = 5) and ApoE/ chlorogenic acid (n = 5). * p < 0.005 vs all other groups at

the two time points using one-way ANOVA analysis with Tukey’s HSD post hoc

analysis.

5.3.6. Vascular eNOS activity, urinary nitrite, nitrate and endothelin-1

At 16 weeks of age, the C57BL and ApoE-/-

mice had comparable

concentrations of urinary nitrite and nitrate (Figure 5.9). While no change in

concentration of urinary nitrite and nitrate was observed for the C57BL mice between

the two time points, the ApoE-/-

control mice excreted significantly lower amounts of

nitrate at 26 weeks of age (Figure 5.9; p < 0.05) compared to the earlier time point. At

16 weeks of age, only quercetin and (-)-epicatechin treatment significantly increased

urinary nitrite concentrations (Figure 5.9; p < 0.05). However, at 26 weeks, there was no

136

C57

BL/ c

ontro

l

Apo

E/ con

trol

Apo

E/ que

rcet

in

Apo

E/ (-)-e

pica

tech

in

Apo

E/ the

afla

vin

Apo

E/ ses

amin

Apo

E/ chl

orog

enic

aci

d

C57

BL/ c

ontro

l

Apo

E/ con

trol

Apo

E/ que

rcet

in

Apo

E/ (-)-e

pica

tech

in

Apo

E/ the

afla

vin

Apo

E/ ses

amin

Apo

E/ chl

orog

enic

aci

d

0

1

2

3

4*

16 week 26 week(A)

*

Diets

Uri

nar

y N

itri

te

( m

ol/

mm

ol

crea

tinin

e)

C57

BL/ c

ontro

l

Apo

E/ con

trol

Apo

E/ que

rcet

in

Apo

E/ (-)-e

pica

tech

in

Apo

E/ the

afla

vin

Apo

E/ ses

amin

Apo

E/ chl

orog

enic

aci

d

C57

BL/ c

ontro

l

Apo

E/ con

trol

Apo

E/ que

rcet

in

Apo

E/ (-)-e

pica

tech

in

Apo

E/ the

afla

vin

Apo

E/ ses

amin

Apo

E/ chl

orog

enic

aci

d

0

5

10

15

2016 week 26 week(B)

* *

*

#

Diets

Uri

nar

y N

itra

te

( m

ol/

mm

ol

crea

tinin

e)

Figure 5.9: (A) Nitrite and (B) nitrate concentrations in the urine from C57BL and

ApoE-/- mice after 10 weeks (16 weeks of age) and 20 weeks (26 weeks of age) of

different dietary treatments: C57BL/ control (n = 5), ApoE/ control (n = 5), ApoE/

quercetin (n = 5), ApoE/ (-)-epicatechin (n = 5), ApoE/ theaflavin (n = 5), ApoE/

sesamin (n = 5) and ApoE/ chlorogenic acid (n = 5). * p < 0.05 vs ApoE/ control at the

same time point using one-way ANOVA analysis with Tukey’s HSD post hoc analysis..

# p < 0.05 vs ApoE/ control mice at 16 week of age using paired t-test.

137

significant difference in nitrite excretion between any of the treatment groups (Figure

5.9). The urinary nitrate concentrations from the polyphenol treated ApoE-/-

mice did

not differ significantly from that of the ApoE-/-

control mice at the earlier 16 weeks time

point (Figure 5.9). At week 26, all five treatments elevated the urinary nitrate

concentration. However, only the quercetin and theaflavin – treated ApoE-/-

mice had

significantly higher urinary nitrate concentrations compared to the ApoE-/-

control mice

(p < 0.05; Figure 5.9).

At 26 weeks of age, the vascular eNOS activity of ApoE-/-

mice in the control

group was significantly lower compared to the C57BL mice (p < 0.05; Figure 5.10).

C57

BL/ c

ontro

l

Apo

E/ con

trol

Apo

E/ Que

rcet

in

Apo

E/ (-)-e

pica

tech

in

Apo

E/ the

afla

vin

Apo

E/ ses

amin

Apo

E/ chl

orog

enic

aci

d

0

200

400

600

800

*

*

*

Diets

Vas

cula

r eN

OS

Act

ivit

y

(pm

ol

L-c

itru

line

per

mg

aort

ic p

rote

in p

er 6

0 m

in)

Figure 5.10: Vascular eNOS activity (pmol L-citrulline per mg aortic protein per 60

min) of C57BL and ApoE-/-

mice after 20 weeks (26 weeks of age) of different dietary

treatments: C57BL/ control (n = 5), ApoE/ control (n = 5), ApoE/ quercetin (n = 5),

ApoE/ (-)-epicatechin (n = 5), ApoE/ theaflavin (n = 5), ApoE/ sesamin (n = 5) and

ApoE/ chlorogenic acid (n = 5). * p < 0.05 vs ApoE/ control using one-way ANOVA

analysis with Tukey’s HSD post hoc analysis.

138

Quercetin and theaflavin significantly increased eNOS activity in the aortic tissues (p <

0.05 vs ApoE-/-

control mice; Figure 5.10), while the other polyphenols showed

insignificant elevations in eNOS activity. The increase in eNOS activity corresponds

with the elevation in excretion of nitrate in the polyphenol-treated ApoE-/-

mice at 26

weeks (Figure 5.10).

At 16 weeks of age, C57BL and ApoE-/-

mice produced similar concentrations

of urinary ET-1, which were unaffected by the dietary polyphenol treatments (Figure

5.11). The production of ET-1 was significantly increased in the ApoE-/-

mice at 26

weeks of age compared to C57BL mice (p < 0.05) and was attenuated with quercetin

and (-)-epicatechin dietary treatments (p < 0.05; Figure 5.11).

C57

BL/ c

ontro

l

Apo

E/ con

trol

Apo

E/ que

rcet

in

Apo

E/ (-)-e

pica

tech

in

Apo

E/ the

afla

vin

Apo

E/ ses

amin

Apo

E/ chl

orog

enic

aci

d

C57

BL/ c

ontro

l

Apo

E/ con

trol

Apo

E/ que

rcet

in

Apo

E/ (-)-e

pica

tech

in

Apo

E/ the

afla

vin

Apo

E/ ses

amin

Apo

E/ chl

orog

enic

aci

d

0

100

200

300

* *

16 week 26 week

#

Uri

nar

y E

T-1

conce

ntr

atio

n

(pg/

mm

ol

crea

tinin

e)

Figure 5.11: Urinary ET-1 concentrations (pg/ mmol creatinine) of C57BL and ApoE-/-

mice after 10 weeks (16 week of age) and 20 weeks (26 week of age) of different

dietary treatments: C57BL/ control (n = 5), ApoE/ control (n = 5), ApoE/ quercetin (n =

5), ApoE/ (-)-epicatechin (n = 5), ApoE/ theaflavin (n = 5), ApoE/ sesamin (n = 5) and

ApoE/ chlorogenic acid (n = 5). * p < 0.05 vs ApoE/ control at the same time point

using one-way ANOVA analysis with Tukey’s HSD post hoc analysis. # p < 0.05 vs

ApoE/ control at 16 week of age using paired t-test.

139

5.4. DISCUSSION

Our study has shown that particular dietary polyphenols are bioactive

molecules that can inhibit the progression of atherosclerosis. All the polyphenol

treatments appeared to reduce lesion formation to some extent, but only quercetin and

theaflavin significantly attenuated the disease progression in ApoE-/-

mice (Table 5.3).

Previous studies have shown that polyphenol-rich beverages, such as red wine167

and

tea266

, can inhibit atherosclerosis in ApoE-/-

mice. These beverages contain a complex

mixture of polyphenolic compounds.

Table 5.3. Effects of specific polyphenols on tested pathways at week 26.

Quercetin Epicatechin Theaflavin Sesamin Chlorogenic

acid

Lesion

formation

↓* -

† ↓ - -

Plasma

cholesterol

- - - - -

Aortic F2-

isoprostanes

↓ ↓ - - -

Urinary F2-

isoprostanes

↓ - ↓ - -

Aortic superoxide

↓ ↓ - - -

Aortic LTB4

↓ - ↓ - -

Plasma soluble P-

selectin

↓ ↓ ↓ - -

Urinary nitrate

↑ - ↑ - -

Vascular eNOS

activity

↑ - ↑ - -

Urinary ET-1

↓ ↓ - - -

*↓ and ↑ represent significant decrease and increase respectively when compared to the

ApoE-/-

control mice. † - represents no significant changes when compared to the ApoE

-/- control mice.

140

In our study, we showed that some individual polyphenols can attenuate

atherosclerosis and these particular polyphenols may represent some of the active

compounds in the polyphenol-rich beverages. Our results also suggested that the

polyphenols, which are able to effectively inhibit atherosclerosis, such as quercetin, do

so through a number of pathways, such as inhibition of oxidative stress, inflammation,

improved endothelial function and platelet function, which may help prevent disease

development (Table 5.3).

Lipid peroxidative damage may be a critical step in the pathogenesis of

atherosclerosis1. The well recognized antioxidant activity of many polyphenols, has led

to the proposal that polyphenol protection against atherosclerosis may involve their

antioxidant properties267

. Quercetin and catechins in red wine and tea have been shown

to inhibit atherosclerosis in ApoE-/-

mice while also reducing LDL susceptibility to

oxidation266, 268

. Quercetin-3-O-glucuronide (a major in vivo quercetin metabolite) was

shown to localise within activated macrophages in human atherosclerotic lesion and

prevent the uptake of oxidised LDL through the down-regulation of scavenger

receptors218

. Black tea consumption decreased lipoprotein oxidation in New Zealand

white rabbits269

and theaflavin, a major polyphenol present in black tea, was shown to

be as effective as catechins as in vitro antioxidants259

. Ingestion of sesame (in which

sesamin is a major lignan) reduced LDL oxidation in postmenopausal women260

.

Chlorogenic acid and its major in vivo metabolite, caffeic acid demonstrated antioxidant

effects in vitro270

, while the consumption of coffee (a major dietary source of

chlorogenic acid) resulted in the incorporation of phenolic acids into LDL and increased

the resistance of LDL to ex vivo oxidation in humans271, 272

. The polyphenols tested in

our study were incorporated into the diet and given at a dose corresponding to

achievable human intake. Some of the compounds tested were able to reduce systemic

oxidative stress in ApoE-/-

mice as indicated by their lowering of urinary F2-

141

isoprostanes, with quercetin and theaflavin showing significant antioxidant activities

(Figure 5.5B). Oxidative stress in the vasculature was effectively attenuated by

quercetin and (-)-epicatechin, as demonstrated by significant reduction of aortic F2-

isoprostanes (Figure 5.5A). Antioxidant effects alone may not be sufficient to reduce

atherosclerotic lesion formation in the ApoE-/-

mice. (-)-Epicatechin did not

significantly reduce aortic lesions while it significantly reduced aortic F2-isoprostanes

but not urinary F2-isoprostanes. Theaflavin, which had no significant antioxidant effect

on aortic F2-isoprostanes, but significantly decreased urinary F2-isoprostanes, was able

to reduce lesion formation (Figure 5.3 and 5.5).

Hypercholesterolemia is well established as a risk factor for atherosclerosis

and it is possible that dietary polyphenols may protect against the disease by exerting

hypocholesterolemic effects. However, animal and clinical studies are not conclusive

118, 167. The polyphenols used in our study had no significant effect on the plasma total

cholesterol concentrations (Figure 5.4). This result is consistent with previous ApoE-/-

mouse studies167, 263, 267

, even though the ApoE-/-

mice in our study were fed a normal

western diet instead of a high-fat atherogenic diet. The absence of hypocholesterolemic

activity suggested that the observed anti-atherogenic effects of quercetin and theaflavin

were independent of the serum cholesterol levels.

Inflammation is recognised as a key process in atherogenesis7. Inflammatory

processes in the vascular wall may be mediated by a range of factors, such as cytokines,

eicosanoids and NO•, which in turn modulate cellular signaling, cell growth and

differentiation and a variety of other cellular processes. There is evidence that LTB4, a

potent chemotactic molecule, is involved in arterial leukocyte recruitment131

. LTB4

signaling through NF-kB-dependent BLT1 receptors on vascular smooth muscle

promotes atherosclerosis and intimal hyperplasia273

while knock down of the BLT1

receptor has been shown to reduce lesion formation in ApoE-/-

mice39

. Selected phenolic

142

acids and some polyphenols have been shown to inhibit eicosanoid pathways89

. We

have previously shown that quercetin and its in vivo metabolites are capable of

inhibiting eicosanoid LTB4 production in vitro in human neutrophils (Chapter 2)253

. (-)-

Epicatechin and related flavonoids have been shown to inhibit the synthesis of pro-

inflammatory cytokines in vitro127

. Theaflavin protected against 12-O-

tetradecanoylphorbol-13-acetate-induced inflammation by inhibiting arachidonic acid

metabolism via both 5-lipoxygenase and cyclooxygenase pathways274

. Sesamin

inhibited lipopolysaccharide-induced interleukin-6 production by suppression of the p38

MAPK signal pathway and nuclear factor-κB activation275

. Our results showed that

quercetin and theaflavin significantly inhibited the ex vivo production of pro-

inflammatory LTB4 in the vasculature of the ApoE-/-

mice and were better inhibitors of

vascular LTB4 production than (-)-epicatechin and sesamin (Figure 5.7). Chlorogenic

acid had no effect on vascular LTB4 production (Figure 5.7), although it has been

reported to exhibit other anti-inflammatory properties in vitro276

. Both quercetin and

theaflavin which were able to significantly reduce vascular LTB4 production, were also

able to significantly inhibit lesion formation, suggesting that this anti-inflammatory

property contributes to the anti-atherogenic effect of these polyphenols.

Leukocyte adhesion to endothelial cells and their subsequent infiltration into

subendothelial spaces are mediated by various adhesion molecules, such as P-selectin

and VCAM-1, which are expressed on leukocytes, platelets and endothelial cells7.

Plasma sP-selectin levels were found to associate with preclinical atherosclerosis in

hypercholesterolemic men277

and elevated plasma levels of sP-selectin were reported in

high-risk patients with hypercholesterolemia278

and hypertension279

. As previously

observed280

, the atherogenic ApoE-/-

mice have significantly higher plasma

concentrations of sP-selectin than normal C57BL mice (Figure 5.8). Dietary treatment

with quercetin, (-)-epicatechin and theaflavin significantly lowered plasma sP-selectin

143

levels. Quercetin and theaflavin inhibited atherosclerosis, as assessed by lesion

formation, so reduction of platelet aggregation and leukocyte infiltration, may

contribute to the anti-atherosclerotic effect of these polyphenols280

. In vivo metabolites

of quercetin and catechins inhibited the adhesion of monocytes to cultured endothelial

cells128

. Black tea consumption lowered plasma concentrations of sP-selectin in healthy

human subjects134

and theaflavins in black tea extract prevented platelet aggregation in a

rabbit-polymorphonuclear leukocyte system by inhibiting platelet-activating factor

synthesis281

. Our results suggest that modulation of sP-selectin may be one of the

mechanisms by which dietary polyphenols inhibit atherogenesis in the ApoE-/-

mouse.

The endothelium regulates vascular tone by balancing the production of

vasodilators, most importantly NO•, and vasoconstrictors, such as ET-1. Disruption of

this balance may result in endothelial dysfunction61

. Polyphenol consumption may help

to reverse endothelial dysfunction142, 144, 282

. Oral administration of pure (-)-epicatechin

to humans increased flow-mediated dilation, closely emulating the acute vascular

effects of flavonol-rich cocoa146

. We have shown that quercetin and (-)-epicatechin (200

mg each) acutely augment NO• status and reduce ET-1 production in healthy men

(Chapter 4)258

. Our data suggests that quercetin and theaflavin in particular may

improve endothelial function by augmenting NO• production measued by urinary nitrate

production and increasing eNOS activity in the ApoE-/-

mice (Figures 5.9 and 5.10).

Quercetin and (-)-epicatechin increased eNOS activity in endothelial cells in-vitro via

the inhibition of NOX242, 243

. Our study showed that quercetin and (-)-epicatechin

inhibited vascular O2•- production in aortic tissue, a product of NOX(Figure 5.6),

supporting the results of the in vitro work. Interestingly, quercetin and (-)-epicatechin

also significantly reduced urinary ET-1 production. (Figure 5.11). Urinary ET-1

production was shown to correspond with plasma ET-1 production in healthy men283

.

ET-1 stimulated O2•- production and vasoconstriction through activation of NOX and

144

uncoupling of NOS in the rat aorta82

. Our study supports this observation that ET-1

inhibition reduces O2•- production by NOX since the specific polyphenols that inhibited

urinary ET-1 also inhibited vascular O2•- production. Quercetin was previously shown to

decrease ET-1 production in thrombin-stimulated cultured human umbilical vein

endothelial cells150

. Red wine polyphenols have also been shown to prevent vascular

oxidative stress by inhibiting NOX activity and/or by reducing ET-1 release248

, in line

with our results.

In conclusion, we have shown that certain dietary polyphenols may have a

range of bioactivities that contribute to the cardioprotective effects of fruits, vegetables,

red wine and tea. Quercetin and theaflavin reduced atherosclerotic lesion formation in

the ApoE-/-

mice and also significantly inhibited markers of oxidative stress,

inflammation and endothelial dysfunction. Quercetin appears to be most effective in

reducing lesion formation and also significantly impacted all pathways investigated.

Interestingly, (-)-epicatechin failed to significantly reduce lesion formation even though

it significantly affected several pathways thought to lead to atherosclerosis. Sesamin

and chlorogenic acid had little effect on any pathway and did not significantly reduce

atherosclerosis. Our results indicate that certain dietary polyphenols may act to prevent

atherosclerosis through inhibiting inflammation and endothelial dysfunction as well as

antioxidant effects, and a combination of these properties may be necessary to

ameliorate lesion formation in atherosclerosis.

145

CHAPTER 6:

CONCLUSIONS AND FUTURE RESEARCH

Experimental studies with cultured cell lines and freshly isolated human cells

provide evidence supporting a role for dietary polyphenols in the prevention of CVD.

However, care should be used when interpreting in vitro studies which are often

conducted using the native or unmodified compounds, instead of in vivo metabolites. In

addition, many studies have been conducted using non-physiologically relevant

concentrations of polyphenols. While experiments of this nature may be instrumental in

identifying the mechanisms of cardioprotective actions, they should be performed with

due consideration to metabolic transformation and bioavailability of the polyphenols.

These in vitro studies using pure authentic in vivo quercetin metabolites have provided

evidence that metabolic transformation exerts profound effects on the bioactivities of

dietary polyphenols (Chapter 2 and 3). While the major in vivo metabolites of quercetin

were shown to significantly inhibit pro-inflammatory eicosanoid production and LDL

peroxidation by human neutrophils at physiologically relevant concentrations, they

exhibited significantly lower antioxidant and anti-inflammatory activities than the

parent aglycone molecule. We are, however, not claiming that non-metabolised

compounds are physiologically irrelevant. These compounds, even though present at

sub-micromolar concentrations, may exert biological effects that can operate beyond

direct free radical scavenging or metal chelation.

Data from epidemiologic observational studies showed an inverse relationship

between the consumption of polyphenol-rich foods and the risk of CVD. A number of

intervention trials using flavonoid-rich foods, such as tea and cocoa, provide evidence

for a beneficial effect on relevant indicators of cardiovascular health, such as flow-

mediated dilatation, blood pressure, and lipid profile. Until recently, no randomized

146

controlled trials had studied the effects of dietary polyphenols on clinical cardiovascular

endpoints. A recently published meta-analysis showed that the cardiovascular benefits

of isolated flavonoids and those of flavonoid-rich products cannot be easily

distinguished because there are less trials with isolated flavonoids284

. Thus, it remains

unclear whether the observed effects on cardiovascular biomarkers can be attributed to a

particular polyphenol or polyphenols, because of the lack of substantial evidence for a

vasoprotective effect of specific polyphenols. More well-designed human trials and

animal studies with isolated pure polyphenols are required to provide clear evidence of

beneficial effects on cardiovascular health and to allow assessment of the potential risks

of high flavonoid intake. I have shown that quercetin and (-)-epicatechin augment NO•

and reduce ET-1 production acutely in healthy men (Chapter 4). One limitation of this

study is that endothelial function was not measured as a clinical endpoint, for example

by measuring flow-mediated dilatation. However, results from this study may help

explain why flavonoid-rich foods improved endothelial function in human intervention

studies. Future studies should examine endothelial function both acutely and after

longer periods of supplementation to see if the effects on NO• are sustained. It is

important to highlight that the dose used for the healthy men is achievable with either a

polyphenol-rich diet or specific supplementation.

Using an established animal model of atherosclerosis, we have shown that

specific dietary polyphenols, such as quercetin and theaflavin, may protect against

atherosclerosis by alleviating the pathological events associated with atherosclerosis

(Chapter 5). Quercetin and theaflavin significantly reduced oxidative stress and

inflammation and restored biomarkers of endothelial function and effectively reduced

atherosclerotic lesion formation in ApoE-/-

mice. The dose used would be attainable in

humans by adopting a polyphenol-rich diet or taking supplements. Although the other

polyphenols tested in the study did not significantly reduce lesion formation, a few of

147

them did exert significant improvement in oxidative stress and inflammation which may

contribute to disease progression. For example, (-)-epicatechin significantly reduced

vascular oxidative stress and urinary ET-1. Long-term supplementation studies with

human subjects may be required to establish the beneficial effects of these polyphenols.

Some dietary polyphenols may have synergistic effects, so combinations of polyphenols

may give better outcomes than a particular polyphenol given alone.

Efficacy of flavonoids is affected by rate and extent of absorption, metabolic

modifications, binding to proteins, levels in target cells, urinary excretion and mode of

action. Growing knowledge of these processes has caused a paradigm shift in

polyphenol research. Dietary polyphenols were thought to exert antioxidant effects by

scavenging reactive radicals and/or chelate metals. However, owing to low

bioavailability and metabolic transformation, this mode of action seems unlikely.

Results from my studies suggested that polyphenols, such as quercetin, may exert

antioxidant effects in vivo by inhibiting prooxidant enzymes, such as NOX and MPO, at

physiologically relevant concentrations and after metabolic transformation (Chapter 2

and 3). Glucuronide metabolites are traditionally viewed as urinary excretion products

of polyphenols, but studies now suggest that they may also represent means of

transporting these compounds in plasma to target cells285

. This is in line with most

bioavailability studies which showed that glucuronides were present as major in vivo

metabolites in human circulation90

. Interestingly, quercetin-3-O-glucuronide has been

shown to localise within activated macrophages in human atherosclerotic lesions and

reduces foam cell formation in vitro by down-regulating the expression of scavenger

receptors SR-A and CD36218

. This suggests that in vitro studies should investigate the

biological properties of these in vivo metabolites, rather than the molecular forms

present in the diet. While dietary polyphenols are thought to increase NO•

bioavailability in vivo by elevating NOS activity149

, they are also likely to retard the loss

148

of NO• by inhibition of NOX activity

255 and hence decreasing a source of ROS which

would react with NO•. The acute human intervention study (presented in Chapter 4)

provided direct evidence to support the current view that certain polyphenols may

augment the bioavailability of NO• in vivo, without significantly altering systemic

oxidative status of the human volunteers. Results of the mouse study (presented in

Chapter 5) showed that these vasoactive molecules inhibited atherosclerosis by

increasing eNOS activity and diminishing localised inflammation (measured as vascular

LTB4 production).

In conclusion, these studies have provided direct evidence that dietary

polyphenols (such as quercetin and theaflavin) may protect against CVD by alleviating

oxidative stress, inflammation and endothelial dysfunction. Much work on the

bioavailability of polyphenols is currently underway and results from these studies will

enable researchers to focus on polyphenols that reach effective concentrations.

Intervention trials should be conducted with either specific polyphenol supplements or

specific foods rich in the polyphenol of interest, using established cardiovascular

endpoints to better understand the mechanisms of protection. As some people are more

or less sensitive to potential beneficial or deleterious effects of polyphenols, it becomes

important to take genetic factors into account in such studies. Small intervention studies

give results to provide the basis for large scale, population-based, intervention studies

which can be used to verify any beneficial effects of dietary polyphenols. A

metabolomic approach could be used to identify new metabolites of dietary polyphenols

and helped establish new markers of polyphenol exposure for application in large

population-based studies286

. Until more is known about the mechanisms by which

polyphenols are absorbed and metabolised, and the effects of the resulting conjugates

and metabolites on cellular processes, it would be unwise to increase polyphenol intake

by supplementation or food fortification or promote them as “health essentials”.

149

Adverse effects were observed in recent intervention trials with supplements of some of

the well recognised antioxidant nutrients, such as β-carotene, vitamin A and α-

tocopherol287, 288

.

150

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Wai Mun Loke B. Sc (Hons) · men was conducted to compare the acute effects on nitric oxide,...

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