The Function of Dietary Phenolic

Acids in Cardiovascular Health

Aidilla Mubarak

This thesis is presented for the degree of Doctor of Philosophy

of

The University of Western Australia

School of Plant Biology

School of Medicine and Pharmacology

2013

ii

Personal Contribution of Author

DECLARATION FOR THESES CONTAINING PUBLISHED WORK AND/OR WORK PREPARED

FOR PUBLICATION

The examination of the thesis is an examination of the work of the student. The work

must have been substantially conducted by the student during enrolment in the

degree.

Where the thesis includes work to which others have contributed, the thesis must

include a statement that makes the student’s contribution clear to the examiners. This

may be in the form of a description of the precise contribution of the student to the

work presented for examination and/or a statement of the percentage of the work that

was done by the student.

In addition, in the case of co-authored publications included in the thesis, each author

must give their signed permission for the work to be included. If signatures from all the

authors cannot be obtained, the statement detailing the student’s contribution to the

work must be signed by the coordinating supervisor.

Please sign one of the statements below.

1. This thesis does not contain work that I have published, nor work under review

for publication.

Student signature…………………Not applicable……………………………………

2. This thesis contains only sole-authored work, some of which has been

published and/or prepared for publication under sole authorship. The

bibliographical details of the work and where it appears in the thesis are

outlined below.

Student signature…...…………… Not applicable ………………………………………………

iii

3. This thesis contains published work and/or work prepared for publication,

some of which has been co-authored. The bibliographical details of the work

and where it appears in the thesis are outlined below.

The student must attach to this declaration a statement for each publication

that clarifies the contribution of the student to the work. This may be in the

form of a description of the precise contributions of the student to the

published work and/or a statement of percent contribution by the student.

This statement must be signed by all authors. If signatures from all the authors

cannot be obtained, the statement detailing the student’s contribution to the

published work must be signed by the coordinating supervisor.

(i) Aidilla Mubarak, Ewald E. Swinny, Simon Y.L. Ching, Steele R. Jacob, Kevin

Lacey, Jonathan M. Hodgson, Kevin D. Croft, and Michael J. Considine.

Polyphenol composition of plum selections in relation to total antioxidant

capacity. Journal of Agricultural and Food Chemistry. 2012; 60: 10256-

10262.

Contribution of Aidilla Mubarak : 80%

(ii) Aidilla Mubarak, Catherine P. Bondonno, Alex H. Liu, Michael J. Considine,

Lisa Rich, Emilie Mas, Kevin D. Croft, and Jonathan M. Hodgson. Acute

effects of chlorogenic acid on nitric oxide status, endothelial function, and

blood pressure in healthy volunteers: a randomized trial. Journal of

Agricultural and Food Chemistry. 2012; 60: 9130- 9136.

Contribution of Aidilla Mubarak: 50%

(iii) Aidilla Mubarak, Jonathan M. Hodgson, Michael J. Considine, Kevin D.

Croft, Vance B. Matthews. Supplementation of a high fat diet with

chlorogenic acid is associated with insulin resistance and hepatic lipid

accumulation in mice. 2012. The Journal of Nutrition (under revision).

Contribution of Aidilla Mubarak : 75%

For any work in this thesis that has been co-published with other authors, I have the

permission of all co-authors to include this work in my thesis.

Student signature

Coordinating Supervisor Signature (Dr Michael Considine)

(Prof Kevin Croft)

iv

Acknowledgements

“By prevailing over all obstacles and distractions, one may unfailingly arrive at his

chosen goal or destination”

(Christopher Columbus)

This Ph.D. journey has certainly involved many obstacles, but most importantly it has

been a joyful road due to immense support from many individuals. My foremost

gratitude goes to my helpful supervisors, Dr Michael Considine, Prof. Kevin Croft and

Prof. Jonathan Hodgson. The completion of this thesis would not have been possible

without their invaluable advice, guidance, patience and academic experience. I am

extremely grateful for the continuous encouragements from them which helped me

pushed myself to the limits in finalizing this thesis. I feel blessed to have them as my

mentor especially in sculpturing the path to my future career.

My sincere thanks also go to Dr Vance Matthews for all the amazing time and advice

which helped me a lot in my study. I also appreciate the kindness of Dr Simon Ching

and Dr Ewald Swinny for sharing the knowledge of some experimental techniques

involved in this thesis. I am also beyond grateful to all the participants in the study, as

without their priceless time and efforts, this thesis would have remained a dream.

The support from the Universiti Malaysia Terengganu and Malaysian Ministry of

Higher Education are also greatly appreciated. I thank them for giving me the

opportunity to further my study in Australia.

My appreciation extends to my fellow friends in the lab, especially Emilie, Adeline, Jeff

and Kelly for all the chats, laughs and the lovely time spent throughout these years. All

of you have certainly made my journey filled with joy, in a way that I can never repay. I

am especially indebted to all my friends for their kind thoughts and words of

encouragement. Special thanks to my best friend Nur Aidya Hanum Aizam for

everything that we have shared together in both of our Ph.D. study. The work on this

thesis would have been a lot different without her motivation, ‘coffee-time’, fight (yes,

fight!), and most importantly her prayers. I can never thank her enough. I must also

v

thank my four-legged mate Jack, for being there for me through my ups and downs. His

love has always made my days a delight.

I have to thank my parents, Salmah Jamal and Mubarak Salim, and to the rest of the

Mubaraks for countless reasons. The past few years would have been impossible

without their endless emotional support and prayers. I thank my parents for raising me,

and providing me with all the love and support which brings me to where I am right

now.

Above all, I thank God for giving me the strength to experience this journey. Thank

You for always being there beside me.

vi

List of Publications, Presentations and

Awards

Publications arising from works in this thesis:

1. Aidilla Mubarak, Ewald E. Swinny, Simon Y. L. Ching, Steele R. Jacob,

Kevin Lacey, Jonathan M. Hodgson, Kevin D. Croft, and Michael J.

Considine. Polyphenol composition of plum selections in relation to total

antioxidant capacity. Journal of Agricultural and Food Chemistry. 2012; 60,

10256−10262.

2. *Aidilla Mubarak,

*Catherine P. Bondonno, Alex H. Liu, Michael J.

Considine, Lisa Rich, Emilie Mas, Kevin D. Croft, and Jonathan M.

Hodgson. Acute effects of chlorogenic acid on nitric oxide status, endothelial

function, and blood pressure in healthy volunteers: A randomized trial.

Journal of Agricultural and Food Chemistry. 2012; 60: 9130−9136.

* Joint first authors.

3. Aidilla Mubarak, Jonathan M. Hodgson, Michael J. Considine, Kevin D.

Croft, and Vance B. Matthews. Supplementation of a high fat diet with

chlorogenic acid is associated with insulin resistance and hepatic lipid

accumulation in mice. The Journal of Agricultural and Food Chemistry.

2013; 61: 4371-4378.

Presentations arising from works in this thesis:

1. UWA Institute of Agriculture 2012 Postgraduate Showcase, ‘Frontiers in

Agriculture’. 2012. Perth. (Oral presentation).

vii

2. Western Australia Australian Society for Medical Research Symposium

2012. Perth. (Oral presentation)

3. International Life Science Institute Symposium on Health Benefits for

Polyphenol-rich Foods and Beverages: Latest Science 2012. Adelaide.

(Poster)

4. Annual Scientific Meeting of High Blood Pressure Research Council of

Australia. 2011. Perth (Oral presentation)

5. International Conference of Polyphenols and Health 2011. Barcelona.

(Poster)

6. Annual Scientific Meeting of Nutrition Society of Australia 2010. Perth.

(Poster)

Awards received:

The University of Western Australia Graduate Research School Travel

Award 2011

Ad Hoc Top-up Scholarship 2012

SLAB/SLAI Scholarship from Ministry of Higher Education, Malaysia

(2009-2013)

Publications arising from work unrelated to this thesis:

Catherine P. Bondonno, Xingbin Yang, Kevin D. Croft, Michael J. Considine, Natalie

C. Ward, Lisa Rich, Ian B. Puddey, Ewald Swinny, Aidilla Mubarak, Jonathan M.

Hodgson. Flavonoid-rich apples and nitrate-rich spinach augment nitric oxide status and

improve endothelial function in healthy men and women: a randomized controlled trial.

Free Radical Biology and Medicine. 2012; 52: 95–102.

viii

Thesis Abstract

Dietary phenolics have been associated with protection against the various forms of

cardiovascular disease (CVD). This thesis outlines three independent studies, from

analytical to intervention, which increase our knowledge on the role of phenolic

compounds in preventative health. Particular attention was paid to phenolics found in

fruit, as they are a rich source and widely available and consumed, and hence make a

large contribution to dietary phenolics intake.

While many fruits are rich in phenolic compounds, it is known that specific cultivars

vary greatly in phenolic composition. In this thesis, the composition of major phenolics

in 29 pre-varietal selections of Western Australian plums was investigated. This

knowledge was essential as the first step in a pathway to develop breeding tools to aid

identification of fruit that may have enhanced health-promoting capacities. Total

phenolics, selected individual phenolic compounds and total antioxidant capacity (TAC)

were quantified. Total phenolic concentration was significantly correlated with TAC.

Neo-chlorogenic acid and quercetin glycosides were found to be the predominant

phenolics. Composition of these predominant phenolic compounds in plums was not

significantly correlated with the TAC. In this study, it is argued that the value of in vitro

TAC assays to breeding programs may be limited. Further, increasing the focus on

individual bioactive phenolic compound was also argued to be more productive to

breeding than TAC assays, as patterns of inheritance of TAC are likely to be very

complex.

ix

Further understanding on the mechanisms of phenolics in preventative health was

sought. There is increasing evidence that specific dietary phenolics can enhance

production of nitric oxide (NO) a mediator in vascular health. A randomized, double-

blind, placebo-controlled, cross-over trial in healthy men and women (n = 23) was

conducted to investigate the acute effects of chlorogenic acid on blood pressure, NO

status and endothelial function. A dose of 400 mg chlorogenic acid (equivalent to 2 cups

of coffee) was chosen as an amount achievable in a usual diet. Relative to control,

systolic blood pressure and diastolic blood pressure were significantly lower after

400 mg chlorogenic acid treatment. This result was observed without concomitant

enhancement in biomarkers of NO status and endothelial function assessed by flow

mediated dilatation. In this study, it was concluded that chlorogenic acid can lower

blood pressure acutely; an effect which if sustained would benefit cardiovascular health.

To further understand the potential benefits of chlorogenic acid on CVD, it is also

important to recognize its effect on metabolic abnormalities which are known to be a

significant risk factor of developing CVD. Benefits of chlorogenic acid on several

features of the metabolic syndrome through coffee consumption were proposed. In this

thesis, an 11-week dietary intervention study was performed to assess whether

chlorogenic acid (1 g.kg-1

diet) had a protective effect on high fat diet induced obesity,

glucose tolerance, insulin sensitivity, fatty acid oxidation and insulin signaling in

C57BL/6J mice. In the study, it was found that supplementation of chlorogenic acid in

the high fat diet did not reduce body weight compared to mice fed the high fat diet

alone. In contrast, chlorogenic acid was found to increased insulin resistance compared

to mice fed a high fat diet only. Furthermore, chlorogenic acid resulted in decreased

phosphorylation of AMPK and ACCβ, a downstream target of AMPK in liver. These

observations were supported by evidence of higher lipid content and more steatosis in

x

the liver of mice fed a high fat diet supplemented with chlorogenic acid, relative to mice

fed a high fat diet only. Therefore, this study demonstrated that chlorogenic acid

supplementation in a high fat diet did not protect against features of the metabolic

syndrome in diet-induced obese mice. It remains to be determined if chlorogenic acid

will have benefits in metabolic disorders in human intervention trials.

The studies in this thesis provide additional knowledge to the available evidence in the

literature on the potential preventative health benefits of chlorogenic acid. Targeting

increased and more consistent consumption of chlorogenic acid-rich food such as fruits

could assist prevention of CVD in the community. In future work, investigation of a

time course effect of chlorogenic acid is crucial to understand the mechanism of

cardiovascular protective effect in more depth. A dose response effect of chlorogenic

acid is also warranted to further understand the protective health benefits.

xi

Table of Contents

Personal Contribution of Author ....................................................................................... ii

Acknowledgement............................................................................................................ iv

List of Publications, Presentations and Awards ............................................................... vi

Thesis Abstract ............................................................................................................... viii

Table of Contents ............................................................................................................. xi

List of Tables and Figures ............................................................................................... xv

List of Abbreviations...................................................................................................... xix

Chapter 1: Literature Review .................................................................... 1

1.1 Introduction ........................................................................................... 1

1.2 Cardiovascular Disease and Atherosclerosis ........................................ 2

1.2.1 Pathogenesis of Atherosclerosis .................................................... 2

1.2.2 Endothelial Function and Nitric Oxide ......................................... 4

1.2.3 Metabolic Syndrome and Cardiovascular Disease ........................ 8

1.3 Phenolic compounds ............................................................................. 13

1.3.1 Classification of Phenolic Compound and Its Dietary Sources .. 13

1.3.2 Fruits as a Major Source of Phenolic Compounds ...................... 20

1.3.3 Bioavailability and Metabolism of Phenolic Compounds .......... 23

1.4 Dietary Chlorogenic Acid and Cardiovascular Protective Effects ... 26

1.4.1 Importance of Studying Chlorogenic Acid ................................. 26

1.4.2 Evidence from Epidemiological Studies and Clinical Trials ...... 27

1.4.3 Effects of Chlorogenic Acid on Hypertension and Endothelial

Dysfunction.................................................................................. 28

1.4.4 Chlorogenic Acid Effect on Metabolic Disorders Associated with

Cardiovascular Disease Risks ...................................................... 33

xii

1.5 Rationale of Thesis ................................................................................ 37

1.5.1 Study 1 – Aim and Hypothesis ................................................... 37

1.5.2 Study 2 – Aim and Hypothesis ................................................... 38

1.5.3 Study 3 – Aim and Hypothesis ................................................... 38

Chapter 2: Phenolic Composition of New Plum Selections in Relation

to Total Antioxidant Capacity ............................................................................ 40

2.1 Introduction .......................................................................................... 40

2.2 Materials and Methods ......................................................................... 43

2.2.1 Chemicals and Apparatus ............................................................ 43

2.2.2 Plant Material .............................................................................. 43

2.2.3 Extraction of Phenolic Compounds for HPLC Analysis, Total

Antioxidant Capacity and Total Phenolic Concentration ........... 44

2.2.4 HPLC-DAD Analyses ................................................................. 44

2.2.5 Total Antioxidant Capacity ........................................................ 45

2.2.6 Total Phenolic Concentration ..................................................... 46

2.3 Results ................................................................................................... 47

2.3.1 Total Phenolic Concentration ...................................................... 47

2.3.2 Quantification of Known Bioactive Phenolic Compounds ......... 48

2.3.3 Total Antioxidant Capacity ......................................................... 52

2.3.4 Relationships of Total Phenolics, Individual Phenolics and Total

Antioxidant Capacity .................................................................. 53

2.4 Discussion .............................................................................................. 54

Chapter 3: Acute Effects of Chlorogenic Acid on Nitric Oxide Status,

Endothelial Function and Blood Pressure in Healthy Volunteers: A

Randomized Trial ................................................................................................... 57

3.1 Introduction .......................................................................................... 57

xiii

3.2 Materials and Methods ......................................................................... 58

3.2.1 Chemicals and Apparatus ............................................................ 58

3.2.2 Participant ................................................................................... 60

3.2.3 Study Design .............................................................................. 60

3.2.4 Measurement of Plasma RXNO, Nitrite, and NOx ..................... 61

3.2.5 Blood Pressure ........................................................................... 62

3.2.6 Flow Mediated Dilatation of the Brachial Artery ...................... 62

3.2.7 Measurement of Plasma Chlorogenic Acid Using Liquid

Chromatography-Tandem Mass Spectrometry ........................... 63

3.2.8 Measurement of Plasma Chlorogenic Metabolites Using Gas

Chromatographic Mass Spectrophotometer ................................ 65

3.2.9 Other Biochemical Analyses ...................................................... 66

3.2.10 Statistical Analyses .................................................................... 66

3.3 Results .................................................................................................... 67

3.3.1 Baseline Data ............................................................................. 67

3.3.2 Blood Pressure, Endothelial Function, and Biomarkers

of NO Status ................................................................................ 69

3.3.3 Plasma Chlorogenic Acid and Its Metabolites ........................... 72

3.4 Discussion ............................................................................................... 75

Chapter 4: Supplementation of a High Fat Diet with Chlorogenic Acid

is Associated with Insulin Resistance and Hepatic Lipid accumulation

in Mice ........................................................................................................................ 80

4.1 Introduction .......................................................................................... 80

4.2 Materials and Methods ......................................................................... 82

4.2.1 Experimental Animal and Diets ................................................. 82

4.2.2 Body Weight, Adiposity and Basal Insulin Measurement .......... 82

xiv

4.2.3 Metabolic Assays ........................................................................ 83

4.2.4 Acute Insulin Signaling Experiment ........................................... 83

4.2.5 Liver and Adipose Tissue Histology ........................................... 83

4.2.6 Hepatic Lipid Analysis ................................................................ 84

4.2.7 Western Blot Analysis of Proteins Associated with Fatty Acid

Oxidation and Insulin Signaling .................................................. 84

4.2.8 Statistical Analyses ..................................................................... 84

4.3 Results ................................................................................................... 85

4.3.1 The Effect of Chlorogenic Acid Supplementation on High Fat

Diet Induced Obesity and Insulin Levels in C57BL/6 Mice ....... 85

4.3.2 Chlorogenic Acid Supplementation Promoted Glucose

Intolerance and Insulin Resistance .............................................. 87

4.3.3 Chlorogenic Acid Resulted in Mild Insulin Resistance in White

Adipose Tissue............................................................................. 88

4.3.4 Chlorogenic Acid Supplementation Reduced Fatty Acid

Oxidation Signaling in Liver and White Adipose Tissue ........... 92

4.4 Discussion .............................................................................................. 97

Chapter 5: Summary and Future Work ...................................................... 102

5.1 Phenolic Composition of New Plum Selections in Relation to Total

Antioxidant Capacity ......................................................................... 102

5.2 Acute Effects of Chlorogenic Acid on Nitric Oxide Status,

Endothelial Function and Blood Pressure in Healthy Volunteers: A

Randomized Trial .............................................................................. 104

5.3 Supplementation of a High Fat Diet with Chlorogenic Acid is

Associated with Insulin Resistance and Hepatic Lipid accumulation

in Mice ................................................................................................. 106

5.4 Conclusion ........................................................................................... 107

References ............................................................................................................... 108

xv

List of Tables and Figures

List of Tables

Chapter 1 Page

1.1 Compounds within major classes of flavonoid and respective

common dietary sources

18

1.2 Compounds of phenolic acid classes and its common dietary

sources

20

1.3 Cultivar variation of phenolic composition in different fruits 22

1.4 Summary of recent studies investigating effect of chlorogenic

acid on metabolic abnormalities

36

Chapter 2

2.1 Quantification of selected phenolics in 29 pre-varietal plum

selections and Black Amber (BA) using HPLC-DAD at

wavelengths 280 and 350 nm

50

Chapter 3

3.1 Baseline characteristics of study subjects (n = 23; Males= 4;

Females= 19)

69

xvi

List of Figures

Chapter 1 Page

1.1 Foam cell formation in atherosclerosis 3

1.2 Progression of atherosclerosis 4

1.3 Synthesis of nitric oxide by the endothelial cells via L-

arginine pathway

7

1.4 Insulin-mediated NO production signaling in healthy

condition and in insulin resistance

10

1.5 Fatty acid oxidation pathway and the involvement of AMPK

and ACC

12

1.6 Classification of chemical structures of the major classes of

phenolic

15

1.7 The chemical structures of chlorogenic acids 19

Chapter 2

2.1 Chemical structures of known bioactive phenolic compounds

detected in the tested plums (R= sugar groups)

42

2.2 Sensitivity of different incubation condition on reaction with

Folin Ciocalteu’s reagent

47

2.3 Total phenolic concentration in 29 pre-varietal plum

selections and Black Amber (BA)

48

2.4 HPLC chromatograms of plum selection #29 (A) and plum

selection #21 (B)

51

2.5 Total antioxidant capacity in 29 pre-varietal plum selections

and Black Amber (BA)

52

2.6 Correlation of total phenolic concentration with total

antioxidant capacity in 29 pre-varietal plum selections and

Black Amber (BA)

53

xvii

Chapter 3

3.1 Participant flow from recruitment through screening and

randomization to trial completion.

68

3.2 Effect of chlorogenic acid on (A) SBP from 60 to 150 min

post-treatment, (B) mean baseline-adjusted SBP post-

treatment, (C) DBP from 60 to 150 min post-treatment, and

(D) mean baseline adjusted DBP post-treatment.

70

3.3 Effect of chlorogenic acid on FMD 120 min post-treatment 71

3.4 Effect of chlorogenic acid on plasma concentrations of (A)

RXNO and (B) nitrite 150 min post-treatment

71

3.5 Typical LC/MS/MS chromatogram trace from the analysis of

chlorogenic acid concentrations at baseline and 150 min post-

treatment in chlorogenic acid-treated participants

73

3.6 Plasma concentration of intact chlorogenic acid (A) and

chlorogenic acid metabolites (B) for control and chlorogenic

acid treatments at 150 min post-treatment

74

Chapter 4

4.1 Effect of chlorogenic acid supplementation on high fat diet

induced weight gain (A), gonadal fat pad weights (B) and

fasting insulin measurements (C) at week 12

86

4.2 Effect of chlorogenic acid supplementation on glucose

intolerance at week 5 (A) and week 10 (B); and insulin

resistance at week 6 (C) and week 11 (D) in mice

88

4.3 Effect of chlorogenic acid supplementation on insulin

sensitivity in white adipose tissue

89

4.4 Effect of chlorogenic acid supplementation on insulin

sensitivity in the liver

90

4.5 Effect of chlorogenic acid supplementation on insulin

sensitivity in the skeletal muscle

91

xviii

4.6 Effect of chlorogenic acid supplementation on AMPK

signaling in the liver

92

4.7 Effect of chlorogenic acid supplementation on high fat diet

induced steatosis (A and B) and lipid content in liver (C).

93

4.8 Effect of chlorogenic acid supplementation on

phosphorylation of ACCβ in white adipose tissue

94

4.9 Effect of chlorogenic acid supplementation on adipocyte

hypertrophy

95

4.10 Effect of chlorogenic acid supplementation on AMPK

signaling in the skeletal muscle (quadricep)

96

xix

List of Abbreviations

Abbreviations Full name

% percent

°C degree Celsius

# number

µ micro

µg.g-1

microgram per gram

µg.kg-1

microgram per kilogram

µg.mL-1

microgram per millilitre

µL microliter

µL.min-1

microliter per minute

µm micrometre

µmol.L-1

micromol per litre

β beta

AAPH 2,2’-azobis(2-amidinopropane) dihydrochloride

ABTS 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid

ACS acyl-CoA synthetase

ACC acetyl-CoA carboxylase

ACCβ acetyl-CoA carboxylase β

AIOR antioxidant inhibition of oxygen radicals

Akt protein kinase B

AMPK AMP-activated protein kinase

ATP adenosine triphosphate

AUC area under curve

BA Black Amber

BH4 tetrahydrobiopterin

BMI body mass index

BP blood pressure

BSTFA N,O-bis(trimethylsilyl)trifluoroacetamide with trimethylchlorosilane

C carbon

Ca2+

calcium

xx

CaMKK calmodulin-dependent protein kinase kinase

CBG cytosolic β-glucosidase

CE collision energy

CGA chlorogenic acid

cGMP cyclic guanosine- 3’,5-monophosphate

CHD coronary heart disease

CI confidence interval

Cmax peak plasma concentration

COMT catechol- -methyltransferases

CPT1 carnitine palmitoyltransferase 1

CPT2 carnitine palmitoyltransferase 2

5-CQA 5- -caffeoylquinic acid

3-CQA 3- -caffeoylquinic acid

4-CQA 4- -caffeoylquinic acid

CVD cardiovascular disease

Cyt C cytochrome c

d day

DAD photodiode array detector

DBP diastolic blood pressure

DPPH 2,2’-diphenyl-1-picrylhydrazyl

ECG electrocardiogram

EDRF endothelium-derived relaxing factor

EDTA ethylenediaminetetraacetic acid

eNOS nitric oxide synthase

ESI electrospray ionization source

ET-1 endothelin-1

FAD flavin adenine dinucleotide

FMD flow-mediated dilatation

FRAP ferric reducing antioxidant power

g gram

g.kg-1

gram per kilogram

GAE gallic acid equivalents

GCE green coffee bean extract

xxi

GC-MS gas chromatographic mass spectrophotometer

GIP glucose dependent insulinotropic peptide

GLP glucagon like peptide

GTP guanosine triphosphate

GTT glucose tolerance test

h hour

HCl hydrochloric acid

HPLC high performance liquid chromatography

ICR Imprinting Control Region

IL-1β interleukin-1 beta

iNOS inducible nitric oxide synthase

IRS-1 insulin receptor substrate

IS internal standard

ITT insulin tolerance test

K2CO3 potassium carbonate

kg kilogram

L litre

L-arg L-arginine

LDL low-density lipoprotein

LKB1 liver kinase B1

LPH lactase phloridzin hydrolase

MAPK mitogen-activated protein kinase

MCD malonyl-CoA decarboxylase

mg milligram

mg.kg-1

milligram per kilogram

MHz megahertz

min minute

MJ.kg-1

megajoules per kilogram

mKATP mitochondrial K + ATPase

mL millilitre

mL.min-1

millilitre per minute

mm millimetre

mmol.L-1

milimol per litre

xxii

mmHg milimetres of mercury

mmol.L-1

millimol per litre

mol.kg-1

mol per kilogram

mol.L-1

mol per liter

mPTP mitochondrial permeability transition pore

MRM multiple reaction monitoring

mTorr millitorr

m/z mass to charge ratio

NADPH nicotinamide adenine dinucleotide phosphate

NaNO2− sodium nitrite

neo-CGA neo-chlorogenic acid

ng.µL-1

nanogram per microlitre

ng.mL-1

nanogram per millilitre

nm nanometre

nmol.L-1

nanomol per litre

NO nitric oxide

NOS nitric oxide synthase

O-G1cNAc O-linked N-acetylglucosamine

OGTT oral glucose tolerance test

ORAC oxygen radical absorbance capacity

P phosphohrylation

PDK-1 phosphoinositide-dependent protein kinase

PI3K phosphatidylinositol 3-kinases

pg.µL-1

picogram per microliter

PPAR-α peroxisome proliferator-activated receptor alpha

Q quercetin

ROS reactive oxygen species

RT room temperature

RXNO S-nitrosothiols + other nitrosylated species

s seconds

SBP systolic blood pressure

SD standard deviation

SE standard error

xxiii

SEM standard error of the mean

Ser473

serotonine 473

sGC soluble guanylate cyclase

SHR spontaneously hypertensive rats

SPE solid-phase extraction

SREBP-1c Sterol Regulatory Element-Binding Protein 1c

SULT Sulfotransferases

TAC total antioxidant capacity

TCA tricarboxylic acid

TE trolox equivalent

TEAC trolox equivalent antioxidant capacity

Thr172

threonine 172

TRAP total radical trapping antioxidant parameter

UGT uridine-5’-diphosphate glucuronosyltransferases

U.kg-1

unit per kilogram

v/v volume per volume

w/w weight per weight

Chapter 1

1

CHAPTER 1

Literature Review

1.1 Introduction

Cardiovascular disease (CVD) is a major concern worldwide and has a large burden on

the healthcare system. Although the death rate from heart disease has fallen from 20%

of all deaths in 2001 to 15% in 2010 1, it remains as the leading cause of death in

Australia. Treatment and prevention strategies have been the key target for controlling

incidence of CVD. This has led to the increased interest among researchers to find

approaches for reducing risks of the disease. Numerous studies have shown an inverse

association between consumption of fruits and vegetables and risk of developing

CVD 2-4

. A number of nutrients in these foods such as fibre, vitamin C and E and β-

carotene are attributed to the protection against the disease. Fruits and vegetables are

also rich in phenolic compounds, including flavonoids and phenolic acids, making them

potential candidates for the health protective benefits. Additionally, phenolic-rich food

and beverages have also been associated with decrease risk of CVD 5-7

. Due to their

potential health effects, phenolics have been under investigation by researchers during

the past few decades. Attention has focused on the flavonoids while on the other hand

knowledge on the potential benefits of phenolic acids such as chlorogenic acid, on CVD

remains unclear. Chlorogenic acid is more correctly termed as hydroxycinnamates

according to its structure, but is referred to as phenolic acid in this thesis.

Chapter 1

2

1.2 Cardiovascular Disease and Atherosclerosis

Cardiovascular disease comprises conditions such as coronary heart disease,

cerebrovascular disease and heart failure. CVD is known to be a complex disease with

multiple causes. However, a major contributor to this disease is the arterial condition

termed atherosclerosis.

1.2.1 Pathogenesis of Atherosclerosis

In recognizing the mechanism by which phenolic compounds might exert beneficial

effects towards reducing risks of CVD, one has to understand the pathogenesis of

atherosclerotic CVD 8. Atherosclerosis is a progressive disease which involves

narrowing of the arteries, resulting in inadequate blood flow and oxygen supply

throughout tissues in the body. The atherosclerotic lesions commonly occur in large and

medium-sized elastic and muscular arteries 9. The development of atherosclerosis

involves complex interactions of vascular cells and the immune system components 9

with the inclusion of several causal events such as vascular inflammation and

endothelial dysfunction 10

.

In the early stage of atherosclerotic development, excess level of low-density

lipoprotein (LDL) is infiltrated and retained in the vascular intima (Figure 1.1). These

lipids undergo oxidation and enzymatic modification resulting in leukocyte adhesion

molecules release by the endothelial cells. Macrophages then upregulate expression of

scavenging receptors to bind the lipoproteins 11

. Increased level of lipids lead to the

formation of lipid-laden macrophages called foam cells which aggregate as fatty streaks

Chapter 1

3

beneath the endothelium, followed by the formation of plaque (Figure 1.2). The plaque

is covered by a cap of smooth muscle cells and a collagens matrix. When the plaque

continues to grow, narrowing of the lumen occurs causing restriction of blood flow. In

the case where the inflammatory responses are overwhelmed, the plaque may become

vulnerable to rupture often due to thinning of the fibrous cap. The ruptured plaque

exposes highly thrombogenic collagen and consequential intraluminal thrombus. This

condition is of clinical significance because it is a major cause of coronary heart

disease, angina pectoris, myocardial infarction, and other cardiac disorders.

Figure 1.1. Foam cell formation in atherosclerosis 12

Chapter 1

4

Figure 1.2. Progression of atherosclerosis (adapted from Libby 2002 13

)

1.2.2 Endothelial Function and Nitric Oxide

Endothelial Function

Endothelium is a single layer of cells contained in the vascular intima, which is between

the lumen and smooth muscle cells of the blood vessels (Figure 1.2). These cells are

called endothelial cells. The endothelium was once known simply as a semipermeable

barrier between blood and interstitium which assists the process of water and small

molecules exchange 14

. It is now recognized to play a crucial role in the control of

vascular tone and maintenance of vascular homeostasis 15

. Endothelium also function in

many biological process such as coagulation, fibrinolysis, leukocyte adherence, platelet

Chapter 1

5

interaction, and myocardial contractility 14, 16

. The endothelium is metabolically active

and able react to hemodynamic forces and hormonal environment to release various

vasoactive mediators to maintain vascular homeostasis 14, 17

. Important vasoactive

mediators released by the endothelium are the vasodilator nitric oxide (NO), and

vasoconstrictor such as endothelin-1 (ET-1). Studies have demonstrated that deleterious

alterations of endothelial normal function, known as endothelial dysfunction, is a key

hallmark of atherosclerosis and CVD and it is also identified as the earliest sign of

atherosclerosis 15, 18, 19

.

The measurement of endothelial function is important to identify early signs of diseases

as well as assessing responses to therapeutic interventions. Several techniques have

been developed for this purpose, including invasive and non-invasive techniques. The

endothelial vasomotor function test is considered to be the gold standard compared to

other tests 20

. However, this invasive method has its restriction since it requires an intra-

arterial catheterization 20

. Ultrasound assessment of flow-mediated dilatation (FMD) of

the brachial artery is an alternative non-invasive technique. The FMD method uses

increased hemodynamic shear stress during reactive hyperemia as stimulus for NO

release 20

. This is the preferred technique for endothelial function measurement due to

its capability to detect asymptomatic atherosclerosis with a non-invasive approach 21.

Nitric Oxide

Endothelial derived NO, earlier known as endothelium-derived relaxing factor

(EDRF)22

is essential in the maintenance of vascular homeostasis and blood pressure

(BP) 23, 24

. Although discovered as a vasodilator, NO is also vital in the regulation of

Chapter 1

6

platelet aggregation, leukocyte adherence and vascular smooth muscle cell

mitogenesis 25, 26

. Endothelium derived NO is synthesised from amino acid L-arginine

catalyzed by the nitric oxide synthase (eNOS) producing L-citrulline as a byproduct.

The required cofactors for NO biosynthesis include flavin adenine dinucleotide (FAD),

nicotinamide adenine dinucleotide phosphate (NADPH), tetrahydrobiopterin (BH4) and

calcium. Acetylcholine and bradykinin are among the agonists for NO synthesis which

acts on membrane receptors resulting in the trigger of cytosolic calcium release and

eNOS activation 24, 27, 28

. Increased shear stress from enhanced blood flow is also a

stimulus for NO production. The synthesized NO diffuses across the endothelial cell

membrane into the vascular smooth muscle cells where activation of guanylate cyclase

occurs. This leads to increase in cyclic guanosine- 3’,5-monophosphate (cGMP)

concentration, resulting in relaxation of the smooth muscle cells (Figure 1.3). cGMP

mediates various biological effects of NO such as regulation of vascular tone and

platelet aggregation 23, 24

. Defects in the L-arginine-NO pathway have been implicated

in a variety of cardiovascular diseases including the onset of essential hypertension 23, 29

.

Chapter 1

7

Figure 1.3. Synthesis of nitric oxide by the endothelial cells via L-arginine pathway 24

.

Abbreviations: Ca2+

, calcium; NOS, nitric oxide synthase; L-arg, L-arginine; NO, nitric

oxide; sGC, soluble guanylate cyclase; and GTP, guanosine triphosphate.

Endothelial Dysfunction

Endothelial dysfunction is often a reversible disorder. It refers to different pathological

conditions associated with disturbance on endothelial normal function. In a condition

where there is an imbalance of vasodilator and vasoconstrictor derived from the

endothelial cells, vascular homeostasis is affected resulting in impairment of

endothelium-dependent vasorelaxation. Although, endothelial cells have various

biological functions, endothelial dysfunction is predominantly referred to the

impairment of endothelium-dependent vasorelaxation caused by loss or reduced NO

activity 18

. Reduced NO bioavailability may result from various factor such as declined

expression of eNOS 30

, decreased level of eNOS cofactors or altered activation of

eNOS 31

and enhanced reactive oxygen species (ROS) production from oxidative

Chapter 1

8

stress 32, 33

. Endothelial dysfunction is an early factor in atherosclerosis and other CVD

complications 34

making it a potential indicator to predict future vascular consequences.

It has been proposed that endothelial dysfunction is an independent vascular risk

factor 26

. Aside from medication, smoking cessation and increased physical activity,

identification of dietary factors such as plant phytochemicals are crucial in reversing

endothelial dysfunction to reduce risk of CVD.

1.2.3 Metabolic Syndrome and Cardiovascular Disease

Metabolic syndrome is normally defined as a condition with a constellation of risk

factors for CVD and diabetes, which includes obesity and central fat distribution,

hypertension, insulin resistance, dyslipidaemia, hyperglycaemia as well as

proinflammatory and prothrombotic factors 35, 36

. Endothelial dysfunction is also

associated with metabolic syndrome 37

. Endothelial dysfunction is therefore regarded as

the missing link between CVD risk factors and its damaging outcomes in cases where

atherosclerosis does not develop despite the existence of CVD risk factors 15

.

Metabolic Abnormalities and Endothelial Dysfunction

Although the relation between metabolic abnormalities and endothelial dysfunction is

established, the exact mechanism has yet to be fully elucidated. Hyperglycaemia was

previously shown to affect endothelial function through impairment of endothelium-

dependent vasodilation 38

. Few mechanisms have been proposed to explain the effect of

metabolic abnormalities on perturbed endothelial function. These include mediation of

oxidative stress 39

, agitated glycocalyx, a protective proteoglycans layer of

Chapter 1

9

endothelium 40

and a direct glycosylation of eNOS by O-linked N-acetylglucosamine

(O-G1cNAc) 41

.

Hyperinsulinemia and insulin resistance is recognized to be associated with endothelial

dysfunction 42, 43

. As an important substance in glucoregulatory function, insulin

increases capillary recruitment and blood flow to aid glucose uptake to the skeletal

muscle 44

. Insulin was found to regulate endothelium-derived NO production and ET-1

release via distinct intracellular signaling pathways 45

. The insulin-mediated NO

production signaling pathway (Figure 1.4) has a different phosphorylation-dependent

mechanism which does not employ the calcium-dependent mechanisms of NO

production by the endothelium 41

. NO production by this pathway requires activation of

insulin receptor kinase induces phosphorylation of insulin receptor substrate (IRS-1)

resulting in activation of phosphatidylinositol 3-kinases (PI3K) followed by activation

of phosphoinositide-dependent protein kinase (PDK-1). The downstream signaling then

involves activation of serine-threonine kinase Akt via phosphorylation. Activated Akt

then phosphorylates eNOS resulting in increased NO production. Insulin also regulates

the production of vasoconstrictor substance, ET-1 by the endothelium through mitogen-

activated protein kinase (MAPK) signaling pathway, producing a balanced vascular

homeostasis. In an insulin resistance condition, however, PI3K–Akt–eNOS signaling is

impaired by decreased activation of Akt, decreased eNOS phosphorylation and

hyperglycaemia-induced eNOS glycosylation. This impairment leads to reduced eNOS-

dependent NO production and mitochondrial dysfunction due to enhanced superoxide

production. Excess NO production from inflammation-induced iNOS (inducible eNOS)

leads to nitrative stress. Meanwhile, the Ras-MAPK pathway can be either less sensitive

or enhanced from this condition 41

.

Chapter 1

10

Figure 1.4. Insulin-mediated NO production signaling in healthy condition and in

insulin resistance 41

. Abbreviations: Akt, protein kinase B; BH4, tetrahydrobiopterin;

Cyt C, cytochrome c; eNOS, endothelial nitric oxide synthase; GlcNAc, N-

acetylglucosamine; iNOS, inducible NOS; MAPK, mitogen-activated protein kinase;

mKATP, mitochondrial K+-ATPase; mPTP, mitochondrial permeability transition pore;

PI3K, phosphatidylinositol 3′-kinase; P, phosphorylation; and SNO, nitrosothiol.

AMP-activated protein kinase (AMPK) is recognized as a metabolic sensor due to its

sensitivity to cellular energy status. AMPK is activated from a number of stimulus

including upstream kinases calmodulin-dependent protein kinase kinase (CaMKK) and

liver kinase B1 (LKB1), metabolic stresses, exercise and glucose deprivation 46

.

Phosphorylation of AMPK activates this protein kinase as a response from intracellular

increase in the ratio of AMP to ATP, resulting in metabolic regulation where it switches

on catabolic pathways that produces ATP, and switches off anabolic pathways that

Chapter 1

11

consume ATP 46

. Role of AMPK in fatty acid oxidation is one of the most profound

functions in metabolic regulation amongst many other affected pathways, especially

with the postulation that abnormal fatty acid metabolism is central to metabolic

syndrome 47

. Fatty acid oxidation pathway (Figure 1.5) primarily involves

transportation of long chain acyl-CoA produced from fatty acids via catalysis of acyl-

CoA synthetase (ACS) into the mitochondria by carnitine palmitoyltransferase 1

(CPT1). Carnitine palmitoyltransferase 2 (CPT2) then converts the acylcarnitine back to

long chain acyl-CoA which then undergo fatty acid β-oxidation pathway and

tricarboxylic acid (TCA) cycle in the mitochondrial matrix to produce energy in the

form of ATP. The function of CPT1 can be inhibited by malonyl-CoA produced from

acetyl CoA synthesized by acetyl-CoA carboxylase (ACC), a downstream target of

AMPK 48

. This action can be regulated by reversible phosphorylation of ACC catalyzed

by malonyl-CoA decarboxylase (MCD) 49

. Elevated fatty acid oxidation observed

together with increased expression of MCD in diet-induced obesity mice supports the

function of this enzyme in relieving CPT1 inhibition by malonyl-CoA 48

. The activity of

ACC can also be inactivated from phosphorylation by AMPK 46, 50

. There is also

suggestion that AMPK may have a direct target on MCD expression which lead to a

decline of malonyl-CoA production 51

, however this mechanism is uncertain.

Obesity is linked to endothelial dysfunction which may be caused by insulin

resistance42

. Elevated level of ROS associated with obesity 52, 53

can decrease

bioavailability of NO 18

. Moreover, increased level of free fatty acid from excessive

adipose tissue was found to disrupt the insulin-receptor mediated phosphorylation of

IRS-1 which in turn compromises the PI3K pathway 54

.

Chapter 1

12

Endothelial dysfunction particularly as a result reduced NO bioactivity provides a link

between CVD risk factors and the clinical manifestations resulting to CVD mortality

and morbidity. Therapeutic approaches towards restoration of this condition, as well as

reducing risks of CVD are therefore a target for reducing the burden disease.

Figure 1.5. Fatty acid oxidation pathway and the involvement of AMPK and ACC (re-

drawn from Folmes & Lopaschuk 2007 48

). Abbreviations: AMPK, AMP-activated

protein kinase; ACC, acetyl-CoA carboxylase; MCD, malonyl-CoA decarboxylase;

ACS, acyl-CoA synthetase; CPT-1, carnitine palmitoyltransferase 1; and CPT-2,

carnitine palmitoyltransferase 2.

Chapter 1

13

1.3 Phenolic Compounds

Phenolics are widely dispersed in plants such as fruits and vegetables with diverse

biological functions. The structures of these compounds vary greatly from simple

molecules such as phenolic acids to highly polymerized molecules such as

proanthocyanidins. Phenolics play a crucial role in numerous plant physiological

activities, including determination of colour of leaves, flowers and fruits of a plant 55

.

Phenolics aid the defence system of plants by protection against microbial or fungal

infection as well as discouraging plant feeding by insects or higher class animals with

its astringent taste. They also protect plants from UV radiation 56

, toxic heavy metals

and oxidation from free radicals 57, 58

. Apart from regulating plant biochemistry and

physiology, phenolics are also found to be useful to help increase the shelf life of food

and inhibit the growth of pathogenic microorganisms due to their antimicrobial property

59. These phytochemicals are also important for regulation of sensory qualities of plant

fruit and beverages especially in the visual appearance (browning and pigmentation)

and taste of fruits 60

, which are significant factor for consumer satisfaction. Although

phenolics are not considered an essential nutrient, there has been increasing interest in

phenolics because of their protective effects against chronic disease.

1.3.1 Classification of Phenolic Compound and Its Dietary

Sources

Phenolics are secondary metabolites of plants that are structurally characterized by the

presence of one or more phenolic hydroxyl groups 61, 62

. With more than 8000 phenolics

Chapter 1

14

identified, these compounds are generally categorized as flavonoid and non-flavonoid

compounds 61

. Phenolics can be divided into different classes depending on the number

of phenol rings in their structure and the components which binds the rings together 63

.

Due to the abundance in plants, phenolics form a vital part of human diet. The richest

dietary sources of phenolic compound are fruits, vegetables, legumes, cereals, nuts and

beverage products of plant extracts such as wine, tea, coffee and cocoa. The type and

level of phenolics vary among different foods. Among the numerous phenolic

compounds, the two main classes with abundance in dietary sources are phenolic acids

and flavonoids. Figure 1.6 summarises phenolic classification and their basic chemical

structures.

Chapter 1

15

Figure 1.6. Classification of chemical structures of the major classes of phenolic.

Flavonoids

Flavonoids are polyphenolic compounds comprising of 15 carbons (C6-C3-C6), with 2

aromatic rings (A and B rings) connected by a 3-carbon bridge (C-ring) (Figure 1.6).

Flavonoids are further divided into its subclasses according to the modifications of the

central C-ring for example by the presence of a carbonyl group at carbon 4, a double

bond between carbon atoms 2 and 3, or a hydroxyl group in position 3 of the C ring 64

.

Chapter 1

16

Generally flavonoids are subdivided into flavonols, flavones, flavanols, flavanones,

isoflavones and anthocyanidins. Other flavonoid groups, which quantitatively are

relatively minor dietary components, are dihydroflavones, flavan-3,4-diols, coumarins

and aurones.

The flavonols are the most widespread flavonoids in plant food, with quercetin,

myricetin and kaempferol being the most common compound. Isorhamnetin, which is

the methylated derivative of quercetin, is also quite common among flavonols

compounds. Flavones are structurally very similar to flavonols and differ only in the

absence of hydroxylation at the 3-position on the C-ring. Flavones are generally

represented in the diet by apigenin and luteolin. They are not quite as widely dispersed

as flavonols. Flavanols are structurally the most complex subclass of flavonoids ranging

from the simple monomers (+)-catechin and its isomer (-)-epicatechin to the oligomeric

and polymeric proanthocyanidins. Proanthocyanidins, also known as condensed tannins

are ubiquitous and present as the second most abundant natural phenolic after lignin 65

.

Their presence in food affects food quality and are capable to regulate food

functionality 66

. The flavanone structure is highly reactive and has been reported to

undergo hydroxylation, glycosylation, and -methylation reactions. Flavanones are

generally represented by naringenin, hesperitin and eriodictyol, and also a number of

minor compounds, including sakuranetin and isosakuranetin. In contrast to most other

flavonoids, isoflavones are characterized by having the B-ring attached at C3 rather than

the C2 position. They have a very limited distribution in the plant kingdom with

significant quantities detected only in leguminous species 67, 68

. Isoflavonoids which are

found in plants include daidzein, genistein, glycitein, puerarin, formononetin and

bichanin. Anthocyanidins are plant pigments and are predominantly evident in fruit and

Chapter 1

17

flower tissue where they are responsible for the diverse colour. There are approximately

17 anthocyanidins found, with only 6 (cyanidin, delphinidin, petunidin, peonidin,

pelargonidin and malvidin) considered to be of dietary importance 69, 70

. These

compounds are involved in photoprotective function in plants 71

. The most widespread

anthocyanin in fruits is cyanidin-3-glucoside 55

. Table 1.1 summarises the compounds

within major classes of flavonoid and respective common dietary sources.

Chapter 1

18

Table 1.1. Compounds within major classes of flavonoid and respective common

dietary sources 63, 72, 73.

Flavonoid

classes

Compounds Common dietary sources

Flavonols Quercetin, myricetin,

kaempferol, isorhamnetin

Onion, kale, leeks, broccoli,

blueberries, red wine, tea

Flavones Luteolin, apigenin Parsley, celery

Flavanols (+)-catechin , (-)-epicatechin,

(+)-gallocatechin,

(+)-epigallocatechin,

(-)-epicatechin-3-gallate,

(-)-epigallocatechin-3-gallate,

proanthocyanidins

Green tea, tea, chocolate, red

wine, apricots, grapes, apples

Flavanones Naringenin, hesperitin,

eriodictyol

Citrus fruits, tomatoes, mint

Isoflavones Daidzein, genistein, glycitein Soybean and soybean products

Anthocyanidins Cyanidin, peonidin,

delphinidin, petunidin,

malvidin, pelargonidin

Red wine, berries, plum,

strawberries

Phenolic Acids

Phenolic acids are the simplest form of phenolics due to the basic structure consisting of

one phenol ring (Figure 1.6) 74

. The nature of phenolic acid structures allows them to

form esters and ether cross-linkages, and therefore makes them important in plant

structural features 75

. Phenolic acids are hence abundant in seeds, skins, stems and

leaves of plants 75

. Phenolic acids can be subdivided into derivatives of benzoic acids

and cinnamic acids. Hydroxybenzoic acids are commonly represented by gallic,

Chapter 1

19

-hydroxybenzoic, vanillic and syringic acids. These compounds are usually present in

the bound form and are usually components of complex structures such as lignins and

hydrolysable tannins. Due to their lower prevalence in plant-based food,

hydroxybenzoic acids has not been widely studied 63

. Hydroxycinnamates are more

common than hydroxybenzoic acids in human dietary sources. The four most common

hydroxycinnamates are caffeic acid, -courmaric, ferulic acid and sinapic acid 76

.

Hydroxycinnamic acids occur frequently in bound forms such as glycosylated

derivatives or esters with quinic acid, shikimic acid and tartaric acid 63

. Chlorogenic

acid (Figure 1.7), is an ester formed between caffeic acid and quinic acid, forming

conjugated structures including caffeoylquinic acid, feruloyquinic acid and

-coumaroylquinic acid 77

. Chlorogenic acid is one of the most abundant

hydroxycinnamate in fruits and vegetables, and in extracted beverages such as coffee.

Major chlorogenic acids found in coffee include 5- -caffeoylquinic acid (5-CQA)

(chlorogenic acid), and its isomers 4- -caffeoylquinic acid (4-CQA) (cryptochlorogenic

acid) and 3- -caffeoylquinic acid (3-CQA) (neochlorogenic acid) 78

. Table 1.2

summarises the compounds of phenolic acids and respective common dietary sources.

Figure 1.7. The chemical structures of chlorogenic acids.

Chapter 1

20

Table 1.2. Compounds of phenolic acid classes and its common dietary sources 63, 79-81

.

Phenolic acid

classes

Compounds Common dietary sources

Hyroxybenzoic

acids

Gallic acid,

-hydroxybenzoic acid,

protocatechuic acid,

vanillic acid, syringic acid

Green tea, black tea, berries,

rosaceous fruits, red wines and

potatoes, onion, herbs

Hyroxycinnamic

acids -courmaric, caffeic acid,

ferulic acid, sinapic acid,

chlorogenic acid

Coffee, wheat bran, blueberries,

kiwi fruits, cherries, plums,

aubergines, apples, pears, chicory,

cider, spinach, broccoli, kale

1.3.2 Fruits as a Major Source of Phenolic Compounds

Knowledge of the plant phenolic composition has aided the identification of plant-based

human dietary sources which are likely to contribute to a healthy nutrition. Fruits are

recognized to have a rich content of phenolics. Fruits are one of the major sources of

phenolics among Australian adults 82

. Furthermore, total phenolic intake from fruit was

identified to be higher than from vegetables in a French population 83

. This suggests the

importance of assessing phenolics levels in fruits to understand its potential in human

health. Among various fruits, apples are one of the fruits which has been explored

widely for its phenolic composition 80

. Phenolic distribution in fruits may vary greatly

due to different factors for instance postharvest storage conditions, processing and

cultivar variation 84-86

. Table 1.3 shows an example of cultivar variation of phenolic

composition in different fruits. The data illustrates that there is more than 6-fold

variation of phenolic composition among grape, more than 9-fold variation among

nectarine cultivars, and more than 10-fold variation among apple cultivars 60, 87, 88

.

Chapter 1

21

Substantial variation of phenolic content among different fruits is also observed 60, 87, 88

.

There are also differences in phenolic content in different tissues of a fruit, for example

in apple flesh and skin 88

. Understanding phenolic composition among different

cultivars of fruit can help fruit breeding programme in developing fruit cultivars with

high phenolic contents. This could also aid the engineering of fruits with elevated level

of phenolics using genetic modification approach, which may contribute to the supply

of natural and effective way towards reducing burden of diseases.

Antioxidant capacity has been used as a measure of antioxidant benefit of food products

such as fruits 73, 89-91

. Many studies have explored the total antioxidant capacity of fruits

using various assays to determine the potential benefits of certain types of fruit, as well

as for health promotion. Some of the various assays commonly used to measure

antioxidant capacity in fruits include oxygen radical absorbance capacity (ORAC), 2,2-

azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), ferric reducing antioxidant

power (FRAP), 2,2’-diphenyl-1-picrylhydrazyl (DPPH), trolox equivalent antioxidant

capacity (TEAC) and total radical trapping antioxidant parameter (TRAP) 89, 92, 93

.

Various assays involving different reactions have been developed in the effort to

improve methods for antioxidant measurement with different sample conditions 94

.

Total antioxidant capacity measure is useful for the in vitro assessment of antioxidant in

foods 94

. However, the relevance of antioxidant capacity measure in reflecting the level

of bioactivity towards promoting health is being questioned 95, 96

. An ideal biomarker to

be applied in breeding programs for developing fruits with enhanced level of health

properties remains unclear. Phenolic composition of fruit could be a good biomarker for

this purpose.

Chapter 1

22

Table 1.3. Cultivar variation of phenolic composition in different fruits 60, 87, 88

.

Abbreviation: Total HC, total hydroxycinnamic

Fruit Cultivar Phenolics (µg.g-1

of fresh weight)

Total

HC

Total

flavanols

Total

flavonols

Total

anthocyanins

Apple Empire Peel

Flesh

182

162

151

0

350

0

208

-

McIntosh Peel

Flesh

169

235

1015

236

300

0

42.9

-

Red

Delicious

Peel

Flesh

50

137

1655

366

244

3.7

149

-

Golden

Delicious

Peel

Flesh

159

165

709

220

220

6.4

0

-

Grape Red Globe

Skin +

Flesh

8 40 61 115

Crimson

Seedless

48 109 54 151

Flame

Seedless

10 41 13 69

Napoleon 10 18 32 76

Nectarine Arctic Star Peel

Flesh

387

103

280

50

74

0

136

0

Fire Pearl Peel

Flesh

78

51

134

23

34

8

173

10

Red Jim Peel

Flesh

430

180

621

215

91

7

261

14

August

Red

Peel

Flesh

329

147

324

123

68

10

34

8

Chapter 1

23

1.3.3 Bioavailability and Metabolism of Phenolic

Compounds

Dietary intake of phenolics from human diet is estimated to be around 1 g.day-1

80, 97

.

However, the maximum plasma concentration of these compounds rarely exceed

1µmol.L-1

following ingestion of 10-100 mg of single phenolic compound 80

. Diversity

of phenolic compounds nature indicates the variation in bioavailability and subsequent

biological effects. For example, one phenolic compound with high abundance in dietary

sources may not be the most biologically active in the body if it is poorly absorbed 63, 80

.

In order to understand the importance of phenolics towards potential health effects in

vivo, whether it is from phenolic-rich food or even isolated phenolic compound, it is

vital to understand the absorption, metabolism, bioavailability and the consequent

biological activities of specific phenolic compounds.

Quercetin absorption was studied in ileostomy subjects showing its ability to be

absorbed in humans 98

, which contradicted an earlier report 99

. The ability of phenolic

compound to be absorbed is further supported by a more recent studies 100, 101

.

Absorption of phenolics depends largely on the chemical structure of each specific

compound. Most phenolic classes including flavonols, flavones, isoflavones and

anthocyanidins typically exist in glycosylated form; conjugated to a sugar group. With

the sugar group attached in the flavonoid glycosides, absorption involves hydrolysis to

release aglycones by action with lactase phloridzin hydrolase (LPH) in the brush-border

of the small intestine epithelial cells or alternatively mediated by cytosolic β-

glucosidase (CBG) within the epithelial cells 102

. The enzymatic deglycosylation can

occur in the food, the gastrointestinal mucosa cells or in the colon microflora after

Chapter 1

24

ingestion 80

. Nonenzymatic deglycosylation does not occur in the human body 103

. The

type of glycosides that the flavonoid contains can determine its rate and site of

absorption 104-106

. For example, polyphenols glycosylated to glucose can be absorbed in

the small intestine following hydrolysis with the β-glucosidases. However, polyphenols

glycosylated to rhamnose may only be cleaved by colonic microflora α-rhamnosidases.

Monomeric flavanols are usually acylated, but this substitution does not affect the

bioavailability of flavanols as much as the glycosylation in flavonols 80

. Flavanols are

believed to be efficiently absorbed without deconjugation or hydrolysis 80

. Polymer

flavanols, proanthocyanidins however, are poorly absorbed compared to the monomers.

This is thought to be associated with its complex structure and larger molecular weight

63, 80.

Absorption of phenolic acid has been studied to a lesser extent than flavonoids, although

there has been recent focus on the bioavailability of hydroxycinnamates from coffee

consumption. Similar to flavonoid aglycones, free phenolic acids can be absorbed into

the small intestine through passive diffusion 63

. However, hydroxycinnamates

frequently occur in food as esters of sugars, organic acids or lipids 80

. Esterification of

caffeic acid to form chlorogenic acid was found to reduce the rate of absorption 107

. This

is supported by findings that a lower proportion of ingested chlorogenic acid is absorbed

in the small intestine, and approximately two thirds of the ingested chlorogenic acid is

metabolized in the colon before absorption 77, 107

.

Upon absorption, phenolic aglycones are then be conjugated forming sulfate,

glucuronide and/or methylated metabolites through actions with sulfotransferases

(SULT), uridine-5’-diphosphate glucuronosyltransferases (UGT) and catechol- -

Chapter 1

25

methyltransferases (COMT) respectively, which takes place in intestine and liver 108-110

.

These conjugations are important in restricting potential toxicity of the aglycones and

augment their biliary and urinary excretion 63

. Once these conjugates reach the blood

stream, they are further metabolized in the liver and kidney. Phenolics which are not

absorbed in the small intestine such as proanthocyanidins 63

and most of ingested

chlorogenic acid 111

which gets through to the colon is metabolized by the colonic

microflora. Metabolized phenolic conjugates can be excreted via urinary or biliary

route. Conjugated phenolics secreted in bile can be further metabolized by the colonic

microflora to release free aglycones which allow reabsorption 106

. Among the

metabolites excreted in urine from chlorogenic acid consumption are caffeic acid,

ferulic acid, isoferulic acids, dihydroferulic acids and dihydrocaffeic acid 77

. However,

renal excretion is not a major elimination route of phenolic acids and its metabolites 112,

113. The metabolites produced from metabolism in small intestine and colon are

suggested to be further metabolized before entering blood circulation 114

. Bioavailability

of phenolic acids is hugely determined by their uptake into the gut mucosa 115

. The

apparent bioavailability of chlorogenic acid varied greatly among subjects with values

ranging from 7.8 to 72.1 % 113

. Given the influence of phenolic acids uptake to

bioavailability, the huge inter-individual variability of this compound in this study

might be explained by the variation of metabolic factors of different individuals.

Generally, absorption of phenolics prior to metabolism is regulated not only by the

chemical structure, but also affected by the food matrix 116

and the nature of intestinal

microflora 117

. The understanding of this area is still an active part of research, and

information of each one of the phenolic compounds remains to be fully elucidated.

Chapter 1

26

1.4 Dietary Chlorogenic Acid and Cardiovascular

Protective Effects

1.4.1 Importance of Studying Chlorogenic Acid

The inverse association between phenolic-rich diets and risk of developing chronic

diseases such as CVD reported from various studies 118, 119

have mainly attributed the

benefits to flavonoids. However, the possibility that health benefits from phenolic-rich

diet is due to complex synergistic effects between various phenolics cannot be

disregarded.

Studies on phenolic acids have been explored at a lesser extent even though there are

reports on health protective effects of coffee which is rich in phenolic acids 120, 121

.

Among phenolic acids, chlorogenic acid is one of the compound that is easily

achievable from the diet especially from coffee, one of the most widely consumed

beverage in the world 120

. Regular coffee consumers may consume up to 1 g chlorogenic

acid daily 107

. The study of chlorogenic acid as a pure compound is important to

understand the potential mechanism it may exert in producing benefits towards reducing

the incidence of CVD. This is crucial realizing that different phenolic compounds may

be metabolized at a differing rates and show differing mechanisms of action.

Chapter 1

27

1.4.2 Evidence from Epidemiological Studies and Clinical

Trials

To date, few studies have explored the effects of pure phenolic acids on CVD risk

factors in clinical trials. However, numerous studies have examined the association of

coffee consumption with CVD. Being a beverage with high content of chlorogenic acid,

observations of studies on coffee could therefore point towards clinical benefits of

chlorogenic acid. Reports on association of coffee consumption with CVD from the

literature are highlighted here to provide an understanding of chlorogenic potential role

on CVD risks factors. However, the potential benefit of coffee consumption is difficult

to be postulated due to some contradictory evidence in the literature.

In earlier years, coffee was reported to elevate risks of developing CVD 122-124

.

However, the underlying factors in forming that view are complex due to involvement

of other confounding factors such as cigarette smoking, inactive lifestyle, unhealthy

dietary routines which may be related to habit of coffee consumption. More recent

studies have reported that coffee consumption may not be related to increased risks of

CVD when these confounding factors are considered. In the Framingham Study,

consisting of 5,209 men and women aged 30 to 62 years, coffee consumption was not

associated with CVD incidence after adjustment of cigarette smoking factor 125

. A

prospective cohort study involving 45,589 men at the age between 40 to 75 years old

who had no history of CVD, reported that total coffee consumption was not associated

with elevated risk of coronary heart disease (CHD) and stroke over 2 years

follow-up 126

. Similar reports were made in another prospective cohort study 127

and a

meta-analysis of 11 prospective studies 128

.

Chapter 1

28

Other studies have reported that coffee consumption has an inverse association with

risks of developing CVD and reduced coronary mortality and morbidity 129-131

.

Additionally, a meta-analysis of 16 randomized controlled trials investigating coffee or

caffeine intake on blood pressure (BP), reported that elevation of BP was bigger from

caffeine when the coffee and caffeine trials were analysed separately 132

. This suggests

that other major component in coffee such as chlorogenic acid may mask the BP

elevation resulting from caffeine.

Contrary to the reports from population studies discussed above, a cross over study

reported that high consumption of chlorogenic acid from coffee raised homocysteine

concentration in plasma, which is a risk for CHD 133

.

It is known that type 2 diabetes influence the higher risks of CVD complications 134

.

Studies have shown inverse association between coffee consumption and risk of

developing type 2 diabetes 121, 135, 136

. Similar associations of caffeinated and

decaffeinated coffee with reduced risk of type 2 diabetes 137, 138

further suggest the

possibility of effect from coffee components other than caffeine.

1.4.3 Effects of Chlorogenic Acid on Hypertension and

Endothelial Dysfunction

Hypotensive Effects of Chlorogenic Acid

The effects of chlorogenic acid on regulation of BP have mainly focussed on reports

from green coffee bean extract (GCE) which has a high content of this phenolic acid.

Chapter 1

29

Suzuki et al. 139

studied an acute oral ingestion of GCE (180 to 720 mg.kg-1

GCE; 28%

chlorogenic acid content) and chronic GCE supplementation over a period of 6 weeks

(0.25 to 1% GCE supplemented in normal diet) on BP in spontaneously hypertensive

rats (SHR). Both measurements showed a dose-dependent decrease of BP in SHR with

no effects seen in control rats. In the same study, a single oral ingestion of 50 to

200 mg.kg-1

5-CQA, a major chlorogenic acid found in GCE was also shown to reduce

BP in a dose-dependent manner 139

. Another study by the same group reported that oral

administration of ferulic acid, a metabolite of chlorogenic acid also reduced BP in

SHR 140

. A BP lowering effect was observed after an acute ingestion of 5-CQA (30-600

mg.kg-1

) and 0.5% dietary supplementation (300 mg.kg-1

per day) for 8 weeks in SHR,

an effect which is not observed in the control rats 141

. These findings support

chlorogenic acid as the active component of GCE treatment in SHR.

Studies of GCE consumption in human clinical trials supports the results from

experiments using animal model as discussed above. A study investigating the effect of

chlorogenic acid intake via GCE consumption was carried out using a randomized

double-blinded controlled trial in 117 mildly hypertensive subjects 142

. They were

allocated to 4 groups receiving placebo, 46, 93 or 185 mg GCE containing 54%

chlorogenic acid respectively for 28 days. The study reported a dose-dependent

reduction of BP following consumption of GCE 142

. Similar findings were also reported

in another study of similar design involving 28 mildly hypertensive subjects 143

. In the

study, BP was reduced after 12 weeks of 140 mg per day chlorogenic acid consumption

from GCE. These studies however focussed on hypotensive effect of chlorogenic acid

from GCE in hypertensive subjects, while the effect of pure chlorogenic acid in

reducing blood pressure in healthy subjects is unclear.

Chapter 1

30

Effect of Chlorogenic Acid on Endothelial Dysfunction: Potential

Mechanism in Improving Hypertension

As discussed earlier, endothelial dysfunction is a reversible condition which may be

benefited by treatment from pharmaceutical or dietary approach towards reducing CVD

outcomes. Effects of chlorogenic acid and its metabolites from coffee intake on CVD

risk may be due to a positive impact on endothelial function.

The study by Suzuki et al. 141

which showed an anti-hypertensive effect of 5-CQA in

SHR as discussed in the previous section, also described that the beneficial effect was

attenuated by N(G)-nitro-L-arginine methyl ester which is an inhibitor of eNOS. This

finding suggests that 5-CQA provide anti-hypertensive actions via enhanced

endothelium activity, which is supported by their report of enhanced urinary NO

metabolites. The authors also reported reduced NADPH-dependent superoxide anion in

aorta after 5-CQA intake. A reduction of superoxide anion may suppress the production

of ROS via reaction with molecular oxygen reduced by uncoupled eNOS 144

. A

reduction of urinary hydrogen peroxide suggests that 5-CQA inhibited oxidative stress

by reducing NADPH oxidase activity 141

. Ferulic acid at 50 mg.kg-1

intravenous

treatment in SHR was reported to induce vasorelaxation of the artery, which was

inhibited by a competitive agonist of acetycholine receptor. This compound was also

reported to enhance NO bioavailability and reduced NADPH-dependent superoxide

anion levels. This result suggests a beneficial effect of the chlorogenic acid metabolite

on enhancing acetylcholine-induced endothelial dependent vasodilation 139

. Similar

effects on enhanced NO bioavailability, reduced hydrogen peroxide and improvement

Chapter 1

31

on endothelial dysfunction was reported in SHR after 8 weeks of 0.5% chlorogenic acid

supplementation in the diet 145

.

The role of chlorogenic acid in normal function of endothelial cells has also been

investigated in vitro. Most of the studies, however, use non-physiological doses to show

beneficial effects of chlorogenic acid. Chlorogenic acid at an in vitro dose of 25 and

50 µM was found to inhibit the expression of adhesion molecules and ROS production

in human umbilical IL-1β-treated endothelial cells. Enhanced expression of adhesion

molecules in the endothelial cells is crucial is pathogenesis of atherosclerosis, where it

leads to recruitment of monocytes to the atherosclerotic sites. Another in vitro study

showed that dihydrocaffeic acid, a metabolite of caffeic acid was reported to stimulate

activity of eNOS at a concentration between 50-200 µmol.L-1

in human endothelial

cells 146

. Results from these studies, may imply beneficial effect of chlorogenic acid and

the metabolites on the healthy function of endothelial cells and enhanced NO production

in prevention of atherogenesis. However, the concentrations used far exceeded

physiological relevance, and the question remains whether a nutritionally relevant

amount of chlorogenic acid would reproduce the effects in vivo, since the peak

concentration of phenolic compounds in human plasma is comparably low after

extensive metabolism (<1 µmol.L-1

) 80

. A direct antioxidant effect of chlorogenic acid,

and in fact, some other phenolic compounds found in in vitro studies is also

questionable not only for its relevance to physiological doses, but also because a causal

association between oxidative damage biomarkers and CVD is yet to be established 95

.

Investigation of chlorogenic acid effects on endothelial dysfunction in humans in scarce.

Evidence on beneficial effects of this compound on promoting healthy endothelial

Chapter 1

32

function is still unclear due to the inconclusive results from human trials. In a cross-

sectional study of 730 healthy women and 663 women with type 2 diabetes from the US

Nurses’ Health Study 147

, higher caffeinated coffee consumption was inversely

associated with plasma level of endothelial dysfunction markers in women with type 2

diabetes, while a similar observation was obtained with higher decaffeinated coffee in

healthy women, suggesting an effect of other coffee components than caffeine, such as

chlorogenic acid, on suppressing endothelial dysfunction. In the study, the authors

suggested that filtered coffee consumption is inversely related to endothelial

dysfunction and inflammation 147

. A randomized double-blinded cross-over study

involving 10 male and 10 female healthy subjects, investigated an acute effect of

caffeinated and decaffeinated espresso coffee with a 5-7 days washout period, on

endothelial function using measurement of FMD of the brachial artery. The authors

reported that caffeinated espresso resulted in decreased FMD, but this effect was not

observed with decaffeinated espresso 148

. In a controlled-clinical trial with 20 healthy

men, ingestion of green coffee extract (140 mg per day caffeoylquinic acid) resulted in

improvement of the reactive hyperemia ratio indicating improvement of vasoreactivity

compared to control subjects 149

.

Despite the potential positive implications of chlorogenic acid and its metabolites on

endothelial dysfunction, the findings from human studies are limited, and have mainly

been obtained from studies investigating effect of chlorogenic acid obtained from

coffee. The variation of chlorogenic acid level from different coffees as a result of

roasting process, types and amount of coffee bean used 78

may influence the effects

reported from different studies. It may also be influenced by major coffee components

other than caffeine. This makes it difficult to conclude the effect of chlorogenic acid on

Chapter 1

33

endothelial function from existing studies. Therefore, effects of pure chlorogenic acid

on endothelial dysfunction and NO status in humans would help to clarify these issues.

1.4.4 Chlorogenic Acid Effect on Metabolic Disorders

Associated with Cardiovascular Disease Risks

Metabolic abnormalities such as hypertriglyceridemia and hyperglycemia both have

adverse effects on the endothelial dysfunction 39, 150

. This would increase the

development of atherosclerosis and other CVD complications. Therefore, treatment of

metabolic abnormalities is necessary to reduced incidence of the diseases. The effect of

chlorogenic acid on reducing or reversing metabolic abnormalities has been stimulated

by evidence showing an inverse association between coffee consumption with risk of

developing type 2 diabetes 121, 151

.

In a randomized cross-over human trial, the effect of chlorogenic acid on glucose

tolerance was tested in 15 non-smoking, overweight men. Subjects were randomized

into 4 treatments: (i) 12 g decaffeinated coffee containing 264 mg chlorogenic acid; (ii)

1 g chlorogenic acid; (iii) 500 mg trigonelline (an alkaloid found in coffee) and (iv)

placebo, with a 6 days washout between the treatments. Chlorogenic acid was found to

reduce early glucose and insulin responses during an oral glucose tolerance test

(OGTT) 152

.

In a recent animal study, supplementation of chlorogenic acid and caffeic acid at

0.2 g.kg-1

in high fat diet on male Imprinting Control Region (ICR) mice resulted in

reduced body weight and fat mass, as well as improved lipid metabolism and hormones

Chapter 1

34

related to obesity. However, the study reported that chlorogenic acid was more potent

than its metabolite, caffeic acid 153

. In another animal study, coffee phenolic

supplementation in high fat diet for 15 weeks was reported to reduce diet-induced

obesity by down-regulation of SREBP-1c (sterol regulatory element binding protein 1c),

which suppress lipogenic enzymes, while increasing fatty acid oxidation via reduction

of ACC-2 mRNA and ACC activity which reduced level of malonyl-CoA 154

. In an

independent animal study, the same group of researchers investigated the effects of

coffee phenolics on postprandial carbohydrate and lipid metabolism, and whole body

substrate oxidation in C57BL/6J mice 155

. In this recent study, coffee phenolics intake

(200 mg.kg-1

body weight) was found to modulate whole body substrate oxidation by

inhibiting postprandial hyperglycaemia, hyperinsulinemia and hyperlipidemia 155

. The

authors suggested that inhibition of postprandial insulin might also explain the

observation of reduced body fat accumulation which they observed in the earlier

study 154

. Investigations using different animal genotypes may however result in

difficult comparison between the observed results from different studies.

In vitro studies have also been performed to investigate the effects of chlorogenic acid

on metabolic abnormalities. This includes a study investigating caffeic acid and

chlorogenic acid on phosphorylation of AMPKα Thr172

and activities of α1- and α2-

isoform-specific AMPK in Sprague-Dawley rat skeletal muscle 156

. The concentrations

used for the compounds were extremely high (1 mmol.L-1

for time-dependent

measurements and between 0.01 to 1 mmol.L-1

for a dose-dependent measurement).

Caffeic acid, but not chlorogenic acid, enhanced AMPK activity (particularly

AMPKα2). The enhancement of the kinase activity was accompanied with insulin-

independent glucose transport and a reduced muscle energy status 156

. This might be

Chapter 1

35

explained by the extensive metabolism of chlorogenic acid 156

. These findings are

limited by the doses used, which are not comparable to physiological concentrations of

the compounds. In addition, the route of administration in some studies (for instance

intaperitoneal injection) 157

might affect the rate of metabolism of the compounds

compared to a dietary supplementation. This would further confound the comparison of

results from different studies. Other recent findings from the studies on chlorogenic acid

and its metabolites effects are summarized in Table 1.4.

36

Table 1.4. Summary of recent studies investigating effect of chlorogenic acid on metabolic abnormalities. Abbreviations: CGA, chlorogenic acid; GIP,

glucose dependent insulinotropic peptide; GLP, glucagon like peptide; AUC, area under curve; PPAR-α, peroxisome proliferator-activated receptor

alpha; AMPK, AMP-activated protein kinase; Akt, protein kinase B.

Study

type

Study design Subject

/strain /cell

CGA

concentration

Findings Reference

Human Randomized,

cross-over

(acute study)

15 overweight

man

1 g CGA - No effect on GIP or GLP secretions

- No improvement on glucose tolerance

Olthof et al.

158

Animal Randomized

cross-over

Male Sprague-

Dawley Rat

120 mg.kg-1

- Decreased AUC for blood glucose, reduced GIP

secretion

- slower glucose appearance from intestine into

Circulation

Tunnicliffe

et al. 159

Animal Randomized,

chronic study

(28 days)

Male Sprague-

Dawley Rat

1 mg.kg-1

/

10 mg.kg-1

in high

cholesterol diet

Hypocholesterolemic, reduced liver fat depositions, increased lipid

unitization in liver via PPAR-α mRNA.

Wan et al. 160

Ex vivo

In vitro

N/A - Skeletal

muscle from

db/db mice

- L6 skeletal

muscle cells

250 mg.kg-1

for

ex vivo

2 mmol.L-1

for

in vivo

In db/db mice: decreased fasting blood sugar from oral glucose tolerance

test, enhanced glucose transport

In L6 myotubes: dose and time-dependent enhanced glucose transport,

CGA phosphorylated AMPK and ACC, enhanced AMPK activity,

activation of Akt; CGA stimulated glucose transport via AMPK activation

Ong et al. 157

Chapter 1

37

1.5 Rationale of Thesis

A diet rich in fruits and vegetables contributes to better health. This health benefit may

in part be attributed to phenolic compounds, which are abundant in plants. If phenolic-

rich food and beverages are to be promoted for health benefits, it is important to

understand the distribution and bioactivity of these compounds.

1.5.1 Study 1 – Aim and Hypothesis

The influence of cultivar variation of fruits to the phenolic content has been illustrated

in previous studies. Health benefits of fruit phenolics have often been attributed to its

antioxidant effect; quenching reactive oxygen species and preventing consequent

cellular damage. Composite assays of total antioxidant capacity have been used as a

measure of antioxidant benefit of food products as well as being used for health

promotion. However, recent studies and commentary seriously question the validity of

total antioxidant capacity, as a reflection of the level of bioactivity in food products.

Yet, there would be great value to horticulture and breeding programs if a simple

biomarker could be applied for the development of fruits with enhanced level of health

properties.

Therefore, in the first study (Chapter 2) of this thesis, the aim was to determine the total

phenolic composition of major known bioactive phenolic compounds in pre-varietal

selections of Western Australian plums. In addition, the antioxidant capacity of the

plums and its association with the phenolic content was measured to determine if

antioxidant ability of phenolics in the plums is a suitable tool for plum breeding. It was

Chapter 1

38

hypothesized that the content of major bioactive phenolic compounds would be a more

valid choice for implementation in the plum breeding program, in order to reflect

potential nutritive value.

1.5.2 Study 2 - Aim and Hypothesis

Chlorogenic acid, a phenolic acid (more correctly termed as hydroxycinnamate) is

easily achieved from dietary sources such as fruits including plums, as well as coffee.

Recent studies have suggested some beneficial effects of chlorogenic acid on

cardiovascular health, although the mechanism of action remains inconclusive.

Therefore, in the second study (Chapter 3) of this thesis, the effects of pure chlorogenic

acid, on blood pressure and endothelial function were investigated in a placebo

controlled clinical trial. NO status was measured to determine if effects seen from

chlorogenic acid, if any, was mediated via enhancement of NO status. It was

hypothesized that chlorogenic acid would acutely increase NO status, which in turn

improves endothelial function and lowers blood pressure in healthy men and women.

1.5.3 Study 3 - Aim and Hypothesis

Coffee phenolics such as chlorogenic acid have been suggested to improve metabolic

abnormalities (fat accumulation, hyperinsulinemia and hyperglycaemia) which are risks

for CVD. Since coffee contains many different compounds, there are advantages to

studying the constituent pure compounds in greater clinical detail.

Chapter 1

39

Therefore, the aim of the third study (Chapter 4) of this thesis was to examine the effect

of pure chlorogenic acid on high fat diet induced obesity, glucose intolerance and

insulin resistance in a mouse model. Effect on fatty acid oxidation and insulin signaling

pathways were also studied. It was hypothesized that chlorogenic acid would exert

improvement on high fat diet-induced obesity, glucose intolerance and insulin

resistance, as well as enhancement of fatty acid oxidation and insulin signaling

pathways in mouse.

Chapter 2

40

CHAPTER 2

Phenolic Composition of New Plum

Selections in Relation to Total

Antioxidant Capacity

(The results from this chapter have been published in Journal of Agricultural and Food

Chemistry. 2012; 60: 10256-10262)

2.1 Introduction

It is widely accepted that dietary intake of fruits and vegetables is inversely related to

the incidence of chronic disease, including cardiovascular disease (CVD), cancers,

obesity, and type 2 diabetes mellitus 161

. Government and intergovernment agencies

have widely promoted consumption of fruits and vegetables to improve health outcomes

and reduce the medical burden. Horticultural breeders and marketers have also seen the

commercial opportunity in crop improvement. Dietary phenolics are principal

candidates to explain the health-protective effects of fruit, vegetables and beverages

derived from them 61, 162

. However, the nexus between in vitro bioactivity and food

phenolic composition is still under debate 95, 163, 164

. Early research focused on the role

of phenolic in attenuating oxidative damage, as they are an abundant form of

antioxidant in the human diet 80, 165

. Crude antioxidant activity was evaluated from a

wide range of foods 166, 167

and found to positively correlate to total phenolic

Chapter 2

41

concentration 168-170

. Yet, this is recognized as a simplification, as knowledge of the

diverse bioactivities of various phenolic compounds has vastly advanced over the past

decade 95, 163, 171

. Furthermore, antioxidant activity per se may be altered by metabolism

and bioavailability 172

. In addition, the activity of quercetin and (-)-epicatechin to

enhance nitric oxide production and reduce endothelin-1 in healthy human subjects is

not necessarily related to their antioxidant activity 173

. Evidence for roles in modulating

gene expression and cell signaling is rapidly accumulating, e.g., the effect of quercetin

on the Nrf2/Keap1 pathway 174, 175

and of proanthocyanidins on microRNA

expression 176

. Increasingly, investigation of cell signaling or other secondary effects of

flavonoids is being applied in clinical trials with whole foods such as apple 177

.

Plums are a rich dietary source of phenolics, are widely consumed 178

, widely available,

and a lucrative horticultural crop 89, 179

. Predominant phenolics in plum include the

hydroxycinnamic acid derivatives, neo-chlorogenic acid and chlorogenic acid, and the

quercetin glycoside rutin 73, 180-182

. Evidence for various bioactivities of chlorogenic

acids and quercetin glycosides, in addition to flavanols, is accumulating 6, 102, 114, 183-

185. However, phenolic composition is determined by (plant) genetic, environmental,

and cultural influences, and hence varies quantitatively and qualitatively among

cultivars of plum and other Rosaceae family fruit 84, 85

. Thus, in order to manipulate

phenolic content for dietary benefit, it is important to understand the cultivar variation

in phenolics that have potential health promoting activity. Understanding this would aid

the agricultural sector in developing and commercializing plum cultivars of high

quality, which meets consumer satisfaction and potentially becomes a prospective

dietary approach toward prevention and treatment of various diseases.

Chapter 2

42

The purpose of this study was to evaluate and quantify the diversity of known bioactive

phenolic compounds (Figure 2.1) in advanced pre-varietal plum genotypes, henceforth

termed selections, as a foundation for crop improvement and further studies into health-

preventative mechanism of whole fruits. The relationships between total phenolics,

individual phenolic compounds, and the total antioxidant capacity (TAC) were assessed,

in view of assessing the relevance of TAC for fruit breeding programs.

Figure 2.1. Chemical structures of known bioactive phenolics detected in the tested

plums (R= sugar groups)

Chapter 2

43

2.2 Materials and Methods

2.2.1 Chemicals and Apparatus

Rutin hydrate, chlorogenic acid, neo-chlorogenic acid, quercetin, (±)-catechin,

(-)-epicatechin, Folin Ciocalteu’s phenol reagent, uroporphyrin I dihydrochloride,

Trolox, 2,2’-azobis(2-amidinopropane) dihydrochloride (AAPH) and Brij solution were

obtained from Sigma-Aldrich (New South Wales, Australia).

Flat-bottom, 96-well microplates used in total phenolic concentration assay were

obtained from Greiner Bio-One (Frickenhausen, Germany). Round-bottom 96-well

microplates used in total antioxidant capacity assay were obtained from Alltech

Australia (New South Wales, Australia).

2.2.2 Plant Material

Twenty-nine pre-varietal plum selections from the Department of Agriculture and Food

Western Australia’s Manjimup Horticultural Research Institute, and one commercial

variety were included in this study. We have referred to individual genotypes as

selections. All selections were harvested at storage maturity between December 2008

and February 2009. All fruit were grade 1-equiv. quality. At least 15 fruit per selection,

sampled from a range of positions on the tree from at least two trees per selection, were

pooled for analysis (one biological replicate). Thus any variance due to environmental

or cultural influences should be negligible. Fruits were transported immediately to the

University of Western Australia. Upon arrival, the flesh and skin but not the seed were

homogenized and stored at -80°C before analysis.

Chapter 2

44

2.2.3 Extraction of Phenolic Compounds for HPLC

Analysis, Total Antioxidant Capacity and Total Phenolic

Concentration

Homogenized whole plums (4 g) were extracted with 10 mL of acidic methanol (0.5%

v/v acetic acid). The mixture was then vortexed and sonicated for 1 min each.

Homogenates were kept on ice between these steps. The homogenates were then left on

the bench at room temperature for 45 min. Homogenates were centrifuged (16 000 × g)

for 15 min at 4°C. Supernatants were removed and then filtered through a 0.45 µm filter

(PALL Life Sciences, Cornwall, UK) and kept at -80°C until analysis. This extraction

was done in duplicate for each plum selection. The extract was directly analyzed by

high performance liquid chromatography (HPLC) within 24 h. The extract was diluted

1:5 with cold phosphate buffered saline buffer (PBS, pH 7) for measurement of TAC.

The extract was also used in total phenolic concentration assay after dilution with

ultrapurified water.

2.2.4 HPLC-DAD Analyses

Selected phenolic compounds were quantified by HPLC coupled with a photodiode

array detector (DAD; Waters 996, Sydney, Australia). The HPLC system consisted of

an autosampler (Waters 717plus) and Waters 600 Solvent Delivery system and

equipped with the Empower Software. A reversed phase column, Alltech C18 (5 μm;

250 × 4.6 mm, Alltech, Sydney, Australia) was eluted with a mobile-phase gradient

program at a flow rate of 1 mL.min−1

. Wavelengths at 280, 350, and 520 nm were used

for detection. Extracts (20 μL) were injected into the HPLC-DAD. Formic acid (2% in

Chapter 2

45

water) (solvent A) and acetonitrile (solvent B) were used in the mobile phase of this

system. The solvents were HPLC grade. Elution started with 90% solvent A and 10%

solvent B which remained isocratic until 15 min. A gradient was then used to reach 80%

A and 20% B between 15 to 35 min. The elution then reached 50% A and 50% B at 35

min, 20% A and 80% B at 38 min, 100% B at 42 min, 90% A and 10% B at 43 min

which was held isocratic up to 61 min. Chromatograms were recorded at 280 nm, 350

nm, 420 and 520 nm. The UV spectra of the different compounds were recorded with a

diode array detector. Compounds were identified by comparison with authentic

standards and UV/Vis spectra. Quercetin glycosides were quantified as quercetin.

2.2.5 Total Antioxidant Capacity

Total antioxidant capacity (TAC) was measured by the antioxidant inhibition of oxygen

radicals (AIOR) method 186

using a Cary Eclipse fluorescence spectrophotometer

(Varian Australia, Victoria, Australia). This assay addresses many incongruous

assumptions of other TAC methods. In this method, peroxyl radicals trigger decrease in

fluorescence of the indicator molecule uroporphyrin I, which is delayed by the presence

of antioxidants. Trolox, the vitamin E analogue, was used as an external standard. Plum

extracts were diluted 1:5 with cold PBS buffer. Triplicate samples (1 μL) were mixed

with 200 μL of uroporphyrin I solution (300 nmol.L-1

) and 40 μL AAPH

(291 mmol.L-1

) and then placed in the fluorescence spectrophotometer and detected for

180 min. TAC data are expressed as mol.kg−1

fresh weight Trolox equivalents.

Chapter 2

46

2.2.6 Total Phenolic Concentration

SPECTRAmax 190 microplate spectrophotometer, Molecular Devices (CA, USA) was

used for measurement of total phenolic concentration. Total phenolic concentration was

measured in plum extracts using 96-well microplate format with Folin Ciocalteu’s

method 187

. For this method, the sensitivity of varied incubation condition was tested in

order to achieve the optimum time and temperature of reaction with the Folin

Ciocalteu’s reagent. This was obtained by comparing the absorbance sensitivity of gallic

acid standard after different incubation condition (45°C for 30 min, room temperature

for 30 min or room temperature for 2 hours) with the reagent. This test demonstrated

that incubation at 45°C for 30 minutes gave the optimum absorbance response for this

assay (Figure 2.2). Therefore, samples and standards were incubated with the reagent

for 30 minutes at 45°C.

Gallic acid with concentration range between 0 and 200 μg.mL-1

was used as standard

in this assay. Plum extracts were diluted to fit in the standard curve linear range.

Standards, blank and extracts prepared were loaded (20 μL) into a flat-bottom 96-well

microplate. Folin Ciocalteu’s reagent (diluted 1:10 with ultrapurified water) was added

(100 μL) to the 96-well microplate and mixed. The microplate was incubated at room

temperature for 5 min. Then, 80 μL of 7.5% sodium carbonate was added to the

microplate and mixed well. The microplate was covered and then incubated at 45 °C for

30 min in dark. After the incubation, absorbance was read at 765 nm using the

microplate spectrophotometer with automix set for 60 s before reading. The area under

the curve was calculated and results were expressed in mg.kg-1

fresh weight gallic acid

equivalents (GAE).

Chapter 2

47

Figure 2.2. Sensitivity of different incubation condition on reaction with Folin

Ciocalteu’s reagent. Abbreviation: RT, room temperature.

2.3 Results

2.3.1 Total Phenolic Concentration

Total phenolics of the 29 pre-varietal plum selections and one commercial variety are

shown in Figure 2.3. The greatest total phenolic concentration was found in plum

selection #4 with 1711.3 mg.kg-1

GAE per unit fresh weight, followed by plum

selections #21, #18 and #27. Plum selection #4 had a 3-fold greater total phenolic

concentration than the commercial variety, Black Amber, and 8-fold higher than

selection #1, which had the least total phenolic concentration (221.6 mg.kg-1

GAE). In

fact, 15 of the pre-varietal plum selections tested had a greater total phenolic

concentration than Black Amber. The mean phenolic concentration from all plums

tested in this study was 684.5 mg.kg-1

.

Chapter 2

48

Figure 2.3. Total phenolic concentration in 29 pre-varietal plum selections and Black

Amber (BA). Data are expressed as mg.kg−1

gallic acid equivalents and are mean of

duplicate extractions of a pool of 15 fruit from two trees for each plum selection.

2.3.2 Quantitation of Known Bioactive Phenolic

Compounds

The most abundant phenolics detected by HPLC in the plum selections tested were neo-

chlorogenic acid and quercetin glycosides. Phenolic abundance varied greatly between

the 30 plum selections tested, both quantitatively and qualitatively (Table 2.1). Total

quercetins (quantified as quercetin) were the most abundant group of phenolics detected

in 23 of the 30 plum selections tested (mean, 48.7 mg.kg-1

fresh weight; range, 9.0 to

239.8 mg.kg-1

). Rutin was the predominant quercetin glycoside detected, being the most

abundant individual phenolic in selection #23, and presenting a mean of 13.1 mg.kg-1

among the plums tested (range, 0 to 63.9 mg.kg-1

). The hydroxycinnamic acid, neo-

Chapter 2

49

chlorogenic was also detected in abundant levels, being the most abundant phenolic

compound detected in 4 of the 30 plums tested (mean, 30.9 mg.kg-1

; range, 0 to 220.5

mg.kg-1

). Chlorogenic acid was also detected in 2 of the 30 plums tested (mean, 2.2

mg.kg−1

; range, 0 to 38.4 mg.kg-1

). Among flavanols, epicatechin was only detected in 5

of the 30 plums tested, but was the most abundant phenolic detected in 1 of the 30 plum

selections (selection #13). The mean epicatechin concentration across all 30 plum

selections was 14.2 mg.kg-1

(range, 0 to 154.2 mg.kg-1

). Catechin was not detected in

any plum selections in the present study. Figure 2.4 shows a typical HPLC

chromatogram for two of the plum selections, showing neo-chlorogenic acid, rutin,

other quercetin glycosides and epicatechin. Interestingly, when only the major detected

phenolic compounds (Table 2.1) were summed, plum selection #5, followed by plum

selection #21, showed the greatest abundance, while plum selection #4, which showed

the greatest total phenolic concentration, had less than 1/10th the sum of either

selections #5 and #21.

Chapter 2

50

Table.2.1. Quantification of selected phenolics in 29 pre-varietal plum selections and

Black Amber (BA) using HPLC-DAD at wavelengths 280 and 350 nm. Data are mean

of duplicate extractions of a pool of 15 fruit from two trees for each plum selections

(mg.kg-1

fresh weight plums). Abbreviations: Neo-CGA, neo-chlorogenic acid; CGA,

chlorogenic acid; Q, quercetin.

Plum

code

Phenolic concentration (mg.kg-1

fresh weight)

Neo-CGA CGA Epicatechin Rutin Q (total)

1 0.0 0.0 0.0 9.6 61.8

2 20.8 0.0 0.0 0.0 42.5

3 20.8 0.0 0.0 0.0 46.7

4 0.0 0.0 0.0 0.0 38.5

5 139.4 0.0 154.3 38.7 239.8

6 0.0 0.0 115.8 10.8 39.7

7 0.0 0.0 0.0 0.0 30.5

8 0.0 0.0 0.0 12.3 45.7

9 0.0 0.0 0.0 0.0 18.5

10 0.0 0.0 0.0 0.0 9.0

11 0.0 0.0 0.0 19.6 81.3

12 0.0 0.0 0.0 18.9 76.0

13 24.2 0.0 36.0 0.00 13.0

14 184.5 0.0 0.0 12.9 37.1

BA 0.0 0.0 0.0 14.5 65.4

15 53.6 0.0 0.0 17.0 78.7

16 0.0 0.0 0.0 0.0 45.0

17 0.0 0.0 0.0 9.5 18.4

18 29.0 0.0 0.0 15.7 57.0

19 27.5 0.0 0.0 0.0 30.3

20 25.5 38.4 0.0 11.9 53.4

21 220.5 0.0 74.2 27.4 37.3

22 22.3 0.0 0.0 0.0 9.9

23 0.0 0.0 0.0 63.9 16.2

24 0.0 0.0 0.0 0.0 30.8

25 24.1 0.0 0.0 11.4 34.5

26 85.4 0.0 0.0 21.4 61.8

27 0.0 27.3 0.0 27.0 60.4

28 16.4 0.0 0.0 29.8 57.7

29 34.2 0.0 44.6 30.8 84.0

Chapter 2

51

Figure 2.4. HPLC chromatograms of plum selection #29 (A) and plum selection #21

(B). Chromatograms recorded at 350 nm (insert shows chromatogram recorded at

280 nm to detect catechins). Peak 1 represents neo-chlorogenic acid; peak 2 represents

rutin; peaks 3-7 represents other quercetin glycosides and peak 8 represents epicatechin

(insert).

Chapter 2

52

2.3.3 Total Antioxidant Capacity

The total antioxidant capacity (TAC) of the 30 plum selections tested showed a 7.5-fold

range; from 4795 to 36 187 mol.kg−1

Trolox equivalents (TE) (mean, 13 281 mol.kg−1

TE; Figure 2.5). Plum selection #4 presented the greatest TAC value, apparently greater

than 2-fold the value found in many other plum selections. Plum selection #4 was

already noted as that with the greatest total phenolic concentration among the 30

selections.

Figure 2.5. Total antioxidant capacity in 29 pre-varietal plum selections and Black

Amber (BA). Data represent total antioxidant capacity (AIOR) in whole plum extracts

expressed as mol.kg−1

trolox equivalent. Data are mean of duplicate extractions of a

pool of 15 fruit from two trees for each plum selections.

Chapter 2

53

2.3.4 Relationships of Total Phenolics, Individual Phenolics

and Total Antioxidant Capacity

The total phenolic concentration of the 30 plums tested was significantly correlated with

TAC (R = 0.95, P < 0.01; Figure 2.6). However, there were no significant relationships

between the abundance of individual monomeric phenolic compounds and TAC (R =

0.29 and R = 0.11 for neo-chlorogenic acid and quercetin glycosides, respectively).

Figure 2.6. Correlation of total phenolic concentration with total antioxidant capacity in

29 pre-varietal plum selections and Black Amber (BA).

Chapter 2

54

2.4 Discussion

It is widely accepted that increased consumption of fruit and vegetables is associated

with reduced incidence of chronic disease. The pharmacological and food science

industries have interacted well to enable a better understanding of the relationship

between food composition and bioactivity, with the goal of improving dietary health

through new products and/or promoting consumption of “healthy” foods. However,

there is evidence for benefits of eating whole foods, particularly fruit and vegetables,

rather than isolated components 188

. Plant breeders have so far been slow to capitalize

on the genetic influence for breeding high phenolic foods. Here we sought to bridge the

gap between plant breeders and the food and pharmacological sciences by investigating

biomarkers in pre-varietal plum breeding germplasm. The knowledge would provide a

foundation for further research toward breeding of elite lines of plum and other

Rosaceae fruit.

We focused our research on phenolic compounds that were abundant in plums and for

which there was pharmacological evidence for their protective dietary function. There is

good evidence that quercetin glycosides have cardiovascular protective effects 6, 173-175,

189.

Quercetins were the most abundant phenolic compound detected in over two-thirds of

the plum selections tested, with rutin being the most abundant glycoside, although not

detectable in all selections. The qualitative and quantitative variation between plum

selections is consistent with other reports 60, 73, 182

.

Hydroxycinnamic acids are an abundant class of phenolic in fruits, vegetables, and

coffee, in particular 61

. Evidence for antihypertensive activities is accumulating 114, 141

,

Chapter 2

55

together with population studies suggesting reduced risk of type 2 diabetes 138

. While

both chlorogenic acid and its isomer neo-chlorogenic were detected among the plum

selections, more than half of the plum selections tested lacked detectable levels of

either. Neo-chlorogenic acid was more abundant across the plum selections than

chlorogenic acid, similar to previous reports in plum and other Rosaceae fruit including

peaches, nectarines and cherries 60, 73, 190

. However, at least one study reported greater

abundance of chlorogenic acid than its isomer in plums 181

.

Flavanols were the third target class of phenolic compound in this study, due to the well

establish protective effects on vascular function and blood pressure 183

. Epicatechin was

only detected in 5 of the 30 plums tested, and catechin was undetectable across the plum

selections. This is consistent with previous reports 60

. Furthermore, levels detected were

low by comparison to those found in tea and cocoa.

In addition to investigating select phenolic compound, we assessed total phenolics and

an in vitro measure of TAC. The “antioxidant capacity” has been a major focus in the

food sciences; “antioxidant-rich” has become a commercial proxy for “healthy”.

Numerous TAC measures are available and their relevance is a subject of continued

debate 95, 163

. Currently, ORAC 191

is the predominant method; this assay does address

some of the technical reservations some researcher have about TAC assays, such as the

percentage inhibition and length of inhibition, but is highly pH- and temperature-

sensitive, and requires separate assays for hydrophilic and lipophilic components 192

.

Further, it is laborious to optimize and impractical for application in a breeding

program. We chose the Antioxidant Inhibition of Oxygen Radicals assay (AIOR) 186

, as

it addresses each of these problems. Nonetheless, our data show strong correlation

between total phenolics (hydrophilic) and the AIOR values. However, we found no

Chapter 2

56

correlation between TAC and any or all of the select phenolics we targeted. While the

debate on the validity and choice of TAC assay continues, the weight of available

evidence suggests that investing further research in developing biomarkers from

phenolic compounds with abundant pharmacological evidence will progress the quest to

breed elite lines of fruit and vegetable more rapidly. The method for quantifying total

phenolic content (Folin Ciocalteu’s method) in this study is not corrected for ascorbic

acid. There is a possibility for variation in ascorbic acid concentration across the

different plum varieties. Therefore, there is a probability for correlation between the

total phenolic content corrected for ascorbic acid, and individual phenolics detected in

the plums.

On the data presented, we suggest that quercetin, epicatechin, and chlorogenic acids

content be used in the breeding program as primary targets for biomarker development

and application. A breeding approach focusing on single or a few target metabolites of a

common biosynthetic pathway is consistent with recent studies identifying genetic

markers for flavonoids, such as in grape 193

and apple 194, 195

.

Our study deliberately sought to minimize variance in phenolics due to environment,

cultural practice, or postharvest storage. This suits our present purpose of seeking

biomarkers for breeding, but other influences will ultimately have to be addressed. We

judge that individual breeding programs may seek a similar process in identifying the

genetic influence first and following up with further study. However, the next line of

study for plum and other Rosaceae fruit should be the heritability of select phenolic

compounds, e.g., quercetin glycosides.

Chapter 3

57

CHAPTER 3

Acute Effects of Chlorogenic Acid on

Nitric Oxide Status, Endothelial

Function, and Blood Pressure in Healthy

Volunteers: A Randomized Trial

(The results from this chapter have been published in Journal of Agricultural and Food

Chemistry. 2012; 60: 9130-9136)

3.1 Introduction

Dietary guidelines recommend a diet rich in plant foods. Phenolic compounds are

components of plant foods that are believed to contribute to human health 196

. The main

classes of dietary phenolics are phenolic acids and flavonoids. There is increasing

evidence that flavonoids can enhance vascular health 197

. Specific flavonoids and foods

and beverages rich in these flavonoids have been found to lower blood pressure 198

,

improve endothelial function 177, 197

, and enhance nitric oxide (NO) status 177, 199

.

Endothelial dysfunction is an early event in the pathogenesis of vascular disease and is

associated with increased risk of cardiovascular events 200

. NO plays a critical role in

Chapter 3

58

maintaining healthy endothelial function and vascular tone 23

. The effects of phenolic

acids on these outcomes are less clear.

Chlorogenic acid is one of the main phenolic acids in the diet with intakes reaching

1000 mg.day-1

or more. It is the primary phenolic acid present in coffee and is also

present in particular fruits 201

. Previous human trials suggest that acute intake of

caffeinated coffee, but not decaffeinated coffee, causes detrimental effects on

endothelial function 148, 202

. Regular consumption of green coffee extract, which is rich

in chlorogenic acid, for 4 weeks resulted in dose-related decreases in blood pressure in

mildly hypertensive subjects 142

. An acute effect of chlorogenic acid on blood pressure

and vascular function has been demonstrated in spontaneously hypertensive rats: effects

that were blunted with a NO synthase inhibitor 141

. These results suggest that

chlorogenic acid may reduce blood pressure by augmenting NO status and improving

endothelial function. The acute effects of chlorogenic acid on NO status, endothelial

function, and blood pressure in humans have yet to be studied. Therefore, our aim was

to investigate the acute effect of 400 mg of chlorogenic acid, an intake equivalent to 2

cups of coffee, on plasma NO status, endothelial function, and blood pressure in healthy

men and women.

3.2 Materials and Methods

3.2.1 Chemicals and Apparatus

Food grade chlorogenic acid (3- -caffeoylquinic acid, CAS Registry No. 327-97-9)

was purchased from Atlantic SciTech Group (Linden, NJ, USA). HPLC analysis 203, 204

of this product was carried out to determine its purity. A single peak was detected which

Chapter 3

59

coelutes with the 5- -caffeoylquinic acid isolated from coffee extract. This compound

is referred as chlorogenic acid hereafter.

1-Hydroxy-2- naphthoic acid 99% was purchased from Aldrich Chem Co. (Milwaukee,

WI, USA), and phloretic acid [3-(4-hydroxyphenyl) propionic acid] was from Lancaster

(Morecambe, UK). Caffeic acid (3,4-dihydroxycinnamic acid), ferulic acid (4-hydroxy-

3-methoxycinnamic acid), and isoferulic acid (3-hydroxy-4-methoxycinnamic acid,

predominantly trans) were purchased from Sigma Chemical Co. (St. Louis, MO, USA).

β-Glucuronidase from Helix pomatia type HP-2 pyridine ≥99% and

N,O-Bis(trimethylsilyl)trifluoroacetamide with trimethylchlorosilane (BSTFA) and

hydrocaffeic acid (3,4-dihydroxyhydrocinnamic acid) were from from Sigma-Aldrich

Pty. Ltd. (NSW, Australia).

Bond Elut C18, 500 mg, 6 mL cartridges were from Varian (Lake Forrest, CA, USA).

Zorbax eclipse XDB C18 2.1 mm × 100 mm/3.5µm column was purchased from

Agilent Technologies (Agilent Technologies, Santa Clara, CA). All solvents were of

high-performance liquid chromatography (HPLC) grade. Water was obtained with

Arium 611 VF water purification system (Startorius stedim biotech, Aubagne, France).

3.2.2 Participants

Twenty-three healthy men and women were recruited from the Perth general population

by newspaper advertisement. All participants were screened prior to enrolment.

Screening and study visits took place at the University of Western Australia, School of

Medicine and Pharmacology located at Royal Perth Hospital. Screening consisted of a

standard medical history questionnaire, routine laboratory analysis of a fasting blood

sample, electrocardiography, height, weight, body mass index (BMI), and blood

Chapter 3

60

pressure measurement. Exclusion criteria included current smoking, BMI <18 or >35

kg.m-2

, systolic blood pressure (SBP) <100 or >160 mmHg, diastolic blood pressure

(DBP) <50 or >100 mmHg, use of antihypertensive medication, history of

cardiovascular or peripheral vascular disease, history of liver disease, renal disease,

chronic gastrointestinal disorder, and any major illness such as cancer, psychiatric

illness, diagnosed diabetes, weight gain or loss >6% body weight within previous 6

months of the study, >30 g.day-1

alcohol consumption, or woman who were pregnant,

lactating, or wishing to become pregnant during the study. All participants were regular

tea (mean ± SD, 1.7 ± 1.5 cups.day-1

) and coffee (mean ± SD, 1.7 ± 1.6 cups.day-1

)

consumers. This study was conducted according to the guidelines laid down in the

Declaration of Helsinki, and all procedures involving human subjects were approved by

the University of Western Australia Human Research Ethics Committee. Participants

provided written informed consent before inclusion in the study. The trial was registered

with the Australian New Zealand Clinical Trials Registry (ACTRN: 12611000467932).

3.2.3 Study Design

A double-blind, randomized controlled cross-over design study was performed.

Participants were allocated to an intervention plan via block randomization using

computer-generated random numbers devised by a statistician. Each subject completed

two visits with a washout period of 1 week in between testing days. The preceding

evening meal and breakfast on the morning of the study day were low polyphenol and

were consistent across all study visits. A food diary was used to verify adherence to the

study protocol. On arrival, a baseline blood sample was taken by venepuncture for

analysis of plasma S-nitrosothiols + other nitrosylated species (RXNO), nitrite, nitric

Chapter 3

61

oxides comprising nitros(yl)ated species + nitrite (NOx), chlorogenic acid, and

chlorogenic acid metabolites. Participants were then provided with the randomly

allocated chlorogenic acid treatment. Treatments were comprised of water (control) and

400 mg of chlorogenic acid dissolved in 200 mL of low nitrate water (chlorogenic acid)

(no difference in taste compared to water). Flow mediated dilatation (FMD) of the

brachial artery using ultrasonography was used to assess endothelial function 120 min

post-treatment. A blood sample was taken by venepuncture 150 min post-intervention

for analysis of plasma RXNO, nitrite, NOx, chlorogenic acid, and chlorogenic acid

metabolites. Blood pressure measurements were taken prior to treatment and then again

at 60, 90, 120, and 150 min post-treatment.

3.2.4 Measurement of Plasma RXNO, Nitrite, and NOx

Plasma was collected from blood samples taken at baseline and 150 min post-treatment

for immediate analysis of RXNO, nitrite, and NOx [nitros(yl)ated species + nitrite]. The

concentrations of RXNO, nitrite, and total NOx in plasma were determined using a

previously described gas phase chemiluminescence assay 205

. Blood was collected into

N-ethylmaleimide (10 mmol.L-1

) and ethylenediaminetetraacetic acid (EDTA) (2

mmol.L-1

), mixed, and centrifuged at 3000 g (5 min, 4 °C). Fresh plasma was kept on

ice in the dark and analyzed within 1 h. For determination of nitroso species, fresh

plasma (300 μL) was treated with sulfanilamide solution [(30 μL, 0.5% in 0.1 mol.L-1

hydrochloric acid (HCl)] for 3 min to remove endogenous nitrite. Antifoam (200 μL)

was added prior to injection into the radical purger containing potassium iodide

(0.125 g) and iodine (0.05 g) in water (2.5 mL)/ glacial acetic acid (7.5 mL) at room

temperature. Plasma S-nitrosothiols (+nitrosylated species) were quantified by the NO

Chapter 3

62

signal peak area of samples pretreated with sulfanilamide against a nitrite standard

(300 μL, 0.5 μmol.L-1

NaNO2−). Quantification of NO released by the redox reactions

occurred by its chemiluminescence reaction with ozone using the Nitric Oxide Analyzer

(CLD66, Eco Physics, Sweden).

3.2.5 Blood Pressure

Blood pressure (BP) was measured using a Dinamap 1846SX/P oscillometric recorder

(Critikon Inc., Tampa, FL, USA) prior to treatment and then at 60, 90,120, and 150

post-treatment. Participants were rested for 5 min in a supine position before BP

measurements were taken. Five blood pressure measurements were taken at 2 min

intervals. The first measurement was discarded, and the mean of the remaining four

measurements was calculated. BP measurements for all time points (60, 90, 120, and

150) were used in the analysis.

3.2.6 Flow Mediated Dilatation of the Brachial Artery

A trained ultrasonographer dedicated to the research protocol and blinded to the

treatments used performed FMD of the brachial artery assessed by ultrasonography. All

measurements were performed according to published protocol 206

. Briefly, subjects

were rested in a supine position in a quiet, temperature-controlled room (21-25 °C). The

left arm was extended and supported comfortably on a foam mat. ECG was monitored

continuously. For the ultrasound, a 12 MHz transducer connected to an Acuson Aspen

128 ultrasound device (Acuson Corp.) was fixed in position with a clamp over the

brachial artery 5−10 cm proximal to the anticubital crease. After a baseline artery

diameter recording of 1 min, a blood pressure cuff was placed around the left forearm

Chapter 3

63

and inflated to 200 mmHg. The cuff was released after 5 min, inducing reactive

hyperaemia. The brachial artery image was recorded for 4 min post-cuff deflation to

assess FMD. Images were downloaded for retrospective analysis. Analysis of FMD of

the brachial artery was performed with semiautomated edge-detection software 206

,

which automatically calculated the brachial artery diameter, corresponding to the

internal diameter. This was gated to the R wave of ECG, with measurements taken at

end diastole. Responses were calculated as the percentage change in brachial artery

diameter from baseline. The FMD was measured at 30 s intervals to 4 min post-cuff

deflation (0, 30, 60, 90, 120, 150, 180, 210, and 240 s). Peak FMD was also assessed.

The analysis was performed by an experienced observer blinded to the treatments used.

3.2.7 Measurement of Plasma Chlorogenic Acid Using

Liquid Chromatography-Tandem Mass Spectrometry

For the analysis of chlorogenic acid, plasma samples were defrosted and were extracted

using a solid-phase extraction (SPE) method. Bond Elut C18 500 mg cartridges were

first conditioned with 2 mL of methanol followed by 2 mL of water. An aliquot of

500 μL of plasma sample was spiked with 10 μL of internal standard (IS) (1-hydroxy-2-

naphthoic acid) working solution (1 ng.mL-1

). Then, plasma was acidified by adding

2 mL of buffer (0.1 mol.L-1

sodium acetate, pH 3) and then vortexed. After

acidification, samples are applied to SPE cartridges and washed with water and hexane

to remove any interfering substances. Compounds were eluted with methanol and

collected in a glass tube. The methanol was evaporated under a stream of nitrogen, and

the residue was then reconstituted with mobile phase (100 μL). Standard stock solutions

were prepared by dissolving a required amount of chlorogenic acid and IS in methanol,

Chapter 3

64

which was then further diluted appropriately with methanol to desired concentration.

The concentration of IS working solution was 100 pg.μL-1

. All solutions were stored at

−20 °C until use. Calibration curves of standards used to quantify chlorogenic acid in

plasma were drawn from the analysis of plasma spiked with 10 μL of IS working

solution (1 ng.mL-1

) and different concentrations of chlorogenic acid standards ranging

from 10 to 1000 ng.mL-1

. Coefficients of linearity for the calibration curve were

typically R2 > 0.99. Chlorogenic acid and IS were analysed on a Thermo Scientific TSQ

QuantunUltra

Triple Quadrupole LC-MS System (ThermoFisher Scientific) equipped

with electrospray ionization source (ESI) operated in negative-ion mode using a Zorbax

eclipse XDB C18 2.1 mm × 100 mm/3.5 μm column. The injection volume was 25 μL.

The mobile phases were as follows: A, 2 mM ammonium acetate, pH 3.6; and B,

methanol at a flow rate of 400 μL.min-1

. Chromatography was achieved using solvent A

(100%) from 0 to 4 min, then ramped to B (100%) from 4 to 12 min, then A (100%)

from 13 to 15 min. Multiple reaction monitoring (MRM) was used to monitor precursor

to product ion transitions of m/z 354.1 [M − 1] to a strong product ion at m/z 191 for

chlorogenic acid (retention time was 6.91 min). IS included m/z 188 [M − 1] and a

strong product ion at m/z 144 (retention time was 10.03 min). The operating conditions

for MS analysis were ion spray voltage, 3500 V; capillary temperature and voltage, 350

°C and 35 V, respectively; sheath gas (nitrogen) and auxiliary gas pressure, 60 and 50

psi, respectively. The mass spectrometer was employed in MS/MS mode using argon as

collision gas (1.2 mTorr). The collision energy (CE) was optimized at 19 eV for

chlorogenic acid and 20 eV for IS.

Chapter 3

65

3.2.8 Measurement of Plasma Chlorogenic Metabolites

Using Gas Chromatographic Mass Spectrophotometer

For the analysis of chlorogenic acid metabolites, plasma samples were defrosted and

were extracted using a liquid−liquid extraction method. Plasma samples (750 μL) were

first spiked with 50 μL of IS 1 ng.μL-1

. The mixture was then acidified with 0.2 M HCl

to pH 5. Then, 30 μL of β-glucuronidase was added and incubated at 37 °C overnight.

The sample was then acidified to pH 3.5 with 2 M HCl and extracted with ethyl acetate.

The mixture was then centrifuged for 5 min, and the ethyl acetate layer was then

recovered and re-extracted with 5% K2CO3. The ethyl acetate phase was then removed

to waste, and the carbonate phase was immediately reacidified to pH 1−2 with 6 M HCl.

This layer was re-extracted with 1 mL of ethyl acetate. The ethyl acetate layer was

recovered and evaporated under a stream of nitrogen. The residue was derivatized with

pyridine and BSTFA and heated at 40 °C for 60 min prior to gas chromatographic mass

spectrophotometer (GC-MS) analysis.

Calibration curves of standards used to quantify chlorogenic acid metabolites in plasma

were prepared from the analysis of mixture 50 μL of IS working solution (1 ng.μL-1

)

and different concentrations of individual metabolite compound ranging from 10 to

500ng.μL-1

. Coefficients of linearity for the calibration curve were typically R2 > 0.99.

A Hewlett-Packard (HP) 6890 Series Gas Chromatograph System coupled with Agilent

5973 Network mass selective detector was used for the analysis. Samples were

separated on a 30 m, 0.25 μm, and 0.25 mm high-resolution gas chromatography

column (HP-1MS J&W Scientific, Folsom, CA, USA). The maximum temperature of

column was 330 °C, and the total run time was 14.17 min. The injection volume was

Chapter 3

66

1.0 μL in the splitless mode. The interface temperature was held at 280 °C. Selected ion

monitoring was used after initial scan of standard compounds, and the following ions

were monitored for detection of chlorogenic acid metabolites and the internal standard:

ions m/e 338.0 and 308.0 for ferulic acid and iso-ferulic acid; ions m/e 179.0 and 310.0

for phloretic acid; 396.0 and 219.0 for caffeic acid; ions 209.0 and 340.0 for

hydrocaffeic acid; and ion 313.0 for IS. The electron impact ionization energy was 70

eV. All compounds were identified by characteristic ions and retention time as

compared to authentic standards.

3.2.9 Other Biochemical Analyses

Routine biochemical analyses on fasting blood samples taken at the screening visit were

carried out in the Path West laboratory at Royal Perth Hospital, Western Australia. A

routine enzymatic colorimetric test with a fully automated analyser (Roche Hitachi 917,

Roche Diagnostics Australia Pty. Ltd., Castle Hill, NSW, Australia) was used to

measure serum total cholesterol, HDL cholesterol, and triglycerides. LDL cholesterol

concentrations were calculated using the Friedewald formula 207

. Serum glucose was

measured using an automated analyzer (Roche Hitachi 917).

3.2.10 Statistical Analyses

The sample size was calculated on the primary end points of plasma RXNO, FMD, and

BP. With α = 0.05, 20 participants provided >80% power to detect a 30% difference in

plasma RXNO, a 20% difference in FMD (a 1.5% absolute difference), and a 3.0 mmHg

difference in SBP. Statistical analyses were performed using SPSS 15.0 (SPSS Inc.,

Chicago, IL, USA) and SAS 9.2 (SAS institute Inc., Cary, NC, USA). Non-normally

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67

distributed data were log-transformed prior to analysis. Participant characteristics are

presented as mean ± SDs. Results in the text and tables are presented as mean (95%

CIs) or geometric mean (95% CIs) for non-normally distributed variables. Results in

figures are presented as mean ± SEM or geometric mean (95% CIs) for non-normally

distributed variables. Outcome variables were analyzed with mixed models in SAS

using the PROC MIXED command. Subject was included as a random factor in all

models. Models for plasma RXNO, plasma nitrite, plasma NOx, peak FMD, plasma

chlorogenic acid, and chlorogenic acid metabolites included fixed effects for group

(control or chlorogenic acid) and order. Models for BP also included fixed effects for

baseline value and time (60, 90, 120, and 150 min).

3.3 Results

3.3.1 Baseline Data

Recruitment began December 8, 2010, and the study ended June 6, 2011. Twenty-three

participants (4 men and 19 women) completed the study (Figure 3.1). The

characteristics of the study participants are shown in Table 3.1.

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68

Figure 3.1. Participant flow from recruitment through screening and randomization to

trial completion.

Chapter 3

69

Table 3.1. Baseline characteristics of study subjects (n = 23; Males= 4; Females= 19)

Mean ± SD Range

Age (years) 52.3 ± 10.6 32 – 65

Weight (kg) 69.9 ± 12.7 50 - 100

Body Mass Index (kg.m-2

) 25.6 ± 4.7 19 – 35

Systolic blood pressure (mmHg) 115.2 ± 10.8 100 - 132

Diastolic blood pressure (mmHg) 67.0 ± 7.1 56 – 80

Mean arterial pressure (mmHg) 85.1 ± 8.2 69 – 97

Pulse pressure (mmHg) 60.9 ± 7.3 37 – 69

Total cholesterol (mmol.L-1

) 4.9 ± 0.9 3.1 – 5.6

Triglyceride (mmol.L-1

) 0.9 ± 0.4 0.4 – 1.7

High density lipoprotein (mmol.L-1

) 1.4 ± 0.3 0.8 – 2.2

Low density lipoprotein (mmol.L-1

) 3.1 ± 0.7 1.8 – 4.5

Fasting plasma glucose (mmol.L-1

) 5.1 ± 0.4 4.1 – 5.8

3.3.2 Blood Pressure, Endothelial Function, and

Biomarkers of NO Status

Mean post-treatment SBP (−2.41 mmHg, 95% CI: −0.03, −4.78; P = 0.05) and DBP

(−1.53 mmHg, 95% CI: −0.05, −3.01; P = 0.04) between 60 and 150 min were

significantly lower for chlorogenic acid relative to placebo (Figure 3.2). There was no

significant difference in peak FMD (0.41%, 95% CI: −1.29, 2.10; P = 0.62) at 120 min

(Figure 3.3). Relative to placebo, chlorogenic acid did not significantly influence

plasma RXNO (13.73 nmol.L-1

, 95% CI: 12.23, 15.42; P = 0.11), plasma nitrite

(2.71 nmol.L-1

, 95% CI: 2.49, 2.91; P = 0.79), and plasma NOx (11.53 nmol.L-1

, 95%

CI: 11.04, 12.03; P = 0.14) at 150 min (Figure 3.4).

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70

Figure 3.2. Effect of chlorogenic acid on (A) SBP from 60 to 150 min post-treatment,

(B) mean baseline-adjusted SBP post-treatment, (C) DBP from 60 to 150 min post-

treatment, and (D) mean baseline adjusted DBP post-treatment. Results are expressed as

geometric mean (95% CIs). A mixed random-effects linear model was used to compare

groups (n = 23); * P < 0.05.

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71

Figure 3.3. Effect of chlorogenic acid on FMD 120 min post-treatment. Results are

expressed as mean (SEM). A mixed random-effects linear model was used to compare

groups (n = 23).

Figure 3.4. Effect of chlorogenic acid on plasma concentrations of (A) RXNO and (B)

nitrite 150 min post-treatment. Results are expressed as geometric mean (95% CIs). A

mixed random-effects linear model was used to compare groups (n = 23).

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72

3.3.3 Plasma Chlorogenic Acid and Its Metabolites

Intact chlorogenic acid was measured in plasma at 150 min. Typical chromatogram

traces from chlorogenic acid-treated participants (pre- and post-treatment) are shown in

Figure 3.5. The concentration of chlorogenic acid in plasma was significantly higher

following 400 mg of chlorogenic acid as compared to placebo (P < 0.001; Figure 3.6A);

however, the concentrations of chlorogenic acid metabolites, isoferulic acid, ferulic

acid, phloretic acid, caffeic acid, and hydrocaffeic acid were not significantly different

according to treatment (Figure 3.6B) (P = 0.09, P = 0.16, P = 0.66, P = 0.77, and

P = 0.72, respectively).

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Figure 3.5. Typical LC/MS/MS chromatogram trace from the analysis of chlorogenic

acid concentrations at baseline and 150 min post-treatment in chlorogenic acid-treated

participants. Relative abundance of chlorogenic acid peak (retention time, 6.9 min)

increased in post-treatment plasma as compared to pre-treatment plasma.

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Figure 3.6. Plasma concentration of intact chlorogenic acid (A) and chlorogenic acid

metabolites (B) for control and chlorogenic acid treatments at 150 min post-treatment.

Results are expressed as mean ± 95% CI. A mixed random-effects linear model was

used to compare groups (n = 10 and n = 23 for intact chlorogenic acid and chlorogenic

acid metabolites measurements respectively); ** P < 0.001.

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75

3.4 Discussion

The aim of this study was to assess whether the consumption of pure chlorogenic acid

can acutely augment NO status, improve endothelial function, and lower blood pressure

in healthy men and women. Enhanced NO status and improved endothelial function

may explain previously reported benefits on blood pressure 142

. While we did observe a

significant decrease in SBP and DBP and a concomitant increase in chlorogenic acid in

the plasma, the effects on NO status and FMD did not reach significance.

An acute intake of 400 mg of chlorogenic acid resulted in significantly lower SBP and

DBP. In a previous study, 4 weeks of regular (chronic) consumption of green coffee

extract, which is rich in chlorogenic acid, led to lower blood pressure in mildly

hypertensive men 142

. A dose-related reduction in SBP and DBP was observed, with the

highest dose of 100 mg.day-1

of chlorogenic acid resulting in lower SBP and DBPs of

approximately 3.5 and 3.0 mmHg, respectively 142

. The blood pressure measurements

were performed in the morning, in the fasting state, and 24 h after the last dose of

chlorogenic acid, and are therefore not due to acute effects of chlorogenic acid 142

. We

have now demonstrated that chlorogenic acid can acutely reduce blood pressure. It is

not known whether these acute effects would be found over and above any chronic

effects.

Coffee consumption has an adverse effect on blood pressure 208

. However, trials with

caffeinated coffee reveal a weaker effect on blood pressure than those with pure

caffeine 132

, suggesting that other coffee components counteract the caffeine effect. It is

possible that chlorogenic acid contributes to blunting of the blood pressure-raising

effect of caffeine in coffee. Plums and other fruit such as berries are also rich in

chlorogenic acid 76

. The blood pressure-lowering effect of chlorogenic acid may

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76

contribute to the growing evidence supporting the protective effect of fruit against

cardiovascular disease and hypertension 196, 209, 210

.

Lower blood pressure was observed without any concomitant significant difference in

NO status or FMD of the brachial artery. FMD of the brachial artery after a period of

ischemia is primarily mediated by NO 211

. Plasma S-nitrosothiols and nitrite are

considered to be reliable measures of endogenous NO production 212, 213

. These

molecules also form part of a circulating NO pool in that they have the potential to be

converted back to NO. A lower FMD is associated with a significantly higher risk of

subsequent cardiovascular events 200

. Thus, augmented NO status and increased FMD

would be consistent with benefits on cardiovascular risk. In our study, FMD was

measured at 120 min post-chlorogenic acid intake, and NO status was measured 150

min post-chlorogenic acid intake. We observed a non-significant trend for both FMD

and RXNO to be higher after the chlorogenic acid in comparison to placebo. These

results are consistent with previous studies showing that FMD did not significantly

improve 1−2 h after decaffeinated coffee intake 148, 202

. Although we found that plasma

concentrations of chlorogenic acid were significantly elevated at 150 min, peak plasma

concentrations of chlorogenic acid may be closer to 30−60 min post-ingestion 214

.

Furthermore, the observed blood pressure difference appeared to be largest between 60

and 90 min and smaller beyond 90 min. These results raise the possibility that acute

effects on NO, FMD, and blood pressure coincide with peak plasma chlorogenic acid

concentrations.

This idea is not supported by the results of Suzuki et al. 141

, who demonstrated an acute

reduction in blood pressure in spontaneously hypertensive rats 9 h after a single dose of

chlorogenic acid. In addition, this effect was inhibited by use of a NO synthase

Chapter 3

77

inhibitor 141

. An improvement in acetylcholine-induced endothelium-dependent

vasodilation in the aorta was also observed 141

. These apparent discordant results could

be due to the use of a very high dose of chlorogenic acid, which may result in a more

sustained elevation in circulating chlorogenic acid and metabolites. The dose of

chlorogenic acid used in our study was equivalent to that obtained from drinking 2 cups

of coffee, an easily achievable amount. This conflicting result could also be due to the

possibility of prominent effects of the chlorogenic acid metabolites at this time point.

Additional studies are needed to investigate the time course of acute vascular effects.

Chlorogenic acid and its metabolites were measured in plasma at baseline and 150 min

post-treatment. A significant increase was seen in the plasma level of chlorogenic acid.

Although some studies have failed to detect chlorogenic acid in the plasma of rats and

human subjects after ingestion of chlorogenic acid as the pure compound or in

coffee 112, 215

, other studies have detected chlorogenic acid in its unchanged form in

urine suggesting absorption of the compound without structural modification 107, 115, 216,

217. The undetectable chlorogenic acid in previous studies may have resulted from the

limit of detection of the assay being used 215

or due to degradation of chlorogenic acid

during sample treatment 106

.

After absorption, chlorogenic acid is metabolized to caffeic acid, quinic acid, and ferulic

acid 107, 218

. Phenolic groups may then be further metabolized by methylation or

conjugation with glucuronic acid or sulfatase in the liver 112

. The existence of

metabolites in plasma of control shown in this study was also observed in baseline

plasma of a previous study 113

. This may be supported by the hypothesis of storage and

recycling of the compounds through excretion and reabsorption 219

.

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78

Previous studies in rats suggest that ferulic acid can reduce blood pressure 139

and

improve vascular function by scavenging superoxide and increasing NO bioavailability

140 or by acting directly on the vascular endothelium

149. In this study, no increase in

ferulic acid or the other metabolites was detected at 150 min post-ingestion of

chlorogenic acid. This does not rule out the possibility that ferulic acid, as a metabolite

of chlorogenic acid ingested, could have an effect on NO, FMD, and blood pressure at a

later time point. According to Stalmach et al. 220

, metabolites such as dihydroferulic

acid had peak plasma concentration (Cmax) values after 4 h of coffee ingestion,

indicating absorption in the large intestine with possible catabolism by the colonic

microflora. Similarly, Renouf et al. 214

reported that dihydrocaffeic acid and

dihydroferulic acid appeared in plasma 6−8 h after coffee ingestion. It is possible that

these metabolites could contribute to a more sustained effect on blood pressure.

The bioactivity of phenolics relies largely on their bioavailability 63

. A study evaluating

pharmacokinetic profile and apparent bioavailability of chlorogenic acid in plasma and

urine of 10 healthy adults after consumption of a decaffeinated green coffee extract

showed that there is a large variation in chlorogenic acid absorption, metabolism, and

kinetics among different individuals 113

. A better understanding of the consumption and

bioavailability of dietary phenolics will lead to the better evaluation of their role in

disease prevention 80

.

The exact molecular mechanisms by which chlorogenic acid or its metabolites could

lower blood pressure and improve vascular function are yet to be elucidated. However,

a number of potential pathways have been proposed. These include direct scavenging of

free radicals; inhibition of NADPH oxidase, an enzyme-producing reactive oxygen

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79

species; and increased NO production and/or inhibition of angiotensin-converting

enzyme 114

. The importance of these and other potential mechanisms in vivo is not clear.

The main limitation of our study may be the timing of blood samples for NO

measurement and the timing of FMD measurement. In addition, we used a

commercially available chlorogenic acid, labelled as 3- -caffeoylquinic acid. Major

dietary sources such as coffee predominately contain 5- -caffeoylquinic acid.

However, HPLC analysis of this product in a solvent system that separates chlorogenic

isomers indicates that this product coeluted with 5- -caffeoylquinic acid. At this stage,

we do not know if different chlorogenic acid isomers have the same biological activity.

However, this study has been able to demonstrate acute intake of pure chlorogenic acid

in a relevant achievable dietary dose can significantly lower blood pressure in healthy

subjects. Another limitation of this study is the imbalanced of sex distribution among

the participants. Level of estrogens during menstrual cycle can significantly affect FMD

response and therefore leading to a higher baseline FMD 221

. Acute improvement in

FMD is thus less likely to occur.

In conclusion, we found an acute blood pressure-lowering effect of chlorogenic acid.

The significant increase of plasma chlorogenic acid with no difference seen in plasma

level of chlorogenic acid metabolites suggests that intact chlorogenic acid absorbed in

the small intestine, rather than its metabolites, could be responsible. Future studies

should investigate both the dose response and the time course of these effects.

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80

CHAPTER 4

Supplementation of a High Fat Diet with

Chlorogenic Acid is Associated with

Insulin Resistance and Hepatic Lipid

Accumulation in Mice

(The results from this chapter have been published in Journal of Agricultural and Food

Chemistry. 2013; 61: 4371-4378)

4.1 Introduction

The metabolic syndrome consists of a group of metabolic abnormalities such as

abdominal obesity, dyslipidemia, hyperglycaemia and hypertension 222

. The condition is

associated with the increased risk of developing cardiovascular disease and type 2

diabetes 222, 223

. Emergence of metabolic syndrome incidence has resulted in an

increased need for therapeutic and prevention strategies. Lifestyle changes 224

and

potential treatment that targets specific molecules for regulation of metabolic

pathways 225

are recommended approaches towards managing metabolic syndrome.

Epidemiological studies have shown inverse associations between coffee consumption

and risk of developing chronic diseases such as type 2 diabetes mellitus 121

and

cancer 226

. These potential benefits are also indicated in individuals consuming

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81

decaffeinated coffee 121, 136

. This suggests that compounds in coffee other than caffeine

may be responsible for the health benefits.

Numerous studies suggest health benefits of phenolics. They include reducing blood

pressure 227

, improvement of endothelial function and nitric oxide status 177

and

augmentation of insulin sensitivity 228

. Chlorogenic acid, is an ester of caffeic acid and

quinic acid 107

. This compound is rich in coffee, as well as other sources such as in fruits

like plums 229

, apples and berries 81

. Coffee consumers can obtain up to 1 g of

chlorogenic acid from daily consumption 107

. In a coffee drinking population,

chlorogenic acid is likely to be one of the major phenolics in the diet 97

. Coffee

phenolics have been shown to have various health protective effects such as suppressing

body fat accumulation 154

, reducing liver damage 230

and inhibiting hyperglycaemia,

hyperinsulinemia and hyperlipidemia 155

. However, these studies either looked at coffee

phenolics, which includes a combination of many hydroxycinnamates including

chlorogenic acid and ferulic acid 154

, or coffee extract which may have been a combined

effect from the various compounds found in coffee. A recent study which looked at

specific coffee phenolics has reported that caffeic acid stimulates AMPK (AMP

activated protein kinase) activity in rat skeletal muscle and enhances insulin-

independent glucose transport, whilst none of the effects were seen with chlorogenic

acid treatment 156

. However, effects of pure chlorogenic acid, which is the most

abundant form of phenolic acid in coffee, on risks of metabolic disorders remain to be

fully elucidated.

The aim of the present study was to test the effect of chlorogenic acid on high fat diet

induced obesity, glucose intolerance and insulin resistance in mice. We also assessed

the effects of chlorogenic acid on fatty acid oxidation and insulin signaling.

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82

4.2 Materials and Methods

4.2.1 Experimental Animal and Diets

C57BL/6 mice were obtained from the Animal Resources Centre, Murdoch, Western

Australia and were maintained on standard chow (14.3 MJ.kg-1

, 76% of energy from

carbohydrate, 5% from fat, 19% from protein; Specialty Feeds, Glen Forrest, WA,

Australia) until 7-weeks of age. The mice were allowed to acclimatize to conditions in

the animal holding room and they were maintained on a 12-hour light/ dark cycle. Mice

were then switched to 3 groups of different diets (n = 10 in each group, 5 mice/cage)

which are standard chow, high fat diet (19 MJ.kg-1

, 36% of energy from carbohydrate,

43% of energy from fat, 21% from protein; Specialty Feeds) and high fat diet

incorporated with 1 g.kg-1

chlorogenic acid (Atlantic SciTech Group, Linden, NJ USA)

for a further 12 weeks. Water and feed were available to the animals ad libitum. All

surgery was performed under methoxyflurane anesthesia, and all efforts were made to

minimize suffering. All animal experiments were approved by the Royal Perth Hospital

Animal Ethics Committee (Approval Number: R510/11-12).

4.2.2 Body Weight, Adiposity and Basal Insulin

Measurement

Body weights of mice were measured weekly throughout the study. The percentage of

starting body weight was then calculated from the acquired data. Gonadal fat pad mass

was also measured. Fasting blood samples were collected via cardiac puncture and

serum was obtained. From this sample, insulin was measured using standard ELISA kits

as previously described 231

.

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83

4.2.3 Metabolic Assays

Glucose tolerance tests (GTT) were performed at week 5 and 10 of the experiment,

while insulin tolerance tests (ITT) were performed at week 6 and 11 of the experiment.

Food was withdrawn from mice 5 hours prior to testing. Blood samples were obtained

by tail bleeding and glucose levels were measured using a glucometer (AccuCheck II;

Roche, Castle Hill, NSW, Australia) immediately before and at 15, 30, 45, 60, 90, and

120 min after an intraperitoneal injection of glucose (1 g.kg-1

body mass) or insulin

(0.5 U.kg-1

body mass).

4.2.4 Acute Insulin Signaling Experiment

For insulin signaling experiments, mice were anaesthetized with methoxyflurane before

an intraperitoneal injection with insulin (2U.kg-1

) or saline. Subsequently, liver, adipose

tissue, and skeletal muscle samples were obtained within 5 min of injection and snap-

frozen in liquid nitrogen, before being analyzed for protein.

4.2.5 Liver and Adipose Tissue Histology

At the end of the experiment, white adipose tissue and liver were dissected and fixed in

4% paraformaldehyde (w/w) overnight before being incubated in 50% ethanol (v/v) and

then promptly embedded with paraffin. The tissues were then cut into 4 μm sections and

stained with haematoxylin and eosin, before being mounted using DPX (Sigma Aldrich,

MO, USA). Sections were then visualized and photographed using the Olympus 1X71

microscope (Olympus, Mt Waverly, Vic, Australia).

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4.2.6 Hepatic Lipid Analysis

Total lipid from the liver was extracted with a chloroform-methanol mixture (2:1)

according to the method by Folch et al. 232

. Empty glass tubes were weighed prior to the

extraction process. After completing the extraction process, extracts were dried under

vacuum using a sample concentrator (miVac, Ipswich UK) and then the tubes were re-

weighed to determine the weight of lipid extracted from the liver samples.

4.2.7 Western Blot Analysis of Proteins Associated with

Fatty Acid Oxidation and Insulin Signaling

Liver, white adipose tissue and skeletal muscle (quadricep) were dissected from mice on

the final day of the experiment, and were snap-frozen in liquid nitrogen. Tissue samples

were lysed in cytostolic extraction buffer containing protease and phosphatase

inhibitors. The lysates were then centrifuged at 15, 493 × g for 10 min at 4°C,

supernatants were collected and the protein concentration was determined using BCA

Protein Assay Reagent (bicinchoninic acid; Thermo Scientific, IL, USA). Samples were

then separated with SDS-PAGE and western blotting was carried out to detect proteins

using primary antibodies from Cell Signaling Technology (Danvers, MA USA) 233, 234

.

4.2.8 Statistical Analyses

Statistical analyses were performed using IBM SPSS Statistics 19 (IBM Corporation,

Armonk, NY, USA) and SAS 9.3 (SAS Institute Inc., Cary, NC, USA). Results are

expressed as the mean ± SEM. Mean values were compared for differences by analysis

of variance with Tukey’s adjustment for multiple comparisons (3-group analyses) or

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85

Student’s t test for unpaired samples (2-group analyses). P<0.05 was considered to be

statistically significant. For data with repeated measures over time (weight, GTT and

ITT), random-effects linear models were fitted in SAS using the PROC MIXED

command to observed data for each variable (weight and glucose). Mouse number was

included as a random factor in all models. The models also contained fixed effects for

treatment group (diet) and time as a categorical variable.

4.3 Results

4.3.1 The Effect of Chlorogenic Acid Supplementation on

High Fat Diet Induced Obesity and Insulin Levels in C57BL/6

Mice

The high fat diet and high fat diet + chlorogenic acid groups displayed a higher rate and

absolute body weight increase compared to the normal diet group (Figure 4.1A). The

rate and absolute body weight increase did not differ between the two high fat diet

groups. Mice fed the high fat diet or high fat diet + chlorogenic acid also displayed

increased gonadal fat pad mass (Figure 4.1B) compared to the mice fed normal diet.

Differences in gonadal fat pad mass between the two high fat diet groups were not

significant (P = 0.23). Both body weight and gonadal fat pad measurements confirmed

that the high fat diet induced obesity in C57BL/6 mice.

We measured fasting insulin levels in basal plasma of mice using ELISA. A trend for

hyperinsulinemia was evident in the high fat diet + chlorogenic acid fed mice (Figure

4.1C) compared to high fat diet fed mice and normal diet fed mice. All subsequent

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86

analysis presented in this chapter will hereafter compare high fat diet fed mice with the

high fat diet fed mice supplemented with chlorogenic acid.

Figure 4.1. Effect of chlorogenic acid supplementation on high fat diet induced weight

gain (A), gonadal fat pad weights (B) and fasting insulin measurements (C) at week 12.

Data expressed as mean ± SEM (n = 10 mice per group for A; n = 5 for B and C);

*P < 0.05.

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4.3.2 Chlorogenic Acid Supplementation Promoted

Glucose Intolerance and Insulin Resistance

We performed intraperitoneal glucose tolerance testing (GTT; at week 5 and 10) and

insulin tolerance testing (ITT; week 6 and 11) after commencement of feeding (Figure

4.2A-D). A tendency for glucose intolerance was observed with chlorogenic acid

supplementation compared to high fat diet alone, particularly at week 10 (Figure 4.2B).

Interestingly, we demonstrated that the chlorogenic acid supplementation group were

markedly more insulin resistant compared to the high fat diet alone mice (P < 0.05) and

this increased with the duration of the diet (Figure 4.2C and 4.2D). We conducted

systematic analysis of insulin stimulation in a number of metabolic tissues to assess in

which compartment insulin resistance was prevailing with chlorogenic acid

supplementation.

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88

Figure 4.2. Effect of chlorogenic acid supplementation on glucose intolerance at week 5

(A) and week 10 (B); and insulin resistance at week 6 (C) and week 11 (D) in mice.

Data expressed as mean ± SEM (n = 9-10 per group); * P < 0.05.

4.3.3 Chlorogenic Acid Resulted in Mild Insulin Resistance

in White Adipose Tissue

Western blot analysis of insulin signaling in white adipose tissue confirmed reduced

phosphorylation of Akt (Ser473

) with chlorogenic acid supplementation (Figure 4.3).

High fat diet fed mice showed a 10.8-fold increase in insulin stimulation compared to

the chlorogenic acid supplemented group which had a 6.9-fold increase in insulin

stimulation. Therefore, there was a propensity for mild insulin resistance in white

adipose tissue from mice fed a chlorogenic acid supplemented high fat diet.

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89

Figure 4.3. Effect of chlorogenic acid supplementation on insulin sensitivity in white

adipose tissue. Representative immunoblots of total and phosphorylated (Ser473

) Akt (A)

and quantification of insulin stimulated phosphorylation of Akt (Ser473

) (B). Data

expressed as mean ± SEM (n = 4 mice per group).

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90

When assessing insulin stimulation in liver, we did not observe an effect due to

chlorogenic acid supplementation. This suggests that chlorogenic acid-induced insulin

resistance is not occurring in the liver (Figure 4.4). Both groups of mice showed a 1.8

fold increase in insulin stimulation.

Figure 4.4. Effect of chlorogenic acid supplementation on insulin sensitivity in the

liver. Representative immunoblots of total and phosphorylated (Ser473

) Akt (A) and

quantification of insulin stimulated phosphorylation of Akt (Ser473

) (B). Data expressed

as mean ± SEM (n = 4 mice per group).

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91

We also assessed insulin stimulation in skeletal muscle (quadricep) (Figure 4.5). From

this analysis, we found no effect of chlorogenic acid supplementation on insulin

signaling in the skeletal muscle (quadricep), where the high fat diet showed a 4.4-fold

increase in insulin stimulation while chlorogenic acid supplemented group displayed a

5.5-fold increase in insulin stimulation. This suggests that chlorogenic acid-induced

insulin resistance did not occur in the quadricep.

Figure 4.5. Effect of chlorogenic acid supplementation on insulin sensitivity in the

skeletal muscle. Representative immunoblots of total and phosphorylated (Ser473

) Akt

(A) and quantification of insulin stimulated phosphorylation of Akt (Ser473

) (B). Data

expressed as mean ± SEM (n = 4 mice per group).

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4.3.4 Chlorogenic Acid Supplementation Reduced Fatty

Acid Oxidation Signaling in Liver and White Adipose Tissue

We examined the phosphorylation of AMP activated protein kinase (AMPK) and acetyl

carboxylase β (ACCβ) as markers of fatty acid oxidation in a number of metabolic

tissues. Analysis of liver highlighted that phosphorylation of AMPK and its downstream

target ACCβ were decreased in liver with chlorogenic acid supplementation (Figure

4.6). This result suggests decreased fatty acid oxidation prevailed in the mice after

chlorogenic acid supplementation.

Figure 4.6. Effect of chlorogenic acid supplementation on AMPK signaling in the liver.

Representative immunoblots (A) and quantification of phosphorylation of AMPK

(Thr172

) (B) and ACCβ (Ser79

) (C) in liver. Data expressed as mean ± SEM (n = 5 mice

per group); * P < 0.05 and **P < 0.001.

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93

We examined liver histology using haematoxylin and eosin staining. Interestingly, we

observed markedly more steatosis in liver of high fat fed mice supplemented with

chlorogenic acid (Figure 4.7B) compared to high fat fed only mice (Figure 4.7A).

Moreover, the hepatic lipid content was higher in mice supplemented with chlorogenic

acid compared to mice fed a high fat diet alone (Figure 4.7C). This is consistent with the

reduced phosphorylation of AMPK and ACC seen in the same group of mice, which

suggests an impaired fatty acid oxidation pathway.

Figure 4.7. Effect of chlorogenic acid supplementation on high fat diet induced

steatosis (A and B) and lipid content in liver (C). Representative haematoxylin and

eosin staining of liver of mice fed a high fat diet (A) and high fat diet + Chlorogenic

acid (B); lipid quantitation (C). Arrows indicate steatosis. Data expressed as

mean ± SEM (n = 10 per group); *P < 0.05.

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94

Fatty acid oxidation signaling was also assessed in white adipose tissue. In this tissue,

chlorogenic acid supplementation resulted in a tendency for decreased phosphorylation

of ACCβ, the downstream target of AMPK, which suggests decreased fatty acid

oxidation in white adipose tissue of mice fed a high fat diet supplemented with

chlorogenic acid (Figure 4.8).

Figure 4.8. Effect of chlorogenic acid supplementation on phosphorylation of ACCβ in

white adipose tissue. Representative immunoblots (A) and quantification of

phosphorylation of ACCβ (Ser79

) (B) in white adipose tissue. Data expressed as

mean ± SEM (n = 4-5 mice per group).

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95

Assessment of white adipose tissue by haematoxylin and eosin staining revealed

adipocyte hypertrophy in mice fed a high fat diet supplemented with chlorogenic acid

compared to mice fed a high fat diet alone (Figure 4.9).

Figure 4.9. Effect of chlorogenic acid supplementation on adipocyte hypertrophy.

Representative haematoxylin and eosin staining of white adipose tissue of mice fed a

high fat diet (A) and high fat diet + Chlorogenic acid (B).

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96

Finally, fatty acid oxidation signaling was also assessed in the skeletal muscle

(quadricep). From the analysis, we found no effect of chlorogenic acid supplementation

on the phosphorylation of AMPK and its downstream target, ACCβ. This suggests that

chlorogenic acid supplementation in high fat diet does not affect the fatty acid oxidation

in skeletal muscle (quadricep) of mice (Figure 4.10).

Figure 4.10. Effect of chlorogenic acid supplementation on AMPK signaling in the

skeletal muscle (quadricep). Representative immunoblots (A) and quantification of

phosphorylation of AMPK (Thr172

) (B) and ACCβ (Ser79

) (C) in quadricep. Data

expressed as mean ± SEM (n = 5 mice per group).

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97

4.4 Discussion

We sought to assess the effect of chlorogenic acid supplementation on diet induced

obesity, glucose intolerance, insulin resistance and signaling pathways associated with

glucose homeostasis and fatty acid oxidation in mice. Our results do not support the

hypothesis that chlorogenic acid can prevent development of features of the metabolic

syndrome.

We demonstrated that 12 weeks of high fat feeding induced an increase in body weight

and hyperinsulinemia in C57BL/6J mice. The same observation has previously been

seen by our group 235

and others 236, 237

. Interestingly, a high fat diet supplemented with

chlorogenic acid increased body weight gain compared to mice fed a normal diet. Mice

fed a high fat diet only and a high fat diet with chlorogenic acid supplementation

showed similar body weights. Despite the similar body weight, fasting insulin was

further elevated in mice fed a high fat diet supplemented with chlorogenic acid

compared to mice fed a high fat diet alone. Gonadal fat pad mass was also increased

2.5-fold in mice fed a high fat diet compared to those fed a normal diet, and was 4-fold

higher in mice fed a high fat diet supplemented with chlorogenic acid when compared to

those fed a normal diet. These results are not consistent with Cho et al. 153

who observed

that chlorogenic acid and its metabolite, caffeic acid reduced weight gain and visceral

fat mass in high fat diet-induced obese Imprinting Control Region (ICR) mice. A recent

study by Murase et al. 155

observed that coffee polyphenols suppressed body fat

accumulation in high fat diet-induced obese mice 155

. It should be noted that our study

used a higher dose of chlorogenic acid (1 g.kg-1

) compared to Cho et al. 153

(0.2 g.kg-1

)

and Murase et al. 155

(0.5 g.kg-1

mixed coffee polyphenols). In addition, the study by

Murase et al. 155

was for 15 weeks which is a longer period of feeding compared to the

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98

present study. Further, Cho et al. 153

employed a different strain of mice in their study.

These differences could explain some of the variation in results compared to our study.

Our study is not the first to demonstrate that higher doses of a particular compound or

chemical may promote detrimental effects on health. A long-term French study

demonstrated that moderate wine drinkers (<60g alcohol/day) had lower risks of deaths

from all causes at all levels of systolic blood pressure. In this same study, there was no

significant reduction in all-cause mortality in heavier drinkers 238

.

When determining the dose of a compound to administer to mice, it is vital to ensure

that it will ultimately equate to physiological concentrations in humans. It has been

highlighted that it would be naive to just use body weight to extrapolate safe doses from

animals to humans. It has been suggested that using body surface area would allow

better estimates as the allometric scaling factor at least approximates to that of clearance

239. Based on body surface area, the dose in humans should be 405 greater than the dose

in mice. Hence, in our current study, a single mouse would consume approximately 3mg

of CGA per day. Based on body surface area, this equates to 1,215mg of CGA in

humans per day. This is a physiological dose in humans as a single cup of coffee

contains approximately 250mg of CGA (1,215mg of CGA= 5 cups of coffee).

To our knowledge, this is the first study of the effect of chlorogenic acid on glucose

tolerance and insulin sensitivity in high fat diet-induced obese mice. We did not observe

any improvement in glucose tolerance in mice fed a high fat diet supplemented with

chlorogenic acid. This is not in accordance with Bassoli et al. 240

who observed a

reduced plasma glucose peak in the oral glucose tolerance test following an acute oral

administration of chlorogenic acid at 3.5 mg.kg-1

body weight in rats. Ong et al. 157

also

found a reduction of blood glucose level after intraperitoneal administration of

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250 mg.kg-1

chlorogenic acid in db/db mice. The different methods of chlorogenic acid

administration and dose used in the experiment may explain some of the variation in

results compared to our study. We believe that performing the glucose and insulin

tolerance tests on mice fed a high fat diet supplemented with chlorogenic acid is a more

robust approach to reflect a natural consumption effect of this compound, rather than

administrating it through intraperitoneal injection or oral gavage. From the insulin

tolerance test, we observed a significant increase in insulin resistance in mice fed a high

fat diet supplemented with chlorogenic acid. We also assessed insulin stimulation in

metabolic tissues by measuring the phosphorylation of Akt (Ser473

). Phosphorylation of

Akt due to insulin stimulation, is required for insulin-stimulated glucose uptake and

anabolic metabolism 241

. Therefore, an increased rate of Akt (Ser473

) phosphorylation

reflects insulin sensitivity in the tissue. We saw a tendency for reduced insulin

stimulated phosphorylation of Akt in the white adipose tissue of mice fed a high fat diet

supplemented with chlorogenic acid, which reflects a mild insulin resistance. This result

does not correspond to some in vitro studies showing improvement of insulin signaling

in 3T3-L1 adipocytes by chicory salad leaf extract containing chlorogenic acid 242

.

However, our result supports the increased insulin resistance observed from the insulin

tolerance test. We did not observe an effect of chlorogenic acid supplementation on

insulin sensitivity in the liver and quadricep muscle, suggesting that insulin resistance

did not occur in these tissues.

We also assessed effects of chlorogenic acid supplementation on signaling pathways

associated with fat oxidation in the liver, white adipose tissue and skeletal muscle. We

and others have highlighted that phosphorylation of ACCβ at its critical site (Ser79

) 243

by AMPK leads to the decrease in activity of ACC resulting in reduced malonyl-CoA

production, which in turn relieves inhibition of carnitine palmitoyl transferase 1

Chapter 4

100

(CPT-1), and increases fatty acid oxidation 50

. In our study, we saw a markedly reduced

phosphorylation of AMPK (Thr172

) and its downstream target ACCβ (Ser79

) in the liver,

suggesting reduced fatty acid oxidation is occurring in this tissue. Haematoxylin and

eosin staining of the liver tissue sections showed that chlorogenic acid supplementation

in the high fat diet resulted in increased steatosis in the liver, which supports the

impaired fatty acid oxidation signaling in this tissue. The chlorogenic acid

supplementation was also found to increase lipid content in the liver by 1.5-fold when

compared to liver of mice fed a high fat diet alone. Our data is not in agreement with

Ong et al. 157

which found that chlorogenic acid increases phosphorylation of AMPK

and ACC in L6 myotubes after incubation with chlorogenic acid in vitro. However,

results from in vitro studies may differ from those found in vivo. In white adipose tissue,

there was a tendency for reduced phosphorylation of ACCβ in mice fed a high fat diet

supplemented with chlorogenic acid compared to mice fed a high fat diet only,

suggesting decreased fatty acid oxidation. Haematoxylin and eosin staining of the white

adipose tissue sections revealed that chlorogenic acid resulted in adipocyte hypertrophy

in mice. This supports the data showing a tendency for impaired fatty acid oxidation

pathways in white adipose tissue. The result from our study is not in accordance to data

shown in Cho et al. 153

where they demonstrated that chlorogenic acid reduced obesity

and improved lipid metabolism in obese ICR mice. However, the lower dose of

chlorogenic acid used may explain some of the variation in results compared to our

study.

We also assessed phosphorylation of AMPK and ACCβ in quadricep. However, we did

not observe an effect of chlorogenic acid on this tissue. Tsuda et al. 156

also did not find

an effect of chlorogenic acid on phosphorylation of these proteins in epitrochlearis

Chapter 4

101

muscle, but observed that caffeic acid, a metabolite of chlorogenic acid, induced the

phosphorylation of AMPK and ACCβ.

In conclusion, we found that chlorogenic acid supplementation in high fat diet increased

insulin resistance and showed a decrease on markers of fatty acid oxidation in mice.

Chlorogenic acid supplementation also resulted in an increase of lipid content and

elevated steatosis in the liver of mice. Our study does not support the hypothesis that

supplementation of chlorogenic acid to a high fat diet will protect against features of the

metabolic syndrome in obese mice. Further work especially on human intervention

studies is required to determine if coffee phenolics are able to protect against metabolic

syndrome and type 2 diabetes in humans.

Chapter 5

102

CHAPTER 5

Summary and Future Work

The benefits of fruit and vegetable consumption on prevention of cardiovascular disease

risk factors are widely accepted 2-4

. Among the numerous plant compounds studied,

phenolic compounds are primary candidates contributing to these benefits. Phenolic

compounds have been reported to have various biological effects in promoting

cardiovascular health 96, 119

. However, the mechanisms of action are inconclusive. This

thesis sought to address the importance of understanding specific phenolics composition

in fruits, as well as to investigate the potential cardiovascular protective effect of

chlorogenic acid.

5.1 Phenolic Composition of New Plum Selections

in Relation to Total Antioxidant Capacity

Fruits are a primary choice of “natural healthy food” by the consumers. They are sold in

a competitive market, which encourages identification of cultivars and practices that are

more ‘healthy’ in some way which will greatly assist marketing efforts. Plums are a

crop with a rich content of phenolic compounds. Therefore, understanding the cultivar

variation of this fruit is important to promote development of enhanced quality of plums

by the agricultural industry, which in turn encourages the production of food sources

with enriched pharmacological properties.

Chapter 5

103

In this thesis (Chapter 2), the diversity of phenolic compounds with abundant

pharmacological evidence in pre-varietal plum genotypes was assessed. Neo-

chlorogenic acid, an isomer of chlorogenic acid, and quercetin glycosides were found to

be the predominant phenolics in the tested plums, with a huge variation amongst the

plum varieties. This knowledge is important for the purpose of crop development,

especially in identification of potential biomarkers for plum breeding. The mounting

evidence of antioxidant activities of plant-based food such as fruits 93, 179, 244

have

encouraged industry advocates and policy makers to implement a measure of

antioxidant capacity in fruit breeding programs, to speed identification and

commercialization of novel cultivars for added value. However, the functional relevance

of antioxidant ability or free radical quenching activities of phenolic-rich foods towards

potential health benefits in human has been challenged 62, 245

. Therefore, in this study,

the relationships between total phenolics, and individual bioactive phenolic compounds

with the total antioxidant capacity (TAC) were evaluated to assess the relevance of TAC

for fruit breeding programs. TAC of the plums were observed to have significant

correlation with total phenolic concentration, but not with the predominant bioactive

phenolic compound detected in the plums. This finding strongly questions the value of

total antioxidant capacity may be limited in fruit breeding programs. Although the TAC

assay used in this study (AIOR) addresses some of the restrictions of other well-

established methods, comparison between the values measured with other studies is

difficult. This can be said of the antioxidant literature as a whole, which is prone to

misinterpretation. Future research could more acutely uncover the validity of TAC

assays. Further, understanding the patterns of inheritance of specific phenolic

compounds in the plums will be necessary to enable translation of this knowledge into

breeding programs.

Chapter 5

104

5.2 Acute Effects of Chlorogenic Acid on Nitric Oxide

Status, Endothelial Function, and Blood Pressure in Healthy

Volunteers: A Randomized Trial

The beneficial effects of phenolics on cardiovascular health outcomes have been

reported in many studies 7, 8, 246

. Most studies however, have focused on flavonoids, with

far less attention to phenolic acids such as chlorogenic acid. Chlorogenic acid, which is

rich in dietary sources including fruits such as plums, and coffee, was suggested to

contribute to reduced incidence of cardiovascular disease (CVD) and type 2

diabetes 121, 143

. This suggestion is derived from limited studies that have mainly

reported effects of coffee consumption, which contains high amount of chlorogenic

acid. This compound was reported to decrease blood pressure (BP) in rats 141

, which

was suggested to be an effect mediated by regulation of NO status.

In this thesis (Chapter 3), the effects of chlorogenic acid on BP, and on markers of

endothelial function through measurements of nitric oxide (NO) status and flow

mediated dilatation (FMD) were investigated. A randomized controlled trial was

performed to investigate the effect of an amount of chlorogenic acid equivalent to two

cups of coffee on healthy volunteers. Chlorogenic acid was shown to reduce BP with a

significant decrease in both systolic and diastolic blood pressure. On the other hand,

chlorogenic acid ingestion did not enhance NO status and FMD significantly. The effect

of chlorogenic acid on BP reduction was found to be greatest between 60 and 90

minutes post ingestion. However, NO status and FMD measurements were carried out at

150 and 120 minutes respectively. These observations suggest that chlorogenic acid

may have peaked in plasma at 60 to 90 minutes after ingestion, resulting in the greatest

Chapter 5

105

reduction of BP. NO status as well as FMD could have been affected by chlorogenic

acid treatment if they were to be measured at the similar time point (60 to 90 minutes

post ingestion). Furthermore, chlorogenic acid in its intact form was detected in plasma

at 150 min post ingestion, but there was no significant increase of chlorogenic acid

metabolite levels in plasma compared to placebo. The metabolites of chlorogenic acid

which are possibly produced at a later time point may have an effect on the endothelial

function markers. The time points chosen for blood sampling for NO status, FMD as

well as chlorogenic acid and its metabolites measurements is a restriction in this study.

The multiple measured outcomes in this study resulted in the difficulty of concomitant

measurement. For example; blood sampling cannot be performed during FMD

measurement because it would interfere with results of FMD assessment. Future studies

addressing the time course of chlorogenic acid treatment is thus warranted. A dose

response study of chlorogenic acid ingestion, at physiologically relevant doses, is also

important to determine the effect of chlorogenic acid at different doses on

cardiovascular health. The imbalanced sex distribution among participants of this study

makes a factor of limitation in this study due to the hormonal differences. Therefore, the

influence of gender on the effect of chlorogenic acid towards the measured outcomes

should also be investigated in future work.

Chapter 5

106

5.3 Supplementation of a High Fat Diet with Chlorogenic

Acid is Associated with Insulin Resistance and Hepatic Lipid

Accumulation in Mice

Chlorogenic acid has been attributed to the protective effects on metabolic

abnormalities 154, 155

which are risk factors of CVD and diabetes 222, 223

. The effects have

mainly been derived from limited studies using coffee phenolics which consist of

different phenolic acids such as chlorogenic acids and ferulic acids. The impact of

dietary supplementation of chlorogenic acid on high fat diet-induced metabolic disorder

is yet to be elucidated.

In this thesis (Chapter 4), the effect of dietary supplemented chlorogenic acid (1 g.kg-1

diet) for 12 weeks on metabolic abnormalities induced by high fat diet consumption in

mice was tested. In this study, chlorogenic acid supplementation in high fat diet did not

reduce body weight compared to mice fed with high fat diet alone. In contrast,

chlorogenic acid increased insulin resistance compared to mice fed a high fat diet only.

Interestingly, chlorogenic acid supplementation in high fat diet was shown to have

resulted in decreased phosphorylation of AMPK and ACCβ in liver, which are markers

of fatty acid oxidation. This result is further supported by higher lipid content and more

steatosis in the liver of mice fed a high fat diet supplemented with chlorogenic acid

compared to mice fed a high fat diet only.

Results shown from this study suggests that chlorogenic acid supplementation in a high

fat diet with the particular dose that was used, does not protect against features of the

metabolic syndrome in diet-induced obese mice. This study uses a higher dose of

chlorogenic acid than other studies that suggests positive effects of chlorogenic acid on

Chapter 5

107

metabolic syndrome. It therefore indicates the need for a dose response investigation in

future work to determine the differences of effects seen. Furthermore, human

intervention studies are also warranted as future work to elucidate the effect of

chlorogenic acid on metabolic syndrome in humans.

5.4 Conclusion

The results from this thesis provide data on the predominant phenolic compounds found

in plums which may have a significant influence on health protection. This thesis also

reports the importance of understanding phenolic compound composition in fruits. This

knowledge is essential to any future work towards developing breeding tools helping

identification of fruit which may have enhanced health-promoting abilities. Study in this

thesis demonstrated the significance of chlorogenic acid on vascular protection in

healthy humans, which may contribute to the cardiovascular protective outcomes from

diets rich in fruits and vegetables. The work in this thesis reported that chlorogenic acid

at a high dose, yet achievable in regular diet, showed no protection on metabolic

syndrome in a mouse model, but deteriorate some of the measured metabolic syndrome

features. More work is therefore needed to verify the protective mechanism of

chlorogenic acid on CVD and the effects of different doses of chlorogenic acid on the

metabolic syndrome especially in human intervention trials.

References

108

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