Olive (Olea europaea L.) leaf

biophenols as nutraceuticals

A thesis submitted to

In fulfilment of requirements for the degree of

Doctor of Philosophy

By

Muhammad Kamran

March, 2016

©Muhammad Kamran, 2016

II

Certificate of Authorship

I hereby declare that this submission is my own work and to the best of my

knowledge and belief, understand that it contains no material previously published

or written by another person, nor material which to a substantial extent has been

accepted for the award of any other degree or diploma at Charles Sturt University

or any other educational institution, except where due acknowledgement is made in

the thesis. Any contribution made to the research by colleagues with whom I have

worked at Charles Sturt University or elsewhere during my candidature is fully

acknowledged.

I agree that this thesis be accessible for the purpose of study and research in

accordance with normal conditions established by the Executive Director, Library

Services, Charles Sturt University or nominee, for the care, loan and reproduction

of thesis.*

------------------------- --------------------

Muhammad Kamran Date

* Subject to confidentiality provisions as approved by the University.

III

Dedication

I dedicate my work to my beloved Father, Mother and my

Family for their Love, Support, Encouragement and Prayers.

IV

Acknowledgements

All praises to ALLAH Almighty, most gracious and merciful, who blessed me with

physical and mental health and gave me strength to complete this work. I am

thankful to ALLAH Almighty for sending Prophet Muhammad (May the peace and

blessings of ALLAH be upon him), who practically told us the way of spending life

and provided guidance to mankind.

I would like to convey my gratitude to those people, without whom this study would

be very difficult to complete and it is my pleasure to mention their names.

Firstly, I would like to express my sincere gratitude to Charles Sturt University for

providing me the Postgraduate Research Scholarship for this study. Special thanks

to Graham Centre for Agricultural Innovation for providing some funds towards the

end of the study. I owe my thanks to Comvita, Olive Leaf Australia, for providing

olive leaf extract capsules for the clinical trial.

I wish to express my deepest gratitude to my supervisor Dr Hassan Kamal Obied

and Co supervisors Dr Christopher James Scott and Dr Adam Scott Hamlin for the

continuous support of my PhD study, for their motivation, immense knowledge and

positive criticism. Their invaluable help of constructive comments and suggestions

throughout the research and thesis works have contributed to the success of this

research.

I would like to express my sincere gratitude to Assoc. Prof. Gaye Krebs, for her

valuable advice and guidance, for being very supportive to me during my

candidature. Special thanks also goes to Dr. S. M. Saqlan Naqvi for invaluable

support towards the end of the study in the absence of my principal supervisor. I

would also like to thank Dr Mahmoud Ahmed (University of Alexandria, Egypt)

for running non-parametric statistical analysis.

V

I am thankful to Technical laboratory staff especially, Mr. Michael Loughlin, Mr

Paul Burton, Therese Moon and Lyn Mathews for providing access to the laboratory

and research facilities, technical support for handling delicate hi-tech instruments.

Without their precious support it would be very challenging to successfully

complete this research.

I would like to thank Mrs Karen MacKney for always being there for any library

related issues and for her support in endnote referencing. I thank my fellow

colleagues Mrs Nusrat Subhan, Mr Arthur Robin, Dr Kah Yaw Ee, Dr Randy

Adjonu, Dr. Adeola Alashi, Dr K M Shamsul Haque, Dr. Saira Hussain and Mr.

Shoaib Tufail for the stimulating discussions, encouragement and support.

Most importantly I would like to extend my warmest thanks to my gorgeous mother

and lovely family, my brothers, my sisters and my wife for their unconditional love,

prayers, support, encouragement and confidence in me not only during the course

of this study but throughout my life. Special feeling of gratitude to my Father Hafiz

Ata Elahi Late, who was my role model, though he is not here now but he left the

fingerprints of grace on my life. Finally, my dearest thanks to my sons Muhammad

Soban Kamran and Muhammad Hammad Kamran for their love, support and for

realizing the importance of quietness and peace I need for writing this thesis.

VI

Table of Contents

CERTIFICATE OF AUTHORSHIP .................................................................. II

DEDICATION ..................................................................................................... III

ACKNOWLEDGEMENTS................................................................................ IV

TABLE OF CONTENTS.................................................................................... VI

LIST OF TABLES ........................................................................................... XIII

LIST OF FIGURES ......................................................................................... XIV

LIST OF SYMBOLS AND ABBREVIATIONS .......................................... XVII

LIST OF PUBLICATIONS ......................................................................... XXIV

ABSTRACT ..................................................................................................... XXV

INTRODUCTION .................................................................................................. 1

CHAPTER 1. LITERATURE REVIEW.......................................................... 4

OLIVE LEAF BIOPHENOLS, CHEMISTRY AND BIOLOGICAL

ACTIVITY .............................................................................................................. 4

1.1. INTRODUCTION ....................................................................................................... 4

1.2. THE OLIVE TREE .................................................................................................... 6

1.3. OLIVE AND HEALTH: THE ROLE OF BIOPHENOLS ............................................... 7

1.4. THE CHEMISTRY OF OLIVE BIOPHENOLS ............................................................. 8

1.4.1. Classification of Olive Biophenols ................................................................... 9

1.4.1.1. Simple phenols (C6-C0-3) ....................................................................................... 9

1.4.1.2. Phenolic acids (C6-C1-3) ......................................................................................... 9

1.4.1.3. Flavonoids (C6-C3-C6) ......................................................................................... 10

1.4.1.4. Secoiridoids ......................................................................................................... 10

1.4.1.5. Lignans ................................................................................................................ 11

VII

1.4.1.6. Isochromans ......................................................................................................... 11

1.5. OLIVE LEAVES ....................................................................................................... 12

1.5.1. Nutritive Value ................................................................................................ 12

1.5.2. Bioactive Phytomolecules of Olive Leaf ........................................................ 13

1.6. OLIVE LEAF BIOPHENOLS .................................................................................... 14

1.6.1. Oleuropein ....................................................................................................... 15

1.6.2. Olive leaf flavonoids ....................................................................................... 17

1.6.3 Factors affecting the biophenol content of olive leaves ................................... 26

1.7. OLIVE LEAVES: NUTRACEUTICAL AND DIETARY APPLICATIONS ..................... 28

1.7.1. Olive leaf extract ............................................................................................. 28

1.7.2. Fortification of olive oil /edible oils/ other food products with olive leaves .. 29

1.7.3. Encapsulation of olive leaf extracts ................................................................ 29

1.7.4. Antimicrobial food packaging ........................................................................ 30

1.8. OLIVE LEAVES: TRADITIONAL AND FOLK MEDICINE ........................................ 30

1.9. PHARMACOLOGICAL ACTIVITIES OF OLIVE LEAVES......................................... 35

1.9.1. Antimicrobial activity ..................................................................................... 35

1.9.1.1. Antibacterial and antifungal activity .................................................................... 35

1.9.1.2. Antiviral activity .................................................................................................. 36

1.9.2. Immunoregulatory effects ............................................................................... 38

1.9.3. Cardiovascular protective activities ................................................................ 38

1.9.4. Antiplatelet activity ......................................................................................... 39

1.9.5. Hypocholesterolaemic effects ......................................................................... 40

1.9.6. Blood pressure regulation ............................................................................... 40

1.9.7. Anticancer and chemopreventive activities .................................................... 41

1.9.8. Renal health promotion ................................................................................... 42

1.9.9. Gastro-protective effects ................................................................................. 43

1.9.10. Antioxidant and free radical scavenging effects ........................................... 43

1.9.11. Anti-inflammatory effects ............................................................................. 45

1.9.12. Anti-diabetic activities .................................................................................. 47

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1.9.13. Anti-Alzheimer’s Disease ............................................................................. 50

1.9.14. Miscellaneous activities ................................................................................ 52

1.10. PHARMACOKINETICS OF OLIVE LEAF BIOPHENOLS ........................................ 53

1.11. TOXICITY AND ADVERSE EFFECTS OF OLIVE LEAF BIOPHENOLS ..................... 53

1.12. POSOLOGY OF OLIVE LEAF/ OLE ...................................................................... 54

1.13. CONCLUSION ....................................................................................................... 56

1.14. AIM AND OBJECTIVES OF THE PROJECT ............................................................ 58

CHAPTER 2. DRYING AT HIGH TEMPERATURE FOR A SHORT

TIME MAXIMIZES THE RECOVERY OF OLIVE LEAF

BIOPHENOLS 59

2.1. INTRODUCTION ..................................................................................................... 59

2.2. MATERIALS AND METHODS .................................................................................. 62

2.2.1. Chemicals and reagents .................................................................................. 62

2.2.2. Collection of olive leaves ............................................................................... 63

2.2.3. Sample treatments ........................................................................................... 63

2.2.4. Moisture content ............................................................................................. 64

2.2.5. Extraction of biophenols ................................................................................. 65

2.2.6. Spectrophotometric measurements ................................................................. 65

2.2.6.1. Total phenols ....................................................................................................... 66

2.2.6.2. Total flavonoids ................................................................................................... 66

2.2.6.3. o-diphenols .......................................................................................................... 66

2.2.6.4. ABTS radical scavenging assay .......................................................................... 67

2.2.6.5. DPPH radical scavenging assay .......................................................................... 68

2.2.7. High performance liquid chromatography (HPLC) ........................................ 68

2.2.7.1. HPLC-DAD-online ABTS .................................................................................. 68

2.2.7.2. HPLC-DAD-MS/MS ........................................................................................... 69

2.2.8. Statistical analysis ........................................................................................... 70

2.3. RESULTS AND DISCUSSION .................................................................................... 70

IX

2.3.1. Moisture content ............................................................................................. 71

2.3.2. Extractable matter (EM) .................................................................................. 71

2.3.3. Total biophenol composition and total antioxidant capacity .......................... 72

2.3.4. Biophenol-antioxidant profile and recovery of individual biophenols ........... 78

2.3.5. Cultivar and seasonal impact on biophenols and oleuropein recovery ........... 82

2.3.6. Sample pre-treatment and biophenol recovery ............................................... 84

2.4. CONCLUSIONS ........................................................................................................ 86

CHAPTER 3. THE IMPACT OF CLIMATIC CONDITIONS, SEASON

AND CULTIVAR ON BIOPHENOL CONTENT OF OLIVE LEAVES ..... 88

3.1. INTRODUCTION ...................................................................................................... 88

3.2. MATERIALS AND METHODS .................................................................................. 91

3.2.1. Chemicals and Reagents ................................................................................. 91

3.2.2. Collection of olive leaves ................................................................................ 91

3.2.3. Drying of olive leaves ..................................................................................... 92

3.2.4. Moisture content ............................................................................................. 92

3.2.5. Extraction of biophenols ................................................................................. 92

3.2.6. Spectrophotometric measurements ................................................................. 92

3.2.6.1. Determination of total phenols............................................................................. 93

3.2.6.2. Determination of total flavonoids ........................................................................ 93

3.2.6.3. Determination of o-diphenols .............................................................................. 93

3.2.6.4. Determination of phenolic classes ....................................................................... 93

3.2.6.5. Antioxidant activity ............................................................................................. 94

3.2.7. High Performance Liquid Chromatography (HPLC) ...................................... 94

3.2.8. Climatic data ................................................................................................... 95

3.2.9. Statistical analysis ........................................................................................... 95

3.3. RESULTS AND DISCUSSION .................................................................................... 96

3.3.1. Dry matter content .......................................................................................... 97

3.3.2. Total phenols (Folin-Ciocalteu) ...................................................................... 98

X

3.3.3. Total flavonoids ............................................................................................ 104

3.3.4. o-Diphenols ................................................................................................... 105

3.3.5. Total Phenolic Compounds (280 nm) ........................................................... 109

3.3.6. Hydroxycinnamic acid derivatives ............................................................... 111

3.3.7. Flavonols ....................................................................................................... 113

3.3.8. Antioxidant activity ...................................................................................... 115

3.3.8.1. ABTS ................................................................................................................. 115

3.3.8.2. DPPH ................................................................................................................. 116

3.3.9. High Performance Liquid Chromatography (HPLC) analysis ...................... 120

3.3.9.1. Oleuropein ......................................................................................................... 121

3.3.10. Correlation analysis .................................................................................... 124

3.4. CONCLUSION ....................................................................................................... 133

CHAPTER 4. COMPARATIVE STUDY OF IN-VITRO ANTIOXIDANT

AND ANTIDIABETIC ACTIVITIES OF AUSTRALIAN OLIVE LEAF

EXTRACTS 136

4.1. INTRODUCTION ................................................................................................... 136

4.2. MATERIALS AND METHODS ................................................................................ 139

4.2.1. Chemicals and reagents ................................................................................ 139

4.2.2. Collection of olive leaves ............................................................................. 140

4.2.3. Drying of olive leaves ................................................................................... 140

4.2.4. Dry matter ..................................................................................................... 141

4.2.5. Extraction of biophenols ............................................................................... 141

4.2.6. Enriched biophenol extract ........................................................................... 141

4.2.7. Extractable matter ......................................................................................... 141

4.2.8. High performance liquid chromatography (HPLC) ...................................... 142

4.2.9. Spectrophotometric measurements ............................................................... 142

4.2.9.1. Total phenols ..................................................................................................... 142

4.2.9.2. ABTS radical scavenging assay ........................................................................ 143

XI

4.2.9.3. Hydrogen peroxide scavenging assay ................................................................ 143

4.2.9.4. Superoxide radical (SOR) scavenging assay ..................................................... 144

4.2.9.5. Copper chelation activity ................................................................................... 145

4.2.9.6. Ferric reducing antioxidant power assay ........................................................... 145

4.2.9.7. Alpha-amylase inhibition assay ......................................................................... 146

4.2.10. Statistical analysis ....................................................................................... 147

4.3. RESULTS AND DISCUSSION .................................................................................. 147

4.3.1. Dry matter ..................................................................................................... 148

4.3.2. Extractable matter ......................................................................................... 148

4.3.3. High performance liquid chromatography (HPLC) ...................................... 149

4.3.4. Total phenols ................................................................................................. 158

4.3.5. Antioxidant activity....................................................................................... 160

4.3.5.1. ABTS radical scavenging .................................................................................. 162

4.3.5.2. Hydrogen peroxide scavenging.......................................................................... 163

3.5.3. Superoxide radical (SOR) scavenging .................................................................. 166

4.3.5.4. Copper chelation activity ................................................................................... 168

4.3.5.5. Ferric reducing antioxidant power (FRAP) ....................................................... 171

4.3.6. Alpha-amylase inhibition .............................................................................. 174

4.4. CONCLUSION ....................................................................................................... 177

CHAPTER 5. AN INVESTIGATION INTO OLIVE LEAF EXTRACT AS

A THERAPEUTIC ANTIOXIDANT TREATMENT OF PREDIABETES 178

5.1. INTRODUCTION .................................................................................................... 178

5.2. MATERIALS AND METHODS ................................................................................ 182

5.2.1. Ethics approval .............................................................................................. 182

5.2.2. Subject recruitment ....................................................................................... 182

5.2.3. Study protocol ............................................................................................... 183

5.2.4. Dietary collection .......................................................................................... 184

5.2.5. Test material (supplement) ............................................................................ 184

5.2.6. Compliance ................................................................................................... 185

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5.2.7. Blood and urine samples ............................................................................... 185

5.2.8. Reagents and standards ................................................................................. 185

5.2.9. High performance liquid chromatography (HPLC) of olive leaf extract

capsules ................................................................................................................... 186

5.2.10. Spectrophotometric measurements ............................................................. 186

5.2.10.1. Total phenols assay .......................................................................................... 186

5.2.10.2. Isoprostanes assay ........................................................................................... 187

5.2.10.3. 8-Hydroxy-2-deoxyguanosine (8-OHdG) assay .............................................. 188

5.2.11. Statistical analysis ....................................................................................... 189

5.3. RESULTS AND DISCUSSION .................................................................................. 189

5.3.1. Nonparametric analysis of clinical data ........................................................ 190

5.3.2. Parametric analysis of clinical data ............................................................... 191

5.3.2. 1. Total phenols .................................................................................................... 195

5.3.2.2. Isoprostanes ....................................................................................................... 196

5.3.2.3. Hydroxy-2-deoxyguanosine (8-OHdG) ............................................................. 197

5.3.3. High performance liquid chromatography (HPLC) analysis of the capsules 200

5.4. CONCLUSION ....................................................................................................... 203

CHAPTER 6. GENERAL DISCUSSION, CONCLUSION AND FUTURE

DIRECTIONS 204

REFERENCES ................................................................................................... 212

APPENDICES .................................................................................................... 246

APPENDIX 1. OLIVE TREES USED FOR LEAVES COLLECTION .................................. 246

APPENDIX 2. LEAFLET INFORMATION OF COMMERCIAL OLIVE LEAF EXTRACTS. 249

APPENDIX 3. ETHICS APPROVAL .............................................................................. 250

APPENDIX 4. INFORMATION STATEMENT FOR CLINICAL TRIAL PARTICIPANTS 252

APPENDIX 5. PRE-STUDY SURVEY .......................................................................... 255

APPENDIX 6. POST-STUDY SURVEY ........................................................................... 260

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List of Tables

Table 1.1: Biophenols reported in olive leaves..................................................... 18

Table 1.2: Worldwide traditional and folk medicinal uses of olive leaves. ......... 31

Table 2.1: Biophenol content and antioxidant activity of Frantoio olive leaves. . 75

Table 2.2: Quantity of major biophenols identified in Frantoio olive leaf. .......... 83

Table 4.1: EC50 and IC50 values of olive leaf extracts for in vitro bioactivity assays.

............................................................................................................................. 161

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List of Figures

Figure 2.1: HPLC chromatograms of olive leaf extracts at 280nm showing the

effect of different drying methods on the phenolic compounds of olive leaves. ...80

Figure 2.2: HPLC chromatograms of olive leaf extracts of oven dried leaves at 105

°C, comparing 280nm and 325nm wavelength and the corresponding ABTS

activity. ...................................................................................................................81

Figure 2.3: Effect of different drying treatments on oleuropein recovery from olive

leaves. .....................................................................................................................84

Figure 3.1: Dry matter of olive leaves 2012 to 2014. .........................................100

Figure 3.2: Climate variation over the experimental timeframe. ........................101

Figure 3.3: Total phenols of olive leaves 2012 to 2014. .....................................102

Figure 3.4: Total flavonoids of olive leaves 2012 to 2014. ................................107

Figure 3.5: o -diphenols of olive leaves 2012 to 2014. .......................................108

Figure 3.6: Phenolic compounds (280 nm) of olive leaves 2012 to 2014. .........110

Figure 3.7: Hydroxycinnamic acid derivatives of olive leaves 2012 to 2014. ...112

Figure 3.8: Flavonols content of olive leaves 2012 to 2014. ..............................114

Figure 3.9: ABTS radical scavenging activity of olive leaves 2012 to 2014. ....117

Figure 3.10: DPPH radical scavenging activity of olive leaves 2012 to 2014. ..118

Figure 3.11: Oleuropein content of olive leaves 2012 to 2014. ..........................122

Figure 3.12: Correlation between total flavonoids and a) ABTS, b) DPPH. ......125

Figure 3.13: Correlation between oleuropein and a) total flavonoids, b) ABTS.

..............................................................................................................................126

Figure 3.14: Correlation between oleuropein and a) total phenols, b) DPPH. ...127

Figure 3.15: Correlation between oleuropein and a) phenolic compounds, b) o-

diphenols. .............................................................................................................128

Figure 3.16: Correlation between total phenols and a) ABTS, b) DPPH. ..........129

XV

Figure 3.17: Correlation between oleuropein and a) hydroxycinnamic acid

derivatives, b) flavonols....................................................................................... 130

Figure 3.18: Trend of antioxidant activity and oleuropein content of olive leaves

2012 to 2014. ....................................................................................................... 132

Figure 4.1: Extractable matter of olive leaf extracts. ......................................... 149

Figure 4.2: Solvent effect on oleuropein content of olive leaf extract. .............. 151

Figure 4.3: Oleuropein content of olive leaf extracts with 50% methanol as the

dilution solvent. ................................................................................................... 152

Figure 4.4: HPLC chromatograms of Hardy’s Mammoth crude olive leaf extract,

comparing 280nm and 325nm wavelength and the corresponding ABTS activity.

............................................................................................................................. 153

Figure 4.5: HPLC chromatograms of Hardy’s Mammoth enriched olive leaf

extract, comparing 280nm and 325nm wavelength and the corresponding ABTS

activity. ................................................................................................................ 154

Figure 4.6: HPLC chromatograms of Comvita olive leaf extract, comparing 280

nm and 325 nm wavelength and the corresponding ABTS activity. ................... 155

Figure 4.7: HPLC chromatograms of Blackmores olive leaf extract, comparing 280

nm and 325 nm wavelength and the corresponding ABTS activity. ................... 156

Figure 4.8: HPLC chromatograms of Swisse olive leaf extract, comparing 280 nm

and 325 nm wavelength and the corresponding ABTS activity. ......................... 157

Figure 4.9: Total phenols of olive leaf extracts as gallic acid equivalents (GAE).

............................................................................................................................. 159

Figure 4.10: Total phenols of olive leaf extracts as oleuropein equivalents (OLPE).

............................................................................................................................. 159

Figure 4.11: ABTS radical scavenging activity of oleuropein and olive leaf

extracts. ................................................................................................................ 164

XVI

Figure 4.12: Hydrogen peroxide (H2O2) scavenging activity of oleuropein and

olive leaf extracts. ................................................................................................165

Figure 4.13: Superoxide radical (SOR) scavenging activity of oleuropein and olive

leaf extracts. .........................................................................................................167

Figure 4.14: Copper chelation activity of luteolin and olive leaf extracts. .........170

Figure 4.15: FRAP values of olive leaf extracts as trolox equivalent. ...............172

Figure 4.16: α-Amylase inhibition activity of oleuropein and olive leaf extracts.

..............................................................................................................................176

Figure 5.1: Fasting blood glucose levels.............................................................193

Figure 5.2: Glycated haemoglobin (HbA1c). .....................................................193

Figure 5.3: Lipid profile. .....................................................................................194

Figure 5.4: Total phenols in urine. ......................................................................195

Figure 5.5: Stat-8- Isoprostane in urine. .............................................................196

Figure 5.6: 8-Hydroxy-2-deoxyguanosine (8-OHdG) in urine. ..........................197

Figure 5.7: Oleuropein content of the capsule with different solvents (mg/capsule).

..............................................................................................................................201

Figure 5.8: HPLC chromatograms of Comvita olive leaf capsule, comparing 280

nm and 325 nm wavelength and the corresponding ABTS activity. ...................202

XVII

List of Symbols and Abbreviations

Abbreviation Definition

8-OHdG 8-hydroxy 2'-deoxy-guanosine

ABTS 2,2′ azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium

salt

AD Alzheimers’s disease

AGE Apigenin-7-O-glucoside equivalent

ANOVA Analysis of variance

AP Alkaline pohsphatase

AP-1 Activator protein-1

ATP Adenosine triphosphate

Aβ β-amyloid

B0 Maximum binding

BME Blackmores extract

C Carbon

CAE Caffeic acid equivalents

CC Column chromatography

Cho:HDL Cholesterol to high density lipoprotein ratio

cm Centimetre

CNPG3 2-Chloro-4-Nitrophenyl-α-maltotrioside

COX-2 Cyclooxygenase-2

Cu Copper

Cu2+ Cupric ion

CYPs Cytochrome P450 enzymes

DAD Diode array detector

XVIII

dL Decilitre

DM Dry matter

DNA Deoxyribonucleic acid

DPPH 2,2′-diphenyl-1-picrylhydrazyl

DSS Dahl salt-sensitive

DTNB 5,5'-dithio-bis-(2-nitrobenzoic acid)

DW Dry weight

EC50 Half maximal effective concentration

ELISA Enzyme-linked immunosorbent assay

EM Extractable matter

ES Electrospray

ESI Electrospray ionization

Fe Iron

Fe2+ ferrous ion

Fe3+ Ferric ion

FRAP Ferric reducing antioxidant power

g Grams

GAE Gallic acid equivalent

GC Gas chromatography

GF/F Glass microfiber filters

GP General health practioner

gp41 Glycoprotein 41

GSH Reduced glutathione

GSSG Oxidized glutathione

GST Glutathione S-transferases

h Hours

XIX

H2O2 Hydrogen peroxide

HbA1c Glycated haemoglobin

HDL High-density lipoprotein

HIV Human immunodeficiency virus

HMCE Hardy’s Mammoth crude extract

HMEE Hardy’s Mammoth enriched extract

HPLC High performance liquid chromatography

HPLC-DAD High performance liquid chromatography with diode array detection

HRP Horseradish peroxidase

HSD Honest significant difference

HSV Herpes simplex virus

i.d. Internal diameter

IC25 Quarter maximal inhibitory concentration

IC50 Half maximal inhibitory concentration

IFG Impaired fasting glycaemia/glucose

IgG Immunoglobulin G

IGT Impaired glucose tolerance

IL Interleukin

IL-1β Interleukin-1 beta

kg Kilograms

kV Kilovolts

L Litre

LC-MS Liquid chromatography–mass spectrometry

LDL Low-density lipoprotein

L–NAME NG-nitro-L-arginine methyl ester

LSD Least significant difference

XX

M Molar

m Metre

mmol Millimole

m/z Mass/charge ratio

m2 Square metre

mAU Milli absorbance unit

mg Milligrams

min Minutes

MJ Mega joule

mL Millilitre

mM Millimole

mm Millimetre

MS Mass spectrometry

MS/MS Tandem mass spectrometry

mVolts Millivolts

MW Molecular weight

n Sample size

NADH Reduced -nicotinamide adenine dinucleotide

NBT Nitrotetrazolium blue

ND Not determined

NF-κB Nuclear factor kappa (B cells)

nm Nanometre

nM Nanomole

NSB Non-specific binding

o ortho

º Degree

XXI

O2- Superoxide anion

ºC Degree Celsius

ODP o-diphenol content

OGTT Oral glucose tolerance test

•OH Hydroxyl radical

OLAE Comvita (Olive Leaf Australia) extract

OLE Olive leaf extract

OLEs Olive leaf extracts

OLPE Oleuropein equivalent

p para

p24 Viral protein

PB Phosphate buffer

PBS Phosphate buffered saline

pg Picograms

pNPP para-Nitrophenyl Phosphate

PPO Polyphenol oxidase

Psi pounds per square inch

PV Pyrocatechol violet

QE Quercetin equivalents

RBL-2H3 Basophilic leukemia cells

RNS Reactive nitrogen species

ROS Reactive oxygen species

Rpm Revolutions per minute

sec Seconds

SGLT Sodium-glucose linked transporter

SOLE Swisse extract

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SOR Superoxide radical

t/ha Tons/hectare

T3 Triiodothyronine

TA Total activity

TC Total cholesterol

TE Trolox equivalent

TEAC Trolox equivalent antioxidant capacity

TFC Total flavonoid content

TGR5 G-protein coupled receptor

TLC Thin layer chromatography

TNB 5-thio-2-nitrobenzoic acid

TNF-α Tumor necrosis factor alpha

TPC Total phenol content

TPTZ 2,4,6-tripyridyl-s-triazine

Trig Triglycerides

Trolox (±)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid

µg Microgram

µL Microliter

µMol Micromole

US$ United States dollars

UV Ultraviolet

UVD Ultraviolet detector

V Volts

v/v Volume per unit volume

VHSV Viral haemorrhagic septicaemia virus

VLDL Very low density lipoprotein

XXIII

w/v Weight per unit volume

w/w Weight per unit weight

α Alpha

β Beta

μm Micrometre

Є Molar extinction coefficient

XXIV

List of publications

Kamran, M., Hamlin, A. S., Scott, C. J., and Obied, H. K. (2015). Drying at high

temperature for a short time maximizes the recovery of olive leaf biophenols.

Industrial Crops and Products, 78, 29-38.

XXV

Abstract

The use of nutraceuticals is increasing at rapid rate around the globe as, they are

convenient and cheaper than expensive medical and surgical procedures. Moreover,

nutraceuticals are surrounded by a halo of propaganda claiming a safer profile only

due to their preparation from natural products. The way forward for giving these

phytomolecules a legal ground to be applied in disease prevention and treatment

will be through proper scientific research. Thus, there is a great need for

comprehensive investigation to evaluate the efficacy of various nutraceuticals and

to optimize their recovery from natural sources.

Olive leaves were selected for this study as olive (Olea europaea L.) leaves are a

significant source of biophenols all over the year. Though in the past olive leaves

were mainly used for animal feeding, during the last two decades their health

promoting properties have been recognized and many studies examined their

potential applications. The superior health benefits of olive leaves are attributed to

biophenols. These health promoting effects are further augmented because of high

bioavailability of these biophenols. The biological activities of olive leaf biophenols

have created a high demand for extensive research to develop the most effective

method for the extraction and separation of olive biophenols, to identify these

biophenols, and to determine their suitability as a nutraceutical or functional food.

The first study investigated the best method for drying olive leaves so as to optimize

the recovery of olive leaf biophenols especially oleuropein. The recovery of

biophenolic compounds was compared between fresh, air-dried, freeze-dried and

oven-dried (at 60 °C and 105 °C) olive leaves. The biophenol content and

antioxidant activity were assessed by gross quantitative methods such as total

XXVI

phenol content (Folin-Ciocalteu’s method), total flavonoid content, total o-diphenol

content and total antioxidant capacity using ABTS+◦ and DPPH◦ scavenging assays.

In addition, the biophenolic composition of extracts was determined by high

performance liquid chromatography (HPLC) equipped with diode array detection

(DAD) with tandem mass spectrometery (MS/MS). The contribution of individual

biophenols to the antioxidant activity of extracts was evaluted by an online ABTS

scavenging assay. Extracts obtained from leaves oven-dried at 105 °C showed the

highest phenol recoveries and antioxidant activities, whereas extracts obtained from

oven-dried leaves at 60 °C had the lowest values. Oven drying of olive leaves at

105 °C for three hours increased oleuropein recovery up to 38 fold as compared

with fresh olive leaves. Results of the study stress the paramount importance of

sample pre-treatment in the preparation and analysis of herbal medicines.

Futhermore, the limitations of sole dependence on gross assessment of total

biophenolic composition and total antioxidant activity in studying plant samples

were highlighted.

Second study was a unique study that covered the complete olive tree cycle. This

study was conducted to investigate the effect of climate, season and cultivar on the

composition of olive leaf biophenols and antioxidant activity in order to determine

the best harvest time for optimal recovery of biophenols from a particular cultivar.

The biophenol content of an Australian cultivar was compared with cultivars of

Italian origin, grown in Wagga Wagga. The correlation between biophenol content

and the antioxidant activity was also determined. A new HPLC method developed

for this study, resulted in rapid and clear separation of olive leaf biophenols without

minimal co-elution. Olive leaf biophenols vary quantitatively during different

stages of the biological cycle of the olive tree, however no qualitative differences

were detected. Significant effects of cultivar/ variety as well as month of collection

XXVII

and season were observed for biophenol composition and antioxidant activity of

olive leaves. Total flavonoids showed moderately positive correlation with

oleuropein and antioxidant activity measured by ABTS and DPPH radical

scavenging assays. Oleuropein showed a moderately positive correlation with

ABTS activity, but a weak correlation with DPPH activity and total phenols. Total

phenol content displayed a weak correlation with ABTS, but no correlation with

DPPH activity. The time of collection is the most influential parameter with regards

to olive leaves suitability for commercialization. None of the cultivars followed a

specific pattern for accumulation of biophenols throughout the collection period.

The summer season, was the best for the optimal antioxidant activity and for

maximum recovery of biophenols, including oleuropein. For the better recoveries

of total phenols, phenolic compunds, o-diphenols, hydrocinnamic acid derivatives

and higher antioxidant activity the Leccino cultivar was most appropriate, whereas

in order to get higher oleuropein and total flavonoid content, the Hardy’s Mammoth

was the best cultivar.

In the third study, in vitro antioxidant and antidiabetic activities of commercial olive

leaf extracts were compared with laboratory prepared extract of Hardy’s Mammoth

olive leaves. An array of antioxidant activity assays involving different mechanism

such as ABTS and superoxide radical scavenging, hydrogen peroxide scavenging,

copper chelation and ferric reducing antioxidant power assay were investigated.

Crude and enriched extracts were prepared from leaves of the Hardy’s Mammoth

cultivar and these extracts were compared with commercially available olive leaf

extracts (OLEs). The oleuropein content of all extracts was determined by HPLC.

Total phenols were also measured, as gallic acid and oleuropein equivalents, to find

the contribution of total phenols towards antioxidant activity. Antidiabetic activity

of OLEs, via α-amylase enzyme inhibition activity, was also investigated. OLEs

XXVIII

demonstrated high potency as a source of antioxidant in all tested assays. They also

confirmed their antidiabetic activity through inhibition of α-amylase in in vitro

assay. Olive leaf biophenols as individual compounds and in a combined form of

extract were excellent antioxidants. These results indicate that olive leaf biophenols

are multi efficient bioactive molecules and have a great potential as nutraceuticals.

Since olive leaf biophenols strongly inhibited the α-amylase enzyme, they could be

beneficial in lowering blood glucose level and might prove to be an effective

antidiabetic agent.

In the fourth study, the potential of olive leaf extract as an antidiabetic agent was

examined in a pilot clinical trial on pre-diabetic subjects. One in three people with

prediabetes will develop type 2 diabetes but it is possible that prediabetics can

return to normal with early pharmacological and intensive diet interventions.

Therefore this study was planned to find out the suitability of OLE as a blood

glucose lowering agent. Eighteen prediabetic adults were recruited, and randomly

allocated to take either OLE or placebo capsules three times per day for 12 weeks.

Reference blood and urine samples were taken at baseline and after 12 weeks to

measure the levels of blood glucose, glycated haemoglobin (HbA1c) and lipid

profile. Total phenols as well as the oxidative biomarkers STAT-8-isoprostane and

8-hydroxy-2'-deoxyguanosine were also measured. For all the studied parameters

no significant differences were observed between the groups (p ˃ 0.05) and neither

supplementation with the olive leaf extract or placebo produced any significant

change compared to their baseline samples (p ˃ 0.05). Some individuals appeared

to respond to the supplement but as a whole group there was no significant response,

which indicated a highly individual response to the supplement. Though it was a

pilot study and the number of participants too small enough to produce any

significant effect but still it gives some valuable information about the baseline

XXIX

levels of blood parameters and effect of olive leaf extract on Australian

prediabeteics.

Olive leaf biophenols have a great potential as a nutraceutical. They demonstrated

good antioxidant and antidiabetic activity in in vitro assays. However, in the pilot

clinical study on prediabetic subjects, supplementation with olive leaf extract did

not produce any significant change. It is suggested that the potential antidiabetic

effect of olive leaf biophenols should be thoroughly investigated in controlled

clinical trials with a larger sample size for the efficient utilization of these valuable

nutraceuticals.

1

Introduction

“Prevention is better than cure”, according to an old proverb. Since ancient times,

it has been known that the purpose of food is much more than to satisfy the

requirements of energy and other essential nutrients for maintenance and

production. If we look at the history of humanity, it has been well documented that

people were aware of the importance of food for the maintenance of health and

disease prevention, as indicated by a quote of Hippocrates (460-370 BC) "Let food

be thy medicine and medicine be thy food". With the advancement in science, many

healthy compounds, at present known as bioactive compounds, have been isolated

form plants and food products, and then used for the prevention and/or treatment of

diseases. Although consumption of these natural compounds has generally involved

no risk and had long lasting effects, they often had a slow action and hence they

have frequently been replaced by synthetic chemicals. Due to advancements in

modern medicine, the disease prevention function of food was largely diminished,

but in the last two decades, focus is again shifting towards natural products. This is

apparent from an increased volume of research on bioactive plant compounds.

People are probably again looking for functional foods and nutraceuticals because

of the adverse effects observed with synthetic medicines. In some instances the

adverse effects of synthetic medicines may be greater than their benefit.

At present, a major focus of nutrition and human health research is an exploration

of the health promoting properties of bioactive compounds from food and non-food

plants, as can be seen from a plethora of research in this area in recent years. Health

promoting foods and extracted bioactive compounds from them are commonly

referred as functional foods and nutraceuticals. Functional foods are those which

possess natural bioactive compounds, while, nutraceuticals are those with added

2

bioactives after extraction from a natural source. Generally functional foods are

those foods that provide supplementary physiological benefit to the consumer, in

addition to simple nutrition, whereas a nutraceutical is a product extracted, isolated

or purified from foods, commonly sold in medicinal forms and usually not

associated with foods (Jones, 2002). A nutraceutical is a part of food that offers

health or medicinal function. The health function may range from prevention to

treatment. It can be explained with the concept that when a functional food offers

prevention, treatment of disease(s) and/or disorder(s) except anaemia, it is called a

nutraceutical (DeFelice, 2002). Being an isolated or purified ingredient a

nutraceutical can have a quicker action than conventionally healthy foods.

Currently nutraceuticals are a point of interest for researchers, industry and the

community. The nutraceutical industry is growing rapidly as demand is increasing,

mainly due to consumers’ awareness and desire for healthy life based on nutrition

rather than treatment with synthetic drugs. Europe has evolved into a nutraceutical

ingredient hub. According to a study in 2015, the global nutraceuticals market was

worth US$182.60 billion and it has been estimated that in 2021 it will have

increased to US$278.96 billion, at the annual growth rate of 7.3%. In that study

global nutraceuticals market is divided into four regions; Europe, North America,

Asia Pacific, and rest of the World. North America was the largest and Asia Pacific

the second largest regional market for nutraceuticals in 2014 (Transparency Market

Research, 2015).

Olive leaves are a highly prized commodity due to its superior value in human

nutrition and their potential in human nutrition should be exploited. Olive leaves

are a valuable nutraceutical resource due to their biophenols, which endow an array

of health promoting properties. A large body of evidence suggests the role of olive

3

leaf biophenols in health promotion (discussed in Chapter 1). The Health promoting

and/or disease prevention and/or therapeutic properties of olive leaf biophenols are

indicative of their potential as a high quality nutraceutical. They can be utilized in

the form of extract or they can be added in daily food via the fortification of food

products with olive leaf biophenols. Increasing demand and interest in olive

biophenols has raised the need for investigation in this area. Moreover, research in

this field would provide more updated information and offer opportunities for

industrial applications of olive leaf biophenols. This research project was designed

to evaluate the best conditions for Australian olive leaves to provide a nutraceutical

source of bioactives such as olive biophenols and to investigate their antidiabetic

effects.

4

Chapter 1. Literature Review

Olive leaf biophenols, chemistry and biological activity

1.1. Introduction

The human-plant relationship and interaction is as old as the creation of human

beings. Plants not only provide food macromolecules such as carbohydrates,

proteins and lipids, they also provide a variety of other compounds which play

diverse roles in health promotion. The function of plants have been beautifully

explained by Janzen (1978) “The plant world is not coloured green; it is coloured

morphine, caffeine, tannin, phenol, terpene, canavanine, latex, phytohaem-

agglutinin, oxalic acid, saponin, L-dopa”. Plants provide us with numerous

bioactive compounds that maintain health or prevent diseases. Among those

bioactive compounds, biophenols have received an enormous amount of interest

due to their multipurpose actions. Their nutraceutical effects have been extensively

investigated over the last three decades and a large number of peer-reviewed papers

have been published on their biological activities. Experimental and clinical

evidence suggests that biophenols may play a role in maintaining health or

preventing disease.

Biophenols are naturally occurring phenolic compounds present in plants. They

mainly act as a (plant) defense mechanism against pathogens (bacteria, fungi,

viruses), competing plants, insect attack, and herbivores due to their bitter taste.

They also act as phytoalexins (Wink, 2003), as well as a protective shield against

UV light (Naczk and Shahidi, 2006). Some are straight phytoalexins, i.e.

hydroxytyrosol, others are phytoanticipins, i.e. secoiridoids. Thus of the olive

biophenol secoiridoids, oleuropein is the most relevant one (Gentile and Uccella,

2014). Furthermore, they are involved in reproduction as signal compounds to

5

attract pollinating animals (Wink, 2003) and are important for communication with

other organisms (Schäfer and Wink, 2009). Biophenols are plant secondary

metabolites, produced during the course of secondary metabolism. They have no

role in the basic physiological processes of plants but nutritionally they may be very

important as they possess a wide range of pharmacological activities including

antimicrobial, antiviral, antioxidant, anti-Alzheimer’s disease, cardioprotective,

gastroprotective, hepatoprotective, anticancer and anti-inflammatory (Korkina,

2007). Some can be assigned to the olive biophenol moiety and others to the

secoiridoid one.

The olive tree (Olea europaea L.) has been cultivated since ancient times for oil

and table olives and is considered as a sign of peace, friendship and happiness. The

olive tree has been regarded as a blessed tree in the holy books including the Old

Testament and the Quran. Olive leaves and leaf extracts have been used for

centuries, as complementary medicine for treatment of disease conditions including

malaria fever, alopecia, colic, hypertension, rheumatic pain, sciatica, paralysis,

arrhythmia and cancer (Benavente-Garcıa et al., 2000; Visioli and Galli, 2002;

Pereira et al., 2007; E. Waterman and Lockwood, 2007; Obied et al., 2012).

More than 100 biophenols have been reported in olive products (fruits, oil, leaves

and processing by-products). The concentrations of these biophenols vary in

different products and specific biophenols are not necessarily present in all olive

products. Olive leaves are a richer source of biophenols than olive oil, and can

contain up to 2% of total phenols on a fresh weight basis (Obied et al., 2012).

To make the maximum use of olive biophenols for health and wellbeing it is

imperative to understand their chemistry and pharmacological properties. In this

chapter, the literature on chemical composition and pharmacological activities of

6

olive leaf biophenols is reviewed. The state-of-the-art research is portrayed and the

challenges and gaps of knowledge are highlighted so as to allow more rational use

of olive biophenols as nutraceutical products.

1.2. The Olive Tree

Evergreen trees belonging to the family Oleaceae, olive trees can grow up to 9 m

tall. They have a lifespan of up to 2,000 years and can produce fruit for over a

thousand years with minimum care (Olive Australia, 2014). There are about 2500

known varieties of olives, but only 250 cultivars have been classified as commercial

cultivars by the International Olive Oil Council. Olives are grown between the

latitudes of 30° and 45° from the equator. Although it is native to Mediterranean

region, southern European countries, Iran and beyond the Caucasus, it can also be

cultivated in similar climate zones. In Australia the main olive producing areas are

between latitudes 31°S and 38°S. Australia’s climatic conditions favour the growth

and processing of olives (Taylor and Burt, 2007). Olive cultivation is better in

neutral to alkaline soils but it can tolerate a wide range of soil conditions. Due to its

excellent drought resistant properties, olive groves can be brought back to

production even after years of neglect (Olive Australia, 2014).

Olives can be grown in cold as well as in warmer climates but for most varieties

some winter frost is preferred. For most of the olive growing regions of the world,

the average maximum temperature in the hottest month is about 30°C; however,

very high temperatures of up to 45°C have very little effect on mature olives. Olive

trees can tolerate temperatures of minus 10°C without any damage, provided there

is sufficient moisture in the soil. Some varieties in good health can survive

temperatures as low as minus 15°C but minus 20°C can be fatal to olive trees. The

optimum temperatures for olive growth are from 15°C to 34°C, with an average

7

monthly minimum winter temperature of less than 10°C required for good fruit

production, although this chilling requirement varies between varieties (Taylor and

Burt, 2007). The use of any particular olive cultivar is determined by its fruit size

and oil content. Those cultivars which have a fruit weight of greater than 4 g and

an oil content of less than 12% are used for table olives, whereas cultivars having

more than 12% oil are used for oil production. In Australia, olive production has

increased significantly in the last two decades and 90% of olives are grown for oil

and 10% for table olives (Taylor and Burt, 2007).

Olive trees have an alternate bearing cycle, also termed as biennial bearing or

periodicity, which means they do not bear a regular and similar fruit year after year.

The tree produces a high-yield crop one year ("on-year"), followed by no fruit or

low-yield the following year ("off-year"). The difference between on and off-year

yields varies between 5 to 30 t/ha (Yanik et al., 2013). The alternate bearing is

beneficial to the tree as it enables it to reserve nutrients not only for vegetative

growth but also for protection against biotic and abiotic stresses that may result in

macronutrient or micronutrient deficiencies (Turktas et al., 2013).

1.3. Olive and Health: The Role of Biophenols

Consumption of olive fruits, oil and leaves has never been restricted to dietary and

culinary applications. The blessings of the holy olive tree have been always

proclaimed as beautifying and healing. Olive oil and leaf extract have been

continuously incorporated in cosmetic and traditional medicine recipes since

antiquity. The active ingredients responsible for the observed biological activities

have been identified as phenolic in nature (Obied, 2013). Olive phenolic

compounds, olive biophenols, are very well prized for their antioxidant properties

(Uccella, 2000). With the introduction of oxidative theory of aging and the role of

8

oxidative stress in disease pathology, biophenols are discovered anew for potential

disease prevention and health promotion (Saija and Uccella, 2000; Obied et al.,

2012).

1.4. The Chemistry of Olive Biophenols

“Polyphenols” is the term used most commonly in literature to describe plant

phenolic compounds. In olive related literature, the term “biophenol” has been

proposed as a more chemically accurate alternative (Uccella, 2000). A chemical

compound having an aromatic ring substituted with one or more hydroxyl groups

is termed a phenol. Recently, the term “biophenols” has been redefined to offer a

more precise and comprehensive description of plant phenols so as to replace the

term “polyphenols” (Obied, et al., 2005a) . Biophenols can be defined as “phenolic

compounds, their derivatives, conjugates, degradation products and metabolites

isolated from plant tissues or products that are derived from shikimate-

phenylpropanoid and/or polyketide pathway(s)” (Obied, 2013). Biophenols

possess at least one aromatic ring with one or more hydroxyl groups. They vary

from simple monohydroxy aromatic, e.g. tyrosol to very complex polymeric

compounds such as tannins. Phenolic compounds are chemically reactive due to the

direct attachment of the hydroxyl group(s) to the electron-rich aromatic benzene

ring. In general, the increase in the number of hydroxyl groups increases phenols’

reactivity, acidity and reducing power (Hemingway and Karchesy, 2012).

Olive biophenols are characterised by multifunctional moieties. They possess a

phenolic structure with alkene, alcoholic or carboxylic functional groups in simple

biophenols; however, when glycoside and monoterpene moieties are attached they

represent more complex phytomolecules such as olive biophenol-secoiridoids

(Uccella, 2000). Olive biophenols can also form supramolecular molecules via

9

complexation with proteins and carbohydrates which affect the function and

reactivity of biophenols (Bianco and Uccella, 2000). Biophenols are present in all

plant parts (root, stem, twigs, leaf, bark, wood, fruit, seed and flower). However,

the nature and the quantity of biophenols present varies significantly.

1.4.1. Classification of Olive Biophenols

There is no standard classification of olive biophenols; however, they can be

divided into several classes/groups. The main groups of biophenols are simple

phenols, phenolic acids, flavonoids, isochromans (not reported in olive leaves),

lignans (also not reported in olive leaves) and secoiridoids (Obied et al., 2012).

1.4.1.1. Simple phenols (C6-C0-3)

Simple substituted phenols have a phenol ring without side chain or with a short

chain of up to three carbon atoms. They can have more than one side chain to form

different combinations and may be conjugated as glycosides or acetates. Examples

of these biophenols are phenyl alcohols (hydroxytyrosol & tyrosol, see Table 1.1)

and catechol, pyrogallol.

1.4.1.2. Phenolic acids (C6-C1-3)

The phenolic acids in olives are mostly hydroxycinnamic acids (e.g. caffeic (Table

1.1), o-coumaric, p-coumaric, ferulic, sinapic acids), hydroxybenzoic acids (gallic,

p-hydroxybenzoic, syringic, vanillic and protocatechuic acids) and phenylacetic

acids (4-hydroxypheylacetic acid and 3,4-dihydroxyphenylacetic acid).

10

1.4.1.3. Flavonoids (C6-C3-C6)

Flavonoids are C15 structures having a flavones skeleton with three rings, of which

two (rings) are phenolic. Variation in the third carbon central unit gives rise to a

variety of different compounds which are either glycosylated, hydroxylated or

methoxylated derivatives. They are mostly found as glycosidic compounds.

Flavonoids can be further divided into many groups. The three most commonly

reported in olives are:

Flavones: the basic flavonoid structures, having no substituents at the C3

central unit. Examples are apigenin, luteolin and their derivatives (Table

1.1).

Flavonols: they have a hydroxyl group at the C3 central unit. Examples are

kaempferol, quercetin and their glycosides (Table 1.1).

Anthocyanidins: They have a flavylium cation and commonly possess

hydroxyl substituents at positions 3 and position 5. They are mostly found

as sugar derivatives (anthocyanins). The B-ring has one or more hydroxy or

methoxy substituents (Quina et al., 2009). Examples are cyanidin (Table

1.1) and delphinidin.

1.4.1.4. Secoiridoids

They constitute a very specific and complex group that are produced as precursors

of various indole alkaloids from the secondary metabolism of terpenes. They are

usually derived from oleoside-11-methyl ester. Oleosides are not biophenols but

they can bond (link) to simple biophenols such as tyrosol or hydroxytyrosol. These

compounds proceed from the acetate/mevalonate pathway. Oleoside-11-methyl

ester combines to biophenols to give olive biophenol secoiridoids (Alagna et al.,

2016). They are abundant in the Oleacea family, with concentrations being

11

significantly higher in olives compared to other plants. Examples are oleuropein,

ligstroside, demethyloleuropein (Table 1.1) and nüzhenide.

1.4.1.5. Lignans

Lignans are formed by a combination of two C6C3 units joined together by a bond

between positions 8 and 8′. Examples are acetoxypinoresinol and pinoresinol,

which have been reported in olive fruit and oil (Ghanbari et al., 2012; Obied et al.,

2012) and hydroxypinoresinol which has been reported in the olive stone (Obied et

al., 2012).

1.4.1.6. Isochromans

They are biomolecules derived from technological reactions in virgin oil, i.e. the

mixing during milling and malaxation. The reaction between aromatic aldehydes

and hydroxytyrosol results in the formation of isochroman derivatives. It has been

suggested that this also can occur in the natural matrix, with oleic acid acting as a

catalyst (Bianco et al., 2002).

12

1.5. Olive Leaves

The olive leaf is thick, coriaceous lanceolate to obovate in shape, having entire

margins and acute tips. The upper surface is greyish-green, shiny and smooth while

the lower surface is paler and has fine white, scale-like hairs. Its length is about 3-

7.5 cm and width is about 1-1.5 cm. The olive leaf is a remarkable source of

bioactive phenolic compounds (olive biophenols), containing a minimum of 5%

oleuropein on a dry matter (DM) basis (Chinou, 2012).

1.5.1. Nutritive Value

The nutritional properties of olive leaves as a food for humans has not been explored

yet; however, a limited number of studies have been performed on small ruminants

to investigate the nutritive value of olive leaves as an animal feed (Molina-Alcaide

and Nefzaoui, 1996; Delgado-Pertıñez et al., 2000; Molina-Alcaide et al., 2003;

Martín-García and Molina-Alcaide, 2008; Molina-Alcaide and Yáñez-Ruiz, 2008).

In humans, only medicinal or pharmacological effects of olive leaves have been

investigated. In order to optimise the use of olive leaves, their nutritive value for

humans should also be evaluated. The nutritive value of olive leaves varies and

depends upon a number of factors including origin, leaf age, climatic conditions,

moisture content, degree of contamination with soil and oils, and storage conditions

(Martín-García and Molina-Alcaide (2008)

The phenolic content as tannins of olive leaves is 8.30 g/kg DM, while the essential

amino acids value is 543 g amino acid-N/kg total N (Martın-Garcıa et al., 2003).

The leaves have a gross energy content of 16.8 MJ/kg DM. Olive leaves are rich in

arginine, leucine, proline, glycine, valine and alanine but low in tyrosine, cysteine,

methionine and lysine (Martın-Garcıa et al., 2003; Martín-García and Molina-

13

Alcaide, 2008; Molina-Alcaide and Yáñez-Ruiz, 2008). The oleuropein content of

fresh leaves is 69.9 g/kg, but this decreases with storage (Delgado-Pertíñez et al.,

1998). Fernández-Escobar et al. (1999) studied seasonal changes of mineral

concentrations in olive leaves during the alternate-bearing cycle and developed

seasonal mineral fluctuations curves for the different leaf minerals. They found that

leaf age affected nutrient content per leaf, observing higher concentrations of

nitrogen, phosphorus, potassium, zinc and boron in younger leaves as compared to

older leaves while in older leaves concentrations of calcium, magnesium,

manganese, copper and iron were higher. In addition, the method of drying impacts

on the nutritive value of olive leaves. Martín-García and Molina-Alcaide (2008)

found that all drying procedures, with the exception of air drying in the shade,

affected all the nutritive parameters of olive leaves.

1.5.2. Bioactive Phytomolecules of Olive Leaf

Various groups of secondary (plant) metabolites, phytomolecules, have been

reported in olive leaves:

Iridoid monoterpenes: including oleuropein, oleuroside, ligstroside,

methyloleuropein, 11-demethyloleuropein, and 7,11-dimethyl ester oleoside

(Gariboldi et al., 1986; Bouaziz and Sayadi, 2005; DerMarderosian and Beutler,

2005) (Table 1.1).

Triterpenes: including oleanolic acid, maslinic acid, and β-amyrin (Dean and

Janićijević-Hudomal, 2007; Chinou, 2012).

Flavonoids: including kaempferol, chrysoeriol, luteolin and apigenin derivatives

(Benavente-Garcıa et al., 2000; Bouaziz and Sayadi, 2005; Dean and Janićijević-

Hudomal, 2007; Dekanski et al., 2009) (Table 1.1).

14

Chalcones: including olivine and olivine-4′-O-diglucoside (Dean and Janićijević-

Hudomal, 2007).

Phenolic acids: including cumaric acid, caffeic acid, ferulic acid, and vanillic acid

(Benavente-Garcıa et al., 2000; Dean and Janićijević-Hudomal, 2007).

Coumarins: including aesculetin, scopoletin, and aesculins (Chinou, 2012).

Alkaloids: Cinchonine and cinchonidine derivatives have been reported earlier

(Ross, 2005).

1.6. Olive Leaf Biophenols

Olive leaves normally possess more complex biophenols such as flavonoids and

secoiridoids glycosides, while the amount of simple phenols is very small. Table

1.1. shows the biophenols commonly reported in olive leaves: oleuropein, rutin,

verbascoside, apigenin-7-glucoside and luteolin-7-glucoside (Benavente-Garcıa et

al., 2000; Japón-Luján and Luque de Castro, 2006), luteolin 4-O-glucoside (Pereira

et al., 2007), luteolin 7, 4-O-diglucoside, apigenin 7-O-rutinoside (Meirinhos et al.,

2005), luteolin 7-O-rutinoside (Bouaziz and Sayadi, 2005), hydroxytyrosol, tyrosol,

caffeic acid, p-coumaric acid, vanillic acid, vanillin, luteolin, (Benavente-Garcıa et

al., 2000; Bianco and Uccella, 2000; Tasioula-Margari and Okogeri, 2001). Olive

leaf extract also contains catechin (Benavente-Garcıa et al., 2000), quercetin, and

chryseriol (Dekanski et al., 2009). The main bioactive compounds of olive leaves

are oleuropein and its hydrolysis products like oleuropein aglycone, Oleoside-11-

methyl ester, hydroxytyrosol and methyl-O-methyl elenolate (Durlu-Özkaya and

Özkaya, 2011). Oleuropein, hydroxytyrosol, tyrosol, rutin, luteolin, catechin and

apigenin, are believed to be responsible for the pharmacological activities of olive

leaf extract (Bowden, 2009).

15

Olive leaves are very rich in biophenols (see Table 1.1); however, the composition

and amount of phenolic compounds in olive leaves varies to some extent between

studies (sources of variation discussed under section 1.6.3.). Pereira et al. (2007)

found oleuropein to be the major compound (about 73%) of the total identified

compounds and caffeic acid as minor compound (about 1% of total phenols with

water extract) whereas Meirinhos et al. (2005) reported flavonoids as the major

compounds (luteolin 4-O-glucoside as the most abundant) with water-methanol

extract.

1.6.1. Oleuropein

Oleuropein, a secoiridoid (monoterpene), is an ester of hydroxytyrosol with the

oleoside skeleton (Table 1.1). It is the chief biophenol in olive leaves (Le Tutour

and Guedon, 1992; Benavente-Garcıa et al., 2000; Soler-Rivas et al., 2000; Bouaziz

and Sayadi, 2005; Japón-Luján and Luque de Castro, 2006; Ranalli et al., 2006;

Pereira et al., 2007; Altıok et al., 2008; Malik and Bradford, 2008; Lee et al., 2009;

Ansari et al., 2011; Durlu-Özkaya and Özkaya, 2011; Hayes et al., 2011; Tayoub et

al., 2012). The pharmacological properties of oleuropein have been extensively

studied and it is considered responsible for most of the health promoting properties

of olive leaves. Initially, oleuropein was detected in olive fruit by Bourquelot and

Ventilesco in 1908 and its structure and the empirical formula C25H32O13 was

proposed in 1960 by Panizzi and co-workers, as a heterosidic ester of

hydroxytyrosol with β-glucosylated Oleoside-11-methyl ester (MacKellar et al.,

1973; Ranalli et al., 2006). Oleuropein gives rise to many compounds found in

olive fruit and leaves via aglycon through opening of the elenolic acid ring (Ansari

et al., 2011). Its quantity is almost negligible in olive oil as compared to olive

16

leaves, because of its extensive enzymatic degradation during oil extraction and low

partitioning in oil (Paiva-Martins and Pinto, 2008).

Oleuropein accounts for about 25% of the total biophenols of olive leaf (Benavente-

Garcıa et al., 2000). In fresh olive leaves, the concentration of oleuropein is 60–90

mg/g DM (Le Tutour and Guedon, 1992; Ryan et al., 2002b; Durlu-Özkaya and

Özkaya, 2011). The concentration of oleuropein is lower in ripe olives due to

hydrolysis and/or conversion to other compounds. During virgin oil extraction,

oleuropein is hydrolysed to a number of compounds including hydroxytyrosol,

elenolic acid, oleuropein aglycone, and glucose based on the hydrolysis conditions.

In addition to oleuropein, olive leaves also contain demethyloleuropein and

oleoside methyl ester (also known elenolic acid glucoside) (see Table 1.1). As the

plant matures, the concentrations of these two compounds increase and the

concentration of oleuropein decreases. This is possibly due to increased activity of

the hydrolytic enzymes during the maturation process, particularly esterases which

are responsible for the hydrolysis of the two ester bonds of oleuropein.

Demethyloleuropein has a free carboxylic group on the secoiridoid ring which

distinguishes it from oleuropein, whereas in the glucoside of the elenolic acid, the

carboxyl that esterifies the dihydroxy-phenyl-ethanol in the oleuropein has the free

functionality which differentiates it from oleuropein. Other compounds present in

olive leaves are ligstroside which has tyrosol residue rather than hydroxytyrosol

(Chinou, 2012); dimethylester of the oleoside in which two acidic functions of the

oleuropein esterified with the residue of methanol also known as glucoside of the

methylester of the elenolic acid (Gariboldi et al., 1986); and oleuropein isomer

known as oleuroside which is different from oleuropein in the exocyclic double

bond position (Kuwajima et al., 1988).

17

1.6.2. Olive leaf flavonoids

Olive leaf is a rich source of flavonoids (Table 1.1). Luteolin 4′-O-glucoside is the

major flavonoid present in olive leaf, ranging from 24–54% of total flavonoids

(Meirinhos et al., 2005). Other abundant flavonoids include apigenin, apigenin-7-

O-glucoside, apigenin-4′-O-rhamnosylglucoside, apigenin 7-O-rutinoside,

diosmetin, luteolin, luteolin 7,4-O-diglucoside, luteolin-7-O-glucoside,

chrysoeriol, chrysoeriol-7-O-glucoside, quercetin, quercitrin, quercetin-3-O-

rhamnoside, and rutin (Heimler et al., 1992; Pieroni et al., 1996; Meirinhos et al.,

2005). Flavonoids act as aldose reductase inhibitors thereby blocking the sorbitol

pathway that is involved in many problems associated with diabetes and they inhibit

cyclooxygenase and lipooxygenase enzymes which offer protection against cancer,

inflammation and cardiovascular diseases (Plazonić et al., 2009).

18

Table 1.1: Biophenols reported in olive leaves

Biophenol MW Structure UV spectrum (nm) Chromatography Reference

Apigenin 270

267,337 HPLC-DAD

LC-MS

Bouaziz and Sayadi (2005)

Apigenin-glucoside 266, 337 HPLC-UVD Rovellini, Azzolini, et al. (1997)

Apigenin-7-O-glucoside 432

266, 337 HPLC-DAD

LC-MS

Le Tutour and Guedon (1992); Bouaziz and

Sayadi (2005); Pereira et al. (2007); Altıok et

al. (2008); Dekanski et al. (2009)

Apigenin-7-glycosides HPLC Baldi et al. (1995)

Apigenin-7- rutinoside 578

267, 339 HPLC-DAD Le Tutour and Guedon (1992)

19

Catechin 288

278 HPLC-DAD Altıok et al. (2008)

Caffeic acid 180

239, 299S, 324 HPLC-DAD Baldi et al. (1995); Pereira et al. (2007); Altıok

et al. (2008); Dekanski et al. (2009)

5-Caffeoylquinic acid 354

324 TLC-densitometer Heimler et al. (1992)

Chlorogenic acid 354

236, 301S, 327 TLC-densitometer Heimler et al. (1992)

20

Chryseriol 300

350 HPLC-DAD Dekanski et al. (2009)

p-Coumaric acid 164

307 HPLC Baldi et al. (1995)

Cyanidin-3-glycosides 449

516 HPLC Baldi et al. (1995)

Demethyloleuropein 526

278 HPLC-DAD Baldi et al. (1995) Le Tutour and Guedon

(1992)

21

Elenolic acid 242

240 HPLC-DAD

LC-MS

Baldi et al. (1995); Agalias et al. (2005)

Hesperidin 610

287, 327 HPLC Baldi et al. (1995)

Hydroxytyrosol 154

280,349 HPLC-DAD

LC-MS

Gariboldi et al. (1986); Baldi et al. (1995);

Agalias et al. (2005); Altıok et al. (2008);

Papoti and Tsimidou (2009)

Ligstroside 524

240, 278 HPLC-DAD

CC

TLC

Gariboldi et al. (1986); Kuwajima et al. (1988);

Le Tutour and Guedon (1992); Limiroli et al.

(1996)

22

Luteolin 286

267, 352 HPLC-DAD

LC-MS

TLC-densitometer

Heimler et al. (1992); Rovellini, Cortesi, et al.

(1997); Bouaziz and Sayadi (2005); Altıok et

al. (2008); Dekanski et al. (2009); Papoti and

Tsimidou (2009)

Luteolin-glucoside HPLC-ES-MS

HPLC-UVD

Rovellini, Cortesi, et al. (1997)

Luteolin-4-O-glucoside 448

350 HPLC-DAD

LC-MS

CC

GC-MS

Le Tutour and Guedon (1992); Komaki et al.

(2003); Pereira et al. (2007); Papoti and

Tsimidou (2009)

Luteolin-7-O-glucoside 448

260, 353 HPLC-DAD

LC-MS

TLC-densitometer

CC

GC-MS

Heimler et al. (1992); Le Tutour and Guedon

(1992); Baldi et al. (1995); Komaki et al.

(2003); Bouaziz and Sayadi (2005); Pereira et

al. (2007); Altıok et al. (2008); Dekanski et al.

(2009); Papoti and Tsimidou (2009)

Luteolin-7-glycoside 350 TLC-densitometer (Heimler et al., 1992)

23

Luteolin-rutinoside 256,343 HPLC-ES-MS

HPLC-UVD

(Rovellini, Azzolini, et al., 1997)

Luteolin-7-O-rutinoside 594

256,343 HPLC-DAD

LC-MS

Le Tutour and Guedon (1992); Bouaziz and

Sayadi (2005)

Oleoside 390

220, 266 HPLC

CC

Gariboldi et al. (1986); Kuwajima et al. (1988)

Oleuropein 540

280 HPLC-DAD

LC-MS

CC

Gariboldi et al. (1986); Kuwajima et al. (1988);

Le Tutour and Guedon (1992); Baldi et al.

(1995); Agalias et al. (2005); Bouaziz and

Sayadi (2005); Pereira et al. (2007); Altıok et

al. (2008); Dekanski et al. (2009); Papoti and

Tsimidou (2009)

24

Oleuroside 540

280 HPLC-DAD

CC

Kuwajima et al. (1988); Le Tutour and Guedon

(1992)

Quercetin 302

255, 305S, 373 HPLC-DAD Dekanski et al. (2009)

Quercitin-3-rhamnoside 448

350 TLC-densitometer Heimler et al. (1992)

Quercitin-3-rutinoside (rutin) 610

233, 256, 300S, 356 HPLC-DAD

LC-MS

TLC-densitometer

Heimler et al. (1992); Le Tutour and Guedon

(1992); Bouaziz and Sayadi (2005); Pereira et

al. (2007); Altıok et al. (2008)

25

Tyrosol 138

275,349 HPLC-DAD

LC-MS

Agalias et al. (2005); Altıok et al. (2008)

Vanillic acid 168

260, 290 HPLC-DAD Altıok et al. (2008)

Vanillin 152

310 HPLC-DAD Altıok et al. (2008)

Verbascoside 624

300S, 334 HPLC-DAD

LC-MS

Gariboldi et al. (1986); Le Tutour and Guedon

(1992); Pereira et al. (2007); Altıok et al.

(2008); Papoti and Tsimidou (2009)

MW: molecular weight; HPLC-DAD: high performance liquid chromatography-diode array detection; LC-MS: liquid chromatography-mass

spectrometry UVD: ultraviolet detector; ES: electrospray; TLC: thin layer chromatography; CC: column chromatography; GC: gas chromatography.

26

1.6.3 Factors affecting the biophenol content of olive leaves

Like other natural products, the biophenol composition of leaves can vary due to

geographical location, plant nutrition, cultivar, physiology, environment, age or

stage of maturity, season, position on the tree, rootstock, agricultural practices and

pathological factors (Ryan and Robards, 1998; Obied et al., 2012), as well as the

extraction solvent and extraction method used. In addition, the biological cycle of

the olive tree, organ/tissue, variety and harvesting time have a significant

quantitative and qualitative impact on the phenolic composition of olive leaves.

Brahmi et al. (2013) found that total phenols, ortho-diphenols and total flavonoids

content were higher in mature leaves and unripe fruit than in growing leaves and

ripe fruit.

Sample preparation as well as sample preservation are very important factors for

accurate estimation of phenolic compounds. In many studies, samples have not been

analysed immediately after collection and this may result in the loss of some

valuable compounds (P. G. Waterman and Mole, 1994). According to Majors

(1999), approximately 30% of analytical error comes from sample preparation and

this step consumes about 60% of analysis time. Therefore, care must be taken in all

steps of extraction such as sample collection, sample preservation, sample

preparation, separation and detection of biophenols in order to achieve optimum

recovery of biophenols. For maximum recovery of biophenols from olive leaves

using the most appropriate extraction method and solvent is critical. The extract

should be uniform, having all components of interest and free from interfering

components.

27

The extraction solvent plays a fundamental role in the recovery process. Different

extraction solvents and extraction techniques were used for the recovery of

biophenols from olive leaves; in most studies extraction of fresh foliage or freeze-

dried material was done either with methanol or ethanol (Le Tutour and Guedon,

1992; Paiva-Martins and Gordon, 2001; Savournin et al., 2001; Briante et al., 2002;

Bouaziz and Sayadi, 2005; Malik and Bradford, 2008; Poudyal et al., 2010; Ahmad-

Qasem et al., 2013). Ethanol is the preferred solvent for food products; however,

80% methanol is considered the most effective solvent for olive leaf biophenols

(Malik and Bradford, 2008). In a few studies boiling water was also used for

extraction of biophenols (Pereira et al., 2007; Malik and Bradford, 2008). It is

believed that most of the phenolic compounds are mainly located in free form and

are easily extracted by alcoholic solvents (Hung and Duy, 2012). Phenolic acids are

mostly bound to cell wall through ester or glycosidic linkages (Ignat et al., 2011).

Thus, organic solvents will not be able to extract them; acid or base hydrolysis is

necessary to liberate these compounds. Lee et al., (2009) extracted olive leaves with

several solvents and evaluated phenolic content and composition of the extracts.

They obtained significant amounts of oleuropein and significantly higher phenolic

contents and total flavonoids in the olive leaves with 80% ethanol, butanol, and

ethyl acetate fractions as compared to hexane, chloroform and water fractions.

Solid–liquid extraction was the most commonly used technique but recently some

advanced techniques such as microwave-assisted extraction, supercritical fluid

extraction and pressurised liquid extraction have been used for the extraction of

biophenols from the olive leaves (Taamalli et al., 2012; Xynos et al., 2012). Brahmi,

et al. (2012a) used hydro-distillation for the extraction of volatile fractions from

fresh and dried olive leaves. Each method has resulted in different composition of

biophenols and even sometimes different compounds. When extracting biophenols

28

from olive leaves by solid-liquid extraction, many factors can contribute towards

the composition of biophenols in the extract including temperature at which

extraction is carried out, liquid–solid ratio, flow rate, time and particle size of the

sample material (Ignat et al., 2011).

Using different extraction solvents and different extraction methods can result in

extracts with different composition, quite variable yield of the biophenols and

subsequently different biological activities. There is no standard procedure

available for the extraction of biophenols that one can use, as phenolic compounds

are not uniformly distributed in plant tissues. They may be associated with

carbohydrates, proteins or structural components, moreover they may be heat stable

or volatile. Polarities of different phenolic compounds vary significantly, so

practically it is impossible to develop an ideal method for extraction of all phenolic

compounds with a single solvent system. Therefore, we should aim for an optimized

method of extraction that provide an optimum recovery that is suitable for the

purpose of the study.

1.7. Olive Leaves: Nutraceutical and Dietary Applications

Olive Leaves possess a myriad of claimed medicinal properties. They have been

consumed as fresh or dry powder, decoction, herbal tea, and liquid or dried extract

(Chinou, 2012) Olive leaves are also used as a food additive in the form of extract

or herbal tea to fortify functional food and to increase the antioxidant

content/functionality of food products (Ghanbari et al., 2012).

1.7.1. Olive leaf extract

Olive leaf extract (OLE) is usually a dark brown liquid prepared from the leaves

after drying and powdering. The use of OLE has increased in recent years, with

29

people consuming it for general wellbeing. It is marketed as a natural/herbal

supplement with wide ranging health benefits. It is rich in functional biophenols

that show some synergistic behaviour (Braun, 2005).

1.7.2. Fortification of olive oil /edible oils/ other food products with olive

leaves

Enrichment/fortification of edible oils with phenolic compounds derived from olive

leaves resulted in increased product overall quality, augmented its antioxidant

capacity, extended its shelf life and improved its sensory properties (Ranalli et al.,

2006; Paiva-Martins et al., 2007; Salta et al., 2007; Bouaziz et al., 2008; Japón-

Luján and Luque de Castro, 2008). Results from earlier studies are promising for

the use of olive leaves as an additive to enrich food products with biophenols. Food

or food products having low or no biophenol content can be enriched with

biophenols from olive leaves, which may result in improved stability and health

attributes.

1.7.3. Encapsulation of olive leaf extracts

Some people do not like to directly consume OLE due to the pungency of

biophenols, particularly oleuropein. Encapsulation increases the palatability and

stability of OLE. Encapsulation of OLE with chitosan microspheres or β-

cyclodextrin has been investigated. Interactions of biophenols with the chitosan

matrix were observed but the activity of phenolic compounds of OLE encapsulated

in the chitosan matrix was found to be retained even after spray-drying (Kosaraju

et al., 2006). When β-cyclodextrin is used, it forms a complex with OLE

components upon mixing in aqueous medium and enhances oxidative stability

30

during subsequent freeze-drying and under oxidative conditions (Mourtzinos et al.,

2007).

1.7.4. Antimicrobial food packaging

Antimicrobial packaging applications of OLE were reviewed by Erdohan and

Turhan (2011). Although usage of OLE as an antimicrobial substance for food

packaging is relatively new, great potential exists as this type of packaging results

in a more protected and safe food. Antimicrobial food packaging prevents the food

from spoilage by reducing the growth and number of food pathogens; it increases

the shelf life and maintains quality and safety of food products by extending the lag

phase. It has been demonstrated that methylcellulose, polylactic acid and

methylcellulose-polylactic acid films prepared with OLE as an antimicrobial food

packaging have a great potential to reduce post-process growth of Staphylococcus

aureus (Erdohan and Turhan, 2011).

1.8. Olive Leaves: Traditional and Folk Medicine

Olive leaves have been widely used in folk medicine in many different countries

(see Table 1.2). Conventionally, the boiling water extract of olive leaves which is

referred to as decoction has been used. Olive leaves first gained medical importance

in the 1800s, when Etienne Pallas, a French colonel reported the beneficial effects

of olive leaf tea. He isolated a crystallisable compound “vauqueline” and suggested

the fever lowering properties of olive leaf were due to this bitter substance (M.

Hansen and Verity, 2002). Spanish physicians prescribed olive leaves as a

“febrifuge” in the early 19th century and during the Spanish war of 1808-1813, the

French officer de Santé used them to treat cases of “intermittent fever”. In 1854,

Daniel Hanbury reported successful use of olive leaves tincture for fever in the

31

Pharmaceutical Journal. Afterwards, scientists isolated a bitter substance from the

leaf and named it oleuropein and suggested that it protects the olive tree from insects

and bacterial damage and gives health promoting properties to the olive (Chinou,

2012). Olive leaf and extracts are considered as natural pathogens killer and are

used in complementary and alternative medicine to treat flu, colds, yeast infections,

and viral infections, such as Epstein-Barr disease, shingles and herpes, which are

difficult to treat (DerMarderosian and Beutler, 2005; Chinou, 2012). Olive leaf

poultices have also been used topically for skin problems such as boils, rashes and

warts. (M. Hansen and Verity, 2002).

Table 1.2: Worldwide traditional and folk medicinal uses of olive leaves.

Country Form of olive leaf

used

Route Indications Reference

Arabic

countries

Dried plant

fumigation

Nasal Abortifacient,

and treatment of

cystitis and sore

throat

(Ross, 2005)

Argentina Decoction of the

dried leaves

Oral Treatment of

diarrhoea,

respiratory and

urinary tract

infections

(Perez and

Anesini, 1994)

Brazil Herbal tea of the

fresh leaves

Oral To induce

diuresis, and

treatment of

hypertension

(Ribeiro et al.,

1986)

32

Bulgaria Herbal tea of the

fresh or dried

leaves

Oral Treatment of

hypertension

(Petkov, 1979)

Canary

Islands

An infusion

prepared from the

fresh or dried

leaves

Oral

Oral

Rectal

Treatment of

diabetes;

hypertension

haemorrhoids

(Darias et al.,

1986; Darias et

al., 1996)

Cuba Herbal tea of the

fresh leaves

Oral

Treatment of

hypertension

(Chinou, 2012)

France powdered dried

leaves as hard

capsules

Oral To promote

urinary and

digestive

elimination

functions

(Martindale,

1996)

Germany extract with

ethanol 96% V/V

as liquid or coated

tablet

Oral Treatment of

atherosclerosis

and

hypertension

(Martindale,

1996)

Greece Hot water extract

of the leaf

Oral Treatment of

hypertension

(Ross, 2005)

Iran Decoction of the

dried leaves

Oral Treatment of

hypertension

and diabetes

(Motamedifar et

al., 2007)

Italy Infusion of the

dried leaf

Oral

Oral

Treatment of

hypertension

and an anti-

(Antonone et al.,

1988; De Feo and

Senatore, 1993;

33

Infusion of the

fresh leaf

Topical

inflammatory;

treatment for

hypertension;

for wound

healing,

emollient for

ingrown nails

and to restore

epithelium

Pieroni et al.,

1996)

Madeira Infusion of leaves Oral Treatment of

hypertension

(Rivera and Obon,

1995)

Mexico Decoction of dried

leaves

Oral Treatment of

diabetes

(Alarcon-

Aguilara et al.,

1998)

Morocco Leaves and

essential oil from

the leaves

Oral

Topical

Treatment of

stomach and

intestinal disease

and as a mouth

cleanser. Also

for treatment of

constipation and

liver pain

As a tonic for

hairs

(Bellakhdar et al.,

1991)

34

Oman Leaves and

poultice from

leaves

Topical Treatment of

skin rash and

ulcers

(Ghazanfar and

Al-Al-Sabahi,

1993)

Reunion

Island

Hot water extract

of the dried leaves

Oral Treatment of

diabetes,

diarrhoea,

rheumatism,

fever and

gastroenteritis in

infants

(Vera et al., 1990)

Serbia/

Yugoslavi

a

Hot water extract

of the dried leaf

Oral Treatment of

Diabetes

(Ross, 2005)

Spain Infusion of the

leaf, extracts of

the leaf and leaves

Oral

Treatment of

hypertension,

gastrointestinal

colic and

diabetes

(Martinez-Lirola

et al., 1996)

Trinidad Hot water extract

of the leaf

Oral To increase milk

supply of

nursing mother

(Simpson, 1962)

Tunisia Extract of the

dried leaf

Oral Treatment of

diabetes and

hypertension

(Boukef et al.,

1982)

35

Ukraine Hot water extract

of dried plant

Oral Treatment of

bronchial

asthma

(Vardanian, 1978)

1.9. Pharmacological Activities of Olive Leaves

1.9.1. Antimicrobial activity

Olive leaf extract is reported to be an effective antimicrobial agent against a variety

of pathogens, i.e. viruses, bacteria, fungi and protozoa. In addition, it can activate

the immune system response against all microbes and directly stimulate

phagocytosis (M. Hansen and Verity, 2002; Bowden, 2009), as well as interfere

with the critical amino acid production necessary for microbes (Balch, 2002; M.

Hansen and Verity, 2002; Bowden, 2009). Olive leaf extract reportedly counteracts

antibiotic resistant strains of bacteria and yeast, it is also useful against

microorganisms that produce chronic fatigue and immune dysfunction syndromes

(Balch, 2002).

1.9.1.1. Antibacterial and antifungal activity

There are contrasting reports of the broad spectrum antimicrobial activity of OLE.

OLE inhibited both gram positive (Bacillus cereus, Bacillus subtilis,

Staphylococcus aureus) and gram negative (Escherichia. coli, Pseudomonas

aeruginosa, Klebsiella pneumoniae) bacteria as well as fungi (Candida albicans

and Candida neoformans) (Pereira et al., 2007). However, Sudjana et al. (2009)

reported the highest activity of OLE was against Campylobacter jejuni,

Helicobacter pylori and Staphylococcus aureus including methicillin-resistant

strain of Staphylococcus aureus, with minimum inhibitory concentrations as low as

36

0.31–0.78% (v/v); least activity was observed against Bacillus subtilis, Candida

spp., Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and

Serratia marcescens, with minimum inhibitory concentrations more than 50%.

When individual biophenols were studied for antimicrobial activity, (Lee and Lee,

2010) found that oleuropein demonstrated strong growth inhibition against

Staphylococcus enteritidis but had no activity against Staphylococcus aureus.

Caffeic acid had moderate growth inhibition against Bacillus cereus, Escherichia

coli, and Staphylococcus enteritidis, but no activity against Staphylococcus aureus.

Rutin and vanillin showed no antimicrobial activity against Bacillus cereus,

Escherichia coli, Staphylococcus enteritidis, or Staphylococcus aureus. Elenolic

acid has also been proposed to possess antimicrobial properties; however, while it

can kill many kinds of viruses, protozoa and bacteria in vitro, after ingestion this

compound immediately binds to proteins in blood serum, making it ineffective in

vivo (Balch, 2002). (Lee and Lee, 2010) found that the combined phenolics mixture

of OLE had greater inhibitory effects against Bacillus cereus and Staphylococcus

enteritidis than the individual phenols, suggesting some synergistic effects between

OLE phytomolecules.

1.9.1.2. Antiviral activity

The exact mechanism of OLE’s antiviral activity is still not clear but several

mechanisms are thought to be involved in either blocking the entry of virus to the

cells by preventing their attachment and absorption or interference with certain

amino acids production, preventing virus budding, shedding or penetration into the

infected cells or preventing the replication process (Lee-Huang et al., 2003; Micol

et al., 2005; Bao et al., 2007; Lee-Huang et al., 2007; Motamedifar et al., 2007).

Olive leaf extract inhibits viruses at the early stages of replication, probably via

37

blocking of viral envelope fusion (Micol et al., 2005). It is effective against

retroviruses and has shown antiviral activities against HIV-1 virus in cell culture

and inhibited HIV-1 replication and cell-to-cell transmission of HIV in a dose

dependent manner (Lee-Huang et al., 2003). Olive leaf extract has been found to be

effective against herpes virus, clearing the lesions and blisters (Privitera et al., 2009)

in 36 to 48 h in three out of six participants in a clinical study, with the remaining

three participants responded better when given a higher dose (Fredrickson, 2000;

Balch, 2002). The time at which OLE is administered impacts on its effectiveness;

OLE showed no antiviral activities when applied to cell culture infected with

Herpes simplex virus-1 (HSV-1) 1 h before challenge whereas it showed anti HSV-

1 activities at concentrations >1 mg/mL when applied to cells 1 h after the virus

infection (Motamedifar et al., 2007). When OLE was added to cell monolayers 36

h post-infection with haemorrhagic septicaemia virus (VHSV), virus titres and viral

protein accumulation was decreased in a dose dependent manner (Micol et al.,

2005).

When individual biophenols were studied for antiviral activity, oleuropein showed

significant antiviral activity against respiratory syncytial virus and parainfluenza

type 3 virus (Ma et al., 2001) and in a U.S. patent it has been claimed to have potent

antiviral activities against herpes virus, hepatitis virus, rotavirus, bovine rhinovirus,

canine parvovirus, and feline leukaemia virus (Fredrickson, 2000). Oleuropein and

hydroxytyrosol are HIV-1 inhibitors, effective against viral fusion and integration.

They act both extra- and intra-cellularly. Both oleuropein and hydroxytyrosol bind

to the conserved hydrophobic pocket on the surface of the central trimeric coiled-

coil of the HIV-gp41 fusion complex domain. Both compounds are active against

multiple stages of the HIV-1 life cycle, inhibiting cell-to-cell HIV-1 transmission

and viral core antigen p24 production (Lee-Huang et al., 2007). Oleuropein has also

38

inhibited in vitro infectivity of the VHSV (Micol et al., 2005). It has been suggested

that the virucidal activity of olive leaves is more likely due to its ability to prevent

the entry of virus into the cells (Motamedifar et al., 2007) and interaction of OLE

and oleuropein with the viral envelope (Micol et al., 2005).

1.9.2. Immunoregulatory effects

There are many claims that OLE boosts the immune system (Walker, 1997; Balch,

2002; M. Hansen and Verity, 2002; Bowden, 2009). TNF-α production reversibly

stimulates the immune cells to produce more mediators, therefore the suppression

of TNF-α production may have a beneficial effect on the anti-allergic reaction

(Nishibe et al., 2001). Oleuropein has been shown to inhibit TNF-α production,

while luteolin 4-O-glucoside has not shown any inhibitory effects on the production

of tumor necrosis factor when rat basophilic leukaemia cells were studied, yet it

showed a strong inhibitory effect on β-hexosaminidase release (Nishibe et al.,

2001). Since both TNF-α production and β-hexosaminidase release are linked to

allergic reactions, there is potential that OLE can be used in the treatment of allergic

reactions and have beneficial effects on the immune system (Nishibe et al., 2001).

1.9.3. Cardiovascular protective activities

It has been established that consuming the “Mediterranean” diet which is rich in

biophenols is associated with a reduced incidence of cardiovascular diseases

(Visioli and Galli, 2002; Visioli et al., 2002; Singh et al., 2008). It has been

proposed that polyphenols impede with biochemical events that are associated in

atherosclerotic disease (Visioli et al., 1994). Biophenols may protect the

cardiovascular system from free radical damage by scavenging reactive oxygen

species (ROS). They are believed to inhibit the oxidation process of low-density

39

lipoprotein (LDL) cholesterol (Diaz et al., 1997) which is responsible for

atherosclerosis. In this way, olive leaf biophenols can play a vital role in decreasing

the risk of cardiovascular diseases. Other cardioprotective effects of OLE are due

to its effects on adrenaline (Somova et al., 2004).

Oleuropein has been shown to increase coronary flow and left intraventricular

pressure in rabbit myocardium (Bruneton, 1995). It has a direct cardioprotective

effect in the acute events that follow coronary occlusion probably because of its

antioxidant properties (Manna et al., 2004). Oleuropein is involved in inhibition of

5-lipooxygenase and 12-lipooxygenase and eicosanoid production (Visioli and

Galli, 2002) and it prevents the membrane lipid peroxidation and protects against

oxidative myocardial injury induced by ischemia and reperfusion. A significant

reduction of oxidised glutathione was observed in ischemic hearts pre-treated with

oleuropein, when the protective effect of oleuropein against the post-ischemic

oxidative burst was investigated by measuring the release of oxidised glutathione

which is a sensitive marker of heart's exposure to oxidative stress in the coronary

effluent (Manna et al., 2004). Hydroxytyrosol has also been found to be effective

in reducing cardiovascular diseases (Fki et al., 2007).

1.9.4. Antiplatelet activity

As a consequence of the antiplatelet effects of olive leaves, blood becomes less

viscous, and thus circulation may be improved, which may offer a degree of

protection from thrombosis and other cardiovascular diseases (Singh et al., 2008).

Oleuropein is involved in inhibition of platelet aggregation by producing

thromboxane A2 (Petroni et al., 1995). When the platelet aggregation and ATP

release function was investigated in healthy fasting, non-smoking, male subjects,

inhibition of platelet aggregation was noticed with olive leaf biophenols possibly

40

via their H2O2 scavenging properties as platelet aggregation is associated with a

burst of H2O2. It was also observed that the platelet inhibition with OLE was greater

than that of oleuropein alone due to the synergistic effect of the other biophenols

found in OLE; catechin can also inhibit platelet function and hydroxytyrosol,

caffeic acid, luteolin and rutin have been noted for their antioxidant activity and

H2O2 scavenging ability (Singh et al., 2008). Hydroxytyrosol has also been reported

to possess antithrombotic activities (Benavente-Garcıa et al., 2000).

1.9.5. Hypocholesterolaemic effects

Olive leaves are believed to possess cholesterol lowering properties. In a Dahl salt-

sensitive (DSS), insulin-resistant rat, OLE prevented the development of

atherosclerosis (Somova et al., 2003). It has been suggested that oleuropein,

oleuropein aglycone and hydroxytyrosol-rich extracts might be responsible for the

hypocholesterolaemic effect of olive leaves (Fki et al., 2007; Jemai et al., 2008). In

rats fed a cholesterol-rich diet for 16 weeks, an increase in the level of high-density

lipoprotein cholesterol and significant reduction in the levels of total cholesterol,

triglycerides and low-density lipoprotein (LDL) cholesterol was observed after the

administration of biophenol-rich olive leaf extracts. (Jemai et al., 2008). In an in-

vitro experiment oleuropein and hydroxytyrosol effectively inhibited CuSO4-

induced LDL oxidation (Visioli et al., 1994).

1.9.6. Blood pressure regulation

The hypotensive effect of olive leaves is said to be because of its vasodilator activity

(Zarzuelo et al., 1991). Olive leaf extract has been shown to lower blood pressure

and increase blood flow in the coronary arteries (Chevallier, 1996; Khayyal et al.,

2002). It also prevented the development of severe hypertension in a DSS, insulin-

41

resistant rat (Somova et al., 2003) whereas in L–NAME (NG-nitro-L-arginine

methyl ester) induced hypotensive rats OLE resulted in normalisation of the blood

pressure (Khayyal et al., 2002). In voltage clamp experiments with cultured

neonatal rat cardiomyocytes, OLE suppressed the L-type calcium channel directly

and reversibly, with an increase in relative coronary flow and a decrease in systolic

left ventricular pressure and heart rate observed (Scheffler et al., 2008). Olive leaves

decoction produced relaxation of the aorta in rats, while oleuropein and

verbascoside reduced blood pressure (Zarzuelo et al., 1991). Oleanolic acid and

ursolic acid isolated from wild African olive leaves have also been reported to have

hypotensive effects (Somova et al., 2004). There is; however, little if any clinical

data on olive leaves as a blood pressure regulatory agent.

1.9.7. Anticancer and chemopreventive activities

Olive leaf extract has an inductive effect on glutathione S-transferases and therefore

may have beneficial effects on cancer. Glutathione S-transferases are a group of

phase II metabolising enzymes that can play a vital role in the detoxification of

chemical carcinogens. They catalyse the conjugation reaction of electrophilic

hydrophobic compounds with reduced glutathione, as reactive forms of chemical

carcinogens are electrophiles (Han et al., 2001). It has been suggested that that

enhancement of the activity of glutathione S-transferases could increase the

capacity of the organism to withstand the neoplastic effects of chemical

carcinogens. In a study on mice, OLE produced an inductive effect on liver

glutathione S-transferases. Oleuropein also produced the same effect in a non-dose

dependent manner but the effect produced by OLE was stronger than isolated

oleuropein, possibly due to synergistic effects (Han et al., 2001). Oleuropein has

demonstrated anti-tumour and cytoskeleton disruptor activities (Babich and Visioli,

42

2003; Hamdi and Castellon, 2005). It also acts as mechanism-based inhibitors of

various cytochrome P450 enzymes (CYPs) (Zhou et al., 2007), which play an

important role in procarcinogen activation. The inhibition of CYPs may decrease

the formation of toxic metabolites (Zhou et al., 2007). The proposed therapeutic

effects of OLE on cancer cells are not only apoptosis but also the induction of

differentiation. It has been shown to have leukaemia protective effects on the human

chronic myeloid leukaemia cells and inhibited the proliferation of K562 cells by

inducing cell cycle arrest, apoptosis, and differentiation toward the monocyte

lineage (Samet et al., 2014).

1.9.8. Renal health promotion

In Europe, olive leaves have been traditionally used as mild diuretic and to treat

cystitis (Chevallier, 1996). Dried as well as fresh olive leaves have been used as

diuretic (Somova et al., 2003), with demonstrated diuretic activity in rats (Ribeiro

et al., 1986; Somova et al., 2003) and humans (Capretti and Bonaconza, 1949). In

a DSS insulin-resistant rat, OLE, oleanolic acid, and ursolic acid all showed potent

diuretic activity and comparable natriuretic and saluretic activity to that of

hydrochlorothiazide. However, no carbonic anhydrase inhibition was detected

(Somova et al., 2003). In a very early study, intake of an olive leaf infusion or

decoction increased urinary output in humans without any effect on blood Na, K or

chloride concentrations (Capretti and Bonaconza, 1949). Although OLE is used as

diuretic in folk medicine and thought to have role in renal health promotion there is

little if any clinical data available.

43

1.9.9. Gastro-protective effects

Gastro-protection is the protection of gastric mucosa from various irritants; it is not

the inhibition of gastric acid secretion. Dekanski et al. (2009) found that gastric

lesions induced by absolute ethanol were significantly attenuated due to pre-

treatment with OLE at all doses tested (40, 80 and 120 mg/kg) and suggested that

the gastro-protective potential of OLE was most probably due to the ability of its

biophenols to scavenge ROS which are produced in ethanol induced gastric injury.

They also suggested the positive response to OLE was possibly related to its ability

to maintain the integrity of the cell membrane through its anti-lipid peroxidative

activity, as well as its ability to strengthen the mucosal barrier.

1.9.10. Antioxidant and free radical scavenging effects

Oxidative stress occurs when there is an imbalance between the production of free

radicals and the biological system’s ability to detoxify these noxious species or

easily repair the resulting damage (Federico et al., 2007; Obied, 2013). By

definition, free radicals are reactive molecular species which have single electron

in an outer orbit in an energy excited state. This is thenreleased upon their reactions

with other molecules like proteins, lipids, carbohydrates and nucleic acids (K.

Rahman, 2007). As a consequence of oxidative stress, cellular proteins, lipids and

nucleic acids are damaged which results in vascular dysfunction (Johansen et al.,

2005). In aerobic organisms, ROS are produced as by-products and can initiate a

chain of damage through autocatalytic reactions and converting the substrate

molecules into free radicals as well. Various stimulants are involved that favour

free radicals production in the human body. These include physical stress, toxic

chemical wastes, occupational exposure to chemically and structurally diverse

environmental pollutants, pesticides, air pollutants and radiation. ROS can be

44

generated through different mechanisms exogenously as well as endogenously. The

major reported mechanisms are from atmosphere as pollutants, UV light irradiation

and by X-rays and gamma rays, metal catalysed reactions, during inflammation by

neutrophils and macrophages and mitochondrial catalysed electron transport

reactions (K. Rahman, 2007). Accumulating evidence suggests a pivotal role for

oxidative stress in aging and age-related degenerative disorders (Baynes and

Thorpe, 1999; Federico et al., 2007; K. Rahman, 2007; Khansari et al., 2009). Free

radicals, mainly ROS and reactive nitrogen species (RNS) are thought to play an

important role in the development of certain forms of cancers, cardiovascular

diseases, diabetes, rheumatoid arthritis, and neurodegenerative diseases like

Alzheimer’s disease, Huntington’s disease and Parkinson’s disease (Baynes and

Thorpe, 1999; K. Rahman, 2007).

Antioxidants can be endogenous (produced in the body) or exogenous (taken as a

dietary supplement). Consuming antioxidant-rich diets may inhibit oxidation and

can delay atherosclerosis by scavenging H2O2. Hydrogen peroxide activates the

enzyme phospholipase C which brings about arachidonic acid metabolism and can

lead to platelet aggregation (Singh et al., 2008). There is a growing interest in the

use of natural antioxidants as bioactive components in food, and such foods have

been termed as functional foods. Many researchers have reviewed and

demonstrated the ability of olive biophenols to modulate oxidative stress through

different mechanisms including free radical scavenging, metal chelating or

stimulating antioxidant enzymes (Visioli et al., 2002; Obied et al., 2009; Servili et

al., 2009; Leopoldini et al., 2011).

The active phenolic compounds in OLE which are known for their capacity to

scavenge H2O2 are part of the secoiridoid family (Singh et al., 2008). The

antioxidant capacity of OLE is higher than that of vitamin C, vitamin E or

45

hydroxytyrosol. Although hydroxytyrosol is a strong antioxidant, the probable

reason for its lower antioxidant activity as compare to OLE may be the synergistic

effects of flavonoids, simple phenols and the high oleuropein content in OLE. The

antioxidant activity of flavonoids such as rutin, catechin and luteolin has been

shown, in vitro, to be almost 2.5 times higher than vitamins C and E (Benavente-

Garcıa et al., 2000). In another study, Le Tutour and Guedon (1992) found OLE,

oleuropein and hydroxytyrosol are more effective than vitamin E in extending the

induction period for oxidation. They concluded that OLE and related ortho-

diphenolic compounds can display a high degree of antioxidant activity and react

with more than two peroxy radicals per molecule, and the higher antioxidat

properties of OLE are due to their high oleuropein content and also to a lesser

amount of flavonoids. However, oleuropein has lower antioxidant activity than

OLE (Benavente-Garcıa et al., 2000; Benavente-Garcia et al., 2002; M. Hansen and

Verity, 2002; Singh et al., 2008). In a study using rats, scientists found that

oleuropein-rich extracts from olive leaves and their enzymatic and acid

hydrolysates, rich in oleuropein aglycone and hydroxytyrosol, significantly

decreased thiobarbituric acid reactive substances content of liver, heart, kidneys

and aorta, increased the activities of superoxide dismutase and liver catalase, as well

as improved the serum antioxidant potential (Jemai et al., 2008). It was suggested

that oleuropein, oleuropein aglycone and hydroxytyrosol-rich extracts can delay the

lipid peroxidation process and increase the activity of antioxidant enzymes.

1.9.11. Anti-inflammatory effects

Oxidative stress plays a vital role in inflammatory processes as it activates nuclear

factor kappa B (NF-κB), activator protein-1 (AP-1) transcription factors and nuclear

histone acetylation and deacetylation in inflammatory diseases (I. Rahman et al.,

46

2006; Obied et al., 2012). In an inflammatory response, cyclooxygenase and

lipooxygenase convert arachidonic acid to compounds such as prostaglandins,

thromboxanes, prostacyclin and leukotrienes that produce inflammatory symptoms

and pain (Obied et al., 2012). Biophenols possess anti-inflammatory activity and

act as modifiers of signal transduction pathways, modulate pro-inflammatory gene

expression and important cellular signalling processes (I. Rahman et al., 2006;

Santangelo et al., 2007). It has been proposed that these anti-inflammatory effects

are due to various mechanisms including: (i) scavenging effect of ROS directly or

via glutathione peroxidase activity and modulation of nuclear redox factor

activation; (ii) inhibition of pro-inflammatory enzymes, such as COX-2,

lipooxygenase and inducible nitric oxide synthase (iNOS); (iii) down regulation of

various pro-inflammatory cytokines such as tumour necrosis factor alpha (TNF-α),

chemokines and interleukins (IL-1β, IL-6, IL-8); (iv) inhibition of NF-κB, AP-1,

phosphoinositide 3-kinase and tyrosine kinases and (v) monocyte chemotactic

protein-1 (I. Rahman et al., 2006; Santangelo et al., 2007; Obied et al., 2012).

Hydroxytyrosol has been shown to lower the production of pro-inflammatory and

prothrombotic mediators and improves plasma antioxidant capacity in laboratory

animals (Fki et al., 2007). In diet-induced obese and diabetic rats, oleuropein and

hydroxytyrosol were found to reverse chronic inflammation and oxidative stress

without changing blood pressure (Poudyal et al., 2010). In rats, OLE showed

protective effects against carrageenan-induced paw oedema (Fehri et al., 1996).

Zaslaver et al. (2005) evaluated the modulator effect of olive leaves on nitric oxide

production and oxidative status in epithelial lung and suggested that olive leaves

may have a therapeutic potential in the treatment of inflammatory diseases.

47

1.9.12. Anti-diabetic activities

Diabetes mellitus is a chronic condition characterised by high level of blood

glucose. It is a metabolic disorder with disturbances in carbohydrate, fat, and

protein metabolism due to relative and/or absolute deficiency in insulin (Rydén et

al., 2007). Insulin is a hormone that is required for the uptake of glucose into tissues

but in diabetic patients glucose is not taken up properly and remains in circulation,

hyperglycaemia (International Diabetes Federation, 2015). Although it does not

have serious acute effects, hyperglycemia has deleterious effects on the long run. It

promotes the production of advanced glycation end products and uncontrollable

free radicals. This in turn creates chronic oxidative stress which leads to all the

serious complications of diabetes e.g. cardiovascular problems, kidney damage,

retinal damage, nerve damage, and blood vessel damage (Nwose et al., 2007).

Prediabetes occurs when blood glucose concentrations are higher than normal but

not high enough to be diagnosed as diabetes. It is also known as impaired glucose

tolerance (IGT) and impaired fasting glucose (IFG) (Twigg et al., 2007; Tabák et

al., 2012). Impaired glucose tolerance is defined as high blood glucose

concentrations after eating; whereas IFG is defined as high blood glucose

concentrations after a period of fasting. Prediabetes is associated with obesity, aging

and shares many characteristics with type 2 diabetes. In prediabetes, insulin is also

not working or not used effectively as in type 2 diabetes (Twigg et al., 2007; Tabák

et al., 2012). Prediabetes is diagnosed from the results of an oral glucose tolerance

test (OGTT) or glycated haemoglobin (HbA1c). It is not true that all people with

IGT will develop diabetes but they are at high risk of developing type 2 diabetes;

they can return to normal blood glucose concentrations. However, about 40–50%

of IGT subjects do develop type 2 diabetes (DeFronzo and Abdul-Ghani, 2011).

Shaw et al. (1999) reported that for more than one-third of people with IGT, their

48

blood glucose concentrations will return to normal over a period of several years.

Prediabetes is not only a risk factor for type 2 diabetes but it is also risk factor for

developing cardiovascular diseases (Tominaga et al., 1999).

The development of type 2 diabetes from prediabetes can be prevented through

lifestyle and pharmacological interventions. Progression from IGT to Type 2

diabetes can be prevented through early pharmacological intervention with oral

antidiabetic agents that improve insulin sensitivity and preserve β-cell function

(DeFronzo and Abdul-Ghani, 2011). There is also evidence that a healthy diet and

physical exercise prevent the progression to diabetes; an intensive diet intervention

soon after diagnosis can improve glycaemic control (Andrews et al., 2011).

Olive leaves have been used as a medicinal herb to treat diabetic hyperglycaemia

for a long time (Komaki et al., 2003; El and Karakaya, 2009; Boaz et al., 2011;

Cumaoğlu et al., 2011; Ghanbari et al., 2012), and the antidiabetic and

hypoglycaemic activities of olive leaves have been investigated in many studies

(Gonzalez et al., 1992; Komaki et al., 2003; Al-Azzawie and Alhamdani, 2006;

Sato et al., 2007; Eidi et al., 2009; Cumaoğlu et al., 2011; Kaeidi et al., 2011;

Wainstein et al., 2012; de Bock et al., 2013; El Amin et al., 2013). In an in-vitro

study, (Cumaoğlu et al., 2011) found that OLE improved the β-cell viability and

offered a degree of protection against cell death after cytokine exposure possibly

through suppression of caspase 3/7 activity. Furthermore, OLE almost completely

restored insulin secretion function whereas oleuropein only partially protected

insulin secretion. It was proposed that the inhibition of cytokine-mediated β-cell

toxicity was through maintenance of redox homeostasis by olive leaf biophenols.

Kaeidi et al. (2011) reported that OLE protected cells against neural damage

associated with high glucose concentrations, inhibited caspase 3 activation.

49

Diabetes-induced thermal hyperalgesia was also suppressed, probably by reduction

in neuronal apoptosis.

After oral loading of starch, olive leaves were found to suppress the elevation of

blood glucose concentrations of the group for borderline diabetes, having fasting

blood glucose concentrations level ranging from 110 to 140 mg/dL (Komaki et al.,

2003). They have also been found to improve insulin sensitivity and pancreatic β-

cell secretory capacity, possibly by reduction in the glucose and insulin excursion

(de Bock et al., 2013) and reduce HbA1c and fasting plasma insulin concentrations

but with no effect on postprandial plasma insulin concentrations (Wainstein et al.,

2012). An ethanol-OLE was shown to inhibit the activities α-amylase from human

saliva; luteolin-7-O-β- glucoside, luteolin-4′-O-β-glucoside and oleanolic acid

purified from OLE possess anti α-amylase activity. (Komaki et al., 2003).

In animal studies, OLE showed hypoglycaemic activity and significantly decreased

the serum glucose concentrations in alloxan induced diabetes (Gonzalez et al.,

1992; Al-Azzawie and Alhamdani, 2006; Jemai et al., 2009), streptozotocin-

induced diabetes (Eidi et al., 2009; Kaeidi et al., 2011; Wainstein et al., 2012; El

Amin et al., 2013), whereas in a DSS, insulin-resistant rat, OLE improved the

insulin resistance (Somova et al., 2003). The serum insulin and antidiabetic effect

of OLE was found to be more effective than antidiabetic drug (glibenclamide) (Eidi

et al., 2009). (Wainstein et al., 2012) found that OLE significantly reduced digestion

and absorption of starch in mucosal as well as serosal side of the intestine in

streptozotocin-induced diabetic rats, and suggested the hypoglycaemic effect of

OLE might be due to reduced digestion and absorption of starch. Biophenols from

OLE, including luteolin-7-O-β- glucoside, luteolin-4′-O-β-glucoside and oleanolic

acid were found to have inhibitory effects on α-glucosidases prepared from rat

50

intestine, and on the postprandial blood glucose increase in diabetic rats (Komaki

et al., 2003).

Oleanolic acid is a highly specific and potent TGR5 (a member of G-protein

coupled receptors) agonist and is suggested to be a potent anti-hyperglycaemic

agent. In mice fed with a high fat diet, oleanolic acid reduced serum glucose and

insulin concentrations and increased glucose tolerance. Oleuropein is also a TGR5

antagonist, and both compounds are considered to be involved in the antidiabetic

effect of olive leaves (Sato et al., 2007).

Olive leaf extract and oleuropein act on diabetes by at least two mechanisms: firstly:

by increasing peripheral glucose utilisation and secondly by improving glucose

stimulated insulin release (Gonzalez et al., 1992). Oleuropein is reported to be

beneficial in inhibiting oxidative stress induced by diabetes and diabetic

complications associated with oxidative stress. The concentrations of blood

glucose, malondialdehyde and antioxidants were restored in diabetic rabbits when

20 mg of oleuropein /kg body weight was given for 16 weeks (Al-Azzawie and

Alhamdani, 2006).

1.9.13. Anti-Alzheimer’s Disease

Alzheimer’s disease is the most common form of dementia in humans with typical

symptoms such as progressive loss of memory, deterioration of intellectual

functions, increased unresponsiveness, reduced speech function, disorientation, and

abnormal gait (Alzheimer's-Association, 2013). It occurs due to changes in the

cerebral cortex, basal forebrain, and other areas of the brain. The most affected

nerve cells are those that produce acetylcholine. Alzheimer’s patient’s brain show

characteristic senile plaques and neurofibrillary tangles (Steele et al., 2007). These

plaques and tangles are like scars and are formed due to protein and cellular

51

deposits. It is believed that accumulation of the protein β-amyloid outside of the

neurons in the brain and the accumulation of the protein tau inside of the neurons

results in the development of Alzheimer’s disease. Alzheimer’s disease appears to

develop to a large extent due to oxidative damage (Praticò and Delanty, 2000), as

β-amyloid increases the level of hydrogen peroxide and free radicals (Yamada et

al., 1999; Nunomura et al., 2006; Ramassamy et al., 2010; Pimentel et al., 2012;

Zhao and Zhao, 2013). Pro-oxidants elevate the β-amyloid concentration, and

antioxidants offer some degree of protection from β-amyloid-induced toxicity

(Yamada et al., 1999; Nunomura et al., 2006; Ramassamy et al., 2010; Pimentel et

al., 2012). Inflammation can cause oxidative damage in Alzheimer’s disease

through microglia which results in excessive production of RNS and ROS which in

turn damage lipid, proteins, and DNA. The role of oxidative stress in Alzheimer’s

disease is also highlighted by mitochondrial dysfunction and damage, with a higher

percentage of damaged mitochondria being observed in neurons (Nunomura et al.,

2006; Butterfield, 2011; Pimentel et al., 2012; Madeo and Elsayad, 2013; Zhao and

Zhao, 2013). The presence of the protein oxidation markers, protein carbonyls and

3-nitrotyrosine, in the brain as well as decreased level of cytochrome c oxidase and

advanced glycosylation end products also support the role of oxidative stress in

Alzheimer’s disease (Butterfield, 2011; Madeo and Elsayad, 2013; Zhao and Zhao,

2013). Therefore, olive leaf biophenols or OLE could potentially be used for the

prevention or at least delaying the onset of Alzheimer’s disease AD.

In a rat model, OLE improved the neurologic deficit scores after transient middle

cerebral artery occlusion and cerebroprotection from ischemic reperfusion as well

as reduced blood-brain barrier permeability, brain oedema and LDL/HDL ratio

(Mohagheghi et al., 2011). Bazoti et al. (2006) reported oleuropein to be a potential

anti-amyloidogenic agent as it forms a noncovalent highly stable complex with β-

52

amyloid and prevents its aggregation. In another study, administering 100 mg of

hydroxytyrosol/kg body weight for 12 days enhanced resistance of dissociated brain

cells to oxidative stress in mice as shown by reduced basal and stress induced lipid

peroxidation (Schaffer et al., 2007). Hydroxytyrosol may be beneficial in

neurodegenerative diseases as it significantly attenuated the cytotoxic effect of both

Fe2+ as well as nitric oxide, when murine-dissociated brain cells were pre-incubated

with hydroxytyrosol (Schaffer et al., 2007).

1.9.14. Miscellaneous activities

An increase in triiodothyronine (T3) concentrations and reduction in circulating

thyroid-stimulating hormone concentrations has been reported in the male rat when

given OLE intra-gastrically. However, no increase in thyroxine concentrations were

found, which may have been due to feedback mechanism (Al-Qarawi et al., 2002).

The smooth muscle relaxant effects of OLE was demonstrated when ileal tissue and

tracheal segments from rats were tested in the presence of calcium channel blockers

including verapamil and nifedipine. It was proposed that OLE modifies calcium

transport possibly though an increase in the intracellular concentration of cyclic

adenomonophosphate (Fehri et al., 1995). In ovariectomised rats, oleuropein

offered protection against inflammation-induced bone loss (Puel et al., 2004).

Oleuropein has been proposed to be a metabolism modulator via pepsin activation

and trypsin inhibition (Polzonetti et al., 2004). Apigenin, apigenin-7-O-glucoside,

apigenin-4′-O-rhamnosylglucoside, chrysoeriol, chrysoeriol-7-O-glucoside,

luteolin, luteolin-4′-O-glucoside, luteolin-7-O-glucoside and quercetin-3-O-

rhamnoside have shown strong inhibition of the classical pathway of the

complement system (Pieroni et al., 1996; PDR for Herbal Medicines, 2007).

53

Oleuropein may also increase life span of human embryonic fibroblasts as in vitro

it showed proteasome stimulatory properties (Katsiki et al., 2007).

1.10. Pharmacokinetics of Olive Leaf Biophenols

Data on the bioavailability of olive leaf biophenols is scarce as very few clinical

studies have been performed; however, it is known that olive leaf biophenols are

readily absorbed through the gastrointestinal tract and their level remains high in

circulation (Dean and Janićijević-Hudomal, 2007). Oleuropein is rapidly absorbed

and plasma concentrations reach a maximum, 2 hours after oral administration (Del

Boccio et al., 2003; Tan et al., 2003; Vissers et al., 2004). Oleuropein is metabolised

and its main metabolite is hydroxytyrosol. Both oleuropein and hydroxytyrosol are

rapidly distributed and excreted in urine, mainly as glucuronides or in very low

concentrations as free forms (Del Boccio et al., 2003). Vissers et al. (2004) reported

that about 15% of an oleuropein-glycoside supplement was excreted in urine as

hydroxytyrosol and tyrosol in healthy human subjects and about 55–60% of

administered ligstroside-aglycone, hydroxytyrosol, tyrosol and oleuropein-

aglycone were absorbed. In contrast, Singh et al. (2008) reported that oleuropein is

poorly absorbed. Given it is a glucoside, it could probably access a glucose

transporter in a similar way as quercetin glycoside which is absorbed through active

sugar transporters.

1.11. Toxicity and adverse effects of olive leaf biophenols

Olive leaves have been used with a great safety profile for centuries in

complementary and alternative medicine. However, when OLE is formulated as a

nutraceutical, food additive or pharmaceutical product, it is a new product that

requires careful examination of its toxicity profile and potential interaction with

54

other co-administered drugs or food. Also, the use of OLE in children, pregnant

women, during lactation, in frail patients and in case of liver and kidney

insufficiency has not been studied. Only a limited number of clinical and animal

studies have been performed on OLE. No long term study on the use of OLE or

biophenol enriched extract or individual compounds has been performed. There is

a small body of experimental evidence to indicate that OLE and olive leaf products

are safe for human and there are no major adverse effects, drug interaction or

toxicity associated with their usage (Perrinjaquet-Moccetti et al., 2008; Wainstein

et al., 2012; de Bock et al., 2013). Orally, it is well tolerated by most patients and

no serious adverse events have been reported. However, intra-ocular use of olive

leaf may irritate the surface of the eye (PDR for Herbal Medicines, 2007). There is

a risk of causing biliary colic through promoting the secretion of bile when OLE is

administered to patients with biliary tract stones (Chinou, 2012). Pollinosis, in the

form of rhinitis or bronchial asthma has also been reported (Rodríguez et al., 2001;

Florido Lopez et al., 2002; Quiralte et al., 2007). Some adverse effects with the use

of OLE in stage-1 hypertension patients were also reported by Susalit et al. (2011).

The most commonly reported effects were coughing (4.6%) and vertigo (5.9%),

while the less common were headache and muscle discomfort. OLE is not

recommended for patients with known hypersensitivity against the herbal

substances, in children and in pregnant or lactating women (Chinou, 2012) due to

the lack of safety studies.

1.12. Posology of olive leaf/ OLE

Standard recommended dose of olive leaves or OLE reported in literature is variable

and for different symptoms the recommended quantity of OLE or leaves is different.

Data on toxic dose of OLE or oleuropein is not available and the researchers still

55

have to answer the question of lethal/toxic dose of OLE/olive leaves in animals and

humans. As a dried powder it has been recommended to use 200-275 mg of leaves

3-5 times a day but not more than 1375 mg (Chinou, 2012). As a herbal tea, Duke

(2010) suggested 2 teaspoons of leaf or 7-8 g dried leaves steeped for 30 minutes

in 150 mL water and taken 3-4 times a day, while as a decoction it is recommended

that 20 g of fresh or 10 g of dried olive leaves should boiled in 300 mL of water and

when the volume reduces to 200 ml, it can be consumed hot after filtration, twice a

day (Van Hellemont, 1986).

In the case of OLE, normally the dose is calculated and recommended on the basis

of its oleuropein content. In commercially available OLE supplements in Australia,

the oleuropein content is standardised to 4.4 to 7.758 mg/mL for liquid OLE with a

recommend dose is 5 mL three times a day, while in capsule form oleuropein is

standardised to 2.4 g and the recommended dose is 2-3 capsules per day for adults

and half of the adult dose in children aged 6 to 12 years.

Hedgcock (2016), recommended daily dose of oleuropein in different age groups

as follows; For Adults, 25 mg in 1-2 doses for health maintenance, 75 mg in 3 doses

for disease prevention and 180 mg in 4-5 doses for acute illness and for children

above 12 years, half of this dose. For children aged 6-12 years the recommended

daily dose of oleuropein is 7 mg in 1-2 doses for health maintenance, 18 mg in 3

doses for disease prevention and 40 mg in 4-5 doses for acute illness. Whereas the

recommended daily dose of oleuropein for children aged form 2-6 years is 5 mg in

1-2 doses for health maintenance, 12 mg in 3 doses for disease prevention and 25

mg in 5 doses for acute illness.

56

1.13. Conclusion

Superior health benefits of olive leaves are attributed to their biophenol content.

Although beneficial effects of OLE in many disease symptoms or disorders have

been demonstrated through in vitro, in vivo and a small number of clinical studies,

there is still no strong enough clinical evidence to support their use in humans as

pharmaceuticals or nutraceuticals to achieve certain clinical outcomes.

Furthermore, their exact mechanism(s) of action is still vague. Although many

studies have shown that olive leaves and OLE possess various capabilities, ranging

from antimicrobial, antioxidant, anti-cancer, antihypertensive, cardioprotective,

neuroprotective, gastroprotective and anti-diabetic, it is still ambiguous whether

they can really perform all of these activities in vivo and what is more intriguing is

how they can exert all of these activities in vivo. In fact, being multi-potent is a

double dagger .i.e. compounds that possess many pharmacological actions will have

high potential to produce some undesirable side effects.

Many researchers propose the various health promoting effects are due to

antioxidant properties of the biophenols. On the other hand, there is a big camp of

researchers who completely reject this idea and consider biophenols as pro-

oxidants. Further research is essential to determine the mechanism(s) of action of

olive biophenols and to understand the fate of these biophenols in the body and their

role in the prevention and treatment of different diseases. More clinical studies are

strongly recommended so as to portray the efficacy and safety of olive biophenols.

Based on the current empirical evidence, especial interest should be given to anti-

diabetic, cardioprotective and neuroprotective potential.

57

An area of primary interest towards commercialization of olive leaf biophenols is

recovery maximization. Careful study of the natural variation of olive biophenol

concentrations in olive leaves and optimization of their recovery is mandatory for

any industrial mass production. Furthermore, it is highly recommended that there

should be some standardisation in procedures for collection, preservation,

extraction and analysis of olive leaves so as to cope with the strict quality control

measures of legal nutraceuticals and food supplements.

After examining relevant literature, the gaps of our current knowledge about olive

leaf supplements have been identified. This research project is designed to address

those gaps with the following aim and objectives.

58

1.14. Aim and objectives of the project

The overall aim of this research project was to evaluate Australian olive leaves as

a nutraceutical resource of bioactive biophenols and to study the antidiabetic effects

of olive leaf biophenols.

To address this overall aim, experiments were designed to address four objectives.

1. To determine the best method for drying olive leaves that allows the

optimum recovery of olive leaf biophenols, especially oleuropein.

2. To determine the effect of season and cultivar on the composition of olive

leaf biophenols.

3. To determine the in vitro antioxidant and antidiabetic activities of olive leaf

biophenols.

4. To gain preliminary evidence of the potential of olive leaf extract as an

antidiabetic agent for pre-diabetic subjects.

59

Chapter 2. Drying at high temperature for a short time

maximizes the recovery of olive leaf

biophenols

2.1. Introduction

Olive leaf extract (OLE) is gaining popularity in the global nutraceutical

market due to a plethora of claimed health attributes. OLE was traditionally

consumed for the treatment of a wide spectrum of ailments such as fever,

malaria, colic, alopecia, paralysis, rheumatism, gout, sciatica, hypertension,

arrhythmia, diabetes and cancer (E. Waterman and Lockwood, 2007;

Flemmig et al., 2011). The functional properties of OLE are essentially due

to its biophenol content (Obied et al., 2012). Biophenols are reactive

phytomolecules that can undergo a myriad of chemical reactions (Obied, et

al., 2005a). Olive biophenols can attenuate oxidative stress and prevent

oxidative damage through diverse mechanisms, including free radical

scavenging, chain reaction breaking, metal chelation and induction of

endogenous antioxidant enzymes (Visioli et al., 2002; Obied et al., 2009;

Servili et al., 2009; Obied et al., 2012).

The literature shows large qualitative and quantitative variation in the

biophenolic composition of OLE (Bouaziz and Sayadi, 2005; Paiva-Martins

and Pinto, 2008; Hashemi et al., 2010; Hayes et al., 2011; Ahmad-Qasem et

al., 2013). Geography, environmental factors, genetics, harvesting time,

agronomic practices, infestation and post-harvest processing can affect the

phenolic composition of olives (Vinha et al., 2005; Gomez-Caravaca et al.,

2008; Obied, , et al., 2008c; Obied, , et al., 2008b). Furthermore, recovery of

olive biophenols is influenced by experimental parameters such as extraction

60

technique, solvent, pH, time and temperature (Obied, et al., 2005b). Sample

handling, processing, clean-up and storage conditions, extract stability,

analytical technique sensitivity and the purity of standards used for

preparation of calibration curves are commonly overlooked factors that can

account for the wide variation in values in the literature. Oleuropein is the

major biophenol found in virtually all studied OLE, with rare exceptions

(Ryan et al., 2002a). Published values for oleuropein recovery from olive

leaves varied massively, from 5.6 to 108.6 mg/g dry weight (Ansari et al.,

2011; Tayoub et al., 2012). In one study, there was a marked variation, with

up to a 10-fold change in oleuropein content based on genetics, season, and

the colour/age of the leaves (Ranalli et al., 2006). Furthermore, it is difficult

to compare results from different studies, as there is no consensus on how to

express the recovery data .i.e. fresh weight, dry weight (DW) or extractable

matter (EM).

Analysis of fresh plant materials is considered an ideal situation, to avoid

artefacts resulting from sample degradation or extensive clean-up procedures.

As it is not always possible and sometimes impossible to analyse fresh plant

samples, several drying techniques have been proposed. Drying has been

always considered a sub-optimal procedure to preserve plant materials and

hence subtle drying techniques are always recommended, such as freeze-

drying (P. G. Waterman and Mole, 1994). Cheaper alternatives such as air-

drying or oven-drying at low temperatures (40-60 °C) have been typically

recommended when cost is an issue. Nonetheless, these conventional

hypotheses and practices have been challenged by sporadic experimental

reports (Vinson et al., 2005; Ahmad-Qasem et al., 2013).

61

As a drug of botanical origin, olive leaves are pharmacopoeially prepared by

air-drying. At the same time, some commercial neutraceutical products

proclaim superiority based on the extraction of fresh olive leaves. Currently,

there are no widely accepted guidelines for the drying of olive leaves. Data

from the literature are insufficient or contradictory. The impact of drying on

the nutritive value of olive leaves as animal feed has been investigated

(Martín-García and Molina-Alcaide, 2008). Though no attention was given

to the phenolic content and antioxidant activity, air-drying was recommended

as a simple and cheap technique that preserved the nutritive value of olive

leaves. Various drying techniques, such as air-drying, freeze-drying, and

oven-drying at low and high temperatures have been used to compare the

effect of drying method on biophenol content (Keinänen and Julkunen-Tiitto,

1996; Julkunen-Tiitto and Sorsa, 2001; Hung and Duy, 2012; Ahmad-Qasem

et al., 2013). Malik and Bradford reported air drying at ambient temperature

(25 °C) as the most suitable method for processing olive leaves for

commercial purposes due to its convenience, economic viability and good

recoveries of biophenols, particularly oleuropein and verbascoside (Malik

and Bradford, 2008). On the contrary, a more recent study identified drying

at 120 °C as the best preserving method for recovery of olive leaf biophenols,

particularly oleuropein (Ahmad-Qasem et al., 2013). Infra-red drying of olive

leaves seemed promising with a three-fold increase in total phenol recovery

at 70 °C, yet the impact of drying on individual biophenols was not

investigated (Boudhrioua et al., 2009). Therefore, we systematically

investigated the impact of different drying conditions on the recovery of

different biophenols from olive leaves, so as to resolve the current

62

controversy and provide a basis for optimizing commercial production of

OLE.

2.2. Materials and methods

2.2.1. Chemicals and reagents

Chemicals and reagents were used without further purification: Folin-

Ciocalteu reagent, 2,2′-diphenyl-1-picrylhydrazyl radical (DPPH•), (±)-6-

hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), 2,2′

azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS),

formic acid, aluminium chloride, sodium nitrate (Sigma-Aldrich, Sydney,

Australia), anhydrous sodium sulphate, sodium hydroxide, sodium carbonate

(Univar, Sydney, Australia); HPLC-grade methanol (Fisher Scientific. New

Jersey, USA); n-hexane, glacial acetic acid, ethanol, hydrochloric acid,

ammonium acetate (Merck, Melbourne, Australia), ethyl acetate and

anhydrous acetonitrile, (UNICHROME, Sydney, Australia).

Gallic acid, chlorogenic acid, caffeic acid, rutin, quercetin, 4-

hydroxymandelic acid, 3,4-dihydroxymandelic acid, luteolin, luteolin-7-O-

glucoside, catechin hydrate, 4-hydroxyphenylacetic acid, syringic acid, 3,4-

dihydroxyphenyl acetic acid, homovanillyl alcohol, 4- hydroxybenzoic acid,

trans-cinnamic acid, catechin, ferulic acid, o-coumaric acid, homovanillic

acid, p-coumaric acid, sinapic acid, hesperetin, neohesperidin and tyrosol

were purchased from Sigma-Aldrich (Sydney, Australia). Apigenin,

apigenin-7-O-glucoside, diosmin, oleuropein and verbascoside were

purchased from Extrasynthese (Genay, France). Hydroxytyrosol was bought

from Cayman Chemical Company (Ann Arbor, MI).

63

Standards were dissolved in 80% aqueous methanol to prepare stock

solutions of 1 mg/mL. Quercetin and luteolin had to be dissolved in absolute

methanol and rutin was dissolved in warm 80% aqueous methanol, as

described previously (Obied, et al., 2005b). Water used in all analytical work

was purified by a Modulab Analytical water system (Continental Water

Systems Corp., Melbourne, Australia).

2.2.2. Collection of olive leaves

Olive leaves were collected from the Charles Sturt University Olive Grove at

Wagga Wagga, NSW, Australia. The mature green leaves were handpicked

at the operator height around the whole perimeter of three trees of the same

cultivar, from summer through to autumn. Leaves of the Frantoio cultivar

were collected in February, 2012. Leaves of the Leccino cultivar were

collected in March, 2012 while leaves of Frantoio and Leccino cultivars were

again collected in April, 2012, in addition to two other cultivars; Hardy’s

Mammoth and Arbequina. Leaves were collected in plastic bags and brought

to the laboratory without any delay. Fresh leaves were analysed or subjected

to various drying conditions within 1 hour of collection.

2.2.3. Sample treatments

A pilot study was conducted for optimizing drying time and moisture content

for different treatments. In oven-drying at 105 °C, measurements were

recorded on an hourly basis for 5 hours. For air-drying, leaves were dried at

room temperature for ten days and the weight was recorded daily. Weight

was recorded after 18 and 24 hours for the freeze drying treatment, while

measurements were taken every 6 hours for 48 hours for oven drying at 60

64

°C. Optimum drying time is the minimum time required to achieve the lowest

moisture content with minimal degradation. Our data showed that optimum

drying time was 24 hour for freeze drying; 48 hours for air drying; 6 hours

for oven drying at 60 °C; and 3 hours for oven drying at 105 °C.

One portion of fresh leaves was ground in a coffee grinder and extracted to

determine the biophenol composition of fresh samples within one hour after

collection. Equal aliquots of fresh leaves were immediately subjected to one

of the following drying treatments.

a) Air drying: leaves were air-dried at room temperature (21 ± 2 °C) for

48 hours.

b) Oven drying at 60 °C: leaves were dried in a forced-air oven at 60 °C

for 6 hours.

c) Oven drying at 105 °C: leaves were dried in a forced-air oven at 105

°C for 3 hours.

d) Freeze drying: leaves were freeze dried in a Christ-Alpha 2–4 LD plus

freeze dryer (Biotech International, Germany) for 24 hours.

Dried leaves were ground in a coffee grinder and stored at –20 °C in air-tight

containers until extracted.

2.2.4. Moisture content

Moisture content was determined using the procedures described by the

United States Pharmacopeia (2002) using the gravimetric method for “articles

of botanical origin” as described previously (Obied, et al., 2005b). Five grams

of fresh leaves were dried in an evaporating dish in a convection oven at 105

65

°C. The leaves were weighed after every hour until the difference between

two successive readings was less than 0.25%.

2.2.5. Extraction of biophenols

Biophenols were extracted according to the method described previously

(Obied, et al., 2005b), with slight modification. Two grams of fresh/dried leaf

powder was extracted with 10 mL of 80% aqueous methanol for 30 min with

continuous stirring using a magnetic stirrer. The extract was filtered through

an Advantech no. 1 filter paper. The filtrate was kept and residues were re-

extracted with additional 10 mL of 80% aqueous methanol for 15 min and

filtered over the first filtrate. The combined extract was defatted thrice with

15 mL of n-hexane. The defatted extract was filtered through GF/F filter

paper and then re-filtered through 0.45 μm nylon syringe filters

(Phenomenex, Australia). All extractions were performed at room

temperature (21 ± 2 °C). The extracts were stored at –20 °C until analysed.

Extraction was performed in triplicate for each treatment.

2.2.6. Spectrophotometric measurements

Colorimetric analyses were performed on a Cary 50 UV/visible

spectrophotometer, using Cary WinUV “version 3” software (Varian,

Australia). Extracts were diluted using an extraction solvent to produce

concentrations within the range of the constructed calibration curves. Crude

extracts of freeze dried and oven dried at 105 °C olive leaves were diluted

(1:100) with 80% aqueous methanol, whereas crude extracts of fresh, air

dried and oven dried at 60 °C were diluted (1:20) with 80% aqueous

methanol. Three extracts of each treatment were analysed in triplicate.

66

2.2.6.1. Total phenols

For the determination of total phenols, 6 mL of water, 100 µL of diluted

extract and 500 µL of Folin-Ciocalteu reagent were added to a 10 mL

volumetric flask (Obied, et al., 2005b). After 1 min, 1.5 mL of aqueous

sodium carbonate solution (20% w/v) was added and mixed. The volume was

made up to 10 mL with water and contents were mixed thoroughly. The flask

was kept for 60 min at room temperature. The absorbance was then recorded

at 760 nm. A seven point (50-600 µg/mL) calibration curve was prepared

using gallic acid. Results were expressed as milligrams gallic acid equivalent

per gram dry weight (mg GAE/g DW).

2.2.6.2. Total flavonoids

Total flavonoid content was analysed by using an AlCl3 colorimetric assay,

as described earlier (Obied et al., 2013). Diluted extract (500 µL) and 150 µL

of NaNO2 (5% w/v) were mixed in a 5 mL volumetric flask. After 5 minutes,

150 µL of AlCl3 solution (10% w/v) was added, mixed for 1 min, and 1 mL

of 1M NaOH was then added. The volume was made up to 5 mL with water,

mixed well and absorbance was recorded at 510 nm. A calibration curve of

eight points (80-1600 µg/mL) was prepared using quercetin. Total flavonoid

contents were expressed as mg quercetin equivalents per gram dry weight of

leaves (mg QE/g DW).

2.2.6.3. o-diphenols

Determination of o-diphenols was performed according to the method

described previously (Qawasmeh et al., 2012), with minor modification.

Diluted extract (250 µL) and 250 µL of sodium molybdate dihydrate solution

67

(5% w/v in ethanol 50%) were added to a 5 mL volumetric flask. After

mixing, the volume was made up to the mark with 50% ethanol. The flasks

were incubated at room temperature for 15 min and the absorbance was

measured at 370 nm. A five-point (50-250 µg/mL) calibration curve was

prepared using caffeic acid. Results were expressed as milligrams of caffeic

acid equivalents per gram dry weight of leaves (mg CAE/g DW).

2.2.6.4. ABTS radical scavenging assay

ABTS•+ scavenging activity was measured as described before (Qawasmeh

et al., 2012). ABTS reagent (7 mM) was added to 2.45 mM potassium

persulphate and incubated in dark at room temperature overnight to generate

ABTS radical cation (ABTS•+). Stock ABTS solution was diluted with water

to an absorbance of 0.70 (±0.02) at 734 nm for analysis. In a plastic macro-

cuvette, 3 mL of the ABTS working solution and 50 µL of diluted extract

were mixed well. Cuvettes were incubated for 30 min in the dark at room

temperature and absorbance of the mixture was measured at 734 nm. Water

was used as blank. The absorbance of the ABTS radical without samples

served as control. A seven point (25-300 µg/mL) calibration curve was

prepared using Trolox and Results were expressed as trolox equivalent (TE)

mM per gram dry weight. The following formula was used to calculate the

percent scavenging of ABTS•+.

% scavenging =[Absorbance (control) − Absorbance (sample)]

Absorbance (control) × 100

68

2.2.6.5. DPPH radical scavenging assay

The stable radical 2,2′-diphenyl-1-picrylhydrazyl (DPPH) was used to

measure the radical scavenging ability of olive leaves, as described earlier

(Obied, et al., 2008a), with minor modification. In macro plastic cuvettes, 3

mL of DPPH working solution in 80% aqueous methanol and 50 µL of diluted

extract were mixed well. Cuvettes were kept in the dark for 60 min at room

temperature and absorbance of the mixture was measured at 517 nm. Aqueous

methanol (80%) was used as blank. The absorbance of the DPPH radical

without sample served as control. A ten point (50-500 µg/mL) calibration

curve was prepared using Trolox and Results were expressed as mM of trolox

equivalent per gram dry weight (mM TE/g). The following formula was used

to calculate the percent scavenging of DPPH radical activity.

% scavenging =[Absorbance (control) − Absorbance (sample)]

Absorbance (control) × 100

2.2.7. High performance liquid chromatography (HPLC)

2.2.7.1. HPLC-DAD-online ABTS

Varian Prostar 240 solvent delivery system, Varian Prostar 335 diode array

detector (DAD) and Varian Prostar Autosampler were used to perform HPLC

analysis. The HPLC system was controlled by a Star Chromatography work

station, version 6.41 (Varian, Australia). The samples were analyzed by

gradient elution on a Gemini C-18 column [150 mm × 4.6 mm i.d., 3 µm,

(Phenomenex, Sydney, Australia)]. Through a T-intersection, the outflow

from the DAD was connected to a reaction coil (3.4 m× 0.178 mm i.d.), which

was maintained at 37°C in a Varian HPLC column temperature controller. A

69

Varian Polaris 210 HPLC pump was used to pump ABTS•+ working solution

to the reaction coil through the T-intersection. A Varian 9050 UV-Vis

detector was used for the detection of ABTS•+ absorbance at 414 nm. The

detector generated positive peaks by reversing the polarity of the analogue

signal. The mobile phases were prepared fresh, degassed under vacuum using

Phenomenex nylon 0.45 µm membranes and sonicated in a Sanophon

ultrasonic bath (Ultrasonic Industries Pty. Ltd, Sydney, Australia) for 10 min

prior to HPLC analysis. Solvent A was a mixture of water/methanol/ acetic

acid (90 + 10 + 1, v/v/v), whereas solvent B was methanol/acetonitrile/acetic

acid (90 + 10 + 1, v/v/v), as previously used (Obied, et al., 2008a). A flow

rate of 0.8 mL/min, injection volume of 5 µL and gradient elution for total

run time of 45 min was used as follows: initial conditions 90% solvent A;

isocratic for 2 min, solvent B was increased to 30% over 3 min; isocratic for

5 min, then solvent B increased to 40% over 5 min; isocratic for 5 min, then

solvent B increased to 50% over 5 min; isocratic for 5 min, then solvent B

increased to 100% over 5 min; and remain isocratic for 10 min. The system

was allowed to equilibrate for 10 min between runs.

2.2.7.2. HPLC-DAD-MS/MS

Analyses were conducted on an Agilent 1200 series liquid chromatographic

equipment (Agilent Technologies, Waldbronn, Germany) by gradient elution

on a Gemini C-18 column [150 mm × 4.6 mm i.d., 3 µm, (Phenomenex,

Sydney, Australia)]. The mobile phase was a gradient of solvent A (0.3%

formic acid in 10 mM ammonium acetate) and solvent B (0.3% formic acid

in acetonitrile/water 50:50, v/v). A flow rate of 0.9 mL/min and an injection

volume of 5 µL were used and gradient elution for total run time of 30 min

70

was used as follows: initial conditions 25% solvent B; isocratic for 2 min,

solvent B was increased to 50% over 3 min; isocratic for 3 min, then solvent

B increased to 80% over 7 min; isocratic for 5 min, then solvent B increased

to 100% over 3 min; isocratic for 4 min, then solvent B decreased to initial

condition (25%) over 3 min. The system was allowed to equilibrate for 5 min

between runs. The DAD was set to record chromatograms at 240, 280, 325

and 510 nm. Through a T-intersection, the outflow from the DAD was

connected to a 6410 triple-quadrupole mass analyzer (Agilent Technologies,

Santa Clara, CA, USA) equipped with an electrospray ionization (ESI)

interface. MS analysis was performed in the negative ion mode (m/z

100−1500) under the following conditions: nitrogen gas; gas temperature,

300 °C; gas flow rate, 12 L/min; nebulizer pressure, 45 psi; capillary voltage,

4 kV; cone voltage, 100 V. Data were analyzed using Agilent MassHunter

workstation version B.01.04 2008 (Agilent Technologies, Waldbronn,

Germany).

2.2.8. Statistical analysis

All assays were performed in triplicate and averaged. Data analysis was

performed by Microsoft Excel and IBM SPSS Statistics package version 20.0

(IBM Corporation, NY). Statistical comparisons were made using one-way

ANOVA and post-hoc LSD test. Results were considered statistically

significant at p ≤0.05.

2.3. Results and discussion

Four cultivars and three collection times were investigated for the effect of

sample pre-treatment. The data from various cultivars and collection times

71

showed a similar pattern. Furthermore, collected cultivars showed similar

phenolic profile and differences among cultivars were mainly quantitative.

We selected the data from the Frantoio cultivar collected during the summer

season (early February) to avoid redundancy and unnecessary details.

2.3.1. Moisture content

The average moisture content of fresh olive leaves was 50.7 ± 0.8 %. The

moisture content of our sample comes in accord with literature values 50-56

% (Cabrera-Gomez et al., 1992; Boudhrioua et al., 2009). Freeze-drying of

olive leaves required 24 h to reach to a constant weight. Meanwhile, it took 6

h at 60 °C and 3 h at 105 °C to achieve complete dryness (constant weight).

On the other hand, air-drying did not result in complete dryness even after ten

days. Hence, we decided to stop air-drying after 48 h as a compromise to

achieve reasonable drying with minimal decomposition of biophenols. After

48 h, the moisture content of air-dried leaves did not exceed 24.8 ± 2.4 %.

2.3.2. Extractable matter (EM)

The highest recovery of extractable matter was 6.6% for oven-drying at 105

°C, followed by 4.5% for freeze-drying, which represents approximately

triple and double of the fresh sample recovery, respectively (Table 2.1). Both

air-drying and oven-drying at 60 °C produced a 50% increase in the recovery

of extractable matter, compared with fresh olive leaves. Overall, drying

enhanced the recovery of extractable matter significantly. Drying of plant

tissues damages cellular structures and produces a loose porous material that

allows facile solvent penetration and solute extrusion (Lewicki, 1998).

Furthermore, heat is able to breakdown cellular structures containing

72

biophenols, like cell wall and vacuoles. Therefore, bound biophenols can be

liberated and recovered (Dewanto et al., 2002). The efficacy of extraction

procedures or extraction solvent can be measured by the amount of residual

solid substance after evaporation of the extraction solvent, referred to as

extractable matter. In food chemistry and nutrition research, recovery of

bioactive compounds is usually expressed as quantity per unit of dry weight

for solid samples, whereas it is common in phytochemistry to represent the

recovery of bioactive constituents as percentage quantity of extractable

matter.

2.3.3. Total biophenol composition and total antioxidant capacity

The need for rapid, sensitive and robust methods to determine the total

phenolic content of plant samples is vital for research and industry. There are

a number of colorimetric assays based on various chemical properties of the

phenolic moiety. Plant phenols are the largest group of plant secondary

metabolites with more than 9,000 identified structures of very widely varying

chemical nature. Therefore, any attempt to measure their total content is a

mere gross approximation that should be only used to compare samples with

similar phenolic composition. Hitherto, no single method is deemed

completely satisfactory and researchers need to acknowledge these

shortcomings when discussing their results. The use of more than one method

for the estimation of total phenols can provide more insight into the

composition of the plant samples and should alert researchers of the

limitations of each method.

Overall, total phenol, total flavonoid and o-diphenol content all varied with

drying method; in descending order (when data are expressed as dry weight):

73

oven-dried leaves at 105 °C > freeze-dried leaves > air-dried leaves > fresh

leaves > oven-dried leaves at 60 °C (Table 2.1). A statistically significant

increase in total phenol composition compared with fresh sample could be

observed for all treatments except for oven-drying at 60 °C where recoveries

dropped well below the fresh sample values (Table 2.1). Total flavonoid

content was the most sensitive parameter; showing up to 230% change, o-

diphenol content was the least affected parameter, where change did not

exceed 50%.

Despite its limitations and lack of selectivity, Folin-Ciocalteu’s reagent is still

the best technique for gross estimation of total phenol content. Folin-

Ciocalteu’s reagent depends on measuring the ability of the sample to reduce

phosphomolybdic/phosphotungstic acid mixture at high pH to form an

intense blue colour. According to Table 2.1, the total phenol content of fresh

olive leaf extract was 51.0 ± 0.7 mg GAE/g DW. A 110% increase was

observed upon oven-drying at 105 °C, while a 90% and 40% increases were

recorded for freeze-drying and air drying, respectively. Oven-drying at 60 °C

produced an almost 57% reduction in total phenol recovery. These results are

consistent with earlier findings, where drying olive leaves at 120 °C produced

higher phenol content compared with drying at 70 °C (Ahmad-Qasem et al.,

2013).

Olive leaf is rich in flavonoids, mainly found as glycosides (Obied et al.,

2012). A number of methods have been proposed to estimate the total

flavonoid content of plant samples, yet none is deemed completely

satisfactory. Flavonoids are the largest group of plant phenols with significant

variation in their molecular structures. In addition, methods based on ortho-

74

diphenol moiety will suffer from considerable interference of non-flavonoid

phenols. We determined the total flavonoid content via measuring the red

colour complex with aluminium (III) after nitrosation with sodium nitrite.

The method is expected to suffer from significant interference from olive

biophenols such as hydroxytyrosol, oleuropein and verbascoside (Papoti et

al., 2011). The total flavonoid content of fresh olive leaves showed an average

value of 263.1 ±10.7 mg QE/g DW. While oven drying at 105 °C resulted in

a 231% increase, freeze-dried and air-dried leaves showed a 158% and 65%

increase, respectively. On the other hand, an 81% reduction in recovery was

recorded for oven-drying at 60 °C (Table 2.1).

Our earlier work demonstrated that o-diphenol content, measured by

complexation with molybdenum (VI), correlated better with antioxidant

activity for olive biophenols (Obied, et al., 2008c). Oven drying at 105°C and

freeze-drying produced an average rise of 50% and 36%, correspondingly.

Air-drying did not significantly affect the recovery of o-diphenols. Consistent

with total phenol results, oven-drying at 60 °C resulted in a 55% decrease in

o-diphenol recovery.

75

Table 2.1: Biophenol content and antioxidant activity of Frantoio olive leaves.

Treatments

Parameters Fresh Air-dried Freeze-dried Oven-dried 60 °C Oven-dried 105 °C

Extractable matter % w/w 2.1 ± 0.2a 3.4 ± 0.1b 4.5 ± 0.2c 3.2 ± 0.3b 6.6 ± 0.1d

TPC (mg GAE/g DW)

(mg GAE/g EM)

51.0 ± 0.7a

118.3 ± 1.6a

71.1 ± 3.2b

134.6 ± 6.0b

91.2 ± 3.7c

201.1 ± 8.2c

21.6 ± 1.4d

67.3 ± 4.3d

106.9 ± 5.9e

161.3 ± 8.9e

TFC (mg QE/g DW)

(mg QE/g EM)

263.1 ±10.7a

610.2 ± 24.8a

435.9 ± 9.9b

826.0 ±18.8b

679.6 ± 40.8c

1497.7 ± 89.8c

49.4 ± 2.4d

153.8 ± 8.2d

871.5 ± 84.1e

1314.3 ± 126.8c

ODP (mg CAE/g DW)

(mg CAE/g EM)

50.8 ± 1.6a

117.8 ± 3.8a

51.7 ± 2.1a

98.1 ± 4.0a

69.0 ± 3.6b

152.1 ± 8.0b

23.0±1.6c

71.7 ± 1.0c

76.2 ± 5.1d

115.0 ± 7.7a

ABTS (mmol TE/g DW)

(mmol TE/g EM)

0.25 ± 0.01a

0.57 ± 0.02a

0.34 ± 0.01b

0.64 ± 0.01b

0.41 ± 0.02c

0.91 ± 0.05c

0.10 ± 0.00d

0.31 ± 0.01d

0.55 ± 0.04e

0.83 ± 0.05e

DPPH (mmol TE/g DW)

(mmol TE/g EM)

0.23 ± 0.01a

0.54 ± 0.02a

0.39 ± 0.02b

0.74 ± 0.09b

0.47 ± 0.02c

1.04 ± 0.04c

0.07 ± 0.00d

0.21 ± 0.08d

0.62 ± 0.04e

0.93 ± 0.06e

Results are presented as mean ± standard deviation. Different superscripts in the same row indicate significant difference at p<0.05. DW: dry weight; EM: extractable

matter; TPC: total phenol content; TFC: total flavonoid content; ODP: o-diphenol content; GAE: gallic acid equivalent; QE: quercitin equivalent; CAE: caffeic acid

equivalent; TE: trolox equivalent; mmol: millimole.

76

Olive biophenols are well prized for their antioxidant properties. Amongst

the different antioxidant mechanisms of action, free radical scavenging

activity is of paramount importance due to the large number of pathological

conditions linked to high level of free radicals (Obied, 2013). Total

antioxidant capacities of extracts, as measured by ABTS•+ and DPPH radical

scavenging assays, were parallel to changes in total phenol composition. All

changes of total antioxidant capacity were statistically significant (Table 2.1).

Samples showed the same ranking in both antioxidant assays and results were

similar to those reported for phenol composition: oven-dried leaves at 105 °C

> freeze-dried leaves > air-dried leaves > fresh leaves > oven-dried leaves at

60 °C. It is important to study antioxidant activity of olive samples along with

their total phenol content as our earlier findings have demonstrated that the

correlation between total phenol content and antioxidant activity is not always

linear (Obied, et al., 2008c).

When the results were expressed as mg equivalents per gram extractable

matter, freeze-drying was superior to oven drying at 105 °C in all assays. This

is most likely due to the higher recovery of extractable matter in 105 °C

(Table 2.1). Apparently, oven-drying at 105 °C increased the recovery of

biophenols as well as non-phenolic matrix components. Thus, freeze-drying

in preparation of olive leaf samples did not only improve the recovery of

biophenols, it had also resulted in significant sample clean-up and improved

the subsequent extraction selectivity.

The present study shows that drying at high temperature such as 105 °C for a

short time or, alternatively, freeze-drying significantly improved the recovery

of antioxidant biophenols from olive leaves. Paradoxically, phytochemistry

77

textbooks and drug compendia frequently recommend drying at temperatures

lower than 60 °C to avoid thermal decomposition (Harborne, 1998; Evans,

2009). Drying at temperatures higher than 50 °C led to significant reduction

in phenolic recovery and total antioxidant capacity in other plants (Julkunen-

Tiitto and Sorsa, 2001; Lim and Murtijaya, 2007; Chan et al., 2009). Drying

of American olive leaves at 60 °C resulted in a considerable loss of most

biophenols, up to 50% (Malik and Bradford, 2008). Similarly, oven-drying at

70 °C resulted in a plunge in total phenol content of Spanish olive leaves (var.

Serrana) (Ahmad-Qasem et al., 2013). On the other hand, the optimum drying

conditions of olive leaves in a tray drier was found to be 51 °C for 5 hours

(Erbay and Icier, 2009).

Freeze-drying produced higher total phenol content and antioxidant activity

than oven-drying at 105 °C for olive mill waste, while, unlike olive leaves,

oven-drying at 60 °C surpassed air-drying (Obied, et al., 2008b). We argue

that the changes in total phenol content and antioxidant activity are both plant

and tissue specific and hence the assumption that one drying method suits all

plants or all tissues from the same plant should be renounced. Air-drying,

moreover, resulted in higher total phenol recovery than oven-drying at 60 °C.

This can be explained by initial enzyme induction via slow drying at medium

temperatures (Evans, 2009). We also found that oven drying at 60 °C resulted

in brown colouring of leaves, while leaves dried at 105 °C were light green.

This browning reaction at lower temperature may be due to the thermal

activation of polyphenol oxidase (PPO) enzymes. Though we could not

retrieve specific data from the literature on the thermal stability of olive PPO,

the optimum temperature for PPO varies widely among different plants and

can reach up to 50 °C. PPO can be thermally inactivated by exposure to

78

temperatures between 70 to 90 °C for varying time periods from 5-60 min

(Queiroz et al., 2008). In the current study, it seems likely that olive leaves

had enough residual catalytic PPO activity to produce considerable

decomposition of biophenols at 60 °C.

2.3.4. Biophenol-antioxidant profile and recovery of individual biophenols

Measuring total phenol content cannot detect degradation of biophenols,

especially when the degradation products are more reactive than their starting

material (aglycones versus glycosides). Thus, analysis of biophenols by

HPLC offers a great advantage to examine the impact of drying on individual

components. Most of the earlier studies did not study the impact of drying on

individual biophenols (Cabrera-Gomez et al., 1992; Boudhrioua et al., 2009;

Erbay and Icier, 2009). In the current study, chromatograms of different

drying treatments are compared at 280 nm in Figure 2.1. The 280 nm, 325

nm and online ABTS scavenging activity chromatograms for leaves oven-

dried at 105 °C are shown in Figure 2.2. A large number of peaks were

detected in the chromatograms at 280 and 325 nm (Figure 2.2). Nine

compounds constituted the major peaks in the chromatograms (Figure 2.1).

The identification of biophenols was based on separation by HPLC equipped

with a DAD, comparison of the chromatographic retention time and UV

absorbance spectra of a compound with those of a reference standard. The

identities of compounds were confirmed by LC-MS/MS. The most abundant

biophenols in olive leaves were: oleuropein (peak 5), verbascoside (peak 2),

luteolin-7-O-glucoside (peak 3), and apigenin-7-O-rutinoside (peak 8). These

results are consistent with earlier findings on olive leaves from the

Mediterranean basin (Bouaziz and Sayadi, 2005). The online ABTS

79

scavenging profile (Figure 2.2) clearly shows that for leaves dried at 105 °C

the main contributors to the radical scavenging activity of olive leaf extracts

are oleuropein (peak 5) > verbascoside (peak 2) > oleuroside (peak 9) >

unknown compound (peak 1) > luteolin 7-O-glucoside (peak 3) > apigenin 7-

O-rutinoside (peak 8).

To study the impact of drying method on the recovery of individual

biophenols, the six identified biophenols were quantified by recording their

peak areas in HPLC chromatogram at 280 nm using calibration curves of

reference standards (Table 2.2). Although different drying treatments had

variable effects on various biophenols, oven-drying at 105 °C generally

produced the highest recoveries of biophenols. Whilst apigenin 7-O-

rutinoside was not significantly changed, luteolin 7-O-glucoside recovery

was nearly doubled in comparison to fresh olive leaves and an almost 40%

increase in rutin recovery was observed. Enhanced recovery of oleuroside (7-

fold) and verbascoside (17-fold) was also observed. The increase in

oleuropein recovery was phenomenal, with a 38-fold increase (Table 2.2)

(Figure 2.3). In a recent study on Spanish olive leaves, (Ahmad-Qasem et al.,

2013) reported that drying olive leaves at 120 °C resulted in the best

processing conditions, particularly for oleuropein. Our results are in

agreement with their findings. Earlier reports also showed good thermal

stability of oleuropein at temperatures up to 130 °C (Malik and Bradford,

2008). Afaneh et al. found that drying olive leaves at ambient temperature

(25 °C) increased oleuropein recovery, while drying at 50 °C decreased its

recovery compared to fresh leaves (Afaneh et al., 2015). Freeze-drying did

not significantly increase the recovery of flavonoid glycosides viz. rutin,

luteolin 7-O-glucoside, and apigenin 7-O-rutinoside.

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Figure 2.1: HPLC chromatograms of olive leaf extracts at 280nm showing the effect of

different drying methods on the phenolic compounds of olive leaves.

(1) unknown (2) verbascoside (3) luteolin-7-O-glucoside (4) rutin (5) oleuropein (6)

flavonoid derivative (7) flavonoid glycoside derivative (8) apigenin-7-O-rutinoside (9)

oleuroside.

81

Figure 2.2: HPLC chromatograms of olive leaf extracts of oven dried leaves at 105 °C,

comparing 280nm and 325nm wavelength and the corresponding ABTS activity.

(1) unknown (2) verbascoside (3) luteolin-7-O-glucoside (4) rutin (5) oleuropein (6)

flavonoid derivative (7) flavonoid glycoside derivative (8) apigenin-7-O-rutinoside (9)

oleuroside.

82

Meanwhile, a considerable increase in the recovery of secoiridoids upon

freeze-drying was observed, with oleuroside increasing three-fold, and

oleuropein, ten-fold compared to fresh leaves. The enhanced recovery of

verbascoside, a 15-fold increase, was the most eminent effect of freeze-drying

(Table 2.2). The effects of air-drying were more incongruous; where rutin

recovery was not significantly affected, verbascoside recovery was dropped

by more than 85% compared to fresh leaves. Luteolin 7-O-glucoside,

apigenin 7-O-rutinoside and oleuroside showed 30-40% increase in recovery

(Table 2.2). On the other hand, oleuropein recovery increased 8-9 fold. Oven-

drying at 60 °C consistently had detrimental effects on the recovery of all

studied biophenols, with losses between 30 and 85%. Apigenin-7-O-

rutinoside and oleuropein were the least affected biophenols by drying at 60

°C, while other compounds were more vulnerable (Table 2.2).

2.3.5. Cultivar and seasonal impact on biophenols and oleuropein

recovery

To confirm reproducibility of our findings, olive leaves from Leccino,

Arbequina and Hardy’s Mammoth cultivars were collected. Furthermore,

Frantoio olive leaves were re-collected in autumn. Samples were treated and

analysed as described above. Results were similar to the findings of the main

study on Frantoio cultivar collected during the summer season. Again the

extracts of oven dried leaves at 105 °C resulted in the highest oleuropein and

other biophenol recoveries and antioxidant activities. This indicates that our

findings are reproducible irrespective of the cultivar used or the season in

which the leaves were collected.

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Table 2.2: Quantity of major biophenols identified in Frantoio olive leaf.

Peak Biophenol Fresh Air-dried Freeze-dried Oven-dried 60 °C Oven-dried 105 °C

2 Verbascoside (mg/g DW)

(mg/g EM)

0.90 ± 0.07a

2.10 ± 0.15a

0.11 ± 0.01b

0.21 ± 0.02b

14.65 ± 0.80c

32.27 ± 1.77c

0.20 ± 0.01d

0.63 ± 0.03d

16.74 ± 1.16c

25.24 ± 1.75c

3 Luteolin-7-O-glucoside (mg/g DW)

(mg/g EM)

2.86 ± 0.07a

6.64 ± 0.16a

3.67 ± 0.03b

6.96 ± 0.05b

3.43 ± 0.14ab

7.56 ± 0.3ab

0.90 ± 0.05c

2.79 ± 0.17c

5.44 ± 0.37d

8.20 ± 0.55d

4 Rutin (mg/g DW)

(mg/g EM)

1.91 ± 0.01a

4.43 ± 0.02a

1.95 ± 0.14a

3.69 ± 0.26a

2.11 ± 0.20ac

4.64 ± 0.45ac

0.50 ± 0.03b

1.55 ± 0.10b

2.66 ± 0.25c

4.01 ± 0.38c

5 Oleuropein (mg/g DW)

(mg/g EM)

3.56 ± 0.02a

8.27 ± 0.05a

32.75 ± 1.47b

62.06 ± 2.79b

37.47 ± 8.43b

82.54 ± 18.56b

2.13 ± 0.34a

6.62 ± 1.06a

139.48 ± 9.49c

210.34 ± 9.48c

8 Apigenin-7-O-rutinoside (mg AGE/g DW)

(mg AGE/g EM)

2.64 ± 0.03a

6.13 ± 0.06a

3.37 ± 0.01b

6.39 ± 0.02b

2.26 ± 0.15ac

4.98 ± 0.33ac

1.85 ± 0.13c

5.76 ± 0.41c

2.79 ± 0.19a

4.20 ± 0.28a

9 Oleuroside (mg OLPE/g DW)

(mg OLPE/g EM)

0.68 ± 0.02a

1.57 ± 0.04a

0.98 ± 0.11a

1.85 ± 0.21a

2.54 ± 0.19b

5.60 ± 0.42b

0.10 ± 0.00a

0.31 ± 0.00a

5.75 ± 0.48c

8.67 ± 0.73c

Results are presented as mean ± standard deviation. Different superscripts in the same row indicate significant difference at p < 0.05. P: peak; DW: dry

weight; EM: extractable matter; AGE: Apigenin-7-O-glucoside equivalent; OLPE: oleuropein equivalent.

84

Figure 2.3: Effect of different drying treatments on oleuropein recovery from olive

leaves.

Different letters indicate significant difference at p<0.05. DW: dry weight; Air: air-

dried; FD: freeze-dried; OD 60: oven-dried at 60 °C; OD 105: oven-dried at 105

°C.

2.3.6. Sample pre-treatment and biophenol recovery

In plants, biophenols are well protected inside intact cells via

compartmentalization. Post-harvest processing can damage cells and expose

biophenols to air and metabolising enzymes. Ideally, plant material should be

analysed while fresh, immediately after collection so as to maintain sample

fidelity (P. G. Waterman and Mole, 1994). However, most of the time this

may not possible or feasible, and sample pre-treatment is mandatory to halt

or slow down biophenol degradation and subsequent deterioration or loss of

quality. Drying is the most commonly applied pre-treatment technique for

preservation of plant samples. Traditionally, air-drying under the sun or in

shade is the cheapest and most convenient method, yet due to prolonged

drying time (days) significant degradation can take place. On the other hand,

lyophilisation (freeze drying) is a subtle and effective preservation technique

85

that is quicker and more effective than air drying for biophenols in plant

samples (Obied, et al., 2008b). However, freeze-drying is an expensive

technique and requires initial freezing of samples. Abascal et al. claimed that

freeze-drying can cause pronounced changes in the composition of medicinal

plants (Abascal et al., 2005). Alternatively, oven drying is faster than the

aforementioned drying techniques. Drying could increase the biophenol

content by releasing bound phenolic compounds. Hot air drying degrades cell

walls and breaks down cellular constituents, while in freeze drying, cells are

damaged by ice crystals. Both drying techniques facilitate the extraction and

result in release of intra-cellular compounds, and increase biophenol recovery

and antioxidant activity (Ahmad-Qasem et al., 2013). Oven drying at

temperatures ≤ 60°C is considered conventionally suitable for medicinal

plants (Youngken, 2012) yet these temperatures may not be high enough to

deactivate metabolising enzymes. Furthermore, some enzymatic activities

can be augmented by heating (Mizobutsi et al., 2010). Oven drying at higher

temperatures rapidly denatures enzymes and shortens the drying time,

however, loss of volatile compounds, thermal degradation, and/or

polymerization are usually expected (Keinänen and Julkunen-Tiitto, 1996).

OLE was an official drug in the French pharmacopeia long before it made its

way to the European and the British Pharmacopoeias. The French

Pharmacopeia states that fresh olive leaves should be used for extraction, but,

shade-dried olive leaves are recommended according to the British and

European pharmacopoeias. The British and European pharmacopoeias

standardize olive leaves based on a minimum of 5% oleuropein. In the French

pharmacopeia (2008), olive leaves are standardized to contain a minimum of

0.015% luteolin-7-glucoside. Moreover, many commercial OLE are

86

advertised as extracts of fresh olive leaves. Based on our results a review of

the pharmacopeial and commercial procedures for preparation of OLE is

highly recommended.

2.4. Conclusions

Sample processing appears to have a paramount effect on the qualitative and

quantitative recovery of biophenols that widely vary based not only on the

plant but also on the tissue involved. Hence, the impact of drying conditions

on recovery of biophenols should not be considered a matter of convenience.

On the basis of above findings, the recommendation can be made that olive

leaves should be dehydrated at 105 °C for 3 h before extraction, in order to

achieve maximum recovery of oleuropein and other biophenols.

Further systematic research is required for the preparation of olive leaf

extract, as most of the commercial olive leaf extracts available in market are

prepared using fresh or air-dried leaves. Our work is of paramount importance

to nutraceutical, pharmaceutical and cosmetic industries utilising olive leaf

extract for its functional properties. As a minor investment in proper drying

of olive leaves can lead to up to 30 fold increase in biophenol recovery and

hence a parallel increase in profitability.

There is not a single method that is completely satisfactory for the accurate

determination of the total phenol content or total phenolic class content in plants.

Lack of specificity of the chemical methods for determination of “total” content has

been undervalued and misused in literature. The use of different methods is

complementary in understanding the phenolic composition of the sample but it

should be always emphasized that these methods do not have simple additive

87

function and cannot directly correlated with each other. Therefore, chromatographic

determination of the phenolic composition and antioxidant activity is becoming

essential for accurate estimation of the phenolic composition.

88

Chapter 3. The Impact of climatic conditions, season and

cultivar on biophenol content of olive leaves

3.1. Introduction

The leaf is a primary site of plant metabolism and a rich source of biophenols.

Biophenols are bioactive phenolic compounds that not only play a vital role in plant

defence mechanisms, but also possess nutritional and biological value for animals

and humans (Pometto et al., 2014). Olive leaves (Olea europaea L.) are a significant

source of biophenols and are available year round, while the life span of an olive

leaf on an adult tree is up to three years (Papoti and Tsimidou, 2009). Moreover,

olive leaves are relatively stable to enzymatic degradation as compared to olive

fruit/drupes, olive oil and olive mill waste (Paiva-Martins and Pinto, 2008). Olive

leaves are mainly used for animal feeding (Ranalli et al., 2005; Molina-Alcaide and

Yáñez-Ruiz, 2008; Boudhrioua et al., 2009), although their higher biophenol

content suggests that they could be a good nutraceutical and can be effectively used

in the preventative medicine, food and cosmetic industries (Bouaziz and Sayadi,

2005; Boudhrioua et al., 2009; Brahmi, et al., 2012b). Olive leaves have been used

as folk medicine since antiquity, and the first medicinal use of olive leaf dates back

to ancient Egypt (Durlu-Özkaya and Özkaya, 2011). However, during the last two

decades olive leaf biophenols have attracted considerable attention and a number

of studies have been performed investigating the health promoting properties of

these biophenols (Obied et al., 2012).

Since it is proposed that oxidative stress is a major cause of aging and age related

diseases, a number of studies have been performed on natural antioxidants to

counteract the adverse effects of oxidative stress. Natural antioxidant sources and

their potential applications as functional food or nutraceutical have been

89

extensively explored. Vegetables, fruits, wine, tea, seeds, onion bulbs, aromatic

plants and olive oil are widely touted for their natural antioxidant capacity

(Dimitrios, 2006), but olive leaves, which could have great potential as a

nutraceutical by preventing/attenuating the adverse effects of oxidative damage or

free radicals have not been explored comprehensively. Biophenols are thought to

be a natural and good source of antioxidants. A linear relationship has been

proposed between the biophenol content and antioxidant activity (Brahmi, et al.,

2012b). Olive leaves are a better source of antioxidants than olive fruits (Brahmi et

al., 2013). The higher antioxidant capacity of olive leaf biophenols is thought to be

central to the health promoting properties of olive leaves. Antioxidant activity of

olive leaves depends upon various factors like variety, age, harvesting time,

tissue/organ, as well as the solvent system used. When the olive leaves at two

different harvesting stages were compared for antioxidant activity, the mature

leaves surpassed the growing leaves and accumulation of biophenols was found to

be highest towards the end of leaf maturity (Brahmi, et al., 2012b; Brahmi et al.,

2013). Contrary to that, another study found no effect of leaf age on biophenol

content (Ryan et al., 2002a).

The biophenolic profile of olive leaf is more complex than those of pulp, seed, or

stone (Ryan et al., 2002a) and olive leaf biophenols vary both qualitatively and

quantitatively during different developmental stages of the biological cycle of olive

trees (Amiot et al., 1989). The biophenol composition of olive leaves can be

affected by several factors such as the cultivar/variety, tissue involved and the stage

of biological cycle (Brahmi et al., 2013), as well as environmental factors like

geographical origin (Papoti and Tsimidou, 2009), UV light, temperature, and

nutrition (Mert et al., 2013) . Furthermore, not only leaves of different cultivars

grown in same orchard under same set of environmental and cultural conditions

90

show variation but also leaves of the same cultivar collected at different time or

season show variation in biophenol contents (Papoti and Tsimidou, 2009; Brahmi

et al., 2013; Talhaoui et al., 2015). Climatic, pathological conditions and

agricultural practices also result in seasonal variations (Obied, et al., 2008c). In

addition to that, moisture content, sampling procedures and conditions and

extraction method may also influence biophenol content (Şahin et al., 2012). It has

been reported that the biophenol content of the plant increased in defence against a

microorganism attack during fruit ripening (Brahmi et al., 2013).

The olive tree has tendency of alternate bearing, which means it produces a high-

yield crop one year ("on-year") and no fruit or a low-yield the following year ("off-

year"). It is thought that the leaves are the ones which receive the initial signal for

alternate bearing, produced by the developing embryo. The leaves then induce

chemical changes in phenolic compounds. An inhibitor is produced by the leaves,

depending upon the intensity of the signal and the environmental conditions. The

changes in phenolic compounds of leaves, seize the flower bud formation during

the physiological initiation period (Ryan et al., 2003; Mert et al., 2013).

Seasonal variation in olive leaf biophenol content is an area which has not been

extensively explored. Although in the three years of my candidature some work has

been published in this area, there is no study which has covered the whole olive

fruit cycle. Moreover, most of the previous studies on seasonal and cultivar

differences were limited to the Mediterranean basin or the United States. To our

knowledge there has been no comprehensive study on cultivars grown in Australia.

This information is vital for any industrial application of olive leaves so that

optimum harvesting times can be chosen and the variability in biophenol recovery

due to cultivar and season is taken into consideration.

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This study was designed to (1) investigate differences in biophenol content of an

Australian cultivar, Hardy’s Mammoth, and from two Italian origin cultivars,

Frantoio and Leccino, grown in Wagga Wagga, Australia; (2) reveal the effect of

season and cultivar on biophenol content and antioxidant activity of olive leaves, in

order to determine the best harvest time for optimal recovery of biophenols from a

particular cultivar; and (3) determine correlation between biophenol content and

antioxidant activity.

3.2. Materials and methods

3.2.1. Chemicals and Reagents

Most of the reagents and standards and other consumables were obtained from the

same sources as described in Chapter 2, with a few exceptions. HPLC-grade

methanol was obtained from Merck, Melbourne, Australia and oleuropein was

obtained from Sigma-Aldrich (Sydney, Australia). All reagents and standards were

prepared in the same way as described in Chapter 2.

3.2.2. Collection of olive leaves

Olive leaves of three different varieties/cultivars (Frantoio, Hardy’s Mammoth and

Leccino) were collected from the CSU Olive Grove, Wagga Wagga, every second

month as well as monthly during the fruit season for, 28 months. The collection was

started in April 2012 and finished in July 2104. The mature green leaves were

handpicked at the operator height around the whole perimeter of three trees of the

same cultivar. Approximately 75 grams of leaves from each cultivar were collected

in each collection. Leaves were collected in plastic bags and brought to the

laboratory without any delay.

92

3.2.3. Drying of olive leaves

Fresh leaves were immediately subjected to drying in a forced-air oven at 105 °C

for 3 hours (Kamran et al., 2015).

3.2.4. Moisture content

Dry matter content was determined according to the method described earlier in

Chapter 2. Five grams of fresh leaves were placed in an evaporating dish. The dish

was transferred to a forced-air oven and dried at 105°C. The leaves were weighed

after every hour until the difference between two successive readings was less than

0.25%.

3.2.5. Extraction of biophenols

Biophenols were extracted according to the method described in Chapter 2. All

extractions were performed at room temperature (20 ± 2 °C) in triplicate for each

cultivar. The crude extracts were stored at – 20 °C until analysed.

3.2.6. Spectrophotometric measurements

Colorimetric analyses were performed on a Cary 50 UV/visible spectrophotometer,

using Cary WinUV “version 3” software (Varian, Australia). For dilution of crude

extracts, an extraction solvent (80% aqueous methanol) was used to produce

concentrations of extracts within the range of the constructed calibration curves.

For the determination of total phenols, o-diphenols, total phenolic compounds,

hydroxycinnamic acid derivatives, and flavonols, the crude extracts were diluted

(1:20). For the determination of total flavonoids, DPPH radical scavenging assay

and ABTS radical scavenging assay, the crude extracts were diluted 1:100. For

93

HPLC analysis, crude extracts were diluted 1:5 and 1:10. Three extracts of each

cultivar were analysed in triplicate. All dilutions were done in the extraction

solvent.

3.2.6.1. Determination of total phenols

Total phenol content was determined by using Folin-Ciocalteu reagent, according

to the method described in Chapter 2. Results were expressed as mg gallic acid

equivalent per gram dry weight (mg GAE/g DW).

3.2.6.2. Determination of total flavonoids

Total flavonoid content was determined with an AlCl3 colorimetric assay, as

described previously (Kamran et al., 2015). Total flavonoids content is expressed

as mg quercetin equivalents per gram dry weight (mg QE/g DW).

3.2.6.3. Determination of o-diphenols

o-Diphenol content was measured according to Kamran et al. (2015). Results are

expressed as mg caffeic acid equivalents per gram dry weight (mg CAE/g DW).

3.2.6.4. Determination of phenolic classes

Different phenolic classes were characterized as described by (Obied, et al., 2005b),

with minor modification. In a 10 mL volumetric flask, 500 µL of diluted extract

was taken and the volume was made up to 10 mL with 2% hydrochloric acid. To

determine total phenolic compounds, the absorbance was measured at 280 nm using

a six point gallic acid (25-400 µg/mL) calibration curve. Hydroxycinnamic acid

derivatives were measured at 320 nm using a five point caffeic acid (20-150 µg/mL)

94

calibration curve as standard, whereas flavonols were measured at 360 nm using a

five point (30-250 µg/mL) calibration curve with quercetin as standard.

3.2.6.5. Antioxidant activity

The antioxidant activity of olive leaf extracts was determined by measuring the

ABTS+ and DPPH radicals scavenging ability. Trolox was used as the standard for

antioxidant capacity. Trolox equivalent antioxidant capacity (TEAC) was defined

as the amount of trolox equivalent to the amount of crude extract that resulted in

equal scavenging of DPPH or ABTS. For DPPH a ten point (50-500 µg/mL)

calibration curve was prepared, whereas for ABTS seven point (25-300 µg/mL)

calibration curve was prepared using Trolox. The following formula was used to

calculate the percent scavenging

% scavenging =[Absorbance (control) − Absorbance (sample)]

Absorbance (control) × 100

ABTS•+ scavenging activity was measured according to a previously described

method (Kamran et al., 2015). Results are expressed as micromoles trolox

equivalent per gram dry weight (µM TE /g DW). The stable DPPH radical was used

to measure the radical scavenging ability of olive leaves as described earlier

(Kamran et al., 2015). Results are expressed as micromoles trolox equivalent per

gram dry weight (µM TE /g DW).

3.2.7. High Performance Liquid Chromatography (HPLC)

HPLC-DAD-online ABTS: For HPLC analysis, the same instruments and column

were used as described in a Chapter 2, but the solvents used for gradient elution

were different. Solvent A was a mixture of water/acetonitrile/ formic acid (50 + 50

+ 0.3, v/v/v), whereas solvent B was 10 mM ammonium acetate with 0.3% formic

95

acid. A flow rate of 0.8 mL/min and injection volume of 5 µL were used with a

gradient elution for total run time of 30 min. The run was as follows: initial

conditions 10% solvent A; solvent A was increased to 25% over 2 min; then solvent

A increased to 45% over 3 min; isocratic for 2 min, then solvent A increased to

80% over 13 min; then solvent A increased to 100% over 2 min; isocratic for 4 min,

solvent gradient was returned to initial conditions over 4 min. The system was

allowed to equilibrate for 5 min between runs.

3.2.8. Climatic data

Monthly rainfall and monthly mean daily global solar exposure data for the Wagga

Wagga Agricultural Institute station and monthly mean maximum temperature for

Wagga Wagga Aeronautical Meteorological Office station were retrieved from the

Australian Bureau of Metrology website(Australian Bureau of Meterology, 2016).

Data were used to find some explanation for observed changes in olive leaves’

composition.

3.2.9. Statistical analysis

All assays were performed in triplicate and averaged. Data analysis was performed

by Microsoft Excel and IBM SPSS Statistics package version 20.0 (IBM

Corporation, NY). Statistical comparisons were made using one-way ANOVA and

post-hoc Tukey HSD test. Results were considered statistically significant at p <

0.05. GraphPad Prism version 5.04 (GraphPad Software, San Diego, USA) was

used for the correlation analysis. Results in the text and Figures are expressed as

mean ± standard error of the mean.

96

3.3. Results and discussion

Sampling from the same cultivars was conducted throughout 28 months, covering

the full production cycle of olive trees, to determine the possible seasonal effects

on biophenol contents. The sampling started in 2012. In the previous year (2011),

a heavy crop load was received. The crop load was very low in 2012 as was

expected because of the alternate bearing nature of olive tree, but surprisingly, in

2013 again the crop load was very low, although a heavy crop load was obtained in

the 2014 season. The causes for low production during year 2013 may be due to

agricultural and environmental factors, though no significant environmental

changes could be observed.

In Australia, there are four seasons and the seasons are defined by grouping the

calendar months. The summer season consists of the three hottest months:

December, January and February. March, April and May represent the autumn

season. The three coldest months; June, July and August comprise the winter

season, and September, October and November are characterized as the spring

season. In Australia, olive trees produce flowers late in spring, while in the orchard

from where leaves were collected, olive trees produced flowers in December, which

is considered early summer. The fruit development and maturation occurred from

summer to autumn and the harvesting was done in early winter. In order to make

our data more meaningful, we divided the entire collection period season-wise and

statistically analysed the data for each parameter studied. The drying treatment was

selected for olive leaves on the basis of previous work (Kamran et al., 2015) and all

extracts analysed were obtained from leaves dried at 105 °C for three hours.

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3.3.1. Dry matter content

Samples were dried at 105 °C to determine the moisture content and hence the dry

matter (DM). The average DM of olive leaves of studied cultivars is shown in

Figure 3.1. There was no significant difference (p ˃ 0.05) in average DM values of

the different cultivars studied over the total period of collection. However,

significant differences (p < 0.05) were observed due to the month of collection and

also in the interaction between the collection time and cultivar. When the studied

cultivars were compared for seasonal variation, no specific pattern was observed

for any season throughout the collection period. Significant differences were

observed for dry matter content due to season (p < 0.05) but no differences were

observed due to cultivars as well as no interaction was observed between season

and cultivar (p ˃ 0.05).

The DM content was lowest in November 2012 in all cultivars, which resulted in

relatively lower DM content in spring. The highest DM content was in April 2013

(56.40 ± 0.13 %) in Hardy’s Mammoth cultivar, whereas lowest DM content was

also obtained in Hardy’s Mammoth in November 2012 (42.2 ± 0.18 %). The values

are consistent with those previously published for other olive cultivars of

Mediterranean and African origins; 44-50% (Gómez-Cabrera et al., 1992; Martín-

García and Molina-Alcaide, 2008; Nourhène et al., 2008; Lafka et al., 2013), apart

from November 2012 samples, where all studied cultivars produced dry matter

within the range of 42 to 43%.

Analysing climatic data in Figure 3.2, some reasonable explanations for seasonal

variation in DM can be deduced. The most significant change in DM was the major

drop in November 2012. Between September and October 2012 (Figure 3.2), there

was an exceptionally low rainfall period followed by a large increase in rainfall in

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November 2012 (the second highest rainfall value for the whole year). Dry weather

conditions are stressful to the olive plantation, thus nutrients (the major part of the

DM composition) are kept in leaves and are not directed to growth of flowering or

aerial parts. Once the heavy rain started in November 2012, the favourable climatic

conditions stimulated olive leaves to move their nutrients to the delayed flowering

process. On the other hand, the drop in DM in November 2013 (Figure 3.1) is not

prominent. This is because November 2013 was the second lowest month in rainfall

for 2013 (Figure 3.2). Similarly, the peak in DM in April 2013 and less prominent

peaking in April 2014 can be explained by a combination of physiological

maturation stage (fruiting) and climatic conditions. Before fruiting, leaves

accumulate nutrients to prepare the plant for the coming stage of increased energy

demand. In 2013, April had the lowest rainfall (climatic stress) so that accumulation

of nutrients in the survival parts of the plant (leaves) is vital. While in April 2014,

the peak is less prominent (Figure 3.1) due to the high rainfall during April 2014

(figure 3.2).

3.3.2. Total phenols (Folin-Ciocalteu)

Total phenol contents of olive leaves are presented in Figure 3.3. The total phenol

content changed significantly over time of collection (p < 0.05) but did not follow

a specific pattern for any month or season throughout the collection period, except

a drop in September each year. Significant differences were observed for total

phenol content among cultivars (p < 0.05), and an interaction between the

collection month and cultivar (p < 0.05) indicating that the cultivars differed in how

they changed over time. This is primarily due to the differences in the Hardy’s

Mammoth cultivar, which showed larger variation than other two cultivars. When

the samples were grouped into seasons, significant differences were observed for

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season and for interaction between season and cultivar (p < 0.05). In summer,

significant differences were seen between all cultivars. Total phenol content of

Frantoio was significantly higher (p < 0.05) than both Leccino and Hardy’s

Mammoth, while Leccino had a higher (p < 0.05) content than Hardy’s Mammoth.

However in autumn, winter and spring seasons no differences (p ˃ 0.05) were

observed among all cultivars. Meanwhile, in a Tunisian olive cultivar, higher total

phenol content in leaves was reported in winter as compared to autumn (Brahmi, et

al., 2012b). The highest quantity of total phenols was in April 2014 for Frantoio

(150.2 ± 2.43 mg GAE/g DW), while Hardy’s Mammoth produced the lowest total

phenols, in June 2012 (59.6 ± 0.80 mg GAE/g DW). The level of total phenols

remained low in all cultivars in 2012, compared to 2013 and 2014. Total phenol

levels increased in late spring and early summer 2013, during flowering, then

decreased again during fruit initiation. The levels increased in autumn 2014, during

fruit growth in all cultivars with the highest levels seen in April 2014. The levels

then again decreased in May 2014, during fruit maturation. Overall the Leccino

cultivar accumulated the highest total phenols (p < 0.05).

Our results are in agreement with the findings of Franco et al. (2014) who reported

a decrease in total phenol content during fruit maturation although this has not

observed in all studied varieties. Our results fall within the reported literature values

59-144 mg/g DW (Orak et al., 2012; Ahmad-Qasem et al., 2013; Salah et al., 2013).

The total phenol contents of our study are relatively high compared to reported

literature values for European, Middle East and African varieties (Abaza et al.,

2011; Brahmi et al., 2013; Lafka et al., 2013; Al-Rimawi et al., 2014).

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Figure 3.1: Dry matter of olive leaves 2012 to 2014.

Same letters on the bars indicate no significant differences (p ˃ 0.05).

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Figure 3.2: Climate variation over the experimental timeframe.

Avr max Temp: average maximum temperature; Solar Exp: solar exposure

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Figure 3.3: Total phenols of olive leaves 2012 to 2014.

Different letters on the bars indicate significant differences (p < 0.05).

103

Drying resulted in up to 2 fold increased total phenol contents on a dry weight basis,

as evident from the study in the previous chapter (Chapter 2). Ahmad-Qasem et al.

(2013) also reported increased total phenol content with drying, though the increase

was not as high as was in the previous chapter of this thesis. In another Australian

study, Goldsmith et al. (2015) reported 236 mg /g total phenols in Frantoio leaves,

where they dried olive leaves at 120 ºC for 90 minutes and then concentrated the

extract by reducing the volume. The higher total phenol content in this study may

be due to concentrated extract.

Studying the phenolic content of olive leaves from three cultivars (carefully chosen

to have wide variation in their genotypes) over three harvesting seasons (28 months)

has not been tried before. This creates an unprecedented wealth of information but

at the same time it produces an extremely complex (real life) profile (Figure 3.3)

due to the interaction of innumerable number of biotic and abiotic factors under

uncontrolled environmental and climatic conditions with the genetic makeup.

Although there is a general tendency that total biophenol level in leaves tends to

upsurge in cold winter season (Talhaoui et al., 2015), this trend was not very evident

in our case. In 2012, there was some accumulation of total phenols towards winter

(July 2012), yet the drop in September (spring) is followed by a gradual increase in

total phenols during the summer season, where highest total phenol content for the

year was reported in December (summer). It worth noting that 2012 was the driest

among the three winter seasons studied (Figure 3.2). In 2013, only Hardy’s

Mammoth leaves showed their highest total phenol peak in winter, while Leccino

and Frantoio had their highest peaks in spring and autumn, respectively. What both

2012 and 2013 had in common is that the lowest recovery was achieved in early

spring (September).

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3.3.3. Total flavonoids

Figure 3.4 represents the total flavonoid content of olive leaves. The level of total

flavonoids varied significantly among cultivars, over time (p < 0.05) and there was

an interaction between cultivar and time (p < 0.05). An interaction between the

collection month and cultivar indicates that the cultivars varied in how the total

flavonoid levels changed over time. This is mainly due to the cultivar Hardy’s

Mammoth, which showed larger changes than the other cultivars. Leccino and

Frantoio cultivars showed almost same behaviour in terms of accumulation of total

flavonoids. No obvious annual rhythms were observed for seasons throughout the

collection period apart from a decrease in September each year that coincides with

our earlier observation about total phenols. When the months were grouped into

seasons, significant differences were observed for season (p < 0.05) and for an

interaction between season and cultivar (p < 0.05). In winter, Hardy’s Mammoth

produced higher (p < 0.05) levels of total flavonoids than Frantoio and Leccino,

with Frantoio and Leccino not different (p ˃ 0.05) from each other. On the other

hand, in summer Frantoio produced significantly higher (p < 0.05) total flavonoids

than Hardy’s Mammoth and Leccino but Hardy’s Mammoth and Leccino were not

different from each other (p ˃ 0.05). No differences were observed among all

cultivars in autumn and spring season (p ˃ 0.05). Leaves of a Tunisian cultivar

collected in winter resulted in higher total flavonoid contents as compared to

autumn leaves (Brahmi, et al., 2012b). In the present study, the summer season

showed the highest and spring the lowest total flavonoids. The level of total

flavonoids in leaves increased during flowering but remained variable during fruit

growth and maturation. Hardy’s Mammoth produced the highest quantity (810.1 ±

8.06 mg QE/g DW) of total flavonoids, in April 2013, whereas the lowest quantity

was produced by Leccino (158.9 ± 4.03 mg QE/g DW), in November 2012. Overall

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the Hardy’s Mammoth cultivar produced the highest total flavonoids (p < 0.05).

Our results are in agreement with those obtained by Goldsmith et al. (2015), who

reported 528.5 mg/g DW of total flavonoids for the Frantoio cultivar. This is similar

to our average total flavonoids content, which ranged from 501 to 533 mg/g dry

weight. Like total phenol contents, total flavonoids also showed higher values than

the values reported for leaves of European, Middle East and African origin (Papoti

and Tsimidou, 2009; Abaza et al., 2011; Orak et al., 2012; Brahmi et al., 2013; Al-

Rimawi et al., 2014; Talhaoui et al., 2015).

3.3.4. o-Diphenols

o-Diphenol contents of olive leaves are shown in Figure 3.5. o-diphenol content

varied significantly over time of collection (p < 0.05) although no specific pattern

was observed for any month or season throughout the collection period, with two

exceptions. Firstly, there was a drop in May each year for Hardy’s Mammoth and

Leccino cultivars, while for Frantoio only in May 2014. The o-diphenol levels

recovered for the June samples, except the Leccino sample of June 2013, which

decreased further. Secondly there was a drop in September each year for Hardy’s

Mammoth cultivar only, which recovered in the November samples. Significant

differences were observed for o-diphenol content between cultivars (p < 0.05), and

an interaction between the collection month and cultivar (p < 0.05), indicating that

the cultivars differed in how they changed over time. This is primarily due to the

differences in the Leccino cultivar, which frequently showed higher levels than the

other two cultivars. The Hardy’s Mammoth cultivar also showed lower o-diphenol

levels than the other two cultivars from May to July 2012. When the samples were

grouped into seasons, significant differences were observed for season and for

interaction between season and cultivar (p < 0.05). In all four seasons, the Leccino

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cultivar produced significantly higher o-diphenols (p < 0.05) than Frantoio and

Hardy’s Mammoth. In the autumn, winter and summer seasons, the Frantoio

cultivar produced significantly higher o-diphenols than Hardy’s Mammoth cultivar

(p < 0.05), while in spring, Frantoio and Hardy’s Mammoth were not different from

each other (p ˃ 0.05).

The level of o-diphenols in leaves increased during flowering then decreased during

fruit initiation, but remained variable during fruit growth and maturation. The

maximum o-diphenol content was obtained in November 2013 with Leccino (97.7

± 0.40 mg CAE/g DW) and Hardy’s Mammoth produced the lowest content, in

January 2013 (34.6 ± 0.27 mg CAE/g DW). Overall, the Leccino cultivar

accumulated the highest o-diphenols (p < 0.05). In the present study, the winter and

spring seasons showed almost similar level of o-diphenols, which were better than

autumn and summer. Furthermore autumn was better than summer with regards to

o-diphenol contents. Similar findings were observed in leaves of a Tunisian cultivar

(Brahmi, et al., 2012b).

Our results for o-diphenols contents are in agreement with our previous study

(Kamran et al., 2015) but were considerably higher than reported literature values

0.88 to 2.42 mg/g DW (Brahmi, et al., 2012b; Brahmi et al., 2013). Drying resulted

in only a 50 % increase in o-diphenols contents as evident from previous work

(Chapter 2). Our data, however indicate a much higher o-diphenol content than

reported values. Limited data was available on o-diphenol content of Tunisian olive

leaves and we were not able to retrieve much data from other geographical

locations.

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Figure 3.4: Total flavonoids of olive leaves 2012 to 2014.

Different letters on the bars indicate significant differences (p < 0.05).

108

Figure 3.5: o -diphenols of olive leaves 2012 to 2014.

Different letters on the bars indicate significant differences (p < 0.05).

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3.3.5. Total Phenolic Compounds (280 nm)

Phenolic content (absorbance at 280 nm) of olive leaves is presented in Figure 3.6.

Phenolic compounds also varied significantly among cultivars and over time (p <

0.05), and there was an interaction between cultivar and time (p < 0.05), which

indicates that the cultivars varied in how the phenolic compounds level changed

over time. This is mainly because of cultivar Leccino showing a spike in phenolic

compound levels in November 2013 only. No specific annual rhythms were

observed for the collection period. When the data grouped into seasons, significant

differences were observed for season (p < 0.05) and for interaction between season

and cultivar (p < 0.05). The differences were season specific, as in autumn, winter

and summer, the Leccino cultivar produced significantly higher phenolic

compounds (p < 0.05) compared to Frantoio and Hardy’s Mammoth, while Frantoio

and Hardy’s Mammoth were not different from each other (p ˃ 0.05). In spring,

Leccino cultivar produced significantly higher phenolic compounds (p < 0.05) than

Frantoio, but the Hardy’s Mammoth was not different from Leccino and Frantoio

(p ˃ 0.05).

The level of phenolic compounds in leaves increased during flowering then

decreased during fruit initiation, remained stable during fruit growth and then again

decreased at maturation. The highest level of phenolic compounds was noticed in

November 2013, with Leccino (89.3 ± 1.8 mg GAE/g DW) and the lowest level

was recorded in November 2012 for the Frantoio cultivar (26.7 ± 0.30 mg GAE/g

DW). Overall, the Leccino cultivar accumulated higher phenolic compounds than

other cultivars (p < 0.05). The winter season showed the highest level of phenolic

compounds, which comes in accord with earlier observations (Talhaoui et al.,

2015).

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Figure 3.6: Phenolic compounds (280 nm) of olive leaves 2012 to 2014.

Different letters on the bars indicate significant differences (p < 0.05).

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3.3.6. Hydroxycinnamic acid derivatives

Figure 3.7 represents the hydroxycinnamic acid derivatives of olive leaves.

Hydroxycinnamic acid derivatives also varied significantly among cultivars, over

time (p < 0.05) and there was an interaction between cultivar and time (p < 0.05),

which indicate that the cultivars varied in how the level of hydroxycinnamic acid

derivatives changed over time. There were no obvious annual rhythms observed

throughout the collection period. When the data were grouped into seasons,

significant differences were observed for season (p < 0.05) and for the interaction

between season and cultivar (p < 0.05). In all four seasons, Leccino cultivar

produced significantly higher hydroxycinnamic acid derivatives (p < 0.05) as

compared to Frantoio and Hardy’s Mammoth. The Frantoio cultivar produced

significantly higher hydroxycinnamic acid derivatives than Hardy’s Mammoth

cultivar in autumn and winter (p < 0.05), but produced the same levels in spring

and summer (p ˃ 0.05).

The level of hydroxycinnamic acid derivatives in leaves increased during flowering

then decreased during fruit initiation, and remained variable during fruit growth and

maturation. The highest level of hydroxycinnamic acid derivatives was recorded in

November 2013 (30.2 ± 0.60 mg CAE/g DW) for Leccino, while the lowest level

was noticed in November 2012 (8.3 ± 0.10 mg CAE/g DW) for Frantoio. Our results

are in accord with the findings of (Mert et al., 2013), who reported from 5 to 32.7

mg/g of 3-hydrocinnamic acid. Our results are also close to the value reported in a

previous study (Obied, et al., 2005b), where levels were reported up to 8.22 mg/g

in olive mill waste. Overall, the Leccino cultivar accumulated more

hydroxycinnamic acid derivatives than the other cultivars (p < 0.05). Generally, the

winter and spring seasons showed almost similar levels, which

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Figure 3.7: Hydroxycinnamic acid derivatives of olive leaves 2012 to 2014.

Different letters on the bars indicate significant differences (p < 0.05).

113

were higher than the autumn, with the lowest levels in summer. Our findings are in

agreement with the study of Mert et al. (2013), who reported higher levels of

hydroxycinnamic acid, p-coumaric acid and caffeic acid in winter and spring as

compared to autumn, with lower levels in summer.

3.3.7. Flavonols

Flavonol contents are shown in Figure 3.8. Flavonol contents changed significantly

over time of collection (p < 0.05) but did not follow a specific pattern for any month

throughout the collection period. Significant differences were observed for flavonol

contents among cultivars (p < 0.05), and an interaction between the collection

month and cultivar (p < 0.05) indicating that the cultivars differed in how they

changed over time. This is primarily due to the Frantoio cultivar, which had higher

flavonol levels during the first 12 months of the collection period, although Leccino

and Hardy’s Mammoth cultivars showed almost the same trend in terms of

accumulation of flavonol contents. When the samples were grouped into seasons,

significant differences were observed for season and for the interaction between

season and cultivar (p < 0.05). In all four seasons, the Leccino cultivar produced

significantly higher flavonols (p < 0.05) compared to Frantoio and Hardy’s

Mammoth, while Frantoio and Hardy’s Mammoth were not different from each

other (p ˃ 0.05). The level of flavonols in leaves increased during flowering then

decreased during fruit initiation. The levels increased during fruit growth and then

again decreased at maturation. Flavonols were noticed at maximum in November

2013 in Leccino (24.9 ± 0.57 mg QE/g DW) whereas the lowest levels were

recorded in November 2012 in Frantoio (8.3 ± 0.10 mg QE/g DW).

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Figure 3.8: Flavonols content of olive leaves 2012 to 2014.

Different letters on the bars indicate significant differences (p < 0.05).

115

Overall the Leccino cultivar produced higher flavonols than other cultivars (p <

0.05). The winter and spring seasons showed similar levels, which were higher than

autumn, which had slightly more flavonols than summer. Our results for flavonol

contents are similar to the findings of Obied, et al., (2005b), who reported 8.85 mg/g

in olive mill waste. Other than this, data on the flavonol contents of olive leaves is

scarce and we were not able to compare our results with other studies.

3.3.8. Antioxidant activity

3.3.8.1. ABTS

The ABTS radical scavenging activity of olive leaves is displayed in Figure 3.9.

ABTS radical scavenging capacity of olive leaves varied significantly among

cultivars (p < 0.05) and over time of collection (p < 0.05) and there was an

interaction between cultivar and time of collection (p < 0.05). An interaction

between the collection month and cultivar indicates that the cultivars varied in how

the ABTS radical scavenging activity changed over time. No noticeable annual

rhythms observed throughout the collection period apart from a decrease in

September each year. When the data grouped into seasons, significant differences

were observed for season (p < 0.05) and for the interaction between season and

cultivar (p < 0.05). In summer, Hardy’s Mammoth cultivar showed significantly

less ABTS radical scavenging activity (p < 0.05) than Frantoio and Leccino

cultivars, which were not different from each other (p ˃ 0.05). In the spring, autumn

and winter seasons, all the three cultivars were not different from each other (p ˃

0.05).

The ABTS•+ radical scavenging activity of olive leaves increased during flowering

then decreased during fruit initiation, remained variable during fruit growth and

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maturation. The highest ABTS•+ radical scavenging activity was noticed in

November 2013 in Leccino (786 ± 6.63 µ Mol TEAC/g DW) while the lowest

activity was in November 2012, in the Frantoio cultivar (331 ± 5.83 µ Mol TEAC/g

DW). Overall, Leccino cultivar showed higher ABTS radical scavenging activity

than other cultivars (p < 0.05). Our results are in agreement with the findings of

Abaza et al. (2011) who reported ABTS activity values of olive leaves up to 1064.25

µMol TEAC/g DW. In conclusion, winter and summer seasons showed the same

capacity, which was higher than the autumn and spring seasons, whereas autumn

was better than spring season in terms of ABTS radical scavenging activity.

3.3.8.2. DPPH

The DPPH radical scavenging activity of olive leaves is presented in Figure 3.10.

The DPPH radical scavenging capacity of olive leaves varied significantly among

cultivars (p < 0.05) and over time of collection (p < 0.05), and there was an

interaction between cultivar and time of collection (p < 0.05).This is mainly due to

great variation in the last 10 months of collection, although all cultivars followed

almost the same general trends. There were no clear annual rhythms observed

throughout the collection period apart from a decrease in September each year.

When the data was grouped into seasons, significant differences were observed for

season (p < 0.05) and for interaction between season and cultivar (p < 0.05). In the

summer season, the Leccino cultivar showed significantly higher DPPH radical

scavenging activity (p < 0.05) than Hardy’s Mammoth cultivar, however Frantoio

and Leccino were not different from each other (p ˃ 0.05).

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Figure 3.9: ABTS radical scavenging activity of olive leaves 2012 to 2014.

Different letters on the bars indicate significant differences (p < 0.05).

118

Figure 3.10: DPPH radical scavenging activity of olive leaves 2012 to 2014.

Different letters on the bars indicate significant differences (p < 0.05).

119

In the spring, autumn and winter seasons none of the cultivars were significantly

different from each other (p ˃ 0.05). The DPPH radical scavenging activity of olive

leaves increased during flowering then decreased during fruit initiation and growth,

then again increased during maturation. DPPH radical scavenging activity was

recorded highest in November 2013 for Leccino (913 ± 8.17 µMol TEAC/g DW),

while the lowest activity was also noticed for Leccino in March 2014 (159 ± 4.40

µMol TEAC/g DW). Overall, the Leccino cultivar showed higher DPPH radical

scavenging activity than other cultivars (p < 0.05), though a cultivar effect on

antioxidant potential of olive leaves has been reported to be partial (Papoti and

Tsimidou, 2009). DPPH radical scavenging activity of olive leaves for seasons

decreased in the following order: summer ˃ winter ˃ spring ˃ autumn.

The results of ABTS and DPPH radical scavenging activity for the same extract

were not the same, as these two assays are different. Differences in the results of

the two assays have also been reported previously (Abaza et al., 2011; Brahmi et

al., 2013). Olive leaves are a good source of antioxidants year-round as indicated

by the results. Papoti and Tsimidou (2009) also reported similar findings and

suggested their suitability for commercialization throughout the year. A good

antioxidant activity has been noticed for all cultivars throughout the collection

period.

OLEs prepared in the study were high in oleuropein content and it has been

proposed previously that the structures of secoiridoids are closely correlated to

antioxidant activity (Goulas et al., 2012). The antioxidant potency of the biophenol

depends on its structure. Goulas et al. (2012) proposed that the number and position

of hydroxyl groups on the aromatic ring as well as the structure of elenolic acid

(open; dialdehydic form or closed; aldehydic form) are important factors for

120

determining the antioxidant activity of olive biophenols. Biophenols that have two

hydroxyl groups linked to an aromatic ring on the ortho position displayed higher

antioxidant activities compared to monohydroxy-substituted ring biophenols and

when the ortho-dihydroxyl groups from aromatic ring are absent, the antioxidant

activity reduced, which is evident in case of tyrosol, which has a 3-fold lower

antioxidant activity as compared to hydroxytyrosol (Goulas et al., 2012).

3.3.9. High Performance Liquid Chromatography (HPLC) analysis

Though a different HPLC method was developed for this study, and different

solvents were used from that of the previous work (Chapter 2), the same biophenolic

compounds were observed for all cultivars as were observed in that work. . The

method developed for this study resulted in rapid and clear separation of olive leaf

biophenols. The HPLC results for the olive leaf extracts revealed only quantitative

differences but no qualitative differences among all studied cultivars throughout the

collection period. Talhaoui et al. (2015) reported similar findings with olive leaves

of six Spanish cultivars at different growth stages. Our results are also in agreement

with the study of Salah et al. (2013) who reported no qualitative differences among

eight cultivars of different origins grown in the same place under same

geographical, geological, climatic and agricultural conditions. On the contrary,

Brahmi et al. (2013) determined the variation in phenolic contents in two Tunisian

cultivars in two seasons and reported significant quantitative and qualitative

differences in phenolic compounds. Furthermore they proposed that these

differences were due to modulation of biosynthetic pathways which are involved in

important ecological and biological functions, like the plant’s defence mechanism

against pathogens. The authors emphasized that the reason for variation was the

plant needs. Geographical differences, in addition to different cultivars, might come

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into account for quantitative variation, as effect of origin, climate and agricultural

practices have also been reported in some studies (Ansari et al., 2011; Şahin et al.,

2012).

3.3.9.1. Oleuropein

The oleuropein content significantly varied over time (p < 0.05) throughout the

collection period (Figure 3.11) but did not follow any specific pattern, apart from

an apparent drop in both September samples, which recovered by November. There

were significant differences in oleuropein content among cultivars (p < 0.05) and a

cultivar-time interaction (p < 0.05). This was due to large changes in all cultivars,

particularly in Hardy’s Mammoth. When the samples were grouped into season,

none of the cultivars significantly differed (p ˃ 0.05) from each other. However

significant differences were observed due to season and an interaction between

season and cultivar (p < 0.05). The level of oleuropein in leaves increased during

flowering and fruiting. The level then decreased during fruit growth and remained

variable during maturation. Frantoio leaves yielded the highest quantity of

oleuropein, in January 2014 (221 ± 11.28 mg /g DW) and the lowest in September

2012 (25.2 ± 0.27 mg /g DW). Both Hardy’s Mammoth and Leccino cultivars

produced their maximum oleuropein yield in July 2013. For the entire collection

period, the highest oleuropein content was observed in Hardy’s Mammoth (p <

0.05) and the lowest in Leccino (p < 0.05). Generally, oleuropein levels were the

highest in summer, whereas autumn and winter seasons showed similar level, which

was higher than spring season. If we look at the yearly data, then the summer of

2013-14 produced higher oleuropein than summer of 2012-13. Overall higher levels

of oleuropein were noticed in 2013 as compared to 2014 (Figure 3.11). In 2014,

significant amount of fruit was produced by the trees, so it was an “on year”.

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Figure 3.11: Oleuropein content of olive leaves 2012 to 2014.

Different letters on the bars indicate significant differences (p < 0.05).

123

Our findings are in agreement with Mert et al. (2013), who reported lower levels of

oleuropein in an “on year” as compared to an “off year” and the lowest level was

found during embryo development and the pit-hardening stage, whereas the highest

level was reported in a dormant period. Furthermore, (Malik and Bradford, 2006)

reported an increasing trend of oleuropein from the flowering to fruiting stage, after

floral bud differentiation and then a decreasing trend during fruit maturation. In this

study, oleuropein levels from the flowering to fruiting were high, while lower levels

of oleuropein during fruit maturation was observed. In a study in the USA, the

Leccino cultivar produced the highest level (148.32 mg/g fresh weight) of

oleuropein (Şahin et al., 2012). The authors reported that the same cultivar showed

significantly lower oleuropein content at different collection times. These findings

coincide with our findings that the time of collection had significant effect on

oleuropein content. Similar findings were observed by Papoti and Tsimidou (2009)

who reported that two Greek cultivars showed higher oleuropein content one year,

but the same cultivars did not show the same trend the next year, and the cultivars

which showed low levels in the first year, accumulated moderate levels the next

year. Şahin et al. (2012) reported that both cultivar and season of collection affected

the oleuropein content, whereas Papoti and Tsimidou (2009) have the opinion that

oleuropein recovery depends on time of collection, irrespective of cultivar.

Caffeic acid and chlorogenic acid are said to play a central role in alternate bearing

(Mert et al., 2013). It has been reported that concentrations of chlorogenic acid are

usually 3 to 4 times higher in the mature leaves of the previous season of fruit-

bearing trees as compared to the leaves of trees without fruit (Ryan et al., 2003).

The changes in the levels of chlorogenic acid occurring during the flowering and

fruit set is involved in alternate bearing (Ryan et al., 2003).

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The changes in the levels of chlorogenic acid occurring during the flowering and

fruit set is involved in alternate bearing (Ryan et al., 2003). However, relatively low

hydroxycinnamic acid derivatives were noted in summer 2012-13, which was

flowering to fruit set period. The lower production might also be linked with

cinnamic acid, which is thought to play a role in crop load (Lavee et al., 1986; Mert

et al., 2013). However, this could not be verified in the current study due to the

absence of these compounds in our sample.

3.3.10. Correlation analysis

Correlation analysis of data revealed moderately positive (r2 = 0.51) correlation

between total flavonoid content and antioxidant activity, measured by ABTS and

DPPH radical scavenging assays (Figure 3.12 a and b). Oleuropein showed

moderately positive correlation (r2 = 0.57) with total flavonoids (Figure 3.13 a) and

ABTS activity (r2 = 0.51, Figure 3.13 b). However oleuropein showed a weak

correlation with total phenols (r2 = 0.25, Figure 3.14 a) DPPH activity (r2 = 0.29,

Figure 3.14 b), and phenolic compounds (r2 = 0.26, Figure 3.15 a). Total phenol

content also displayed a weak correlation (r2 = 0.32) with ABTS activity (Figure

3.16 a) but surprisingly, total phenol content showed no correlation (r2 = 0.004)

with DPPH activity (Figure 3.16 b). On the other hand, oleuropein also

demonstrated no correlation with o-diphenol contents (r2 = 0.0014, Figure 3.15 b),

hydroxycinnamic acid derivatives (r2 = 0.0014, Figure 3.17 a) and flavonols (r2 =

0.053, Figure 3.17b). The trend of oleuropein content and antioxidant activity

throughout the collection period is presented in Figure 3.18. Oleuropein displayed

almost the same trend as the ABTS and DPPH scavenging activity for most of the

months, with only a few exceptions. This trend is consistent with the correlation of

oleuropein content and antioxidant activity (Figure 3.13b and 3.14b).

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Figure 3.12: Correlation between total flavonoids and a) ABTS, b) DPPH.

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Figure 3.13: Correlation between oleuropein and a) total flavonoids, b) ABTS.

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Figure 3.14: Correlation between oleuropein and a) total phenols, b) DPPH.

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Figure 3.15: Correlation between oleuropein and a) phenolic compounds, b) o-diphenols.

129

Figure 3.16: Correlation between total phenols and a) ABTS, b) DPPH.

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Figure 3.17: Correlation between oleuropein and a) hydroxycinnamic acid derivatives, b)

flavonols.

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Usually it has been commented that higher antioxidant capacity of olive leaves is

credited to high oleuropein content (Salah et al., 2013). Correlation analysis of our

data displayed weak to moderate correlation of oleuropein and flavonoids with

ABTS and DPPH radical scavenging activity. Though Salah et al. (2013) reported

no correlation between oleuropein and anti-radical activity, they still suggested that

high anti-radical activity of some varieties relates to high oleuropein content. Our

extracts also exhibited high antioxidant activities and oleuropein content but still

we found only moderate correlation between them. Almost no correlation was

found between total phenols and DPPH activity, whereas a weak correlation was

found between total phenols and ABTS. Though the antioxidant activity of olive

leaves was correlated with biophenols, both phenolic and flavonoid compounds are

responsible for the high antioxidant activity of olive leaves (Lee et al., 2009;

Brahmi, et al., 2012b; Salah et al., 2013). Abaza et al. (2011) suggested that the

trolox equivalent antioxidant activity depends more on flavonoids than on total

phenols. Nevertheless, it has been suggested that in addition to oleuropein, other

secoiridoids and flavonoids also contribute to the antioxidant potential of olive leaf.

On the same level of total phenols, variation in antioxidant activity has also been

reported and the synergistic behaviour of olive leaf biophenols is also a very well

documented phenomenon (Benavente-Garcıa et al., 2000; Singh et al., 2008; Papoti

and Tsimidou, 2009).

The results of this study present relatively high biophenol contents compared to

reported values. The probable reason for that is the drying treatment, as reported in

the Chapter 2. The drying resulted in higher biophenol recoveries and antioxidant

activities from oven-dried leaves at 105 °C as compared with fresh olive leaf

extracts. Moreover, the quantitative differences in biophenol contents reported in

the literature depend on genetic and environmental factors, as outlined before.

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Figure 3.18: Trend of antioxidant activity and oleuropein content of olive leaves 2012 to 2014.

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Variation in the composition of olive leaf extracts might be due to developmental

stages of the tree rather than the cultivar itself, and results cannot be comparable,

particularly when the sampling is done in different seasons and from the trees of

different geographical regions (Papoti and Tsimidou, 2009; Şahin et al., 2012). In

our study, only mature leaves were collected at each collection time from the trees.

Leaf age has demonstrated a significant effect on biophenolic composition (Papoti

and Tsimidou, 2009; Brahmi, et al., 2012b; Brahmi et al., 2013). Mature olive

leaves are reported to have 1.5 and 3.5 fold higher oleuropein content as compared

to yellow and new leaves, respectively (Papoti and Tsimidou, 2009).

A controversy exists regarding the time or season of olive leaf collection for optimal

biophenol contents. A few studies have reported that the cold season is better than

the warm season for collection of olive leaves in order to get higher recovery of

biophenol content (Hashemi et al., 2010; Brahmi et al., 2013). Talhaoui et al. (2015)

proposed summer and winter months (June and December) for leaf collection, as

they noticed higher concentrations in those months, and lower in August. On the

other hand, Şahin et al. (2012) reported a trend for olive leaf biophenols to decrease

in hot summer days, with higher content in spring. They recommended sampling in

spring. Nonetheless, Mert et al. (2013) reported higher phenolic content in olive

leaves when no fruit was produced on the trees (off year) as compared to an on year.

A probable reason for this contradiction may be differences in geography, climate,

cultivars and cultural practices.

3.4. Conclusion

Our result showed no reproducible pattern for the distribution of biophenolic

compounds in a particular season of different years. From these results it can be

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assumed that accumulation of biophenols in olive leaves under uncontrolled

conditions is a very complex process, where a suite of factors interact with each

other to determine the final outcome. Shorter term studies where one or two seasons

are examined can lead to oversimplification. Brahmi, et al. (2012b) reported a

seasonal pattern of increasing total phenols, o-diphenols and flavonoid contents

between the leaves harvested at two different stages (autumn and winter). Their

findings cannot be compared with the results of our study due to the fact that they

only used two harvesting times and sampling was done once, not in the following

year.

Harvesting time has a very significant effect on the phenol content and antioxidant

activity of olive leaves. We support the earlier conclusion (Papoti and Tsimidou,

2009) that the level of individual biophenols depends more upon the sampling

period than on cultivar or age of leaves. We also confirm that individual compounds

peak at different times(Heimler et al., 1996). Season and cultivar had an impact on

biophenol content, as can be seen from the results, and similar observations have

been reported previously by other authors (Hashemi et al., 2010; Şahin et al., 2012;

Brahmi et al., 2013; Talhaoui et al., 2015).

The present study pointed out that the time of collection is the most influential

parameter with regards to olive leaves suitability for commercialization. This study

demonstrated that for maximum recoveries of total phenolic compounds and higher

antioxidant activity, Leccino cultivar was the most appropriate of the cultivars

tested, whereas in order to get higher total flavonoids and oleuropein content,

Hardy’s Mammoth was the best cultivar. As far as season is concerned, the results

indicate that the summer season was better for optimal antioxidant activity and for

maximum recovery of biophenols including oleuropein. Oleuropein is the major

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contributor to antioxidant activity of olive leaves, yet there is a significant

synergism between oleuropein and other antioxidant biophenols.

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Chapter 4. Comparative study of in-vitro

antioxidant and antidiabetic activities of

Australian olive leaf extracts

4.1. Introduction

Metabolic syndrome is a condition where risk factors for developing cardiovascular

disease and type 2 diabetes cluster together. Although in scientific publications, the

definition of metabolic syndrome may differ to some extent, its characteristic

features or components are the same. These include obesity, insulin resistance,

impaired glucose tolerance, hypertension, and dyslipidemia as major changes. In

addition, a prothrombotic state, increased fibrinogen, increased blood viscosity,

elevated microalbuminuria, increased uric acid, decreased plasminogen activator

and elevated plasminogen activator inhibitor-1 are minor changes ( Hansen, 1999).

Metabolic syndrome is thought to be related with oxidative stress, and high

oxidative damage has been noticed in patients with metabolic syndrome

(Panagiotakos and Polychronopoulos, 2005; Roberts and Sindhu, 2009). Obesity is

the major risk factor or contributor to metabolic syndrome and it has been reported

that oxidative stress is involved in the pathogenic mechanism of obesity, while fat

accumulation is correlated with oxidative stress in humans and mice (Furukawa et

al., 2004). Oxidative stress thought to be responsible for DNA damage, lipid

peroxidation, protein modification and may also result in the development of

cancer, cardio-vascular diseases, atherosclerosis, Alzheimer's disease, and

Parkinson's disease (Jomova and Valko, 2011). Oxidative stress is also thought to

be the one of the main culprits in the pathogenesis of diabetes mellitus and many

age related diseases.

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Free radicals initiate or take part in oxidative reactions and a single radical can start

a sequence of electron transfer reactions. A free radical has a single electron in its

outer orbit and it gets electrons from surrounding molecules to produce an electron

pair, as a result of which the donor molecule becomes free radical, the process can

be ongoing until a free radical reacts with antioxidant. Antioxidants donate an

electron to the free radicals, to prevent oxidation. When the antioxidants donate an

electron to the free radicals, the chain reaction is broken because the antioxidant

forms a stable product (Nwose et al., 2007). According to Ou et al. (2002) the

antioxidants could be classified into two systematic categories; (1) preventive

antioxidants, which inhibit formation of reactive oxygen species and (2) chain-

breaking antioxidants, which break radical chain sequences by scavenging oxygen

radicals. The second type of antioxidants donate hydrogen or electron to radical as

a result stable antioxidant radical is produced.

Synthetic antioxidants like butylated hydroxyanisole and butylated hydroxytoluene

are available in the market, but studies in a rat model showed carcinogenic activity

and reported them to be toxic (Jayalakshmi and Sharma, 1986). Exploitation of

plants as a source of natural antioxidants has been a major focus of nutritional and

biomedical research in the last decade. Various plants of different species and

genera have been explored for this reason. Diets high in fruits and vegetables are

thought to be good for health due to their protective effects. The Mediterranean diet

seems to be effective in reducing the prevalence of the conditions associated with

oxidative stress as evidenced by lower cardiovascular disease cases(Esposito et al.,

2004). Olives and olive oil are the main components of the Mediterranean diet. It is

suggested that antioxidant activity of olive biophenols is involved in prevention of

diabetes mellitus, cancer and coronary heart diseases either directly or indirectly

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(Gonzalez et al., 1992; Komaki et al., 2003; Sato et al., 2007; Obied et al., 2009).

Biophenols are chain breaking antioxidants and they break radical chain sequences

by acting as free radical scavengers or metal chelators (Ou et al., 2002). The ability

of biophenols to modulate oxidative stress through different mechanisms, including

radical scavenging, metal chelating or increasing antioxidant enzymes, has been

demonstrated and reviewed using different bioassays in many studies (Visioli et al.,

2002; Obied et al., 2009; Servili et al., 2009; Leopoldini et al., 2011).

Olive leaf extract has attracted a lot of attention, not only in the research community

but also in the nutraceutical industry, where it has emerged as a valuable

nutraceutical due to its acclaimed bioactivities. Olive leaf is a rich source of

numerous potentially bioactive compounds. Among those compounds, biophenols

are the most abundant physiologically active compounds. Studies on olive leaves

have demonstrated their activities as antioxidant (Benavente-Garcıa et al., 2000;

Somova et al., 2003; Bouaziz and Sayadi, 2005) and hypoglycaemic agent (Komaki

et al., 2003; Sato et al., 2007; Wainstein et al., 2012). Olive leaf extract is reported

to have inhibitory activity against α-amylase from human saliva (Komaki et al.,

2003).

The purpose of this study was to investigate the biological activities of Australian

olive leaves and extracts as natural antioxidants. Australian olive leaves have been

studied for their pharmacological activities. In addition, there are many commercial

preparation of olive leaf extract with varying potencies and patented pharmaceutical

preparation procedures. In this chapter, I will compare the composition and relative

potencies of Hardy’s Mammoth olive leaf extracts versus some commercially

available extracts in a set of in vitro antioxidant and anti-diabetic assays.

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4.2. Materials and methods

The study was conducted to ascertain the bioactivities of experimental as well as

commercial olive leaf extracts. Three commercially available olive leaf extracts;

Comvita olive leaf extract, Blackmores olive leaf extract and Swisse olive leaf

extract were purchased from local pharmacies. In addition, crude extract as well as

phenolic enriched extracts of locally grown olive leaves were also used. On the

basis of the results of the study reported in chapter 3 (Effect of season and cultivar

on biophenol content of olive leaves), Hardy’s Mammoth cultivar was selected for

this experiment.

4.2.1. Chemicals and reagents

Reagents and chemicals were used without further purification. Folin-Ciocalteu

reagent, (±)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox),

2,2′ azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS),

2,4,6-tripyridyl-s-triazine (TPTZ), ferric chloride, pyrocatechol violet (PV), sodium

acetate trihydrate, copper sulphate, anhydrous sodium sulphate, formic acid,

nitrotetrazolium blue chloride (NBT), potassium phosphate monobasic, phenol red

(ACS reagent), reduced -nicotinamide adenine dinucleotide disodium salt (NADH),

phenazine methosulfate, horseradish peroxidase (HRP) (type IV), α-amylase (EC

3.2.1.1), 2-Chloro-4-Nitrophenyl-α-maltotrioside (CNPG3) and 3-(4,5-

dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide were purchased from

Sigma-Aldrich (Sydney, Australia). Sodium carbonate (Univar, Sydney, Australia);

HPLC-grade methanol (Fisher Scientific. New Jersey, USA); n-hexane, ammonium

acetate, hydrochloric acid, sodium chloride, (Merck, Melbourne, Australia), and

anhydrous acetonitrile, ethyl acetate UNICHROME (Sydney, Australia). Ferrous

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sulfate (FeSO4) and hydrogen peroxide (H2O2) were obtained from Biolab (Sydney,

Australia).

Phenolic standards were oleuropein, caffeic acid, rutin, luteolin, (Sigma-Aldrich).

Most standards were dissolved in 50% methanol to prepare stock solutions of 1

mg/mL. The exceptions were luteolin, which was dissolved in absolute methanol

and then diluted with water to achieve 50% methanol, and rutin, which was

dissolved in warm 50% aqueous methanol as described earlier (Obied, et al.,

2005b). Syringe filters (0.45 µm and 0.22 µm) were purchased from Millex GP

(Sydney, Australia) and membrane nylon filters (0.22 µm) were purchased from

Sigma-Aldrich. Water used in all analytical work was purified (0.22 µm) by

GenPure water purification system (Thermofisher Scientific, Melbourne,

Australia).

4.2.2. Collection of olive leaves

Olive leaves of Hardy’s Mammoth cultivar were collected from the CSU Olive

Grove, Wagga Wagga, in May 2013. Mature green leaves were hand picked at

operator height around the whole perimeter of three trees. Approximately 500 gram

of leaves was collected in plastic bags and brought to the laboratory without any

delay.

4.2.3. Drying of olive leaves

One portion of fresh leaves was immediately dried in a forced-air oven at 105 °C

for 3 hours, as described in Chapter 2.

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4.2.4. Dry matter

Dry matter content of olive leaves was determined according to the method

described in Chapter 2.

4.2.5. Extraction of biophenols

Biophenols were extracted according to the method of Chapter2.

4.2.6. Enriched biophenol extract

In a 250ml separatory funnel, 50 mL of crude extract, 2g of sodium chloride and

5ml of formic acid were extracted with 50 mL of ethyl acetate. To separate the

organic layer, water was added in such a way that after adding 10 mL of water, the

contents of the flask was swirled. Fifty mL of water was found to be enough to

separate the liquid into two layers. The supernatant was dried over anhydrous

Na2SO4 and the lower portion was again extracted with 50 mL of ethyl acetate. The

process was repeated again twice, but in the 2nd and 3rd extraction step only ethyl

acetate was used for separation. Three ethyl acetate extracts (supernatants) were

dried over anhydrous Na2SO4. The combined extract was transferred to a round

bottom flask and ethyl acetate was evaporated under vacuum at 50°C in a rotary

evaporator. The residues were dissolved in 80% aqueous methanol to make the

volume up to 50 mL, filtered through 0.45 μm nylon nonsterile syringe filters

(Phenomenex) and stored in amber coloured glass vials at −20°C in aliquots of 10

mL until used for analysis.

4.2.7. Extractable matter

For determination of the extractable matter of crude, enriched and commercial

extracts, 2 mL of extract was added to an evaporating dish, which was placed in a

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boiling water bath for evaporation. Then the evaporating dish was transferred to a

hot air oven and the contents were dried at 105°C for 3 hours. The dried residues

were weighed and extractable matter was calculated as mg/mL.

4.2.8. High performance liquid chromatography (HPLC)

For HPLC analysis, the same instruments and column were used as used in Chapter

2. The solvents and method used for gradient elution were same as described in

chapter 3. For HPLC analysis, the commercial extracts were diluted 1:10, whereas

the crude and enriched extracts were diluted 1:20. The identification and

quantification of oleuropein was done according to previously reported method in

chapter 2.

4.2.9. Spectrophotometric measurements

Colorimetric analyses were performed on Cary 50 UV/visible spectrophotometer,

using Cary WinUV “version 3” software (Varian, Australia) and a microplate

reader (FLUOstar Omega UV-Vis plate reader; BMG, GmbH, Offenburg,

Germany). Aqueous methanol (50% v/v) or working buffer were used for dilution

of extracts and as a blank. Extracts were diluted to produce concentrations within

the range of the constructed calibration curves. All the extracts and oleuropein

(luteolin in copper chelation) were analysed in triplicate on three different times.

4.2.9.1. Total phenols

Total phenols were determined using Folin-Ciocalteu reagent as described in

Chapter 2. A six point (200-2200 µg/mL) calibration curve was prepared using

oleuropein, and results were expressed as milligrams oleuropein equivalent per mL

of extract (mg OLPE/mL). Results were also expressed as milligrams gallic acid

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equivalent per mL of extract (mg GAE/mL) and a seven point (50-600 µg/mL)

calibration curve was prepared using gallic acid.

4.2.9.2. ABTS radical scavenging assay

ABTS Radical Scavenging activity was measured according to the method of

chapter 2, with modifications. The assay was performed in 96-well flat bottom

microtiter plates instead of macro cuvettes. A stock solution of ABTS reagent (7

mM) was prepared in water. Into the ABTS reagent, 2.45 mM potassium persulfate

was added to produce ABTS radical cation (ABTS•+). The mixture was kept at

room temperature for 16 hours. For analysis, stock ABTS solution was diluted with

water to an absorbance of 0.70 (±0.02) at 734 nm. In each well, 275 µL of ABTS

working solution and 5 µL of diluted extract were added. The plate was covered

with aluminium foil and placed on a plate shaker, incubated and shaken at speed 1

for 30 minutes at room temperature. The absorbance was measured at 734 nm.

Water was used as blank. The absorbance of the ABTS radical without diluted crude

extracts served as control. The absorbance of all extracts without ABTS radical was

also measured and extract colour values were subtracted from the readings to get

colour corrected values. All extracts were analysed in triplicate at three different

times. The following formula was used to calculate the percent scavenging of

ABTS•+.

% scavenging =[Absorbance (control) − Absorbance (sample)]

Absorbance (control) × 100

4.2.9.3. Hydrogen peroxide scavenging assay

The previously described method of Obied et al. (2009) was used to determine the

H2O2 scavenging capacity of olive leaf extracts. Hydrogen peroxide solution was

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prepared in 100 mM phosphate buffer (pH 7.4), fresh for each set of experiments.

As the hydrogen peroxide was liquid, its concentration was determined by

measuring its absorbance at 230 nm using єH2O2, 230 nm= 81/ M .cm. All reagents

and commercial extract dilutions were made with assay buffer while crude extract

and phenolic enriched extracts were diluted in 50% methanol. Horseradish

peroxidase (0.3 mg/mL) was mixed with 4.5 mM phenol red to prepare the enzyme

mixture. The reaction was carried out in a 96-well flat bottom microplate, with 50

μL of extract/ buffer and 50 μL of 2 mM H2O2 solution. The microplate was

incubated at room temperature (20 ± 2 °C) for 20 minutes. Then 100 μL of enzyme

mixture was added to the reaction mixture, mixed for 1 minute on plate shaker at

speed 1 and incubated for 10 minutes at room temperature. The absorbance was

measured at 610 nm. The % H2O2 scavenging activity was calculated with the

following formula:

% Scavenging =[Absorbance (control) − Absorbance (sample)]

Absorbance (control) × 100

4.2.9.4. Superoxide radical (SOR) scavenging assay

The SOR scavenging activity of olive leaf extracts was measured by the method of

(Obied et al., 2009) with some modifications. Flat bottom 96 well microplates were

used for the assay and all reagents and extract dilutions were made with 100 mM

phosphate buffer (pH 7.4). In each well, 50 μL of extract/buffer, 50 μL of NBT (200

μM), 50 μL of NADH (624 μM), and 50 μL of phenazine methosulfate (80 μM)

were sequentially added. The plate shaker was set at speed 1 for 1minute and after

that the plate was incubated at room temperature for 5 minutes. The absorbance was

measured at 560 nm. Assay buffer was used as a negative control. Due to the

sensitivity of the phenazine methosulfate solution to light, it was prepared fresh in

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an amber coloured vial for every set of experiment. The SOR scavenging activity

was calculated as follows:

% Scavenging =[Absorbance (control) − Absorbance (sample)]

Absorbance (control) × 100

4.2.9.5. Copper chelation activity

Metal ion chelating activity of olive leaf extracts was evaluated by a cupric ions

chelation assay using pyrocatechol violet (PV), according to (Sánchez-Vioque et

al., 2013), with modifications. In a 96-well flat bottom microplate, 20 µL of

sample, 75µL of 50 mM sodium acetate buffer (pH 6.0), and 50 µL of 0.5 mM

CuSO4 in acetate buffer were added and mixed on a plate shaker for 2 minutes.

After 10 minutes of incubation at room temperature, 50 µL of 0.4 mM PV in acetate

buffer was added to all control, sample and standard wells. The plate was again

shaken for 1 minute and after 10 minutes of incubation at room temperature the

change in colour of the solution was measured at 632 nm. Luteolin was used as the

standard in this assay, as oleuropein did not show metal chelation activity. Luteolin

was dissolved and diluted in 80% methanol, while all other extracts were diluted in

50% methanol. The percentage of inhibition of PV–Cu2+complex formation was

calculated as

% Chelation =[Absorbance (control) − Absorbance (sample)]

Absorbance (control) × 100

4.2.9.6. Ferric reducing antioxidant power assay

The ferric reducing antioxidant power (FRAP) assay was conducted to determine

the reducing capacity of olive leaf extract. A previously reported method (Benzie

and Strain, 1996) was used with modifications. The assay buffer was 300mM

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sodium acetate buffer (pH 3.6) and all extracts were diluted in this buffer. The

FRAP reagent was prepared by mixing 2.5 mL of 5 mM 2,4,6-tripyridyl-s-triazine

(TPTZ), 2.5mL of 5mM ferric chloride (FeCl3) and 25mL of assay buffer. TPTZ

was dissolved in 40 mM HCl solution at 50 ºC. The FRAP reagent was prepared

fresh and placed at 37 ºC for at least 15 minutes before use. In a 96-well flat bottom

microplate, 10 µL of diluted extract, 200 µL FRAP reagent and 90 µL of water were

added and mixed on a plate shaker for 1 minute. The microplate was incubated at

room temperature (20±2 ºC) for 4 minutes, then the absorbance was measured at

593 nm. The antioxidant capacity of each extract was determined based on

increasing in Fe2+-TPTZ absorbance. The difference in absorbance was used to

measure the reducing capacity of olive leaf extracts. A standard curve was made

using six concentrations (10 to 1000 µmol/mL) of Trolox. Results were expressed

as millimoles of Fe2+ as TE per mL of olive leaf extract.

4.2.9.7. Alpha-amylase inhibition assay

The α-amylase inhibitory activity was determined by the previously described

spectrophotometric method of Ashok Kumar et al. (2011), with modifications. The

commercial extracts were diluted in assay buffer, while crude extract and phenolic

enriched extracts were diluted in 50% methanol. Acarbose was used as reference

compound; a five point calibration curve (10 µg to 2000 µg/ mL) was prepared. In

a macro plastic cuvette, 1000 µL of 40 mM phosphate buffer (pH 6.9), 60 µL of

extract and 20 µL of α-amylase (EC 3.2.1.1) dissolved in the above buffer (0.5

U/ml), were added. The cuvettes were incubated at 37 ºC for 10 minutes then 100

µL of CNPG3 (2.25mM) was added, mixed and again incubated at 37 ºC for 10

minutes. After incubation, 1820 µL of absolute ethanol was added to the cuvettes

to stop the reaction, and absorbance was measured at 405 nm. In the control buffer

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or 50% methanol was used in place of extract. The enzyme and substrates were

prepared fresh for each set of experiment. The percentage inhibition was calculated

by the following equation:

% Inhibition =[Absorbance (control) − Absorbance (sample)]

Absorbance (control) × 100

4.2.10. Statistical analysis

All assays were performed in triplicate at three different times and averaged. Seven

concentrations of the samples and standards were used in triplicate at three different

times in all assays. A blank, having the extract and assay buffer, was added for all

tested concentrations in all assays, to avoid the effect of extract colour on the assay

results. All the readings were corrected for extract colour absorbance, if there was

any. Results in the text, Tables and in Figures are expressed as mean ± standard

error. Data analysis was performed by Microsoft Excel and IBM SPSS Statistics

package version 20.0 (SPSS, IBM Corporation, New York). Statistical comparisons

were made using one-way ANOVA and post-hoc Tukey test. Results were

considered statistically significant at p < 0.05. GraphPad Prism version 5.04

(GraphPad Software, San Diego, USA) was used to calculate IC50 values of the

extracts.

4.3. Results and discussion

Hardy’s Mammoth cultivar was used for the preparation of crude and enriched

extracts. This cultivar was selected because of three main reasons. Firstly, it is an

Australian cultivar. Secondly, it produced higher levels of oleuropein and total

flavonoids compared to the other cultivars studied in Chapter 3. Moreover, its other

biophenol contents were also comparable to other cultivars. Thirdly, it has not been

148

investigated before for its biological activities. Ethyl acetate was used for the

preparation of the enriched extract as it has previously been reported to increase

biophenol content of the crude extract by removing not only highly polar

nonphenolics but also large molecular weight hydrophilic polymers (Obied, et al., ,

2005b; Obied, et al., 2008a; Obied et al., 2009).

4.3.1. Dry matter

The dry matter content of olive leaves collected for the preparation of crude and

enriched extract was 48.60 ± 0.15%. This was in accord with reported literature

values (Gómez-Cabrera et al., 1992; Martín-García and Molina-Alcaide, 2008;

Nourhène et al., 2008; Lafka et al., 2013).

4.3.2. Extractable matter

The extractable matter of the prepared and commercial olive leaf extracts is

presented in Figure 4.1. The extractable matter of Comvita and Blackmores olive

leaf extracts were significantly higher (p < 0.05) than other extracts; both showed

similar recovery of extractable matter (p ˃ 0.05), crude and enriched extracts had

the same recovery of extractable matter (p ˃ 0.05), which was significantly lower

than the Swisse extract (p < 0.05). The results indicate that commercial extracts

contain lot of biomass, which may have more non-phenolic matrix components as

compared to crude and enriched extracts. These results are in fact most probably

due to the added pharmaceutical excipients required to prepare and stabilize the

nutraceutical product.

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Figure 4.1: Extractable matter of olive leaf extracts.

HMCE: Hardy’s Mammoth crude extract, HMEE: Hardy’s Mammoth enriched extract,

OLAE: Comvita (Olive Leaf Australia) extract, BME: Blackmores extract, SOLE: Swisse

extract. Different letters on the bars indicate significant differences (p < 0.05).

4.3.3. High performance liquid chromatography (HPLC)

HPLC of commercial, crude and enriched extracts was performed in order to

estimate the oleuropein content of the extract. Three different solvents; water and

aqueous methanol 50% and 80% were compared for the commercial extracts in

order to determine the most suitable dilution solvent for them (Figure 4.2).

Oleuropein is thought to be contribute greatly towards the antioxidant activity of

olive leaf extract (Generalić Mekinić et al., 2014), as the major biophenol in the

extract. Blackmores extract had a higher oleuropein content (p < 0.05) than the

other two commercial extracts. For the Blackmores extract, 80% aqueous methanol

showed a higher oleuropein content (p < 0.05) than 50% methanol and water, while

c c

a a

b

0

100

200

300

400

500

600

700

HMCE HMEE OLAE BME SOLE

Extr

act

ab

le m

att

er m

g/m

L

Olive Leaf Extrcts

150

water showed more oleuropein than 50% methanol (p < 0.05). The Comvita extract

had higher oleuropein than the Swisse extract (p < 0.05) but there was no difference

in oleuropein content between the solvents (p ˃ 0.05). The Swisse extract showed

higher oleuropein in 50% methanol (p < 0.05) than in 80% methanol and water,

while 80% methanol had more oleuropein than water (p < 0.05). The crude extract

showed higher oleuropein content as compared to all other extracts (p < 0.05).

Crude extract was prepared in 80% aqueous methanol and it showed higher

recovery of oleuropein with 80% aqueous methanol than 50% aqueous methanol (p

< 0.05). Nevertheless a lower recovery of oleuropein has been reported when water

was used as solvent (Generalić Mekinić et al., 2014). The enriched extract had

higher oleuropein content than the commercial extracts (p < 0.05) and a higher

oleuropein content was noticed with 80% aqueous methanol than 50% aqueous

methanol (p < 0.05). The enriched extracts had solubility issues with water, as it

was prepared in ethyl acetate which has a very low solubility in water. As the

oleuropein level decreased with increasing water portion (from 80% methanol to

50% methanol) the crude and enriched extracts were not diluted with water. Water

was not a good choice as far as the crude and enriched extracts were concerned, but

the commercial extracts also revealed that it did not have any advantage for them

as a dilution solvent for in vitro assays. Though results indicate that 80 % aqueous

methanol was the best solvent for dilution of extracts for oleuropein content, it was

not preferred, due to the very high portion of organic solvent, which might have

compatibility issues with assay conditions. Therefore, 50% aqueous methanol was

preferred although the oleuropein level was compromised to some extent.

151

Figure 4.2: Solvent effect on oleuropein content of olive leaf extract.

HMCE: Hardy’s Mammoth crude extract, HMEE: Hardy’s Mammoth enriched extract,

OLAE: Comvita (Olive Leaf Australia) extract, BME: Blackmores extract, SOLE: Swisse

extract. Different letters on the bars within each extract indicate significant differences (p

< 0.05).

The oleuropein content of extracts in 50% aqueous methanol is presented in Figure

4.3. All extracts were significantly different from each other in terms of oleuropein

content (p < 0.05). The level of oleuropein decreased in the following order: crude

extract ˃ enriched extract ˃ Blackmores ˃ Comvita ˃ Swisse. The enriched extract

showed lower content of oleuropein than crude extract. Removal of some phenolic

compounds particularly hydrooxytyrosol glucoside in ethyl acetate extract is also

reported in literature (Obied et al., 2009).

ac

c

b

b

ab

b

a

a

aa

a

0

5

10

15

20

25

HMCE HMEE OLAE BME SOLE

Ole

uro

pei

n m

g/m

L

Olive Leaf Extracts

Water 50% Methanol 80% Methanol

152

Figure 4.3: Oleuropein content of olive leaf extracts with 50% methanol as the dilution

solvent.

HMCE: Hardy’s Mammoth crude extract, HMEE: Hardy’s Mammoth enriched extract,

OLAE: Comvita (Olive Leaf Australia) extract, BME: Blackmores extract, SOLE: Swisse

extract. Different letters on the bars indicate significant differences (p < 0.05).

The chromatograms of crude, enriched, Comvita, Blackmores and Swisse olive

leaf extracts in 50% aqueous methanol, at 280 nm, 325 nm and their corresponding

ABTS activity are shown in Figure 4.4, 4.5, 4.6, 4.7, and 4.8, respectively. In all

extracts, oleuropein was eluted at 15.5 minutes. Though commercial extracts were

low in oleuropein content, they had a greater number of compounds, as indicated

by the number of peaks (Figures 4.4-4.8).

a

b

dc

e

0

2

4

6

8

10

12

14

16

HMCE HMEE OLAE BME SOLE

Ole

uro

pei

n m

g/m

L

Olive Leaf Extrcts

153

Figure 4.4: HPLC chromatograms of Hardy’s Mammoth crude olive leaf extract,

comparing 280nm and 325nm wavelength and the corresponding ABTS activity.

Crude extract at 280 nm

Oleuropein

Crude extract at 325 nm

Crude extract ABTS activity

154

Figure 4.5: HPLC chromatograms of Hardy’s Mammoth enriched olive leaf extract,

comparing 280nm and 325nm wavelength and the corresponding ABTS activity.

Enriched extract at 280 nm

Oleuropein

Enriched extract at 325 nm

Enriched extract ABTS activity

Oleuropein

155

Figure 4.6: HPLC chromatograms of Comvita olive leaf extract, comparing 280 nm and

325 nm wavelength and the corresponding ABTS activity.

Comvita extract at 280 nm

Oleuropein

Comvita extract at 325 nm

Comvita extract ABTS activity Oleuropein

156

Figure 4.7: HPLC chromatograms of Blackmores olive leaf extract, comparing 280 nm

and 325 nm wavelength and the corresponding ABTS activity.

Blackmores extract at 280 nm

Oleuropein

Blackmores extract at 325 nm

Blackmores extract ABTS activity Oleuropein

157

Figure 4.8: HPLC chromatograms of Swisse olive leaf extract, comparing 280 nm and 325

nm wavelength and the corresponding ABTS activity.

Swisse extract at 280 nm

Oleuropein

Swisse extract at 325 nm

Swisse extract ABTS activity

Oleuropein

158

4.3.4. Total phenols

The total phenols of olive leaf extracts was calculated on the basis of both gallic

acid equivalent basis (GAE) and oleuropein equivalent (OLPE). When the total

phenol results were compared on the basis of GAE (Figure 4.9), all extracts showed

a significant difference from each other (p < 0.05). The total phenol content

decreased in the following order; Swisse ˃ enriched extract ˃ Comvita ˃

Blackmores ˃ crude extract. When results were compared on OLPE basis (Figure

4.10), the total phenols showed the same trend, with all extracts significantly

different from each other (p < 0.05), and the total phenol content decreasing in the

same order. The Swisse extract had the highest total phenols among all extracts but

at the same time it had lowest oleuropein content. Conversely, the crude extract was

high in oleuropein content but was lowest in terms of total phenol content. On the

other hand, the enriched extract had lower oleuropein than the crude extract but was

high in total phenols. It is clear from the results that the crude extract was a

concentrated source of oleuropein while the commercial extracts were high in other

biophenolic content. Nevertheless, the extraction of crude extract with ethyl acetate,

removed non phenolics and high molecular weight polymers, which eventually

increased the biophenolic content of the enriched extract. The results of our study

are in agreement with the previous studies of (Orak et al., 2012; Ahmad-Qasem et

al., 2013; Salah et al., 2013), who reported the values of total phenol content of

olive leaf extracts from 59-144 mg/g DW.

159

Figure 4.9: Total phenols of olive leaf extracts as gallic acid equivalents (GAE).

Figure 4.10: Total phenols of olive leaf extracts as oleuropein equivalents (OLPE).

HMCE: Hardy’s Mammoth crude extract, HMEE: Hardy’s Mammoth enriched extract,

OLAE: Comvita (Olive Leaf Australia) extract, BME: Blackmores extract, SOLE: Swisse

extract. Different letters on the bars indicate significant differences (p < 0.05).

e

b

cd

a

0

50

100

150

200

HMCE HMEE OLAE BME SOLE

Tota

l p

hen

ols

mg G

AE

/mL

Olive Leaf Extrcts

e

b

cd

a

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300

400

HMCE HMEE OLAE BME SOLE

Tota

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Olive Leaf Extrcts

160

4.3.5. Antioxidant activity

There is no standard method with which to measure the therapeutic activity of

antioxidants, especially plant/natural antioxidants. Therefore, the results obtained

by one type of in vitro antioxidants assay cannot be considered as total antioxidant

activity. The results of the antioxidant activity measured by a single assay cannot

be generalized, as these results reflect the chemical reactivity under the specific

conditions suitable for that assay (Huang et al., 2005). The same antioxidant can

have different activities with other types of assay, as every assay works at its

specific conditions. This may also occur due to the interfering compounds present

in plant extracts which interfere with the assay method. Moreover, the results of an

in vitro assay may not be true in vivo, as the in vivo environment is much more

complex and variable, where dietary interactions may also come into account. In

the literature it is recommended that antioxidant activity should be measured by a

number of methods, or at least two independent assays, as no single method

measures all possible antioxidant mechanisms (Wong et al., 2006; Obied et al.,

2009). Huang et al. (2005) divided antioxidant capacity assays into two categories

on the basis of the chemical reactions involved. One type of assays are hydrogen

atom transfer reactions, which mostly involve monitoring of competitive reaction

kinetics. The other type of assays are single electron transfer reactions, which

involve a redox reaction with the oxidant as an indicator of the reaction endpoint

(Huang et al., 2005).

The half maximal effective concentration (EC50) values were used to determine

antioxidant capacity of olive leaf extracts, except for the FRAP assay. The half

maximal inhibitory concentration (IC50) values were used to determine α-amylase

inhibition. The EC50 values measure the potency of a drug/substance, and IC50

161

measures the efficiency of a substance, how it inhibits a specific biochemical

function. The EC50 value of an olive leaf extract is defined as the concentration of

olive leaf extract that produce half maximal response of the desired activity;

scavenging radical or reduction in absorbance. The extracts with lower EC50 values

had higher antioxidant capacity or potency. In the FRAP assay antioxidant capacity

was expressed as FRAP value mM TE/mL of extract.

Table 4.1: EC50 and IC50 values of olive leaf extracts for in vitro bioactivity assays.

Assay

ABTS

Radical

Scavenging

Hydrogen

peroxide

Radical

Scavenging

Superoxide

Radical

Scavenging

Copper

Chelation

activity

α-amylase

Inhibition

EC50 EC50 EC50 EC50 IC50

HMCE 0.99 ± 0.06 0.27 ± 0.06 1.29 ± 0.02 4.76 ± 0.06 4.87 ± 0.03

HMEE 0.62 ± 0.08 0.34 ± 0.05 0.81 ± 0.07 3.27 ± 0.09 2.71 ± 0.04

OLAE 8.14 ± 0.07 3.22 ± 0.05 7.60 ± 0.03 17.29 ± 0.04 67.61 ± 0.04

BME 6.74 ± 0.06 2.77 ± 0.05 7.81 ± 0.01 15.52 ± 0.03 58.17 ± 0.05

SOLE 3.95 ± 0.07 1.30 ± 0.03 3.46 ± 0.01 3.59 ± 0.06 24.42 ± 0.02

Oleuropein 0.43 ± 0.04 0.47 ± 0.09 0.90 ± 0.02 ND 2.03 ± 0.04

Luteolin ND ND ND 0.12 ± 0.03 ND

EC50 and IC50 values (mg extractable matter/mL of extract) are presented as mean

± standard error. HMCE: Hardy’s Mammoth crude extract, HMEE: Hardy’s

Mammoth enriched extract, OLAE: Comvita (Olive Leaf Australia) extract, BME:

Blackmores extract, SOLE: Swisse extract, ND; not determined.

162

4.3.5.1. ABTS radical scavenging

The ABTS scavenging assay is a hydrogen atom transfer reaction, in which ABTS

is oxidized to its radical cation, ABTS•+ by peroxyl radicals. Upon oxidation, it

produces an intense green colour. When an antioxidant reacts with the ABTS•+

radical, the colour intensity is decreased. This is measured at 734 nm to determine

the capacity of that antioxidant. The ABTS radical scavenging activity of olive leaf

extracts and oleuropein is shown in Figure 4.11 and the EC50 values are presented

in Table 4.1. On the basis of the ABTS EC50 values, the antioxidant capacity was

decreased in order: oleuropein > enriched extract > crude extract > Swisse extract

> Blackmores extract > Comvita extract.

Oleuropein was found to be the most effective and showed the highest antioxidant

capacity in terms of ABTS radical scavenging compared to the olive leaf extracts.

Among the olive leaf extracts, the enriched extract was a better scavenger of ABTS

radicals than the crude and commercial extracts. On the basis of these results it can

be assumed that the biophenols in the combined form as an extract, did not show a

significant synergistic effect in this assay. Nonetheless, if there was any synergism,

it would be not as potent as oleuropein was in this particular assay. On the other

hand it is clear from results that oleuropein alone was not responsible for the ABTS

scavenging based antioxidant activity, as the enriched extract, which had less

oleuropein than the crude extract, showed greater potency than the crude extract.

Similarly, the Swisse extract had less oleuropein than the other commercial extracts

but had greater potency. The EC50 values for ABTS in this study were much lower

than the reported IC50 values for ABTS radical by El Babili et al. who reported

38.65 mg/ litre. The higher potency of our extracts may be due to the higher

concentration of biophenols achieved by the drying method. Moreover other genetic

163

and environmental factors may also be involved, in addition to extraction solvent

and extraction method.

4.3.5.2. Hydrogen peroxide scavenging

For the measurement of hydrogen peroxide scavenging activity, a peroxidase based

system was used, as very high interfering absorbance was reported for olive

biophenols with direct measurement at 230 nm (Obied et al., 2009). Hydrogen

peroxide radical scavenging activity of olive leaf extracts and oleuropein is

presented in Figure 4.12, and Table 4.1 displays the EC50 values. The antioxidant

capacity on the basis of the EC50 values for H2O2 scavenging was decreased in

following order: crude extract > enriched extract > oleuropein > Swisse >

Blackmores > Comvita extract.

In the H2O2 scavenging assay, the crude extract showed greater potency than

oleuropein and other extracts. The results suggest an additive and/or a synergistic

effect of biophenols for H2O2 scavenging activity. In this particular assay

oleuropein was not the most active antioxidant. The results are in accord with the

findings of Obied et al. (2009), who reported a decrease in H2O2 scavenging activity

with esterification of hydroxytyrosol to secoiridoid residues.

164

Figure 4.11: ABTS radical scavenging activity of oleuropein and olive leaf extracts.

(a) oleuropein (b) crude extract (c) enriched extract (d) Comvita extract (e) Blackmores

extract (f) Swisse extract.

165

.

Figure 4.12: Hydrogen peroxide (H2O2) scavenging activity of oleuropein and olive leaf

extracts.

(a) oleuropein (b) crude extract (c) enriched extract (d) Comvita extract (e) Blackmores

extract (f) Swisse extract.

166

3.5.3. Superoxide radical (SOR) scavenging

Phenazine methosulfate acts as an electron transfer catalyst between NADH and O2

to produce superoxide radical in non-enzymatic phenazine methosulfate/NADH/O2

system. In the reaction, free SOR reduces water soluble yellow coloured

nitrotetrazolium blue (NBT2+) radicals to water insoluble blue coloured

diformazan. Biophenols, or their phenoxide ions produce H2O2 by reducing SOR

in aqueous solutions at physiological pH. Higher absorbance readings for the

yellow NBT2+ indicates higher SOR scavenging activity (Obied et al., 2009; Liang

and Kitts, 2014)

SOR scavenging activity of olive leaf extracts and oleuropein can be seen in Figure

4.13, with the EC50 values in Table 4.1. The antioxidant capacity as depicted by

SOR EC50 values was decreased in the following order: enriched extract >

oleuropein > crude extract > Swisse extract > Comvita extract > Blackmores

extract. The order of SOR scavenging activity was different from that of H2O2

scavenging activity. The enriched extract was more potent than oleuropein and

other extracts in terms of SOR scavenging. The crude extract, which showed greater

potency in the H2O2 scavenging assay, showed less SOR scavenging activity. These

findings are in agreement with the study of Obied et al. (2009), who reported that

good H2O2 scavengers showed less SOR scavenging activity. They also reported

good SOR scavenging activity of ethyl acetate extract, comparable to that of pure

compounds. Furthermore an additive and a synergistic effect of biophenols was also

suggested for the SOR assay. Obied et al. (2009) suggested a major role of the

nonphenolic component in determining the SOR scavenging activity.

167

Figure 4.13: Superoxide radical (SOR) scavenging activity of oleuropein and olive leaf

extracts.

(a) oleuropein (b) crude extract (c) enriched extract (d) Comvita extract (e) Blackmores

extract (f) Swisse extract.

168

4.3.5.4. Copper chelation activity

The chelating activity of the olive leaf extracts was measured by using PV. Upon

reaction of Cu2+ with PV, a Cu2+–PV complex is formed which shows a blue colour.

With the addition of an equivalent amount of sample or standard, the complex

dissociates and the blue colour turns to yellow. The chelating activity is then

calculated by measuring the rate of colour reduction at 632 nm. Figure 4.14 and

Table 4.1 display the copper chelation activity of olive leaf extracts and luteolin and

EC50 values, respectively. For this assay luteolin was used instead of oleuropein,

for reasons discussed below. The copper chelation activity on the basis of EC50

values was decreased in order: luteolin > enriched extract > Swisse extract > crude

extract > Blackmores extract > Comvita extract. Among the extracts the enriched

extract showed higher potency as a metal chelator, but the Swisse extract

surprisingly was found to be a better chelating agent than the crude extract, although

the crude extract was better than the other two commercial extracts. Though the

Swisse extract was the highest in total phenol content, it did not show any activity

higher than the crude extract for any other assay. Moreover, it has been suggested

that copper chelating activities depend upon the structure of the molecules and on

specific biophenols or other compounds of the extract. These may be

polysaccharides or peptides, rather than total phenolic content (Saiga et al., 2003;

Wong et al., 2006; Sánchez-Vioque et al., 2013). It has been also reported that

chemical groups in the molecule can effect metal chelating activity, and chelating

activity depends on the presence of catechol groups. The presence of a catechol

structure in the B benzene ring or 4-keto group with a 3- and/or 5-hydroxyl group

are considered strong chelating sites for metal ions (Megías et al., 2009; Sánchez-

Vioque et al., 2013).

169

Chelation of metal ions is one of the antioxidant mechanisms. Transition metals like

iron and copper are redox active. They catalyze redox cycling reactions and

generate reactive radicals in biological systems, such as hydroxyl radical (•OH),

superoxide anion (O2-) and nitric oxide. Both iron and copper are important

catalysts but they generate highly reactive hydroxyl radicals (•OH) by the Fenton

reaction, which in turn augments lipid peroxidation (Saiga et al., 2003; Wong et al.,

2006; Sánchez-Vioque et al., 2013). Metal chelators prevents the metals from

taking part in these redox reactions. Chelating agents change redox potentials of

metal ions and make metal ions inactive by binding with them and halt further

oxidative damage (Sánchez-Vioque et al., 2013). Biophenols can prevent oxidative

damage by chelating the transition metals iron and copper (Megías et al., 2009).

The results of this study are in complete agreement with the above studies, as olive

leaf biophenols effectively chelated metal ions.

Luteolin was used instead of oleuropein for copper chelation activity because when

oleuropein was mixed with assay reagents it did not produce results. The crude and

enriched extracts which were high in oleuropein showed relatively high IC50 values for

copper chelation in comparison to other antioxidant capacity assays. Even the crude extract

showed a higher IC50 value than the Swisse extract in this assay. A similar finding was

observed by Generalić Mekinić et al. (2014) who reported the lowest chelating activity for

those extracts which had higher total phenols and oleuropein content, and the best activities

for the extracts that had lower contents of total phenols and oleuropein. Some earlier reports

suggests chelating action of oleuropein and it was indicated that it effectively inhibited

copper sulfate-induced-oxidation of LDL in a dose-dependent manner (Visioli et al., 1994;

Andrikopoulos et al., 2002). In our study however we did not find significant copper

chelation activity for oleuropein. This might be due to poor miscibility or low activity of

oleuropein for copper chelation. A possible reason for this may be some complex

intermolecular interactions or interference of oleuropein with an assay reagent, since those

170

studies used different assay method with a different mechanism. It has been proposed that

biophenols with a specific chemical structure participate in the chelation and the chelation

activity depends upon compounds that have a more favourable chemical structure for metal

chelation. Luteolin has a more favourable structure for metal binding and is responsible for

very high chelating activity (Sánchez-Vioque et al., 2013).

Figure 4.14: Copper chelation activity of luteolin and olive leaf extracts.

(a) luteolin (b) crude extract (c) enriched extract (d) Comvita extract (e) Blackmores extract

(f) Swisse extract.

171

4.3.5.5. Ferric reducing antioxidant power (FRAP)

FRAP is a redox assay. Its mechanism is a single electron transfer and it is a

reasonable tool for the determination of redox status in cells or tissues. (Prior et al.,

2005). In the FRAP assay FeCl3 reacts with TPTZ, and Fe3+ is reduced to Fe2+,

when an antioxidant donates a single electron. A blue colour is produced as a result

of the Fe2+–TPTZ complex, which absorbs strongly at wavelength 593 nm. The

more Fe3+ is reduced to Fe2+ greater the intensity of colour. Different concentrations

of oleuropein, crude, enriched and commercial extracts were investigated for

antioxidant activity measured as FRAP value. Figure 4.15 represents the FRAP

value of oleuropein and olive leaf extracts at a concentration of 100 µg of

oleuropein/ mL or extractable matter per mL of extracts. At the same concentration

of oleuropein and all extracts, oleuropein showed a significantly higher FRAP value

than all extracts (p < 0.05). Among the extracts, the enriched extract was

significantly higher than all other extracts (p < 0.05) in terms of FRAP values,

followed by crude extract and then the commercial extracts. All the commercial

extracts were not different from each other (p ˃ 0.05) in terms of FRAP values.

The results of the FRAP assay showed same trend of antioxidant capacity for

oleuropein and extracts as was observed for the ABTS scavenging activity. Though

different mechanisms were involved, the results were the same. Oleuropein found

to be more potent as an antioxidant than the extracts in the FRAP assay too. Like

the other antioxidant activity assays, the FRAP assay also revealed that the enriched

and crude extracts prepared from dried olive leaves showed significantly higher

values compared to the commercial extracts.

172

Figure 4.15: FRAP values of olive leaf extracts as trolox equivalent.

TE: trolox equivalent, HMCE: Hardy’s Mammoth crude extract, HMEE: Hardy’s

Mammoth enriched extract, OLAE: Comvita (Olive Leaf Australia) extract, BME:

Blackmores extract, SOLE: Swisse extract. Different letters on the bars indicate significant

differences (p < 0.05).

The FRAP assay is widely used for assessing the antioxidant capacity. The

antioxidant activity of a compound would be totally dependent on its ability to

reduce iron (Ou et al., 2002). However the accuracy of FRAP value as an

antioxidant is questioned in the literature for a number of reasons. Firstly it has been

commented that all reducing agents are not antioxidants, as any compound which

has a redox potential lower than the redox potential of Fe3+/Fe2+ (0.77) can

contribute to the FRAP value. Secondly, not all antioxidants reduce Fe3+ to Fe2+ at

a fast rate (4-8 minutes), as some biophenols may require a longer time for reaction.

Thirdly, the FRAP assay is carried out at an acidic pH (3.6) to maintain iron

solubility, but low pH may prevent electron transfer (Ou et al., 2002; Prior et al.,

2005; Wong et al., 2006; Konczak et al., 2010). However, in the present study it

c

b

d d d

a

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5

10

15

20

25F

RA

P V

alu

e m

illi

mole

s T

E/

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Olive Leaf Extracts

173

seems to be a good representative of antioxidant capacity, as it showed the same

trend as was seen by ABTS radical scavenging assay. Prior et al. (2005) reported

that redox potential of Fe3+-TPTZ (0.77) is comparable with that of ABTS•+ (0.68)

and the compounds which react with FRAP also react with ABTS, even though both

assays were performed at different reaction conditions, as the ABTS is performed

on neutral pH while FRAP is performed at acidic pH.

Biophenols perform an antioxidant function by a number of potential pathways.

Free radical scavenging is one of the most important, where biophenols most likely

break the free radical chain reaction. The backbone structure of biophenols may

have different substituents which endow them with antioxidant properties,

especially their hydrogen-donating capacity (Carrasco-Pancorbo et al., 2005). In the

antioxidant capacity assays, the crude and enriched extracts showed higher

antioxidant capacities, clearly indicating they were more potent than the

commercial extracts. The enriched extract was more potent than the crude extract

for all assays except the H2O2 scavenging assay, where the crude extract was more

potent. The results of the present study are in accord with the findings of Obied et

al. (2009). They also reported a higher potency for ethyl acetate extract in

comparison to crude extract. Furthermore, individual biophenols; oleuropein and

luteolin in case of copper chelation, showed greater potency than extracts. The only

exceptions were the SOR and H2O2 scavenging assays. It is evident from the results

that sample treatment and extraction solvent play a vital role in the recovery of

biophenols and thereby in the antioxidant activity. The antioxidant potency of the

extract can be increased with sample treatment and by selecting most suitable

solvent for extraction.

174

On the other hand, it is clear from the results that oleuropein was not the only

biophenol responsible for the antioxidant activity, as the enriched extract, which

had lower oleuropein levels than the crude extract, showed greater potency than the

crude extract, except for H2O2 radical scavenging activity. Moreover, the

antioxidant capacity of the extracts was not very well correlated with total phenol

content, as the Swisse extract was the highest in total phenol content but it did not

show the highest activities. The lower activities of commercial extracts in contrast

to crude and enriched extracts may be due to more non phenolic matter in the

commercial extracts, as they showed higher recoveries of dry and extractable

matter. Although the commercial extracts contained a greater biomass in the same

volume, their lower potency indicates that in addition to biophenols, other non-

active compounds were also present.

4.3.6. Alpha-amylase inhibition

The α-amylase inhibition of olive leaf extracts and oleuropein can be seen in Figure

4.16 and the IC50 values in Table 4.1. The enzyme inhibition, as denoted by IC50

values, was decreased in following order: oleuropein > enriched extract > crude

extract > Swisse extract > Blackmores extract > Comvita extract.

Oleuropein was found to be a more potent α-amylase inhibitor than the extracts,

which indicates a poor or no synergistic effect of biophenols in extracts. The

enriched and crude extracts, which had a higher oleuropein content, also showed

greater potency than the commercial extracts for enzyme inhibition. These findings

suggest that oleuropein is the main biophenol responsible for the antidiabetic effect,

but other biophenols may also involved. Our findings are in agreement with the

findings of (Gonzalez et al., 1992; Al-Azzawie and Alhamdani, 2006; Sato et al.,

175

2007; Jemai et al., 2009). They all reported that oleuropein is responsible for the

antidiabetic effect of olive leaves. A reduction in post-prandial hyperglycaemia is

considered one of the therapeutic approaches for diabetes mellitus. The decrease in

post-prandial hyperglycaemia can be achieved by inhibition of enzymes like α-

amylase, which hydrolyse carbohydrates (Haveles, 2014). α-amylase is involved in

the hydrolysis of long chain carbohydrates to oligosaccharides. Inhibitors of this

enzyme can interfere with carbohydrate digestion and absorption, thereby reducing

the post-prandial hyperglycaemia. Acarbose is an α-amylase inhibitor and is

considered as effective drug for treating diabetes mellitus. Inhibition of α-amylase

enzyme by olive leaf extracts suggests that olive biophenols can interfere with

carbohydrate metabolism/digestion and absorption. Hence they can play a positive

role in controlling glucose absorption and managing type 2 diabetes mellitus. On

the basis of the results it can be suggested that olive biophenols may be able to halt

the progression of prediabetes to type 2 diabetes by reducing postprandial

hyperglycaemia. Our results are in agreement with the findings of (Komaki et al.,

2003), who reported a good inhibition of human salivary and pancreatic α –

amylases with olive leaf extract, showing IC50 values of 4.0 and 0.02 mg/ml,

respectively in an in vitro assay. An inhibitory effect of olive leaf biophenols on the

postprandial increase in blood glucose in diabetic rats was also reported in that

study. The findings are also in accord with (Bechiri et al., 2015), who also reported

an inhibition of α-amylase with an aqueous extract of olive leaves, in an in vitro

assay.

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Figure 4.16: α-Amylase inhibition activity of oleuropein and olive leaf extracts.

(a) oleuropein (b) crude extract (c) enriched extract (d) Comvita extract (e)

Blackmores extract (f) Swisse extract.

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4.4. Conclusion

The present study made a multidimensional evaluation of antioxidant activity and

α-amylase enzyme inhibition activity of olive leaf extracts in comparison to

standards. The antioxidant activities of olive leaf extracts and biophenols were

demonstrated in array of diverse bioassays.

It is evident from the results that olive leaf biophenols as individual compounds,

and in a combined form of extract, are excellent antioxidants. Hence the role of

olive leaf biophenols can be elucidated as potential nutraceuticals, being a multi

efficient bioactive molecules.

The results clearly indicate that olive leaf biophenols strongly inhibited the α-

amylase enzyme, so it could be beneficial in lowering blood glucose levels and can

be utilized as an antidiabetic agent. The scope of olive leaves is promising, as the

side effects of synthetic drugs necessitate the need for natural inhibitors of

carbohydrate hydrolysing enzymes from plant sources.

It is suggested that the interference of test substances with assay or assay reagents

as well as the limitations of the assay should be taken into consideration before

selecting an assay for antioxidant activity, in order to increase the accuracy and

credibility of the assay.

It can be concluded that olive leaves are an excellent source of natural antioxidants

and their biophenols are capable of inhibiting metabolic enzymes, especially α-

amylase, which suggests their potential as nutraceuticals.

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Chapter 5. An investigation into olive leaf extract

as a therapeutic antioxidant treatment

of prediabetes

5.1. Introduction

Diabetes mellitus is a massive challenge to the world, with high mortality rates. In

2015, on average one person died every six seconds from diabetes (International

Diabetes Federation, 2015). According to International Diabetes Federation (2015),

five million people died due to diabetes, and the annual expenditure on health care

of diabetic patients has reached to US$ 673 billion. It has been estimated that about

415 million people, were living with diabetes in 2015 worldwide. This number is

expected to rise to 642 million by 2040. In 2015, about 20.9 million cases of

gestational diabetes were reported (International Diabetes Federation, 2015). The

International Diabetes Federation (2015) also estimates that about 193 million

people are unaware that they have diabetes. In Australia, 1.7 million people have

diabetes and diabetes is increasing at a faster rate than other chronic diseases such

as cancer and heart disease, with about 280 cases of diabetes recorded every day

(Diabetes Australia, 2015). The total annual cost impact of diabetes in Australia

estimated at $14.6 billion (Diabetes Australia, 2015) and Australia had the highest

diabetes related spending on healthcare per person (US$ 7652 to US$ 14,498)

worldwide (International Diabetes Federation, 2015).

The commonly used terms for prediabetes are impaired glucose tolerance (IGT) and

impaired fasting glycaemia (IFG) (Twigg et al., 2007; Tabák et al., 2012). A person

is said to be prediabetic with IFG when the fasting blood glucose level is between

6.1 to 6.9 mM/L. IGT is diagnosed following an oral glucose tolerance test when

blood glucose level 2 hours after glucose intake is from 7.8- 11 mM/L, with the

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Australian definition (Diabetes Australia, 2015). It is possible that one can have

both IFG and IGT. Worldwide 318 million people (6.7%) are living with

prediabetes, meaning one in 15 adults (International Diabetes Federation, 2015). In

Australia, 2 million people have prediabetes and one in three people with

prediabetes will develop type 2 diabetes. Obesity, lack of exercise, high blood

pressure, high cholesterol and family history of type 2 diabetes and heart disease

are risk factors for prediabetes (Diabetes Australia, 2015).

Imbalance in glycaemic control occurs when β-cells fail to compensate for the

insulin resistance and β-cell failure leads to type 2 diabetes from prediabetes/IGT

(DeFronzo and Abdul-Ghani, 2011). From a medical and economic point of view

it is imperative to treat the individuals with prediabetes to prevent the progression

towards the type 2 diabetes. Recent research from the studies on people with IGT

suggested that early intervention in type 2 diabetes is beneficial and can cure it

(Andrews et al., 2011; DeFronzo and Abdul-Ghani, 2011). Weight loss, exercise,

nutritional management and pharmacological intervention are the common

strategies used to prevent the progression of prediabetes to type 2 diabetes. An

intensive diet intervention soon after the diagnosis is effective in managing blood

glucose levels, however exercise in this particular study did not result in additional

benefits (Andrews et al., 2011).

Oxidative stress can affect biological macromolecules such as DNA, lipids and

proteins. The oxidation of those macromolecules by oxidative stress is considered

to be a critical step in the pathophysiology of many degenerative and age related

diseases such as atherosclerosis and cardiovascular disease (Favier, 2003). There is

accumulating evidence that oxidative stress plays a significant role in the aetiology

and pathology of diabetes mellitus. The cardiovascular complications in diabetes

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are reported to begin at the prediabetes stage and involve hyperglycaemia-induced

oxidative stress (Nwose et al., 2007). This gives a broader aspect for the effect of

oxidative stress on the diabetic patient. Oxidative stress plays a crucial role in the

pathogenesis of type 2 diabetes mellitus and its complications, as it plays an

important role in the development of insulin resistance (Eriksson, 2007; Nwose et

al., 2007; Al-Aubaidy and Jelinek, 2010). Hyperglycaemia results in the generation

of ROS, which then cause oxidative damage to the erythrocytes (Nwose et al., 2007)

and free radical production increased in individuals suffering from prediabetes due

to high blood glucose. This in turn modulates most of the tissue damage that results

in the progression to type 2 diabetes and complications associated with diabetes

(Nwose et al., 2008), as reactive oxygen species (ROS) and reactive nitrogen

species (RNS) can damage macro molecules such as proteins, lipids, and nucleic

acids (Al-Aubaidy and Jelinek, 2011a). Type 2 diabetic patients had significantly

higher oxidative DNA damage as evidenced by elevated levels of 8-hydroxy 2'-

deoxy-guanosine (8-OHdG), which is a common biomarker for the measurement

of oxidative DNA damage, total systemic oxidative stress and for the diagnosis of

diabetes (Al-Aubaidy and Jelinek, 2011a, 2011b). It is proposed that 8-OHdG is a

product of oxidative DNA damage produced by specific enzymatic cleavage after

ROS-induced 8-hydroxylation of the guanine base in mitochondrial and nuclear

DNA (Al-Aubaidy and Jelinek, 2011b).

The major antioxidant defence system in the body is reduced glutathione (GSH),

superoxide dismutase, thioredoxins, catalase and antioxidant nutrients (Powers and

Jackson, 2008). GSH, a cellular antioxidant found in erythrocytes, acts as a

substrate for GSH peroxidase-1 (Al-Aubaidy and Jelinek, 2011b). It has been

noticed that diabetic patients have more of the oxidized form of glutathione rather

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than reduced form, which may result in poor defence against oxidative stress (Al-

Aubaidy and Jelinek, 2010).

This has lead to the hypothesis that administration of botanical antioxidant

supplements may correct this oxidative stress and consequently treat diabetes

mellitus. Olive leaves have also been known for improving metabolic disorders,

possessing anti-diabetic properties and are widely recognized as a traditional

remedy for diabetes and hypertension in the Mediterranean basin (Komaki et al.,

2003). The hypoglycaemic activity of OLE has been demonstrated in animal studies

and oleuropein is known to act on diabetes, as discussed in Chapter 1 (Gonzalez et

al., 1992). Multiple studies in animals and humans have shown a range of disease

ameliorating activities of OLE, which can be very useful in treating diabetes

mellitus, such as glucose lowering, cholesterol lowering, sensitising tissues to

insulin, reduction of high blood pressure, cell and neuron protection and anti-

obesity properties. Most of these studies are preclinical (in vitro, ex vivo, and animal

models), so more randomized controlled clinical trials are needed to confirm these

findings.

The effect of olive leaf biophenols on glucose metabolism, particularly by

attenuating the oxidative stress, has not been thoroughly investigated, with only a

few studies conducted and very limited data existing. The current study was a pilot

study undertaken in people who are diagnosed as pre-diabetic, to develop

preliminary evidence on whether early intervention with dietary supplementation

of OLE can produce a measurable effect on plasma oxidative stress and slow or

revert the progression of the disease in pre-diabetic patients.

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5.2. Materials and methods

This was a single-centre, randomised, double blinded, prospective pilot study on

the effect of dietary supplementation with olive leaf extract capsules. Prediabetic

male and female subjects (n=18) were randomized into two groups and received

daily doses of placebo, or olive leaf extract capsules. Their blood glucose level,

lipid profile (total cholesterol, triglycerides, LDL-cholesterol, VLDL-cholesterol,

HDL-cholesterol and cholesterol/HDL) and glycated haemoglobin (HbA1c) were

measured by the pathology lab that collected the blood and urine. In addition to that

total phenol contents and oxidation markers; STAT-8-isoprostane and 8-hydroxy-

2'-deoxyguanosine were also measured to assess the impact of supplementation.

5.2.1. Ethics approval

The study protocol was approved by the Charles Sturt University Ethics in Human

Research Committee (Approval number: 2012/049) (see APPENDIX 3). An

information statement (see APPENDIX 4) was provided to all subjects. The study

was verbally explained to them and written consent was obtained from all

participants.

5.2.2. Subject recruitment

Prediabetic subjects were recruited from the community through announcements in

the local newspaper, television and on Charles Sturt University’s newsle between

February 2014 and March 2014. Complete details, email addresses and postal

addresses were collected from those who wished to participate. Emails were sent,

providing a thorough explanation of the study and expectations of the participants.

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The inclusion criteria for subjects were as follows: (1) diagnosed with impaired

glucose tolerance, based on an oral glucose tolerance test; (2) aged from 20 to 67

years; (3) not taking any medication for diabetes; (4) not taking any medication that

may have effect on blood glucose level directly or indirectly; (5) not taking

corticosteroids and beta blockers; (6) not having any chronic disease like

cardiovascular diseases or Alzheimer’s disease; (7) female participants should not

be pregnant or lactating.

5.2.3. Study protocol

After recruitment, 18 participants (8 male and 10 female) were randomised into two

groups; control and treatment group with 9 subjects in each (Group A and B). To

avoid any researcher bias in assigning participants to supplement protocols, the

randomisation was prepared and assigned using computer random number

generation and both researchers and subjects were ‘blinded’ to the contents of

capsules being taken. The code was released after statistical analysis.

Although adverse effects of olive leaf extract were not documented, subjects were

made aware that it may produce symptoms like headache and fatigue. Subjects were

advised to discontinue the supplementation protocol immediately if symptoms

occurred and report to research team and general health practioner (GP).

Before treatment, reference blood and urine samples of each subject were taken by

a pathology lab collection service. The blood glucose level, lipid profile (total

cholesterol, triglycerides, LDL-cholesterol, VLDL-cholesterol, HDL-cholesterol

and cholesterol/HDL) and glycated haemoglobin (HbA1c) were measured by the

pathology laboratory. The oxidative biomarkers; STAT-8-isoprostane and 8-

hydroxy-2'-deoxyguanosine and total phenols were measured by the researcher in

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the research laboratory. At the end of the study (after 12 weeks), again blood and

urine samples were taken to assess the same parameters. Values at the start and at

the end of experiment were compared, to determine the effect of olive leaf extract

and placebo on blood glucose levels and oxidative stress.

5.2.4. Dietary collection

Participants were asked to fill in pre-study and post-study surveys (see APPENDIX

5 and 6) about their food intake, feeding habits, life style and health status that may

influence the results. Participants recorded their consumption of the foods high in

biophenol content like tea, green tea, red wine, fruits and vegetables as serves per

week. In order to minimize error in the study results, the participants were advised

to maintain their current dietary intake and not to make changes to their diet and

physical activity levels for the duration of the study.

5.2.5. Test material (supplement)

Olive leaf extract capsules and empty bottles for the placebo were obtained from

Comvita, Olive Leaf Australia. The placebo was prepared by The Melbourne

Compounding Centre, Seddon, Victoria. The placebo was matched with the

capsules as closely as possible in look, feel and taste, and consisted of a 1:9.5:15

mix of vinegar, water and glycerol, with yellow, red and green food colour in a

3:1:1.5 mix. As preservatives can have an antioxidant effect, it was necessary to

avoid preservative(s) in the placebo formula. Therefore glycerol was used to

stabilise the placebo. The treatment group was provided with olive leaf extract

capsules (2400 mg) and asked to take three capsules per day for 12 weeks, while

the control group was provided with placebo capsules and asked to take three

capsules per day for 12 weeks. Taking capsules was not restricted to meals.

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5.2.6. Compliance

All participants were required to return the remaining capsules and empty capsule

bottles at the end of the 12 weeks period. Compliance was measured by the allotted

number of daily doses dispensed minus the number of daily doses returned.

5.2.7. Blood and urine samples

About 6 mL of blood sample was collected in a fasting state from each subject from

the median cubital vein in the morning. A urine sample from each subject was also

collected by the pathology collection service. The blood sample was separated as

serum by the pathology laboratory. Both serum and urine samples of the subjects

were provided by the pathology laboratory at baseline and follow-up. The urine

samples then were centrifuged at 4500 rpm for 10 minutes at 4 ºC in a Beckman

Coulter (Allegra X-30R) centrifuge and the supernatant was collected in aliquots.

Aliquots of serum and urine samples were stored at -80° C until analysed for

oxidation markers.

5.2.8. Reagents and standards

Here only those chemicals are listed which were used by the researcher to perform

total phenols and oxidative biomarkers assays. Reagents, chemicals and standards

were used without further purification: Folin-Ciocalteu reagent, gallic acid,

metaphosphoric acid, triethanolamine and phosphate-buffered saline were from

Sigma-Aldrich (Steinheim, Germany). The STAT-8-isoprostane Kit (500431) was

purchased from Cayman Chemical Company, Ann Arbor, MI, USA, The 8-

hydroxy-2-deoxyguanosine (8-OHdG) kit (KOG-200S/E) from the Japan Institute

for the Control of Aging (JaICA, Fukuroi, Shizuoka, Japan) was purchased from

186

Life Sciences Research, Australia. Water used in all analytical work was purified

(0.22 µm) by a GenPure water purification system (Thermofisher Scientific,

Melbourne, Australia).

5.2.9. High performance liquid chromatography (HPLC) of olive leaf

extract capsules

For HPLC analysis, the same instruments and column were used as in Chapter 2.

The solvents and method used for gradient elution were same as described in

chapter 3. All the contents of the capsule were transferred to a beaker and 10 mL of

water or 50% aqueous methanol or 80% aqueous methanol was added. The mixture

was sonicated for 20 minutes and then centrifuged at 4000 rpm for 10 minutes. The

supernatant was collected and filtered through a 0.45 µm syringe filter. The

prepared extracts were diluted 1:10 for HPLC analysis. The identification and

quantification of oleuropein was done according to the method of chapter 2.

5.2.10. Spectrophotometric measurements

Colorimetric analyses were performed on a microplate reader (FLUOstar Omega

UV-Vis plate reader; BMG, GmbH, Offenburg, Germany).

5.2.10.1. Total phenols assay

Total phenol content of urine samples was measured by a modified version of the

method described in Chapter 2. The assay was performed in a 96 well flat bottom

microtiter plates instead of macro cuvettes. Urine samples were centrifuged at

10,000 rpm for 10 minutes and diluted (1:10) with water. To each well of the

microplate, 100 μL of water, 20 μL of sample or standard or 50% aqueous methanol

and 50 μL of Folin-Ciocalteu reagent was added. The plate was shaken for 1min

187

and then 90 μL of aqueous sodium carbonate solution (20% w/v) was added. The

microplate was shaken for 1 hour at room temperature. The absorbance was read at

760 nm in a plate reader. A seven point calibration curve (10-120 µg/mL) was

prepared using gallic acid. Results were expressed as milligrams gallic acid

equivalent per ml of urine (mg GAE/g).

5.2.10.2. Isoprostanes assay

A competitive enzyme-linked immunosorbent assay (ELISA) STAT-8-Isoprostane

kit was used to measure isoprostanes. All reagents were prepared and used as

recommended by the manufacturer. Urine samples were centrifuged at 10,000 rpm

for 10 min and diluted 1:2 and 1:10 with tris buffer supplied with kit. A stock

standard solution was diluted to eight different concentrations (23.4-3000 pg/mL)

for standard curve preparation. In a 96-well flat bottom microplate which was pre-

coated with mouse anti-rabbit IgG, 50 μL of sample or standard was added. In

maximum binding wells (B0), 50 μL, and in non-specific binding (NSB) wells, 100

μL of Tris buffer was added. Then 50 μL of STAT-8-isoprostane AP (alkaline

pohsphatase) tracer was added to each well except total activity (TA) and the blank

wells. After mixing, 50 μL of STAT-8-isoprostane EIA antiserum was added,

except to TA, NSB and the blank wells. After side by side mixing, the plate was

covered and incubated at room temperature for one hour on a plate shaker. After

incubation, the contents of the plate were discarded and the plate was washed with

washing buffer. The plate was then inverted on lint free paper towel to remove

residual washing buffer. The washing was repeated four times more with washing

buffer. Then 200 μL of freshly prepared para-Nitrophenyl Phosphate (pNPP) was

added to all wells and 5 μL of tracer was added only to TA wells. The plate was

covered and incubated on a plate shaker in the dark for 90 minutes and the

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absorbance was read at 420 nm. The concentration of 8-isoprostane in samples was

measured with reference to an eight point standard curve (23.4-3000 pg/mL).

5.2.10.3. 8-Hydroxy-2-deoxyguanosine (8-OHdG) assay

For the measurement of oxidative DNA damage, a competitive ELISA kit was used

for 8-OHdG (JaICA, Fukuroi, Shizuoka, Japan). All reagents and samples except

the chromatic solution were brought to room temperature (20-22 ºC) before use.

Urine samples were centrifuged at 10,000 rpm for 10 min and diluted 1:2 with

phosphate buffered saline (pH= 7.4). All reagents were prepared and used according

to manufacturer instructions. The primary antibody (Anti 8OHdG monoclonal

antibody (clone N45.1)) was reconstituted with phosphate buffered saline. In a pre-

coated 96- well plate 50 μL of samples or standard 8-OHdG solution (0.5, 2, 8, 20,

80, 200 ng/mL) and 50 μL of reconstituted primary antibody was added to each

well. The outermost wells of the plate were not used, to avoid edge effects, and

were filled with water to maintain a uniform volume and temperature. The plate

was sealed with an adhesive strip and placed on a plate shaker, and the contents

were thoroughly mixed for 30 sec. Then the plate was incubated at 37°C for 1 hour

in a water bath. After removing the contents of the well, 250 μL of washing solution

(5x phosphate buffered saline, diluted with water) was added to each well. The plate

was thoroughly agitated, and the washing solution disposed of. The plate was then

inverted on lint free paper towel to remove residual washing buffer. The washing

was repeated twice more. Then 100 μL of the secondary antibody (HRP conjugated

anti mouse IgG) was added in each well. The plate was sealed with an adhesive

strip and contents were thoroughly mixed for 30 seconds on a plate shaker. Then

the plate was incubated at 37°C for 1 hour in a water bath and the washing procedure

was repeated in the same manner as before. Enzyme substrate solution was prepared

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freshly by diluting one part of chromatic solution (3,3',5,5' tetramethyl benzidine)

to 100 with diluting solution (hydrogen peroxide/citrate phosphate buffered saline).

Then 100μL of substrate solution was added in each well. The plate was then

incubated at room temperature in the dark for 15 min after 30 sec shaking on a plate

shaker. The reaction was stopped by adding 100μL of 1M phosphoric acid. After

30 sec shaking on a plate shaker, the plate was read at 450 nm. The concentration

of oxidative DNA in samples was measured with reference to a six point standard

curve (0.5-200 ng/mL).

5.2.11. Statistical analysis

All assays were performed in at least triplicate and averaged. Results are expressed

as mean ± standard error. Data analysis was performed by Microsoft Excel and IBM

SPSS Statistics package version 20.0 (SPSS, IBM Corporation, New York).

Statistical comparisons were made using repeated measures one-way ANOVA and

post-hoc Tukey test. Non-parametric statistical analysis was performed with the

Statistical Package for Social Sciences (SPSS) version 23 software program (IBM

Corporation, New York). The Wilcoxon signed-rank test was performed to compare

the clinical values of pre- and post-intervention in both treatment and control

groups. The mean value was used to complete the missing data. Results were

considered statistically significant at p < 0.05.

5.3. Results and discussion

Eighteen subjects (eight males and ten females) started the study. Twelve of them

completed the study (six male and six female participants, out of them five were on

placebo and seven were on olive leaf extract capsules supplementation).While four

of them did not given the final blood and urine samples for oxidation markers. Two

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of the subjects withdrew at the beginning of study without providing baseline blood

and urine samples due to personal commitments. Two other subjects started taking

the supplements and provided baseline samples but quit the trial for personal

reasons. A further two subjects left within two weeks after start, due to adverse

reactions; one reported some skin rashes and the other reported gastric reflux. The

data of pre- and post-study surveys, regarding the dietary intake, feeding habits, life

style and health status was not analysed, as sample size was eventually too small

and data were not different. There was no value to subdivide this small data, as it

would lose its statistical power.

The levels of blood glucose, glycated haemoglobin (HbA1c) and lipid profile

including total cholesterol, triglycerides, LDL-cholesterol, VLDL-cholesterol

HDL-cholesterol and cholesterol/ HDL ratio were measured at baseline and at the

end of the study.

5.3.1. Nonparametric analysis of clinical data

Due to the limitations of the sample size, non-parametric statistics should be applied

to these data. However, non-parametric data will not allow comparison between the

control and treatment group. Hence, we will report both parametric and non-

parametric data analysis to maximize the benefit from our data. Although we have

seen a drop and increase in base values, non-parametric data analysis using

Wilcoxon signed-rank test revealed that these change are not statistically

significant. The only two changes which were close to be statistically significant

are total blood cholesterol decrease in the treatment group (p-value = 0.059) and

the increase in the glycated haemoglobin in the control group (p-value= 0.066).

Therefore, there is a high chance of getting more promising data upon increasing

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the sample size. Logistic ordinal regression was performed to predict the effect of

confounding factors, age and gender, but none showed statistical significance.

5.3.2. Parametric analysis of clinical data

The average fasting blood glucose levels of both the control and treatment groups

at baseline and at the end of study are shown in Figure 5.1. Blood glucose levels

were not significantly different between the groups (p ˃ 0.05) and no significant

reduction in the glucose level was achieved with the supplementation of olive leaf

extract or placebo (p ˃ 0.05). Glycated haemoglobin (HbA1c) levels are presented

in Figure 5.2. Both groups showed the same levels of HbA1c (p ˃ 0.05) at the start

of the study and again no significant reduction of HbA1c was achieved with the

supplementation of olive leaf extract or placebo (p ˃ 0.05).

The lipid profile (total cholesterol, triglycerides, LDL-cholesterol, VLDL-

cholesterol HDL-cholesterol and cholesterol/ high density lipoprotein ratio) of the

participants at the baseline and at the end of the study can be seen in Figure 5.3.

The results of the lipid profile also showed no difference between groups (p ˃ 0.05)

and no significant advantage of olive leaf extract supplementation or placebo (p ˃

0.05).

Our results are in agreement with the study of de Bock et al. (2013), who also

reported no significant effect on lipid profile with olive leaf biophenol

supplementation in overweight, middle-aged men at risk of developing the

metabolic syndrome. However in other studies, supplementation with olive leaf

extract resulted in a significant reduction of triglyceride level in patients with stage-

1 hypertension (Susalit et al., 2011) and decreased cholesterol level in borderline

hypertensive monozygotic twins (Perrinjaquet-Moccetti et al., 2008). Though in

some subjects apparently reduced values were noticed, statistically there were no

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differences. There are a number of possible reasons for this. The dose rate may have

been too small to bring any change. The small sample size, differences in age among

the participants could also be reasons for the variation among individuals. This was

a pilot study and only eighteen subjects were recruited, but only eight completed

the study. Thus the sample size made it very difficult to be able to detect statistical

differences. In another study, performed on overweight, middle-aged men at risk of

developing the metabolic syndrome, olive leaf biophenol supplementation for 12

weeks significantly improved insulin sensitivity (15%) and pancreatic β-cell

secretory capacity (28%) in comparison to placebo (de Bock et al., 2013).

The sample size of that study (n = 46) was good enough to predict the change.

Moreover the dosage rate was higher than our study. The age of the participants

could be another factor for differences in results as the average age of the

participants in their study was 46.5 years, whereas in our study it was 55.1 ± 1.45

years. In another study on patients with type2 diabetes mellitus, olive leaf extract

supplementation for 14 weeks significantly lowered HbA1c and fasting plasma

insulin levels but not the postprandial plasma insulin levels (Wainstein et al., 2012).

In this study again the sample size was large (n = 79) as well as the participants

were diabetic and were receiving the drugs for the reduction of blood glucose level,

might the olive leaf extract provided additional benefits with drugs.

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Figure 5.1: Fasting blood glucose levels.

Figure 5.2: Glycated haemoglobin (HbA1c).

Pre-Control: control group at baseline; Post-Control: control group at the end of

study; Pre-Treatment: treatment group at the baseline and Post-Treatment:

treatment group at the end of study. Same letters on the bars indicate no significant

differences (p ˃ 0.05).

a a a a

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

Pre-Control Post-Control Pre-Treatment Post-Treatment

mM

/ li

tre

Treatment Groups

a

a

a

a

0.0

2.5

5.0

7.5

Pre-Control Post-Control Pre-Treatment Post-Treatment

Hb

A1c

%

Treatment Groups

194

Figure 5.3: Lipid profile.

Pre-Control: control group at baseline; Post-Control: control group at the end of

study; Pre-Treatment: treatment group at the baseline and Post-Treatment:

treatment group at the end of study. TC: total cholesterol; Trig: triglycerides; HDL:

high density lipoprotein; VLDL: very low density lipoprotein; LDL: low density

lipoprotein; Cho/HDL: cholesterol to high density lipoprotein ratio. Within each

lipid, there was no significant difference (p ˃ 0.05) between groups.

0.0

1.0

2.0

3.0

4.0

5.0

6.0

TC Trig HDL VLDL LDL Cho/HDL

Valu

es m

M/

lite

r

Lipid Profile

Pre-Control

Post-Control

Pre-Treatment

Post-Treatment

195

5.3.2. 1. Total phenols

The antioxidant status of participants was also measured by determining total

phenols in urine using the Folin-Ciocalteu reagent. Total phenols of urine samples

at baseline and 12 weeks later are presented in Figure 5.4. The results of total

phenols showed no difference between groups (p ˃ 0.05) and no significant

advantage of olive leaf extract supplementation or placebo (p ˃ 0.05). Total phenols

gave us an estimate of the change in phenolic levels. Although total phenols are

generally used as an estimate for phenolic content, these values are also an estimate

of antioxidant status, as this method measures total reducing capacity of the samples

(Huang et al., 2005; Prior et al., 2005).

Figure 5.4: Total phenols in urine.

Pre-Control: control group at baseline; Post-Control: control group at the end of

study; Pre-Treatment: treatment group at the baseline and Post-Treatment:

treatment group at the end of study. Same letters on the bars indicate no significant

differences (p ˃ 0.05).

a

a

a

a

0

5

10

15

20

25

30

Pre-Control Post-Control Pre-Treatment Post-Treatment

mg/

mL

of

uri

ne

Treatment Groups

196

5.3.2.2. Isoprostanes

The STAT-8-isoprostane marker was used for the determination of lipid

peroxidation. The STAT-8-isoprostane values at baseline and follow up are

presented in Figure 5.5. Both groups showed the same status of oxidation (p ˃ 0.05)

and again no significant change in oxidation status was observed with the

supplementation of olive leaf extract or placebo (p ˃ 0.05). The isoprostanes are

biomarkers of oxidative stress and antioxidant deficiency. They are produced by

the random oxidation of tissue phospholipids by oxygen radicals. Though

isoprostanes are present in urine and plasma in normal conditions, their levels are

elevated in oxidative stress.

Figure 5.5: Stat-8- Isoprostane in urine.

Pre-Control: control group at baseline; Post-Control: control group at the end of

study; Pre-Treatment: treatment group at the baseline and Post-Treatment:

treatment group at the end of study. Same letters on the bars indicate no significant

differences (p ˃ 0.05).

a

aa

a

0

5

10

15

20

25

Pre-Control Post-Control Pre-Treatment Post-Treatment

pg/

mL

of

Uri

ne

Treatment Groups

197

5.3.2.3. Hydroxy-2-deoxyguanosine (8-OHdG)

The 8-OHdG marker was used for the measurement of DNA oxidation. The 8-

OHdG values at the baseline and follow up are presented in Figure 5.6. Again both

groups showed the same status of oxidation (p ˃ 0.05) and again no significant

change in oxidation status was observed with the supplementation of olive leaf

extract or placebo (p ˃ 0.05). 8-OHdG is a biomarker used for the quantitative

detection of the oxidative DNA adduct. It has been reported that diabetic patients

with hyperglycaemia show elevated levels of urinary 8-OHdG and the level of

urinary 8-OHdG in diabetes correlated with the severity of diabetic nephropathy

and retinopathy (Wu et al., 2004).

Figure 5.6: 8-Hydroxy-2-deoxyguanosine (8-OHdG) in urine.

Pre-Control: control group at baseline; Post-Control: control group at the end of

study; Pre-Treatment: treatment group at the baseline and Post-Treatment:

treatment group at the end of study. Same letters on the bars indicate no significant

differences (p ˃ 0.05).

a

a

a

a

0.0

1.0

2.0

3.0

4.0

5.0

Pre-Control Post-Control Pre-Treatment Post-Treatment

pg/m

L U

rin

e

Treatment Groups

198

The total phenols by Folin-Ciocalteu reagent and Stat-8- Isoprostane and 8-OHdG

oxidation marker tests were performed on urine samples. Therefore the results

obtained represent the whole body oxidative status (Kendall et al., 2009). But

measurement of oxidative markers as mass per unit volume in urine samples can

present a problem as individuals do not produce the same volume of urine at the

same time every day. Normal values of the oxidation markers used in this study

have not been established and the values varied from study to study. A baseline of

levels of oxidation markers should be established for these common oxidation

markers.

The results of oxidation markers indicate no significant change in prediabetic

participants with the supplementation of olive leaf extract. Similar findings were

reported earlier by (Kendall et al., 2009) when they investigated the effect of olive

leaf extract on the oxidative status of healthy young Australians, and reported that

none of the oxidative markers produced the appreciable change. They suggested

that the incompatibility of these oxidative markers with oxidative status in the

cohort may be one of the possible reasons (Kendall et al., 2009). The possible

reason for no change may be the individuals’ response to olive leaf supplement or

the dose given. Some individuals appeared to respond better to the supplement than

others but as a whole there was no significant response. It is worth mentioning that

the standard deviations for these markers were relatively high, which indicates a

high variability in the individual responses to the supplement/placebo.

Another possible reason for the lack of effect of treatment may be that the dosage

of the supplement was not high enough to produce any change. Although the dosage

was used according to manufacturer’s recommendation, this dose was for general

wellbeing and the participants of the study were prediabetic. They may need a

199

higher dosage to produce substantial change. As discussed earlier in this chapter

and in Chapter 1, higher glucose levels in the blood could increase free radical

production and hence increase oxidative stress (Nwose et al., 2008; Wright Jr et al.,

2006).

It has been suggested that the capsule form of olive leaf extract may have low

bioavailability or physiologically low activity and the olive leaf extract in liquid

form could have more bioavailability (Kendall et al., 2009). In that study,

compliance for the capsule was much greater than liquid extract, yet it showed the

same results as the liquid extract. Furthermore it was also proposed that liquid olive

leaf extract supplement at the recommended dosage could have produced a change

in oxidative status.

Identification of the placebo was another issue which is also worth mentioning here.

Though the placebo capsules looked almost the same to extract capsules in

appearance and packed in the same bottles, still some of the subjects identified it,

as they were familiar with the commercial product. Two of the subjects who quit at

the start were from the control group.

This was a pilot study, limited by the cost of the wide array of tests to be conducted.

This limited the sample size, limited the statistical power of the study and made the

influence of individual variability very great.

200

5.3.3. High performance liquid chromatography (HPLC) analysis of the

capsules

HPLC analysis of olive leaf capsules was performed in order to estimate the

oleuropein content of the capsules. Three different solvents; water and aqueous

methanol 50% and 80% were used to dissolve the content of the capsule, in order

to determine the most suitable solvent for maximal recovery of oleuropein content

(Figure 5.7). Oleuropein is considered as the main biophenol involved in the

hypoglycaemic effect of olive leaves (Al-Azzawie & Alhamdani, 2006; Gonzalez

et al., 1992), as in addition to its high activity it is also the major biophenol in the

extract. The results of Chapter 4 also showed good inhibition of α-amylase enzyme.

The extract of capsules prepared in 50% aqueous methanol showed significantly

higher oleuropein content than water and 80% aqueous methanol (p < 0.05), and

water showed significantly higher oleuropein content than 80% aqueous methanol

(p < 0.05). The chromatograms of Comvita olive leaf extract capsules in 50%

aqueous methanol, at 280 nm, 325 nm and their corresponding ABTS activity are

shown in Figure 5.8. A difference in oleuropein content of Comvita olive leaf

capsules was observed for different solvents , however when the Comvita olive leaf

extract was diluted in these solvents no differences were observed due to solvents

(Chapter 4). In this study we used different solvents to dissolve the contents of the

capsule for HPLC analysis, whereas in Chapter 4, solvents were used only for

dilution of the olive leaf extract. In this study, solvent may also play some part in

dissolving the solid content of the capsule. Due to that, 50% aqueous methanol

produced higher contents than other solvents. Moreover the concentration of

oleuropein was also different in the capsule from the liquid extract obtained from

the same company and analyzed in Chapter 4. For the liquid extract, we observed

201

the same oleuropein content as was mentioned in the product specification, but in

the case of the capsule, only the quantity of olive leaf in the capsule was mentioned

but not the oleuropein content. We determined that the oleuropein content of the

capsule was twice that of the liquid extract.

Figure 5.7: Oleuropein content of the capsule with different solvents (mg/capsule).

Different letters on the bars indicate significant differences (p < 0.05).

b

a

c

0

3

6

9

12

15

Water 50 % Methanol 80 % Methanol

Ole

uro

pei

n m

g/

cap

sule

Solvents

202

Figure 5.8: HPLC chromatograms of Comvita olive leaf capsule, comparing 280 nm and

325 nm wavelength and the corresponding ABTS activity.

Comvita Olive Leaf Extract Capsule at 280 nm

Oleuropein

Comvita Olive Leaf Extract Capsule at 325 nm

Comvita Olive Leaf Extract Capsule ABTS activity

Oleuropein

203

5.4. Conclusion

As to our knowledge, no study has been performed on the effects of olive leaf

extract on the prediabetic population. The present study did not demonstrate any

significant effects of olive leaf capsule supplementation on prediabetic subjects.

The blood glucose level, glycated haemoglobin, lipid profile and oxidative markers

did not show any significant change either over time or in comparison to the control

group. Though it was a pilot study and the number of participants ended up too

small to produce any significant conclusion, this study has been a practical exercise

of the numerous challenges that face clinical trials for nutraceuticals. For the future

studies it is recommended that more careful consideration be given to the size of

cohorts, placebo, form and dosage of the supplement or extract in order to make the

judicious use of the oxidative biomarkers. The number of participants is a very

important factor as greater numbers will minimize the effect of individual variation.

It is also suggested that for any future studies the dietary intake during the study

period should be monitored. This is important for at least couple of reasons, firstly

it will ensure that the change/effect is due to supplement or food and secondly, there

may be some food and supplement interactions may involve, and if there is any that

must be accounted for the validity of results.

204

Chapter 6. General discussion, conclusion and

future directions

The concept of medicinal food and phytotherapy for health promotion and disease

prevention is gaining popularity across the globe. It is being adopted as it is more

economical and convenient than expensive medical and surgical procedures. The

way demand for nutraceuticals is increasing, it seems that in the future they would

play a vital role in disease therapy. At the same time, increasing demand and

consumer preference for nutraceuticals over synthetic drugs necessitates more

detailed and comprehensive research on nutraceutical sources. This research project

was designed for this reason, with an aim to evaluate Australian olive leaves as a

nutraceutical source of bioactive biophenols, and to study the antidiabetic effects of

olive leaf biophenols. To address this aim four experimental studies were designed.

In the first study the objective was to find out the best method for drying olive

leaves, one that allows the optimum recovery of olive leaf biophenols,

especially oleuropein. For the commercialization of olive leaves as

nutraceuticals, it is necessary to determine the best processing method, ideally

one that could increase the recovery of biophenols from the same biomass.

For this purpose, different drying treatments were tested on olive leaves and

the extracts from dried leaves were compared for biophenol contents with

extracts from fresh leaves. Olive leaf biophenolic composition and

antioxidant activity was determined by both spectrophotometric and HPLC

methods. Results of this study confirmed that the drying of olive leaves at

105 °C for 3 h before extraction results in a prodigious increase in biophenols

and antioxidant activity, in comparison to fresh leaves. The most phenomenal

increase was for oleuropein recovery, where a 38-fold increase was observed.

205

Therefore the drying of olive leaves at105 °C for 3 h before extraction

increases the suitability of olive leaves for commercial utilization in

nutraceutical industry.

For commercial purposes, it is strongly recommended that olive leaves should be

dried at a higher temperature for short time to increase the recovery of oleuropein,

other biophenols and antioxidant activity. It is worthwhile to put a little more effort

and expenditure on drying, as it pays a lot at the end in terms of more biophenols

from the same biomass. More recovery of oleuropein and other biophenols can be

easily produced on an industrial scale by drying the leaves prior to extraction and

drying in a hot air oven can be easily achieved and adopted on industrial scale. It is

likely that the consumer would be happy to pay more for a higher quality product.

Another important factor for the recovery of biophenols from plant material is the

extraction solvent. The selection of extraction solvent for olive leaf biophenols

seems not to be a complicated question anymore in the light of our results with 80%

aqueous methanol, as well as other published work (Obied, et al., 2005b; Malik and

Bradford, 2008; Abaza et al., 2011; Brahmi, et al., 2012b). It is worth mentioning

that 80% aqueous methanol has proven to be a good solvent for olive biophenols

and has demonstrated a good recovery of biophenols from olive leaves, but it may

not be as good with other plants and their different tissues/organs.

After achieving the first objective, next step was to determine the effect of season

and cultivar on the composition of olive leaf biophenols. Though it was reported in

the literature that genetic and environmental factors can greatly influence the

composition of biophenols, the variability in biophenols due to cultivar/variety and

environmental factors have not been studied extensively, particularly in Australia.

206

Therefore, it was of prime importance to study these factors, so that best time of

harvest could be identified, for optimal recovery of biophenols. For this purpose a

unique study was designed, which monitored biophenolic contents and antioxidant

activity of olive leaves for 28 months covering the complete production cycle of the

olive tree, beginning at the start of the fruiting season of one year and finishing at

the end of the 3rd year fruiting season. To our knowledge none of the previous

studies have examined biophenols for such long duration. In this study, analysis

was performed on extracts prepared from leaves dried at 105 °C for 3 h, as the

previous study confirmed that this treatment was best for highest biophenol

recovery.

Olive leaf biophenols varied quantitatively during different developmental stages

of biological cycle of olive tree, but no qualitative differences were detected. In all

the samples throughout the collection period the same biophenolic compounds were

observed. The study demonstrated that cultivar/ variety, month of collection and

season had significant effects on biophenol composition and antioxidant activity of

olive leaves. None of the cultivars followed a specific pattern for accumulation of

biophenols throughout the collection period. For the optimal antioxidant activity

and for maximum recovery of biophenols, including the oleuropein, the summer

season was found to be best. In comparing the cultivars, it became apparent that the

Leccino cultivar produced higher total phenols, phenolic compounds, o-diphenols,

hydroxycinnamic acid derivatives and higher antioxidant activity, whereas the

Hardy’s Mammoth cultivar produced higher oleuropein and total flavonoid content.

Correlation analysis revealed that biophenol contribution towards antioxidant

activity was not uniform. Biophenols responded differently to the different

antioxidant assays. Even oleuropein, which was always the major biophenol in the

207

extract, responded differently to the antioxidant assays performed. Therefore it is

recommended that antioxidant activity cannot be relied on one type of assay.

The results suggest that the time of collection is the most influential parameter with

regards to the suitability of olive leaves for commercial collection. For the recovery

of required biophenol for nutraceutical purpose, the selection of cultivar is also

important. Data generated in the study provide guidelines for seasonal and varietal

differences. Further studies on the quantification of individual biophenols and on

the bioavailability of these biophenols are required to establish the best time of

harvest for a particular biophenol recovery and to optimize their usage as a

functional food or nutraceutical.

That study answered the question regarding the best time of collection for a

particular cultivar. The next step was to find out the antioxidant and antidiabetic

activities of olive leaf biophenols, in order to determine their suitability for

nutraceutical industry. To address this objective, another study was planned, to find

out the in vitro antioxidant and antidiabetic activities of olive leaf biophenols. On

the basis of our results in the previous study it was decided that the antioxidant

activity of olive leaves should be determined by examining a number of different

mechanisms involved in antioxidant activity. The results of one single type of

antioxidant assay would not be appropriate for an actual estimate of an extract or

individual biophenol’s antioxidant capacity. Antioxidant activity should be

measured by considering different mechanisms. For the generalization of

antioxidant capacity of olive leaf biophenols, measurement should be done with a

few different assays. Moreover the antioxidant results obtained by the in vitro

assays may not reflect what would happen in vivo because of the complexity of

biological systems and due to interactions of nutritional and other factors within the

208

biological system. It was observed in the study that total phenols were not very well

correlated with antioxidant activity and it was not a given that an extract that had

higher level of total phenols would also possess higher antioxidant activity.

Olive leaf biophenols demonstrated excellent antioxidant capacity in all studied

antioxidant assays and strongly inhibited the α-amylase enzyme. This was the case

both for individual compounds and in a combined form of extract. Olive leaf

biophenols are multi efficient bioactive molecules, could be beneficial in lowering

blood glucose level. They have a great scope and have potential to be efficiently

utilized in nutraceutical industry as an antioxidant and antidiabetic agent.

Oleuropein is the major biophenol in olive leaf and appears to be mainly responsible

for olive leaf bioactivities. It possess excellent antioxidant capacity, acts as a free

radical scavenger and can be helpful in the prevention of oxidative stress as well as

in prevention of conditions associated with oxidative stress. Despite this, oleuropein

alone was not responsible for the antioxidant activity measured in the assays. It

appears that other biophenols are also involved in the antioxidant action of olive

leaves.

An excellent potency for the inhibition of α-amylase enzyme has been demonstrated

by oleuropein as an individual compound and in combination with other olive leaf

biophenols in the form of an extract. Olive leaf biophenols might therefore be

considered as a hypoglycaemic agent, may prevent hyperinsulinemia by reducing

blood glucose. This highlights the need to study the health promoting effects of

olive leaf biophenols beyond their antioxidant action.

The commercial extracts showed lower antioxidant and α-amylase inhibition

activity as compared to the crude end enriched extracts prepared in the laboratory.

209

This reflects the importance of drying of olive leaves at high temperature for a short

time before extraction, considering the remarkable increase in biophenol recovery

with drying, which was demonstrated in the first study. Furthermore, for the

preparation of the crude and enriched extracts, the leaves of the Hardy’s Mammoth

cultivar were collected at a time when oleuropein content was high. This reinforces

the earlier conclusion that drying and time of collection are important factors which

need to be considered to get extracts having more biophenols and higher potency.

This study demonstrated the in vitro antioxidant and antidiabetic activities of olive

leaf biophenols. The next step was to verify the results of in vitro study in an in vivo

experiment. For this purpose a study was planned to gain preliminary evidence of

the potential of olive leaf extract as an antidiabetic agent for pre-diabetic subjects.

To avoid the complicating effects of diabetic medication, it was decided to conduct

the study on prediabetic participants who were not taking any medication that may

have effect on blood glucose levels directly or indirectly. The intention was to

recruit at least 20 participants for the study so that the control and treatment groups

would both have at least 10 participants. Unfortunately we only found 18

participants and only 12 participants completed the study.

The study did not demonstrate any significant effects of supplementation with olive

leaf capsules on prediabetic subjects. The blood glucose level, glycated

haemoglobin, lipid profile and oxidative markers did not show any significant

change in comparison to the control group, probably due to highly individual

variability. Though some individuals did appear to respond to the supplement, as a

whole group there was no significant response.

210

To establish the in vivo antioxidant and antidiabetic activities of olive leaf

biophenols further investigation at the clinical level with a larger sample size is

needed. A small sample size can drastically effect results because of the individual

variation effect, which can be minimized in a larger population. Monitoring dietary

intake and lifestyle during a clinical study on diabetic or prediabetic subjects is of

vital importance and these factors must be considered while analysing results.

Although this data was collected, the eventual sample size of the study removed

any value from doing such an analysis.

A body of evidence suggests that olive leaf biophenols offers protection against

diseases and symptomatic conditions. Nevertheless these results are mostly

generated at the in vitro and subclinical level and only few clinical studies have

been performed. Therefore it is crucial to investigate the health promoting effects

of olive leaf biophenols at clinical levels/ in vivo for the efficient utilization of these

valuable nutraceuticals. Data generated in this research project provides a

reasonable basis for the utilization of olive leaves as a source of biologically active

compounds for health and wellbeing.

The superior biological activities of olive leaf biophenols do suggest the judicious

use of olive leaves as a supplement or nutraceutical to combat oxidative stress and

conditions associated with oxidative stress like diabetes mellitus. Olive leaves can

be consumed in a number of ways such as tea, extract, or they can be added to food

or food products to get their nutritional benefits. Fortification of food products with

olive leaves seems to be a good option to increase the value of food as it not only

increases the biophenol content but also prevents its oxidation and increases the

shelf life of food.

211

The ever increasing demand for nutraceuticals is asking for more comprehensive

investigation at the subclinical, clinical and molecular level. Moreover, questions

regarding the safety, dosage, toxicity and efficacy of these nutraceuticals must be

answered realistically by further research. This project is of utmost importance to

the nutraceutical, pharmaceutical and cosmetic industries. Olive leaf biophenols are

important bioactive molecules which bestow diverse bioactivities to olive leaves

and these bioactivities indicate that olive leaf biophenols have the potential to be

high value nutraceuticals, which might be effectively used for pathological

conditions. Olive leaf biophenols demonstrated strong inhibition of the α-amylase

enzyme, which suggests a potential role as an antidiabetic agent through reduction

in glucose uptake into the body. Though treatment with olive leaf extract did not

show an appreciable change in prediabetic patients in the pilot clinical trial due to

aforementioned constraints, it is still suggested that their antidiabetic effect should

be thoroughly investigated in controlled trials with a larger population, so that

individual variation can be minimized and more realistic results can be achieved. It

is concluded that olive leaf biophenols have a great potential as a nutraceutical, due

to their very high biophenol content, improving the quality of life by maintaining

health. This suggests that olive leaves or their isolated biophenols can be

successfully used in preventative medicine, nutraceutical, food and cosmetic

industries.

212

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Appendices

Appendix 1. Olive trees used for leaves collection

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248

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Appendix 2. Leaflet information of commercial olive leaf extracts

Comvita olive leaf extract:

Each 500mL is equivalent to 500 grams of fresh olive (Olea europaea) leaf.

Ingredients: Olive leaf extract, glycerine (vegetable), no alcohol, no added sugar.

Main active component: Oleuropein standardised to 4.4mg/mL.

Extraction: Non alcoholic

Adult Dose: 5mL three times a day.

Minerals/Trace elements: No information provided.

Blackmores olive leaf extract:

Each 5mL is equivalent to 5 grams of fresh olive (Olea europaea) leaf.

Ingredients: Olive leaf extract, alcohol free, no added gluten, milk derivatives, salt,

artificial colours, flavours or sweeteners. Contains potassium sorbate.

Main active component: Oleuropein standardised to 4.4mg/mL.

Extraction: Non alcoholic

Adult Dose: 5mL two to three times a day.

Minerals/Trace elements: No information provided.

Swisse olive leaf extract:

Each 7.5mL is equivalent to 7.5 grams of dry olive (Olea europaea) leaf.

Ingredients: Olive leaf extract and, no added gluten, lactose, egg, yeast, salt, soy

preservatives, artificial colours and flavours.

Main active component: Oleuropein 7.5mg/mL.

Extraction: No information provided.

Adult Dose: 7.5mL diluted in water twice daily.

Minerals/Trace elements: No information provided.

Comvita olive leaf extract capsule:

Each capsule is equivalent to 2.4 grams of fresh olive (Olea europaea) leaf.

Ingredients: Concentrated pure olive leaf extract. Free of animal products, gluten

and not genetically modified.

Main active component: Oleuropein

Extraction: No information provided.

Adult Dose: Two to three capsules per day.

Minerals/Trace elements: No information provided.

250

Appendix 3. Ethics approval

251

252

APPENDIX 4. Information statement for clinical trial participants

253

254

255

APPENDIX 5. Pre-study survey

Investigation into olive leaf extract as therapeutic

antioxidant

Study Protocol No. 2012/049

The Principal Investigator: Muhammad Kamran

School of Biomedical Sciences

PhD Student,

Charles Sturt University (02) 6933 4569

Co-Investigators:

Dr. Hassan Obied PhD (Charles Sturt University, 02 6933 2161)

Dr. Christopher Scott PhD (Charles Sturt University, 02 6933 4133)

Dr. Adam Hamlin PhD (Charles Sturt University, 02 6933 2339)

Pre survey for participants of clinical trial

Participant Name:..................................................

No:...........................................

Thank you very much for participating in the trial

256

Please circle the most appropriate option and write in the spaces

provided if needed. If you are not sure about any question please ask

the research team to explain, Thanks.

1. How old are you?

a. Under 35 years

b. 35 - 44 years

c. 45 - 54 years

d. 55 - 64 years

e. 65 years or over

2. Gender

a. Female

b. Male

3. Your height (cm)

4. Your weight (Kg)

Your waist measurement taken below the ribs (the correct place to measure your

waist is halfway between your lowest rib and the top of your hipbone, roughly in

line with your navel. Breathe out normally and measure directly against your skin

while standing.)

Less than 80 cm

80 - 90 cm

91 - 101 cm

102 - 110 cm

More than 110 cm

5. Are you of Aboriginal, Torres Strait Islander, Pacific Islander or Maori

descent?

a. No

b. Yes

257

6. Where were you born?

a. Australia

b. Asia

c. Middle East

d. North Africa

e. Southern Europe

f. Other

7. Have either of your parents or any of your brothers or sisters or close

relative(s) been diagnosed with diabetes (type 1 or type 2)?

a. No

b. Yes

Have you ever been found to have high blood glucose level, in a?

a. Health examination

b. Disease or illness

c. Pregnancy

d. No high level of blood glucose

Have you tried to modify your diet since you diagnosed with high blood glucose

levels?

a. No

b. Yes (Please specify how)

Have you tried to modify your life style (become physically active/more active)

since you were diagnosed with high blood glucose levels

c. No

d. Yes (Please specify how)

Have you tried to reduce alcohol consumption since you were diagnosed with high

blood glucose levels

e. No

f. Yes (Please specify how)

Have you ever been diagnosed with high cholesterol?

g. No

258

h. Yes

Have you ever been diagnosed with high blood pressure?

a. No

b. Yes

Are you currently taking medication for high blood pressure?

a. No

b. Yes

Are you currently suffering from any heart disease including angina?

i. No

j. Yes

Are you currently suffering from any kidney/renal disease?

k. No

l. Yes

Do you smoke cigarettes or any other tobacco products on a daily basis?

a. No

b. Yes ( 5 or less per day)

c. Yes (more than 5 but less than 10 per day)

d. Yes (more than 10 but not more than 20 per day)

e. Yes ( more than 20 per day)

How often do you eat vegetables or fruit?

a. Every day

b. Thrice a week

c. Twice a week

d. Once a week

e. No

Are you currently taking any medication?

a. No

b. Yes ( please write name of medicine(s))

8. Are you currently taking any herbal supplement(s) for general

wellbeing?

259

a. No

b. Yes ( please write name(s))

9. Are you a regular drinker of tea, green tea, coffee or any herbal infusion?

a. No

b. Yes ( please write name of drink and number of cups/day)

10. Does your diet contain fats from animal sources like cheese, butter etc?

a. No

b. Yes (up to seven serves a week)

c. Yes (more than 7 but less than15 serves a week)

d. Yes (more than 15 but less than 21 serves a week )

e. Yes (more than 21 serves a week )

Comments:

Thanks for your cooperation in filling in the questionnaire

260

Appendix 6. Post-study survey

Investigation into olive leaf extract as therapeutic

antioxidant

Study Protocol No. 2012/049

The Principal Investigator: Muhammad Kamran

School of Biomedical Sciences

PhD Student, Charles Sturt University

Phone No. 02 6933 4569

Co-Investigators:

Dr. Hassan Obied PhD (Charles Sturt University, 02 6933 2161)

Dr. Christopher Scott PhD (Charles Sturt University, 02 6933 4133)

Dr. Adam Hamlin PhD (Charles Sturt University, 02 6933 2339)

End of study survey for participants of clinical trial

Participant Name:..................................................

No:...........................................

Thank you very much for participating in the trial

261

Please circle the most appropriate option and write in the spaces provided if needed.

If you are not sure about any question please ask the research team to explain, Thanks.

Your weight (Kg)

Your waist measurement taken below the ribs (the correct place to measure your

waist is halfway between your lowest rib and the top of your hipbone, roughly in line

with your navel. Breathe out normally and measure directly against your skin while

standing.)

Less than 80 cm

80 - 90 cm

91 - 101 cm

102 - 110 cm

More than 110 cm

Have you modified your diet during the course of the research trial?

No

Yes (Please specify how)

Have you modified your life style (became physically active/more active) during

the course of the research trial?

No

Yes (Please specify how)

Have you reduced alcohol consumption during the course of the research trial?

No

Yes (Please specify how)

During the course of the research trial how many times did you eat vegetables or

green salad?

I did not eat vegetables or green salad

1-3 times per week

4-6 times per week

1 time per day

2-3 times per day

Did you forget to take your capsule?

No

Yes ( occasionally)

Yes ( more than 5 per week)

Yes (more than 10 per week)

262

Yes (more than 60 per month)

Have you been well throughout this trial?

Yes

No ( please specify)

Have you found yourself under physical or mental stress during the course of the

research trial?

No

Yes

Have you taken any medication, vitamins supplement, traditional or herbal

medicine during the course of the research trial?

No

Yes ( please specify)

Did you feel discomfort with the herbal supplement given to you?

No

Yes ( please specify)

Did you feel any difference in general wellbeing during the research trial?

No

Yes (positive)

Yes (negative)

Comments:

Thanks for your cooperation in filling in the questionnaire