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Handbook of Olive Oil: Phenolic Compounds, Production and Health Benefits
 9781536123579

Table of contents :
Contents
Preface
Chapter 1
Extra Virgin Olive Oils: Production and Polyphenolic Composition
Abstract
Introduction
References
Chapter 2
Olive Varieties and Phenolic Compounds in Olive Oils
Abstract
1. Olives and Olive Oil Composition
2. Phenolic Compounds
3. Olive Oil Stability and Ripeness Index
4. Organoleptic Characteristics of Olive Oil
References
Chapter 3
Biomedicinal Aspects and Activities of Olive Oil Phenolic Compounds
Abstract
Introduction
Antioxidant Activity
Anti-Inflammatory Activity
Antimicrobial Activity
Cardiovascular Effects
Antioxidant Activity
Anti-Inflammatory Activity, Endothelial Dysfunction, and Platelet Aggregation
Dyslipidemia
Atherosclerosis
Hypertension
Endocrine Effects
Diabetes Mellitus
Obesity
Liver Disease
Osteoprotective Effects
Central Nervous System Effects
Anticancer Effects
Bioavailability and Metabolism
Synergistic Effects of Combinations of Phenolic Compounds and Their Combinations with Drugs
References
Chapter 4
An Overview of Sample Treatment and Analytical Methodologies for the Determination of Phenolic Compounds in Olive Oils
Abstract
Introduction
Sample Preparation
Sample Treatment Prior to Isolation
Extraction of Phenolic Compounds from VOO
Liquid-Liquid Extraction (LLE)
Solid Phase Extraction (SPE)
Super Critical Fluid Extraction (SFE)
Phenolics Determination
Assays for Total Phenolic Content Determination
Assays for o-Diphenols Determination
Separation Analytical Techniques for Analysis of Phenolic Compounds
Gas Chromatography (GC)
Capillary Electrophoresis (CE)
Liquid Chromatography (HPLC)
Analytical Difficulties and Challenges
References
Chapter 5
The Natural Variation of Phenolic Compounds in the Fruits and Oils of Olive (Olea europaea L.)
Abstract
Introduction
Health and Sensory Properties
Natural Variation of Phenolic Compounds
Conclusion
References
Chapter 6
Hydroxytyrosol and Tyrosol, Phenolic Compounds of Virgin Olive Oil, Could Act Like Anti-Inflammatories in Chronic Inflammation
Abstract
Introduction
Materials and Methods
1. Chemicals
2. Cell Culture and Treatment
3. Cytotoxicity Assay
4. Raybio® Human Cytokine Antibody Array in M1 State THP-1 Macrophages
5. TNFα Production
6. NFκβ Detection in M1 State THP-1 Macrophages
7. NO Production in M1 Type THP-1 Macrophages
8. Statistical Analysis
Results and Discussion
1. Effects in Cytotoxicity
2. RayBio® Human Cytokine Antibody Array in M1 State THP-1 Macrophages
3. TNFα Production
4. NFκβ Production
5. NO Production
Conclusion
References
Chapter 7
Oleocanthal: The New Promising Compound of Extra Virgin Olive Oils
Abstract
Olive Oil: From Ancient Times to Today
Healthy Effects of Extra Virgin Olive Oil Phenolic Compounds
Oleocanthal: A Multiterapeutic Agent
Alzheimer’s Disease
Cancer
Inflammatory Diseases
Future Considerations
References
Chapter 8
The Health Benefits of Olive Oil Phenolic Compounds
Abstract
Introduction
1. Phenolic Compounds Present in Extra Virgin Olive Oil
2. Health Benefits of EVOO Phenolic Compounds
2.1. Antioxidant Activity
2.2. Anti-Inflammatory Activity
2.3. Prevention of Atherosclerosis or Cardiovascular Diseases
2.4. Prevention or Treatment of Certain Cancers
2.5. Prevention or Treatment of Some Neurodegenerative Diseases
2.6. Anti-Allergic Effect
2.7. Antimicrobial Activity
2.8. Phenolics Compounds and Obesity
2.9. Phenolics Compounds and Diabetes
2.10. Phenolics Compounds and Infertility
Conclusion
References
Chapter 9
Extra Virgin Olive Oil and Hepatoprotective Effects
Abstract
1. Introduction
2. Types of Liver Damage
2.1. Drugs
2.2. Toxic Substances
2.3. Alcohol
2.4. Virus
2.5. Obesity and Related Metabolic Alterations
3. Liver Damage and EVOO Protection
3.1. EVOO and Hepatic Steatosis
3.2. EVOO and Non-Alcoholic Steatohepatitis
3.3. EVOO and Cirrhosis
3.4. EVOO and Hepatocellular Carcinoma
3.5. EVOO and Ischemia-Reperfusion Injury
4. Molecular Mechanisms Involving the Protective Effect of EVOO
(i) Activation of Nuclear Transcription Factor Nrf2,Inducing a Cellular Antioxidant Response, by the Gene Expression of Antioxidant Enzymes or of Enzymes Involved in Cell Detoxification
(ii) Inactivation of Nuclear Transcription Factor NF-kB, Preventing the Cellular Inflammatory Response
(iii) Inhibition of the PERK Pathway, Preventing the Reticulum Stress, through the UPR System, and Autophagy
Conclusion
Abbreviations
References
Chapter 10
The Health Benefits of Oleocanthal and Other Olive Oil Phenols
Abstract
Introduction
Antioxidant and Anti-Inflammatory Effects
Antitumor Effects
Cardiovascular Protective Effects
Neuroprotective Protective Effects
Antimicrobial Effects
Bioavailability and EVOOLS Metabolism
Experimental Concentration Considerations
Future Research
References
Chapter 11
Health and Economic Impact: Organic Olive Oil
Abstract
Introduction
Agriculture: A New Vision
Literature
Links between Health and Nutrition
References
Chapter 12
Potential of Virgin Olive Oil against Cancer: An Overview of In Vitro and In Vivo Studies
Abstract
Introduction
In Vivo and In Vitro Studies Highlighting the Potential Effect of Olive Oil on Cancer
Relative Potential Compounds in Olive Oil That Reduce Cancer Risks
Oleuropein
Hydroxytyrosol
Oleocanthal
Oleic Acid
Apoptotic Pathways
Nuclear Factor-Kappa B (NF-KB) Signaling Pathway
Conclusion
References
Chapter 13
Hydroxytyrosol and Tyrosol, Main Phenols of Virgin Olive Oil, as Healthy Natural Products in Human Breast Cancer Prevention: A Review
Abstract
Introduction
Hydroxytyrosol and Tyrosol Synthesys in Olives and Bioavailability
Health Benefits of Hydroxytyrosol and Tyrosol in Breast Cancer
Hydroxytyrosol
Tyrosol
References
Chapter 14
Consumption of Extra Virgin Olive Oil and the Components of the Metabolic Syndrome
Abstract
1. Introduction
2. Mediterranean Diet
3. Olive Oil and Its Properties
4. Fatty Acid Composition
5. Antioxidant Content
6. Beneficial Effects of Olive Oil to Counteract the Metabolic Syndrome
6.1. Obesity
6.2. Hypertension
6.3. Dyslipidemia
6.4. Insulin Resistance
Conclusion
References
Chapter 15
Environmental Factors Affecting the Phenolic Profile of Virgin Olive Oil: Compositional, Biochemical and Molecular Aspects
Abstract
1. Introduction
2. Effect of Temperature
3. Effect of Light
4. Effect of Water Deficit
5. Effect of Salinity
6. Effect of Olive Fly Infestation
Conclusion
References
Chapter 16
Networking Entrepreneurship in High Quality Agri-Food: The Case of Olive Oil Production in Apulia
Abstract
Introduction
Literature: Network Relevance
Light and Shade of Olive Oil Sector in Apulia
Analysis
Results
Conclusion
References
Chapter 17
The Growing Import and Domestic Production of Olive Oil in Japan: The Application of the Gravity Model
Abstract
1. Introduction
2. Japan’s Market of Olive Oil
2.1. Import and Market Share
2.2. Import and Market Share
3. Methdology
3.1. Model
3.2. Data
4. Empirical Results and Discussion
4.1. Empirical Results of the Estimation of the Gravity Equation
4.2. Discussion
Conclusion
Acknowledgments
References
Chapter 18
The Effects of Agronomic and Technological Aspects on the Phenolic Profile of Virgin Olive Oil
Abstract
Introduction
1. Phenolic Molecules in Virgin Olive Oil (VOO)
1.1. Definition
1.2. Terminology
1.3. Chemistry of Phenolics
2. Factors Affecting the Phenolic Profile of Virgin Olive Oil (VOO)
2.1. Agronomic and Climatic Aspects
2.1.1. Cultivars
2.1.2. The Degree of Fruit Ripeness
2.1.3. Irrigation
2.1.4. Growing Area and Seasonal Conditions
2.2. Technological Aspects
2.2.1. Olive Fruit Storage
2.2.2. Crushing
2.2.3. Malaxation
2.2.4. Separation System
2.2.5. Oil Storage
References
Chapter 19
An Overview on Olive Oil Mill Wastes Management and Bio-Valorization
Abstract
Introduction
Olive Oil Extraction and Waste Characteristics
Olive Oil Wastes Management Technologies
Physicochemical Processes
Biological Processes
Biovalorization of Olive Mill Wastes
Recovery of Phenols and Other Biologically Active Compounds
Production of Microbial Metabolites
Generation of Biofuels
Bioconversion to Animal Feed
Production of Fertilizers by Composting
Biosorbent for Heavy Metals
Conclusion
References
Chapter 20
The Recovery of Added-Value Compounds from Olive Mill Wastewater
Abstract
Status of Olive Oil Industry: A Problem of Environmental Sustainability
Possible Treatment Procedures for the Management of OMW
The Next Step: What If We Recover Some Added-Value Components from OMW?
Conclusion
Acknowledgments
References
Chapter 21
Patents of Invention on Hydraulic Presses in the Historical Archive of the Spanish Office of Patents and Trademarks for Olive-Oil Production: An Overview (1826-1966)
Abstract
Introduction
Material and Methods
Hydraulic Press
Invention Privileges and Patents
Results and Discussion
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Conclusion
Funding
Acknowledgment
References
Chapter 22
Chemometric Analysis of Geographic Location and Lipophilic Antioxidants in Tunisian Extra Virgin Olive Oils (Olea europaea L.)
Abstract
Introduction
Materials and Methods
Oil Sampling
Measurement of Radical Scavenging Capacity (RSC)
HPLC Analysis of Tocopherols
Oxidation Stability Analysis
Determination of Iodine Value (IV) and Oxidative Susceptibility
Statistical Analysis
Results and Discussion
1. Characterization of VOOs
2. Chemometrics
Correlation among Studied Traits
Classification of Voos from Different Production Regions
References
Index
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Citation preview

FOOD SCIENCE AND TECHNOLOGY

HANDBOOK OF OLIVE OIL PHENOLIC COMPOUNDS, PRODUCTION AND HEALTH BENEFITS

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FOOD SCIENCE AND TECHNOLOGY Additional books in this series can be found on Nova’s website under the Series tab.

Additional e-books in this series can be found on Nova’s website under the eBooks tab.

FOOD SCIENCE AND TECHNOLOGY

HANDBOOK OF OLIVE OIL PHENOLIC COMPOUNDS, PRODUCTION AND HEALTH BENEFITS

JOZEF MILOŠ EDITOR

Copyright © 2017 by Nova Science Publishers, Inc. All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. We have partnered with Copyright Clearance Center to make it easy for you to obtain permissions to reuse content from this publication. Simply navigate to this publication’s page on Nova’s website and locate the “Get Permission” button below the title description. This button is linked directly to the title’s permission page on copyright.com. Alternatively, you can visit copyright.com and search by title, ISBN, or ISSN. For further questions about using the service on copyright.com, please contact: Copyright Clearance Center Phone: +1-(978) 750-8400 Fax: +1-(978) 750-4470 E-mail: [email protected]. NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers’ use of, or reliance upon, this material. Any parts of this book based on government reports are so indicated and copyright is claimed for those parts to the extent applicable to compilations of such works. Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS. Additional color graphics may be available in the e-book version of this book.

Library of Congress Cataloging-in-Publication Data ISBN:  H%RRN

Published by Nova Science Publishers, Inc. † New York

CONTENTS Preface Chapter 1

ix Extra Virgin Olive Oils: Production and Polyphenolic Composition Isabel López-Cortés, Domingo C. Salazar-García, Alba Mondragón-Valero, Gilberto Hernández-Hernández and Domingo M. Salazar

Chapter 2

Olive Varieties and Phenolic Compounds in Olive Oils Gassan Hodaifa and Santiago Rodriguez-Perez

Chapter 3

Biomedicinal Aspects and Activities of Olive Oil Phenolic Compounds Cvijeta Jakobušić Brala, Monika Barbarić, Ana Karković Marković and Stanko Uršić

Chapter 4

Chapter 5

Chapter 6

An Overview of Sample Treatment and Analytical Methodologies for the Determination of Phenolic Compounds in Olive Oils L. Molina-García, M. L. Fernández-de Córdova and E. J. Llorent-Martínez The Natural Variation of Phenolic Compounds in the Fruits and Oils of Olive (Olea europaea L.) Ana G. Pérez, Angjelina Belaj, Mar Pascual and Carlos Sanz Hydroxytyrosol and Tyrosol, Phenolic Compounds of Virgin Olive Oil, Could Act Like Anti-Inflammatories in Chronic Inflammation Cristina Sánchez-Quesada and José J. Gaforio

1

19

47

87

115

133

vi Chapter 7

Contents Oleocanthal: The New Promising Compound of Extra Virgin Olive Oils C. Carrasco and A.B. Rodríguez

145

Chapter 8

The Health Benefits of Olive Oil Phenolic Compounds Hedia Manai-Djebali and Sonia Ben Temime

163

Chapter 9

Extra Virgin Olive Oil and Hepatoprotective Effects Sandra A. Soto-Alarcón, Rodrigo Valenzuela and Alfonso Valenzuela

193

Chapter 10

The Health Benefits of Oleocanthal and Other Olive Oil Phenols Roberto Ambra, Sabrina Lucchetti and Gianni Pastore

215

Chapter 11

Health and Economic Impact: Organic Olive Oil Marilene Lorizio

237

Chapter 12

Potential of Virgin Olive Oil against Cancer: An Overview of In Vitro and In Vivo Studies Houda Nsir, Amani Taamalli and Mokhtar Zarrouk

Chapter 13

Chapter 14

Chapter 15

Chapter 16

Chapter 17

Chapter 18

Hydroxytyrosol and Tyrosol, Main Phenols of Virgin Olive Oil, as Healthy Natural Products in Human Breast Cancer Prevention: A Review Cristina Sánchez-Quesada and José J. Gaforio Consumption of Extra Virgin Olive Oil and the Components of the Metabolic Syndrome Hady Keita and María del Rosario Ayala Moreno Environmental Factors Affecting the Phenolic Profile of Virgin Olive Oil: Compositional, Biochemical and Molecular Aspects David Velázquez-Palmero, M. Luisa Hernández and José M. Martínez-Rivas

251

277

287

303

Networking Entrpreneurship in High Quality Agri-Food: The Case of Olive Oil Production in Apulia Antonia Rosa Gurrieri and Sabrina Spallini

323

The Growing Import and Domestic Production of Olive Oil in Japan: The Application of the Gravity Model Kenichi Kashiwagi

341

The Effects of Agronomic and Technological Aspects on the Phenolic Profile of Virgin Olive Oil Sonia Ben Temime and Hedia Manaî

361

Contents Chapter 19

Chapter 20

Chapter 21

Chapter 22

Index

An Overview on Olive Oil Mill Wastes Management and Bio-Valorization Mohamed Neifar, Fatma Arous, Wafa Hassen, Habib Chouchane, Ameur Cherif and Atef Jaouani The Recovery of Added-Value Compounds from Olive Mill Wastewater J. M. Ochando-Pulido and A. Martinez-Ferez Patents of Invention on Hydraulic Presses in the Historical Archive of the Spanish Office of Patents and Trademarks for Olive-Oil Production: An Overview (1826-1966) José Ignacio Rojas-Sola and Manuel Jesús Hermoso-Orzáez Chemometric Analysis of Geographic Location and Lipophilic Antioxidants in Tunisian Extra Virgin Olive Oils (Olea europaea L.) Amani Taamalli, Hedia Manai, Maria del Mar Contreras and Mokhtar Zarrouk

vii

395

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463

475

PREFACE This book examines the latest research in olive oil. Topics included in this book include biomedicinal activities of olive oil phenolic compounds, including antioxidant, anti-inflammatory, antimicrobial, cardiovascular, endocrine, anticancer and central nervous system effects. Also, some insights related to bioavailability and synergistic activities are presented; a summary and critical analysis of the available information about phenolic compounds in VOO; the beneficial effects of phenolic compounds, contained in extra virgin olive oil, which have been reported in the last few years; an overview of different analytical approaches, including the most recent advances, and the difficulties regarding phenolic compounds determination in olive oil; olive oil wastes (OMW) characteristics, bio-valorization potentialities and treatment options with regard to the economic feasibility, environmental regulations and challenges of existing waste disposal practices in olive-growing countries are discussed; the health effects of olive oil, including for the liver; a summary of the knowledge of the in vitro and in vivo effects of oleocanthal comparing, where available; the determinant factors that affect Japan’s olive oil imports; research on oleocanthal and its promising applications as a preventive and/or therapeutic agent for several diseases; and an examination of the organic olive oil sector, demonstrating it’s importance in terms of wealth and economic impact. Chapter 1 - Olive oil and especially EVOO (Extra Virgin Olive Oil), in addition to being a basic food component of the Mediterranean Diet, plays an essential role in human health due to its polyphenols, sterols and antioxidants composition. The alarming growth in the rates of obesity and diabetes is a problem in Western civilization child health. One of the causes of these pathologies is without a doubt the inadequate high-fat diet, in which components that include fats of animal origin and tropical vegetable origin are used. Currently, this causes the use of hydrogenated fats and others resistant to rancidity. This situation leads to the aforementioned pathologies and therefore makes it absolutely necessary to incorporate organoleptically pleasant EVOO in infant food.

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This is why the objective of this chapter is to know the composition of compounds which play an important health role, such as sterols, tocopherols and polyphenols, all of them present in the main varieties of EVOO. The study was conducted during five consecutive harvests of the composition of the oils, the production and yields of Western Mediterranean varieties. The biometric study of the different varieties has also been carried out, as well as its relationship pulp/stone and the relationships between these parameters are studied in order to establish a broader biochemical characterization of these olive varieties. It is known that the type of crop, rainfed (with the sole contribution of precipitation) and different irrigation doses influence acidic composition, this is why the samples of the main varieties were studied in both conditions, and in trees located in two different areas of cultivation. Thus, the authors were able to establish a characterization and modeling of the composition of EVOO. It is therefore established a better knowledge in light of the current focus on varietal oils and as a basis for formulated bi or polivarietals, to suit consumer demands and in particular to know the role of minority components in EVOO. All materials have been previously characterized by CPVO-TP/099/1. Chapter 2 - The olive stage of ripeness influences in the olive composition i.e., phenolic compounds and concentration vary along ripeness process. Phenolic compounds are key in the oil quality as they are natural antioxidants that contribute to protecting the oil stability against oxidation. Phenols are present in the olive oil in a percentage from 0.1% to 0.3% depending on the olives state of ripeness from which olive oil was extracted. Olives and olive oil contain polyphenols such as oleuropein, hydroxy-tyrosol, tyrosol, rutin, and quercetin, as well as caffeic, vanillic, and o- and p-coumaric acids. Hydroxy-tyrosol significantly inhibits the lipid oxidation of olive oil and has a positive effect on human health. Previous studies have suggested that polyphenols present in the olive oil could improve the oxidative stability of canned products and reduce the concentration of carcinogenic compounds such as heterocyclic amines in fry processes. In fact, diets rich in natural foods and food-derived components such as phenolic compounds receive a great deal of attention because they are perceived as ‘safe’ and ‘non-medicinal’ which some are known to function as chemopreventive agents against oxidative damage, cerebrovascular disease, and aging. The phenolic composition and concentration are different for each olive variety, being the variety “Picual” the one of the highest phenolic concentration. The most abundant polyphenol among the varieties studied was hydroxy-tyrosol, with concentrations of 1.4–12 mg L−1. In general, reduction in polyphenols content along ripeness process was observed. This reduction has been noted in the hydroxy-tyrosol concentration over the ripeness process and varied according to the olive variety, by 10, 2.8, and 1.6 mg L−1 for the varieties ‘Hojiblanca’, ‘Picual’, and ‘Arbequina’, respectively. The evaluation of the influence of the degree of ripeness on oxidative stability of olive oils is important for decisions on producing oils

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with a certain overall quality. The practice of harvesting ripe olives as soon as possible to produce oils of high phenolic contents and thus high oxidative stability implies bitter and piquant oils, this being admissible for oils of some varieties but detrimental to others appreciated in the market for being more fruity and sweeter. Chapter 3 - Olive oil is a basic component of the Mediterranean diet. Mediterranean populations experience reduced incidence of cardiovascular disease, atherosclerosis, diabetes mellitus, metabolic syndrome, neurodegenerative diseases, certain types of cancer and higher life expectancy. Following impressive number of various biomedicinal studies related to the phenomena that accumulate for decades, these health benefits could be at least partially attributed to the olive oil, and more specifically the phenolic compounds naturally present in olive oil. While a number of reports have linked the health benefits of olive oil with its phenolic content, there is a great number of in vitro and in vivo studies that have demonstrated positive effects of olive oil phenolic compounds following analysis of certain physiological parameters. Thus, the phenolic compounds are deemed to be of central importance for beneficial antioxidant, antiatherogenic and anti-inflammatory, antimicrobial, cardiovascular, anticancer, and neuroprotective effects that can be ascribed to the consumption of extra virgin olive oils. The aim of this chapter is to review the biomedicinal activities of olive oil phenolic compounds, including antioxidant, anti-inflammatory, antimicrobial, cardiovascular, endocrine, anticancer and central nervous system effects. Also, some insights related to bioavailability and synergistic activities are presented. Chapter 4 - The increasing popularity of olive oil is mainly attributed to its high content in phenolic compounds, which corresponds with the minor components fraction. Some polar olive oil phenols are not generally present in other fats, and this is one of the reasons that make this product unique. Phenolic compounds comprise a large family of secondary metabolites of plants and present a wide variety of health benefits. These compounds act as natural antioxidants and play an important role in the prevention of human diseases. Due to the chemical diversity of phenolic compounds, and to the fact that some of them are found at very low concentrations, their analysis is relatively complex. Briefly, assays for phenols in olive oil can be classified as those determining the total content of polyphenols, and those allowing the determination of the individual phenolic profile. Most of the analytical methods used for the quantitative determination of total phenols in olive oil are based on colorimetric assays. The analysis of o-diphenolic compounds is also carried out by this type of determination. Qualitative and quantitative composition of phenols in olive oil varies because of the strong dependence on variety and ripeness of the fruit, agronomic factors, and the processing system used for its extraction. Therefore, the identification and quantification of the individual components of olive oil are of great interest. Extraction, chromatographic separation, and characterization are the three basic steps involved in the analysis of the phenolic profile

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of olive oil. The extraction procedures are mainly based on liquid-liquid extraction (LLE) and solid-phase extraction (SPE) using, in most cases, methanol as solvent. Regarding the analytical separation, it can be accomplished by capillary gas chromatography (GC) and, mainly, reverse phase high-performance liquid chromatography (RP-HPLC) with different detectors. Capillary electrophoresis (CE) has also been used for this purpose, achieving the same aims than HPLC but higher resolution, and reducing sample volume and analysis time. Nevertheless, nowadays liquid chromatography coupled to mass spectrometry (HPLC-MS) is widely accepted as the main tool in identification, structural characterization, and quantitative analysis of phenolic compounds in olive oil. Nuclear magnetic resonance (NMR) spectroscopy is also a powerful complementary technique for structural assignment in the cases where mass spectral data are insufficient to establish a definitive structure for phenolic compounds. In recent years, the hyphenation of HPLC with the most information-rich spectroscopic technique NMR has been proposed for structure elucidation of phenolic compounds in olive oil. Several studies have been carried out regarding the development of efficient and accurate analytical methods for the qualitative and quantitative analysis of phenolic compounds in olive oil. This chapter pretends to show an overview of different analytical approaches, including the most recent advances, and the difficulties regarding phenolic compounds determination in olive oil. Chapter 5 - Different scientific evidences suggest that the long term dietary consumption of virgin olive oil (VOO) seems to be related to an attenuation of the inflammatory response and reduction of the associated risk of chronic inflammatory disease states. VOO phenolic compounds are claimed to be the main responsible for these positive health benefits. They are mainly synthetized from phenolic glucosides present in the olive fruit by the action of glucosidases occurring when they come together once the olive fruit is crushed during olive oil extraction. The genetic variability of the major phenolic compounds was studied in a representative sample of olive cultivars (Olea europaea L.) from the World Olive Germplasm Collection established at IFAPA Centre “Alameda del Obispo” in Cordoba, Spain. The most abundant phenolic components found in VOO are the secoiridoid derivatives resulting from the enzymatic hydrolysis of oleuropein, ligstroside and demethyloleuropein present in the fruit, which showed to be on average the main phenolic glucosides. The mean content of phenolic compounds in the oils was 494.51 µg/g oil, displaying a variability range of 63.74-1432.04 µg/g oil. The mean content of phenolic compounds in the fruits was 12384.23 µg/g fruit with a range of 3754.13-30696.39 µg/g fruit. Total phenolic compounds in the fruits and the oils were significantly correlated (r = 0.66). Thus, the composition and biochemical status of the olive fruit seem to be the most important variables determining the synthesis of the VOO phenolic compounds during the oil extraction process. On the other hand, the content of oleuropein and derivatives in the oils and fruits showed a correlation coefficient (r = 0.64) lower than that observed for ligstroside and derivatives (r = 0.73). These findings

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might be related to the higher oxidation rates of the former due to the action of oxidative enzymes during the oil extraction process as a consequence of the orthodiphenolic structure they possess. Data on phenolic composition would be of interest for the selection of optimal parents in olive breeding programs with the aim of obtaining new cultivars with improved nutritional quality. Chapter 6 - Hydroxytyrosol (HT) and tyrosol (TY) are two phenols present in virgin olive oil (VOO), the principal fat used in Mediterranean countries. It is well known the benefits of Mediterranean diet in the prevention and development of certain illness as cancer or cardiovascular diseases as well as the natural protection exerted by VOO in these same diseases. When there is an injury in the body, inflammation is one of the early response to the damage; immune cells drive the immunologic response to clean and heal the damage. An uncontrolled response of the immune system could derive in the appearance of several illness. Macrophages are the main cells that control this process and their appearance and activity is the key of the development of cancer or Crohn’s diseases, among others. In this chapter, the effects in macrophages of two natural compounds (HT and TY) present in VOO are studied. Results showed that both compounds are able to manage M1 macrophage response into an anti-inflammatory state, which could be very useful in the treatment of several diseases as Crohn’s or inflammatory bowel diseases. Even more, there could be able to prevent chronic inflammation, which in turn is one of the reason of certain disease appearances. Chapter 7 - Traditionally, the healthy properties of extra virgin olive oil have been attributed to its monounsaturated fatty acid high content. However, increasing evidence points out the participation of different minor antioxidant components, such as phenolic compounds. For the last decade, increasing research efforts have been made to explore the beneficial effects of these phenolic compounds on several physiological and physiopathological processes. Depending on the grade of bioavailability of each phenolic compound, they seem to carry antioxidant, anti-inflammatory and antimicrobial properties. One of the newest phenolic compounds discovered in the extra virgin olive oil food matrix is oleocanthal. The discovery of this molecule opens new perspectives on the biomedical applications of this natural compound with similar properties to those of socalled nonsteroidal anti-inflammatory drugs (NSAIDs). Oleocanthal has also exhibited antitumor properties on several tumor cell lines via different molecular mechanisms. Moreover, it has been proposed as an effective agent for the treatment of Alzheimer´s disease. Therefore, it is necessary to increase research efforts about oleocanthal and its promising applications as a preventive and/or therapeutic agent for several diseases. Chapter 8 - It has been known for decades that olive oil has beneficial health effects. However, only recently the biological properties of its constituents have been investigated. Recent studies confirmed that phenolic compounds of olive oil have several beneficial effects on human health. Olive oil is a source of at least thirty six phenolic compounds. The major phenolic compounds in olive oil are hydroxytyrosol, tyrosol and

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oleuropein and their derivatives. The olive oil phenols includes numerous classes, such as phenolic acids like caffeic, gallic, vanillic and coumaric acids, phenolic alcohols including tyrosol and hydroxytyrosol and more complex compounds such as secoiridoids (oleuropein, ligstroside…), lignans (acetoxypinoresinol and pinoresinol), flavonoids and finally hydroxyl-isochromans. All these classes of phenols have potent antioxidant, antiinflammatory, antimicrobial and antiviral activities. High consumption of extra virgin olive oils, which are particularly rich in these phenolic antioxidants should afford considerable protection against ageing, cardiovascular, cerebrovascular and neurodegenerative diseases, diabetes mellitus, metabolic syndrome and several cancers by inhibiting oxidative stress. A reduction in total mortality has also been confirmed after consumption of olive oil phenols. Some phenolic compounds of olive oil have also been demonstrated that they act as anti-allergic and an infertility treatment agents.This work summarizes the beneficial effects of phenolic compounds, contained in extra virgin olive oil, which have been reported in the last few years. Chapter 9 - The liver plays a vital rol in the body; performs metabolic, digestive, immunological, reservoir and homeostatic functions. However, is an organ susceptible to multitude injuries, which can be caused by viruses, drugs, toxic substances, alcohol and by obesity. Extra virgin olive oil (EVOO) is considered the gold standard of edible oils, has a composition rich in monounsaturated fatty acids and other minor components, including many phenolic compounds which may have beneficial effects on human health. EVOO has positive impact on different stages of liver damage, including hepatic steatosis (reducing the number and size of fat globules and the accumulation of triglycerides), nonalcoholic steatohepatitis (reducing inflammation and oxidative stress), cirrhosis (inducing less formation of fibrous tissue), hepatocellular carcinoma (due to its antioxidant properties reduces ROS production, decreasing DNA damage and promotes cellular apoptosis) and ischemia-reperfusion injury (reducing the liver damage and ROS levels). EVOO in liver is involved in the activation of various metabolic pathways in order to prevent inflammation, oxidative stress, endoplasmic reticulum stress, mitochondrial dysfunction and insulin resistence, key situations in the onset and progression of hepatic tissue damage. Among the most important molecular effects of EVOO in the prevention of liver damage are: i) activation of nuclear transcription factor Nrf2, inducing a cellular antioxidant response by the positive regulation of gene expression of antioxidant enzymes and/or of enzymes involved in cell detoxification; ii) inactivation of nuclear transcription factor NF-kB, preventing the cellular inflammatory response and; iii) inhibition of the PERK pathway, preventing reticulum stress and autophagy. The present chapter reviews the main scientific evidence about the hepatoprotective action of EVOO, discussing the molecular mechanisms involved in this protection. Chapter 10 - Different phenolic compounds are present in extra-virgin olive oil (EVOO). The biological effects of the more characterizing for EVOO, i.e., secoiridoids,

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have been studied since their discovery and their health beneficial properties, that go beyond merely antioxidant activities, are becoming more and more recognized. Oleocanthal is the secoridoid responsible for the oral irritation induced by EVOO and perceptually it gives an oropharyngeal sensation similar to that produced by ibuprofene. Oleocanthal similarity with ibuprofene is also at the pharmacological level since it induces a dose dependent inhibition of COX enzymes even greater than the drug. Furthermore, oleocanthal is able to inhibit in vivo the formation of neurofibrillary aggregates responsible for Alzheimer’s disease. This review summarizes the knowledge of the in vitro and in vivo effects of oleocanthal comparing, where available, data with related EVOO phenols, and focusing on its anti-inflammatory, chemotherapeutic, neuroprotective and antimicrobial activities, discussing also bioavailability and experimental concentration issues. Chapter 11 - Agriculture not only responds to meet food needs, but it must also respond to the needs and institutional context. The agricultural sector supports and creates employment opportunities and also the economic viability of rural areas with low settlement. In this scenario, organic olive oil area is one of the most important of the agricultural sector because olive oil is the link between health and nutrition. The aim of the paper is to investigate organic olive oil sector, thus to demonstrate it’s importance in terms of wealth and economic impact. Chapter 12 - Mediterranean diet (MD) represents the gold standard in preventive medicine due to its association with lower overall mortality patterns. Its role in human nutrition is one of the most important areas of investigation, therefore a wide range of epidemiological studies found an inverse correlation between the MD consumption and the incidence of certain cancers in populations living in the Mediterranean area, compared with populations living in Northern Europe or the USA, probably because of the harmonic combination of many elements with antioxidant and anti-inflammatory properties. Extra virgin olive oil (EVOO) stands for the main source of fat in MD, characterized by bioactive components particularly phenolic compounds, it has a potential preventive and functional action on Cancer disease, liver, breast, colon which present the second cause of death after cardiovascular diseases worldwide. The beneficial effects of EVOO have been attributed mostly to its phenolic fraction and its anti-proliferative properties causing apoptosis of human cancer cells. In this regard, this chapter presents an overview of benefits of olive oil against cancer, assessing and discussing the mechanism of action undersigning these effects as well as case studies. Chapter 13 - Hydroxytyrosol (HT) and tyrosol (TY) are two of the main phenolic compounds present in several plants of the vegetable kingdom, but with major presence in the product of olive trees. These two compounds appear from the secoiridoid hydrolysis of virgin olive oils during storage. While the concentration of TY is always higher than HT, the quantity of both depend on the olive tree variety, climatic and agronomic conditions. Multiple health claims are attributed to these two compounds (as

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cardioprotectives, antioxidants, protection against DNA…). Therefore, recent studies show us that they may play a key role in the development and prevention of different kind of cancers, among them breast cancer. This chapter explain and describe the more notably beneficial effects of these two compounds in the prevention, appearance and development of breast cancer. In vivo and in vitro studies demonstrate that these two phenols could aid in chemotherapies against breast cancer and in the prevention of it. Nevertheless, although they are natural compounds, special attention should be paid to the concentration administrated, this is the main reason why more deeply studies are needed for asseverate their preventive and antitumoral activities. Chapter 14 - Chronic-degenerative diseases are currently a public health problem worldwide, including dyslipidemias, alterations in glucose metabolism, arterial hypertension and abdominal obesity, which together characterize the so-called metabolic syndrome. The risk of developing cardiovascular disease is estimated to be approximately double in subjects with metabolic syndrome and three times the risk of developing type 2 diabetes, which represent the main causes of death in the population. Although metabolic syndrome is a complex and multifactorial entity, the alarming increase in the components of metabolic syndrome is mainly associated with two etiological factors, the decrease in physical activity and the dietary pattern in the population, particularly the high consumption of energy from simple carbohydrates and fats. The authors know that maintaining a healthy diet is essential to prevent metabolic alterations, not only in terms of total caloric intake, but also the “quality” of fats consumed, i.e., the type of fatty acids that characterize the individual’s diet is determinant for the development of the disease. The Mediterranean diet has become in recent decades a reference icon for healthy eating, and its beneficial effects have been recognized by scientists, doctors, nutritionists and international organizations such as the World Health Organization (WHO), the organization of the United Nations for Food and Agriculture (FAO), among others. It is now known that extra virgin olive oil is one of the fundamental components of the Mediterranean diet, and that it is characterized by a high content of essential fatty acids and phenolic components, whose biological effect on the body gives it preventive properties and/or the ability to reduce the incidence of chronic-degenerative diseases associated with metabolic syndrome. The results summarized in this chapter have been obtained from population studies, as well as experimental animal models, which demonstrate some of the mechanisms of action of the main components of extra virgin olive oil, on metabolic processes related to the pathophysiology of metabolic syndrome, that confer the beneficial effects to this oil. The above highlights the importance of the Mediterranean diet on the prevention and/or control of metabolic syndrome. Chapter 15 - The phenolic content and profile of virgin olive oil is determined by the composition and biochemical status of the olive fruit. In this sense, the presence of phenolic compounds in virgin olive oil is directly related to the content of phenolic glycosides initially present in the olive fruit tissues and the activity of hydrolytic and

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oxidative enzymes acting on these glycosides during olive fruit processing. Recently, the first olive fruit genes and enzymes involved in the biosynthesis and transformation of the phenolic compounds primarily detected in the olive fruit mesocarp have been characterized. In the present review the authors will focus on the effect of environmental factors which can alter the phenolic composition of virgin olive oil, such as temperature, light, water deficit, salinity, and olive fly infestation. In addition, the regulation of the mentioned genes and enzymes in response to those environmental factors is also discussed. These recent studies represent an important step towards the understanding of the molecular mechanisms that regulate the phenolic composition of virgin olive oil. Chapter 16 - Networking entrepreneurs stimulate the growth of the group through the reinforcement of the relations among the firms involved. The opportunity to work in clusters is particularly necessary for SMEs, which always have a low power, especially for those who work in a low technological sector. Italy is characterized by a massive presence of small and medium enterprises, organized in districts or networks. Despite the economic crisis, some of these groups are able to survive and score positive performances both locally and abroad. In this work the authors investigate the olive-oil biological sector, which is able to tow the Apulian economy based on its vocation. This sector shows a trend in network formation. Moreover, from an entrepreneurial point of view, such a strategy brings advantages both for the single unit and the group. This study aims at investigating the effect of the network on firm performances, considering the identity of the network and its social interactions. The authors believe that the entrepreneur’s ability to innovate and the locational advantages in supporting network necessity are relevant factors for a network identity. The common factors are the entrepreneurial culture and social contacts of the team. Chapter 17 - As the size of the global olive oil market has been rapidly growing, new markets emerged outside Europe, particularly in the Asia-pacific region. Japan, as an export destination, is an emerging market for olive oil in East Asia. This chapter investigates the determinant factors that affect Japan’s olive oil imports. Based on unbalanced panel data of Japan’s olive oil imports from 15 trade partners from 1988 to 2013, the commodity-specific gravity model is estimated. The results suggest that an increase in the GDP of Japan and its trade partners has a positive effect and the distance between them is a resistance factor. The difference in factor endowments has a negative impact on olive oil imports, whereas increasing domestic production has a positive effect on the flow of imports. Together with the fact that Japan increased its olive oil exports, these results indicate the development of an intra-industry olive oil trade and support the concept of a new trade theory rather than the traditional Heckscher-Ohlin discussion. These findings imply that promotion of the export of varieties of olive oil through product differentiation would be more relevant to explore Japan’s emerging market, rather than producing a large quantity sold at a lower price.

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Chapter 18 - Virgin olive oil (VOO) has excellent nutritional, technological and sensory characteristics that make it a unique and basic ingredient of the Mediterranean diet and accumulating evidence suggests that it may have health benefits which include reduction of risk factors of coronary heart disease, prevention of several types of cancers, and modification of immune and inflammatory responses. VOO can be considered as example of a functional food, with a variety of components that may contribute to its overall therapeutic characteristics. The importance of VOO is mainly attributed both to its high content of oleic acid, a balanced contribution quantity of polyunsaturated fatty acids, and its richness in phenolic compounds, which act as natural antioxidants and may contribute to the prevention of several human diseases. The main classes of phenols in virgin olive oil are phenolic acids, phenolic alcohols, hydroxy-isocromans, flavonoids, secoiridoids and lignans. The concentration and composition of phenolic compounds in virgin olive oil is strongly affected by many agronomical and technological factors, such as olive cultivar, place of cultivation, climate, degree of maturation, irrigation, crop season and production process. The aim of this review paper is to summarize and critically analyze the available information about phenolic compounds in VOO. Chapter 19 - The olive oil industry has experienced continuous growth mainly due to its nutritious and economic importance, particularly in Mediterranean countries. This is accompanied by the disposal of large amounts of wastes produced by different phases of olive oil extraction technologies (traditional pressing or centrifugation). The composition of olive mill wastes (OMW) varies considerably, owing to geographical and climatic conditions, olive tree variety and age, agricultural practices, olive type, extraction technology, use of pesticides and fertilizers, harvest time and stages of maturity. OMW are difficult to treat due to their high organic load composed of sugars, tannins, phenols, polyalcohols, pectins, proteins, oil emulsion, etc. For this reason, several management strategies have been investigated for the treatment and valorization of OMW, including physical, chemical and biological processes as well as combination of thereof. On the other hand, OMW can be used in a wide range of biotechnological applications which could enhance the economic viability of the various systems used for OMW treatment. In this chapter, OMW characteristics, bio-valorization potentialities and treatment options with regard to the economic feasibility, environmental regulations and challenges of existing waste disposal practices in olive-growing countries are discussed. Chapter 20 - Olive oil industry is actually one of the main engines of the economy of the Mediterranean Basin countries, of which Spain, Italy and Greece cope with the highest total production worldwide, but neither should be disregarded the rapid widespread of this industry in other countries such as Syria, Argelia, Turkey, Morocco, Tunisia, Portugal, France, Libya, Lebanon, Serbia and Montenegro, Macedonia, Cyprus, Egypt, Israel, Jordan, as well as in the USA, Argentina, Australia and China, the last country incorporated to this industrial sector and with an enormous growth potential.

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In the past decades, extraction of olive oil by means of the traditional batch press method was substituted by more efficient continuous production processes to meet the growing demand of olive oil worldwide. In olive oil mills two main liquid effluents stand out, one derived from the washing of the fruit (OWW) and the other from the extraction of the olive oil (OVW, a mixture of the proper olive-fruit humidity along with processadded water). These effluents are commonly referred to as olive mill wastewater (OMW). Only in Spain, a total volume of more than 9 million m3 of OMW per year is generated. Many obstacles are met in regard to the management of OMW. As pointed, small size of olive oil mills in addition to their geographical dispersion poses the main evident difficulty from an economical point of view, neither forgetting seasonality of olive oil production. Moreover, OMW physico-chemical composition is also dependent on several factors highlighting not only the extraction process, but also edaphoclimatic and cultivation parameters, as well as the type, quality and maturity of the olives, and thus the pollutants load of this wastewater is extremely variable. Uncontrolled disposal of OMW is at the present time a patent environmental hazard, since it gives rise to underground leakage, soil contamination and water body pollution. Major organic pollutants presence is confirmed including phenols, organic acids, tannins and organohalogenated contaminants, mostly phytotoxic compounds recalcitrant to biological degradation. The management of olive mill wastewaters (OMW) is a task of global concern not anymore constrained to a specific region, and represents an ever-increasing problem still unresolved. OMW is one of the main wastes generated during the production of olive oil and represents the main environmental problem of this production process. On another hand, the principal components of OMW are polysaccharides, sugars, polyphenols, polyalcohols, proteins, organic acids, fatty acids and oil. Among them, phenolic compounds represent one of the major factors of the environmental problems caused by OMW. They are present in considerable concentration and have different negative effects such as phytotoxicity, toxicity against aquatic organisms, suppression of soil microorganisms and difficulty to decompose. Despite that fact, they possess high antioxidant activity that makes them interesting for the food, pharmaceutical and cosmetic industry. Because of that, their recovery represents an important objective for olive oil industry that can help obtain interesting extracts and diminish the volume of these effluents. This chapter will give a focus on the management of OMW, including not only the treatment possibilities, but also the recovery of added-value components, particularly polyphenolic compounds. Chapter 21 - The evolution of techniques and procedures for milling and pressing olives in the batch system of olive-oil production, over the centuries, has been marked by physical or geometrical considerations, and by the evolution of technology.

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In pressing milled olives, the primary aim has always been to apply the highest pressure to get the greatest amount of olive oil in the first pressing, since this renders oil of the highest quality. However, the greatest advances in the field of mechanical engineering and applied physics occurred in the 17th and 18th centuries, for example, with the introduction of the wheel-driven presses using gears that multiply the pressure, and with the introduction of the Pascal’s Principle in the hydraulic presses. These types of presses are the subject of this historical research because they were the last great impetus to the oil-production process, before the implementation of the continuous system by centrifugation. The research presented in this chapter constitutes a work of industrial archaeology, with a dual purpose: first, to describe the technological improvements of the hydraulic presses and, secondly, to display the graphic information such as drawings as documentation of olive-oil industrial heritage. Thus, the authors have documented privileges and patents of invention in the Historical Archive of the Spanish Office of Patents and Trademarks for the period 1826-1966. Specifically, 14 historical inventions are described, showing an overview of significant technological improvements to the optimization of olive-oil production, and presenting an overview of the technological evolution of the hydraulic press. Chapter 22 - In this work, extra virgin olive oils (EVOOs) from the Tunisian cultivar ‘Chemlali’ grown in different regions were studied. Several characteristics such as lipophilic antioxidants α-, β- and γ-tocopherols, stability to oxidation, monounsaturated fatty acids, iodine value, stability susceptibility and radical scavenging capacity. Chemometric analysis was applied to explore correlations among studied parameters and classify the VOO samples according to the production origin. Significant differences were observed between the studied samples. Principal Component Analysis and Hierarchical Cluster Analysis permitted a good classification of the VOOs according to their production region. Three main groups could be distinguished. According to the studied VOOs, Oueslatia, AinZena, El Bhayer and Bir Ali Ben Khelifa production zones appear of a great interest for the ‘Chemlali’ cultivar.

In: Handbook of Olive Oil Editor: Jozef Miloš

ISBN: 978-1-53612-356-2 © 2017 Nova Science Publishers, Inc.

Chapter 1

EXTRA VIRGIN OLIVE OILS: PRODUCTION AND POLYPHENOLIC COMPOSITION Isabel López-Cortés1,*, Domingo C. Salazar-García2, Alba Mondragón-Valero1, Gilberto Hernández-Hernández1 and Domingo M. Salazar1 1

Department of Vegetal Production, Universitat Politècnica de València, Valencia, Spain 2 Department of Archaeogenetics, Max-Planck Institute for the Science of Human, History, Jena, Germany Ikerbasque Basque Foundation for Science, Vitoria-Gasteiz, Spain

ABSTRACT Olive oil and especially EVOO (Extra Virgin Olive Oil), in addition to being a basic food component of the Mediterranean Diet, plays an essential role in human health due to its polyphenols, sterols and antioxidants composition. The alarming growth in the rates of obesity and diabetes is a problem in Western civilization child health. One of the causes of these pathologies is without a doubt the inadequate high-fat diet, in which components that include fats of animal origin and tropical vegetable origin are used. Currently, this causes the use of hydrogenated fats and others resistant to rancidity. This situation leads to the aforementioned pathologies and therefore makes it absolutely necessary to incorporate organoleptically pleasant EVOO in infant food.

*

Corresponding author: Professor Isabel López-Cortés Department of Vegetal Production. Universitat Politècnica de València. Camino de Vera S/N Valencia 46022. Spain. [email protected].

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I. López-Cortés, D. C. Salazar-García, A. Mondragón-Valero et al. This is why the objective of this chapter is to know the composition of compounds which play an important health role, such as sterols, tocopherols and polyphenols, all of them present in the main varieties of EVOO. The study was conducted during five consecutive harvests of the composition of the oils, the production and yields of Western Mediterranean varieties. The biometric study of the different varieties has also been carried out, as well as its relationship pulp/stone and the relationships between these parameters are studied in order to establish a broader biochemical characterization of these olive varieties. It is known that the type of crop, rainfed (with the sole contribution of precipitation) and different irrigation doses influence acidic composition, this is why the samples of the main varieties were studied in both conditions, and in trees located in two different areas of cultivation. Thus, we were able to establish a characterization and modeling of the composition of EVOO. It is therefore established a better knowledge in light of the current focus on varietal oils and as a basis for formulated bi or polivarietals, to suit consumer demands and in particular to know the role of minority components in EVOO. All materials have been previously characterized by CPVO-TP/099/1.

Keywords: polyphenols, production, yields, antioxidants, sterols

INTRODUCTION The oil obtained from the olive tree has been considered to be a food ingredient, raw material for lighting, medicinal ointment and liquid body "revitalizer" by Phoenicians, Greeks and Carthaginians, basically all Mediterranean cultures. They were able to wisely pass this knowledge onto the younger generation integrated in the physical framework of the Mare Nostrum [36]. This meant that over the years, the extra virgin olive oil (EVOO) obtained from the fruit of the olive has gradually been regarded as a high value luxury food in the nutrition sector. Valenzuela (1940) stated in a small section of his work that he named Olive oil in the human health that “the nutritional value of olive oil is very high in calories and in proper compounds, but it can provide excess calories, olive oil facilitates the biliary secretion transforming food and facilitating their absorption like no other food. It is recognized as a morphological and growing food, a vitamin supplement, it helps body development and offers energy like no other food, because if Andalusian peasants … and live for a long time it is because of their diet based on bread and oil in its various preparations such as migas, salmorejo, cascaflore, churros, croutons and gazpachos. Modern medicine uses it orally in weak subjects, injections in therapeutic applications, it is recommended to correct narrowness of esophagus and pylorus, in the treatment of stomach ulcer, constipation, biliary lithiasis and anemia.”

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Olive oil is unlike other vegetable oils in two clear characteristics, it comes from a fruit and it is edible (refining is not needed) at the time of production when they come from good quality raw material. The presence of olive oil in a healthy diet has been proved on multiple occasions and thanks to different clinical studies and in recent years, its great antioxidant role has been ensured. We have studied the composition of the oils obtained from the different varieties present in our working area. In order to obtain the oils of this work, samples were taken of olives of homogeneous plots in two different areas, both set in an altimetry of 550 m and in the same climatic and cultivation conditions, these plots are located in the area of Valencia (Spain). The olives were collected in the phenological stage 85, which allows for a comparison of the obtained results. As may be observed in Table 1, these varieties show great variability or dispersion in terms of fruit weight and endocarp and, consequently, in its pulp/endocarp relation, the latter being an appropriate study given that some of these varieties can be interesting as double aptitude. Table 1. Weight and pulp/endocarp relation of olives according to olive oil variety origin Nº 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Olive oil v ariety Alfafara Aguilar Arbequina Baix fulla Blanqueta Blanqueta roja Blanqueta reina Blanquiroja Borriolenca Cabaret Callosina Carrasqueña Casellera Cornicabra Cuquillo Changlot Real Choco De la Cueva De la Lloma Del Pomet Dotó Egipcia Empeltre Farga

Olive weight (g) 6.43 ± 2.04 1.95 ± 0.28 1.51 ± 0.86 4.10 ± 0.75 1.65 ± 0.25 2.13 ± 0.49 3.24 ± 1.03 1.77 ± 0.32 4.12 ± 0.58 3.60 ± 0.70 4.67 ± 1.27 3.60 ± 0.77 6.06 ± 0.27 4.01 ± 0.29 1.03 ± 0.36 3.36 ± 0.68 1.54 ± 0.27 2.51 ± 0.96 2.24 ± 0.45 2.54 ± 0.99 1.52 ± 0.17 2.81 ± 0.63 2.79 ± 0.54 1.57 ± 0.33

Endocarp weight (g) 0.68 ± 0.18 0.31 ± 0.02 0.24 ± 0.07 0.52 ± 0.05 0.26 ± 0.05 0.27 ± 0.04 0.30 ± 0.05 0.28 ± 0.04 0.46 ± 0.07 0.51 ± 0.08 0.64 ± 0.08 0.46 ± 0.10 0.45 ± 0.09 0.45 ± 0.09 0.22 ± 0.03 0.45 ± 0.10 0.42 ± 0.06 0.47 ± 0.09 0.47 ± 0.08 0.34 ± 0.04 0.32 ± 0.05 0.64 ± 0.04 0.32 ± 0.02 0.36 ± 0.06

Pulp/endoc. Rel. 89.42 84.10 84.11 87.32 84.24 87.32 90.74 84.18 88.83 85.83 86.30 87.22 92.57 88.78 78.64 86.61 72.73 81.27 79.02 86.61 78.95 77.22 88.53 77.07

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I. López-Cortés, D. C. Salazar-García, A. Mondragón-Valero et al. Table 1. (Continued) Nº 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53

Olive oil v ariety Figuereta Genovesa Godellera Grossal Hojiblanca Jandra Llumero Manzanilla Marfil Mas Blanc Millareja Moixentina Monteaguda Morons Morruda Negra Patronet Picual Piñonera Quartera Rotja Rotgeta Rufina Seniero Serrana Valentins Vallesa Verdal Villalonga

Olive weight (g) 1.49 ± 0.15 6.15 ± 0.87 3.52 ± 0.69 4.09 ± 1.09 8.54 ± 1.40 3.52 ± 0.71 1.93 ± 0.30 2.14 ± 0.39 2.08 ± 0.36 2.61 ± 0.27 4.67 ± 0.93 3.83 ± 0.53 2.44 ± 0.51 3.01 ± 0.58 2.60 ± 0.61 2.21 ± 0.35 1.76 ± 0.29 3.67 ± 0.72 2.18 ± 0.48 4.58 ± 0.95 2.81 ± 0.97 1.98 ± 0.37 4.19 ± 0.77 2.30 ± 0.56 2.71 ± 0.76 3.75 ± 0.79 3.61 ± 0.83 1.90 ± 0.32 3.96 ± 0.94

Endocarp weight (g) 0.22 ± 0.03 0.60 ± 0.11 0.50 ± 0.09 0.47 ± 0.11 1.47 ± 0.20 0.48 ± 0.10 0.39 ± 0.20 0.41 ± 0.02 0.36 ± 0.06 0.27 ± 0.02 0.51 ± 0.08 0.46 ± 0.05 0.48 ± 0.08 0.43 ± 0.08 0.50 ± 0.09 0.40 ± 0.08 0.35 ± 0.08 0.51 ± 0.06 0.37 ± 0.07 0.82 ± 0.15 0.45 ± 0.12 0.37 ± 0.08 0.63 ± 0.13 0.41 ± 0.09 0.46 ± 0.15 0.58 ± 0.21 0.57 ± 0.15 0.30 ± 0.03 0.46 ± 0.16

Pulp/endoc. Rel. 85.23 90.24 85.80 88.51 82.79 86.36 79.79 80.84 82.69 89.66 89.08 87.99 80.33 85.71 80.77 81.90 80.11 86.10 83.03 82.10 83.99 81.31 84.96 82.17 83.03 84.53 84.21 84.21 88.38

From all the olives studied, the ones which have a higher weight are the Hojiblanca, Alfafara and Casellera varieties, especially Hojiblanca, with an average weight of over 8 grams, while those that have a lower weight are the Cuquillo, Figuereta and Arbequina varieties. On the other hand, as we already mentioned, the pulp/stone relation is important to know the suitability of the olives, since a high flesh to stone ratio indicates that these varieties have better conditions for double aptitude, in this parameter the Casellera and Genovesa varieties stand out. We must take this into account because the polyphenols content can be taken from the oil but also from marinated olives, in which content can increase, this happens in some traditional marinades with thyme and rosemary. Obtaining quality oil can be done using different extraction systems, but depending on the different industrial yields obtained, we will be able to classify oils as from an industrial point of view or economic classification.

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Table 2. Industrial and dry yields according to oil variety origin Olive oil variety Alfafara Aguilar Arbequina Baix fulla Blanqueta Blanqueta roja Blanqueta reina Blanquiroja Borriolenca Cabaret Callosina Carrasqueña Casellera Cornicabra Cuquillo Changlot Real Choco De la Cueva De la Lloma Del Pomet Dotó Egipcia Empeltre Farga Figuereta Olive oil variety Genovesa Godellera

Ind. Y. 25.38 22.63 23.50 24.53 23.47 22.53 24.60 28.82 28.86 22.15 24.56 26.34 23.19 26.33 28.45 28.28 28.74 26.29 29.32 24.32 21.45 26.59 13.72 29.63 28.91 Ind. Y. 21.81 23.68

Dry Y. 60.78 50.93 51.34 60.18 56.87 54.64 63.49 51.69 60.14 48.62 51.43 57.30 50.18 55.63 54.31 56.58 53.32 41.61 65.43 44.56 48.54 41.48 44.04 57.43 54.00 Dry Y. 45.28 49.72

Olive oil variety Grossal Hojiblanca Jandra Llumero Manzanilla Marfil Mas Blanc Millareja Moixentina Monteaguda Morons Morruda Negra Patronet Picual Piñonera Quartera Rotja Rotgeta Rufina Seniero Serrana Valentins Vallesa Verdal Olive oil variety Villalonga

Ind. Y. 29.42 29.47 25.12 28.31 23.71 22.63 25.94 21.56 22.57 23.74 31.90 29.78 27.97 24.65 21.90 20.50 16.74 31.35 23.18 20.75 24.95 26.17 37.09 30.29 23.61 Ind. Y. 28.02

Dry Y. 56.46 53.27 41.89 56.14 48.72 47.07 48.06 49.49 51.23 61.58 52.08 57.74 54.51 51.84 51.84 50.67 41.42 52.30 49.63 48.69 59.35 43.25 60.63 54.46 41.98 Dry Y. 46.67

Higher industrial yield olives are the Valentins, Vallesa, Rotja and Morons varieties, all of them with a performance higher than 30%, whereas the lowest performance varieties are Empeltre, Del Pomet and Quartera, with parameter values lower than 20%. Performance can also be measured in dry, resulting in differences between the varieties studied. The ones with a greater performance are De la Lloma, Blanqueta Reina, Monteaguda, Alfafara, Valentins and Baix fulla varieties, all of them with performances above 60%, while the minimum values for this measure are the Pomet and Serrana varieties with performances under 40%. It is well known that fats are necessary for our normal development, they are basic structural elements for our cells [36]. Nevertheless, control over the level of consumption is necessary, since they contribute to the evolutionary process of atherosclerosis and some types of thrombosis.

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The oil from the olive tree, depending on its quality level, has always been used in multiple ways, amongst which we must emphasize the following:    

  



Sacralization (in practically all religions in the Mediterranean). Present in many religious and sociopolitical ceremonies Marinating in food preparation Food preservation, especially in order to avoid its oxidation and the growth of bacterial colonies EVOO as food incorporated many years ago to the mediterranean diet, more recently considered not only the healthiest fat product but also nutraceutical and preventive of a broad group of human pathologies. Used in lighting Hygiene, cleaning and cosmetics (perfumes, ointments, creams) Pharmacological, used as raw material for certain active components or as an excipient, especially since it was associated its consumption and serum levels of cholesterol with coronary heart disease and other pathologies Component of embalming compounds

Evidence shows that its protective effect is due to fatty acids, especially monounsaturated, but also other components such as vitamin E and other antioxidants must be taken into account [1, 14]. The knowledge of the sterol composition (Table 3) and tocopherols is required to know and describe, as the Community Regu which we have worked. Like polyphenols, tocopherols of which there are four and their respective four tocotrienols in EVOO, although α-tocopherol is the clear majority. And has more biological activity than the rest, are potent antioxidants that in addition to controlling the fluidity of the membranes due especially to the phenolic group that it contains, also increases immune response and regulates platelet aggregation with its power to inhibit the cyclooxygenase reducing the formation of Thromboxanes (postgandines). It also participates in the inhibition of cell proliferation [26] by inhibiting the action of the proteinquinasa [2] so it may delay cancer but not reverse a carcinogenic process that has already begun. α-tocopherol also plays an important role in cardiovascular pathologies since it minimizes the development of atheromas (artery blockage plaques), decreases triglycerides plasma levels and prevents the oxidation of LDL because when this oxidation takes place, the atherosclerotic process begins [6, 11, 17] producing this synergistic action with ascorbic acid and, in general with all flavonoids [27]. Tocopherols also contribute significantly to the improvement of type II diabetic patients, which has been proven by various authors, first in pregnant and newborn rats, and subsequently in pigs, where it was shown that tocopherols are able to minimize the

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potential derived teratogenic effects, although it is multifactorial, it is possible in cases of females with mellitus insulin dependent diabetes, because there is an increase of free radicals and a decrease of antioxidants [28]. It has also been verified that EVOO with a high monounsaturated content and rich in α-tocopherol improves the response of diabetic patients as well as improving their lipid profile [23]. It is important to point out that EVOO also plays other roles in the field of health, such as its role in osteoporosis both for its monounsaturated fat content (oleic acid) and polyphenols, which are associated with increases in bone mass and the reduction of the risk of osteoporotic fracture by inhibiting prostaglandins [22, 38]. Table 3. Proportion of each sterol according to oil variety origin Olive oil variety Alfafara Aguilar Arbequina Baix fulla Blanqueta Blanqueta roja Blanqueta reina Blanquiroja Borriolenca Cabaret Callosina Carrasqueña Casellera Cornicabra Cuquillo Changlot Real Choco De la Cueva De la Lloma Del Pomet Dotó Egipcia Empeltre Farga Figuereta Genovesa Godellera Grossal Hojiblanca Jandra Llumero Manzanilla

Sterols (%) β-sitosteroles 96.37 ± 0.38 94.61 ± 0.25 96.70 ± 0.35 94.98 ± 0.34 94.01 ± 0.35 94.55 ± 0.30 94.60 ± 0.52 94.28 ± 0.43 91.93 ± 0.34 93.69 ± 0.29 94.09 ± 0.48 94.58 ± 0.40 94.50 ± 0.96 96.57 ± 0.17 96.58 ± 0.26 95.63 ± 0.88 98.41 ± 0.12 95.89 ± 0.19 93.25 ± 0.56 94.93 ± 0.30 94.06 ± 0.17 97.39 ± 0.16 96.28 ± 0.21 95.36 ± 0.50 95.24 ± 0.41 96.09 ± 0.12 95.63 ± 0.09 94.98 ± 0.22 96.61 ± 0.22 94.41 ± 0.18 95.42 ± 0.63 96.53 ± 0.18

Campesterol 2.97 ± 0.32 3.12 ± 0.13 2.88 ± 0.29 4.58 ± 0.31 4.34 ± 0.31 3.48 ± 0.27 3.59 ± 0.40 3.57 ± 0.18 4.36 ± 0.23 3.72 ± 0.09 2.94 ± 0.31 3.51 ± 0.08 3.31 ± 0.92 2.79 ± 0.15 3.06 ± 0.24 2.76 ± 0.19 1.24 ± 0.05 3.19 ± 0.18 3.79 ± 0.28 2.66 ± 0.13 3.69 ± 0.21 1.72 ± 0.07 2.43 ± 0.13 2.96 ± 0.20 3.22 ± 0.18 2.68 ± 0.08 1.99 ± 0.04 3.36 ± 0.11 2.94 ± 0.09 2.65 ± 0.07 3.47 ± 0.41 2.91 ± 0.23

Stigmasterol 0.62 ± 0.08 0.95 ± 0.09 0.41 ± 0.11 0.53 ± 0.11 1.61 ± 0.12 1.98 ± 0.08 1.80 ± 0.16 2.13 ± 0.46 3.68 ± 0.18 2.58 ± 0.24 1.46 ± 0.42 1.90 ± 0.38 2.18 ± 0.60 0.65 ± 0.09 0.39 ± 0.05 1.51 ± 0.34 0.50 ± 0.09 0.92 ± 0.05 3.02 ± 0.44 2.41 ± 0.23 0.95 ± 0.07 0.89 ± 0.16 1.29 ± 0.11 1.67 ± 0.38 1.36 ± 0.09 0.41 ± 0.09 0.43 ± 0.08 1.22 ± 0.09 0.46 ± 0.08 2.94 ± 0.21 1.11 ± 0.38 0.56 ± 0.13

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I. López-Cortés, D. C. Salazar-García, A. Mondragón-Valero et al. Table 3. (Continued) Olive oil variety Marfil Mas Blanc Millareja Moixentina Monteaguda Morons Morruda Negra Patronet Picual Piñonera Quartera Rotja Rotgeta Rufina Seniero Serrana Valentins Vallesa Verdal Villalonga

Sterols (%) β-sitosteroles 96.48 ± 0.35 96.99 ± 0.12 95.60 ± 0.59 93.57 ± 0.43 96.88 ± 0.21 95.45 ± 0.39 96.18 ± 0.35 96.12 ± 0.57 94.65 ± 0.28 96.39 ± 0.99 95.14 ± 0.80 97.47 ± 0.31 95.87 ± 0.28 92.35 ± 0.22 94.61 ± 0.37 96.73 ± 0.51 95.63 ± 1.42 96.48 ± 0.39 92.63 ± 0.56 96.53 ± 0.41 94.50 ± 2.48

Campesterol 3.03 ± 0.37 2.58 ± 0.08 3.52 ± 0.31 3.59 ± 0.39 2.81 ± 0.21 3.93 ± 0.09 2.44 ± 0.32 2.91 ± 0.13 3.86 ± 0.10 2.93 ± 0.30 3.86 ± 0.10 2.36 ± 0.28 3.17 ± 0.25 3.92 ± 0.10 3.25 ± 0.16 2.74 ± 0.41 3.11 ± 0.64 2.65 ± 0.23 4.36 ± 0.26 2.91 ± 0.25 3.31 ± 0.93

Stigmasterol 0.45 ± 0.06 0.43 ± 0.13 0.88 ± 0.34 1.84 ± 0.10 0.52 ± 0.13 0.92 ± 0.18 1.46 ± 0.11 0.86 ± 0.11 1.51 ± 0.23 0.35 ± 0.15 0.93 ± 0.19 0.21 ± 0.13 0.96 ± 0.07 3.73 ± 0.13 2.14 ± 0.28 1.19 ± 0.37 1.26 ± 0.85 1.65 ± 0.18 2.73 ± 0.15 0.56 ± 0.21 2.18 ± 0.60

Sterols are represented as a percentage of each of them in their composition, the maximum values of β-sitosterol are found in the Chocó, Egyptian and Quartera varieties, exceeding 97% in all cases. In most varieties the most important second sterol is campesterol, the varieties that have a higher content are Baix fulla, Vallesa, Borriolena and Blanqueta with values above 4%. Finally, the sterol found in smaller proportion is stigmasterol, whose highest values can be found in Rotgeta, Borriolenca and De la Lloma, exceeding 3% of the total sterol content. Currently, there are two European Parliament Regulations [32] and Council [33], which establish the characteristics that must be met by food to be defined and considered as healthy. The terminology used in these foods must be based on enough scientific evidence and grounds that are identified and reproducible. Certainly due to the numerous studies on EVOO, it is now considered not only healthy for humans but it also prevents certain health problems, and its role has been clearly revalued in numerous recent researches. At this juncture, EVOO should be regarded as a functional food, although it should not be viewed as a drug since its use as a therapeutic agent has not been sufficiently studied yet. At least with regard to its relationship with cancer because there is not sufficient data to clearly determine its role in tumor regression, as there is in the

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regression of certain cardiovascular disorders that at least justifies its use in control diets after cardiovascular clinical procedures. The influence of oil in health was evaluated in the first human epidemiology studies [18] and they showed a direct relationship between a low incidence of cardiovascular diseases and nutritional habits in the countries of the Mediterranean area, where fat nutrient input was even above 35%, but these inputs came from the olive tree. Throughout history, EVOO has been recognized as a potent pharmacological agent, even back in ancient Greece Hippocrates came to mention 60 health benefits of using EVOO. The highest cardiovascular benefits of EVOO and its role in secondary factors in inflammatory diseases and hypertension [5, 7]. In addition to its composition in fatty acids, it is necessary to point out the phenolic compounds composition of EVOO, therefore we must consider the interaction between phenolic derivative components and other intrinsic components of food, and how they interact to benefit human health [29]. The oxidative stress to which our cells are subjected is a serious health problem, since the hydroxyl radical (OH), the superoxide radical (O2-) and hydrogen peroxide (H2O2) that are formed in the aerobic metabolic processes are highly reactive and potentially cytotoxic inducing vital molecule breakage such as phospholipids (deteriorating membranes), proteins and nucleic acids (by altering the genetic material and implementing carcinogenic processes). Currently, diet type, together with hereditary factors and certain contaminants are considered to be important. It has been proven that olive oil has a protective effect in some types of induced cancer, this effect is attributed to the high content in squalene and various olive oil polyphenols. The importance of the consumption of antioxidants is necessary as a preventive from the beginning of the appearance of cellular oxidative stress that highly intensifies in transition fetus- newborn since in those times generate in free radicals that have negative implications in the processes of degenerative and inflammatory nature and therefore influence cardiovascular diseases and especially problems such as cancer and ageing [37]. The most frequent oxidative lesions are the mutational transition G-C to A-T as a result of the oxidation of guanine to 8HdGuanine that induces changes in the electronic properties of this base and results in errors in DNA replication, it is also necessary to consider the mutagenic potential change from cytosine to 5H-cytosine, which is defining in carcinogenesis, all evident in tissues with oncogenic development in which these oxygen changes are detected, and the presence of oxidizing enzymes that do not occur in regular cells [31], all of this has been proven in lung, stomach, ovarian, prostate, duodenum (duodal and colorectal), and breast tissue. Obviously stimulated by other external factors such as solar radiation, ionizing radiation, bromides, nitroacetates,

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nitroquinoleinas, and other compounds that have been partially reduced “in vitro” and “in vivo” in rats with massive doses of polyphenols, from remains prepared with olive leaves and pulps, and even with high doses of olive oil [4]. Many authors have proven the protective role of polyphenols in the oxidative modification of the genomic material, since the oxidizing agents that lead to the oxidation of bases clearly contribute to the genetic instability of tumor cells and thus reduce their metastatic potential [10], [19, 25]. There are mechanisms of self-control using endogenous antioxidants that work against the effects of peroxidases, catalases and superoxides, but there are also valuable exogenous antioxidants that come from food, such as vitamin C, found in many fruits, vitamin E, β -carotene and especially polyphenols and α-tocopherol, found in olive oils, which also contain flavonoids, effective antioxidant substrates and this is why olive oil is considered to be a clear reducer in the incidence of premutagenic oxidation as a result of the role of these compounds in the improvement of the enzymatic activity on the reactive species, since unsaturated fatty acids are reactive species per se. The phenolic compounds contained in EVOO posess distinct anti-inflammatory qualities, the anti-inflammatory response has been demonstrated in obese individuals, who are common consumers of vegetable oils, which is clearly reduced after the ingestion of EVOO, rich in polyphenols [30]. We must not forget that, according to the World Health Organization, in the period between 1980 and 2014 the obese population has doubled, and there are 600 million obese people, with a high incidence in children under 18 years of age. EVOO consumption is a good method of obesity control, we must not forget that in the new millennium, obesity will be clearly an epidemic, and 95% of its causes have an exogenous or nutritional origin. EVOO, wastewater from the mills and olive tree leaves are sources of abundant polyphenols with antioxidant activity such as the hydroxytyrosol and oleuropein [40]. Phenolic compounds, both oleuropein and hydroxytyrosol, found in olive oil, are nowadays considered to be clear stabilizers of the hydroxyl radical and, in particular, are considered to be the protectors of the digestive tract and facilitators of its motility especially with a protective effect against colon adenocarcinoma. Specifically, hydroxytyrosol is a good antioxidant capable of blocking the cytotoxic effects of the peroxinitritre. Back in 2009, [16] it was found that oleuropein concentrations of 200 μg/mL induce death of cancer cells. Over the years, these studies have been widened [13] by determining, back in the year 2006, the action of oleocanthal and its selective effect in the reduction of human melanoma. Hydroxytyrosol is a polyphenol most present in the olive tree, it is an exceptional antiradical and its action is logically high during the oxidation process. Widely known are its therapeutic benefits against psoriasis and studies carried out to decrease the fat in the bloodstream [15].

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Table 4. Polyphenol and α-tocopherol content according to oil variety origin Olive oil variety Alfafara Aguilar Arbequina Baix fulla Blanqueta Blanqueta roja Blanqueta reina Blanquiroja Borriolenca Cabaret Callosina Carrasqueña Casellera Cornicabra Cuquillo Changlot Real Choco De la Cueva De la Lloma Del Pomet Dotó Egipcia Empeltre Farga Figuereta Genovesa Godellera Grossal Hojiblanca Jandra Llumero Manzanilla Marfil Mas Blanc Millareja Moixentina Monteaguda Morons Morruda Negra Patronet Picual Piñonera Quartera

Polyphenol (mg/kg) 462.81 ± 3.18 161.34 ± 3.64 203.00 ± 1.98 321.35 ± 3.53 403.05 ± 2.71 303.21 ± 3.18 448.32 ± 6.93 458.32 ± 4.37 142.91 ± 2.58 234.93 ± 3.43 318.73 ± 2.54 173.43 ± 2.95 140.26 ± 2.51 474.53 ± 6.34 203.65 ± 4.23 309.93 ± 2.32 167.43 ± 1.89 137.94 ± 1.45 218.64 ± 2.12 305.25 ± 2.21 345.51 ± 1.62 306.53 ± 1.95 316.91 ± 0.97 243.95 ± 0.95 184.65 ± 1.22 164.73 ± 0.24 187.49 ± 0.98 432.69 ± 3.24 418.23 ± 2.98 173.98 ± 6.23 132.64 ± 1.22 307.40 ± 0.93 198.73 ± 1.21 187.34 ± 0.93 126.83 ± 0.94 191.23 ± 1.20 237.93 ± 1.14 167.94 ± 1.36 325.83 ± 2.31 421.36 ± 1.63 296.60 ± 1.87 328.04 ± 1.42 198.08 ± 1.18 167.11 ± 1.02

α-tocopherol (mg/kg) 206.76 ± 8.54 139.71 ± 1.15 184.09 ± 8.12 164.93 ± 1.72 170.77 ± 6.58 89.21 ± 3.87 122.41 ± 3.05 157.02 ± 4.55 160.97 ± 2.03 97.14 ± 1.51 276.49 ± 5.36 154.42 ± 1.37 126.13 ± 9.21 233.32 ± 6.55 343.01 ± 6.98 183.89 ± 5.10 98.41 ± 0.12 169.71 ± 2.43 183.38 ± 0.51 99.51 ± 0.53 193.65 ± 2.53 201.10 ± 3.37 166.64 ± 1.83 240.21 ± 1.83 168.31 ± 1.23 176.33 ± 0.56 181.63 ± 0.43 199.58 ± 1.17 178.46 ± 2.85 231.19 ± 2.19 139.21 ± 2.63 288.72 ± 0.86 270.14 ± 0.94 86.84 ± 0.56 181.77 ± 1.69 98.97 ± 2.07 236.98 ± 1.21 167.93 ± 1.23 158.64 ± 0.58 253.47 ± 1.04 113.75 ± 0.71 132.51 ± 0.79 167.21 ± 1.23 171.05 ± 1.06

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I. López-Cortés, D. C. Salazar-García, A. Mondragón-Valero et al. Table 4. (Continued)

Olive oil variety

Polyphenol (mg/kg)

α-tocopherol (mg/kg)

Rotja Rotgeta Rufina Seniero Serrana Valentins Vallesa Verdal Villalonga

145.74 ± 1.33 293.65 ± 2.03 318.63 ± 2.67 206.48 ± 1.63 245.21 ± 1.75 173.93 ± 1.52 139.83 ± 1.42 165.87 ± 1.32 202.12 ± 1.54

284.49 ± 0.61 153.46 ± 0.74 191.54 ± 0.56 149.67 ± 1.91 186.97 ± 2.58 143.91 ± 0.49 161.43 ± 0.52 288.72 ± 0.74 116.11 ± 2.93

These phenolic compounds contained in EVOO may play a protective role in the excessive accumulation of fat associated with oxidative stress, however evidence in animal models will be necessary [29]. At present, these polyphenols are sought in the pharmaceutical industry as a nutraceutical supplement and in cosmetic formulations, and of course for its use in diets. They play a clear role in protection against diseases and in current diet and lifestyle [21]. We should also mention verbascoside, present in oil, although it is really found in greater amounts in the leaves of the olive tree. As we have already discussed, olives are rich in polyphenols, but only 2% of the total phenolic composition of the fruit makes it to the oil phase, since most of these polyphenols remain in the water (53%) solid waste phase (45%) so that polyphenols reach around 2-8 g depending on the chosen procedure [34]. Since the 2000s, polyphenols are extracted from the different parts of the olive tree, mainly from leaves and are marketed for its multiple benefits. Also the flavonoids contained in olive oil are vasodilators and help prevent arterial hypertension as cardiovascular and anticancer protectors. Cornicabra, Alfafara, Blanquiroja and Blanqueta reina are the varieties with the greatest concentration of polyphenols, for all of them such concentration exceeds 400 mg/kg, while the minimum values are obtained in Millareja and Llumero, De la Cueva, Barriolenca, Vallesa and Rotja varieties, with values of lower than 150 mg/kg. The table above also shows the concentration of α-tocopherol, which is higher in Cuquillo, Manzanilla, Verdal, Rotja, Callosina and Marfil varieties, with values higher than 270 mg/kg, on the other hand, varieties with lower values are more Blanc, Moixentina, Choco, Del Pomet, Blanqueta Roja and Cabaret.

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Figure 1. Distribution of analysed varieties according to their polyphenol content.

As shown in graph 1, there are three distinct groups based on their polyphenols content, on the one hand, varieties that have a concentration lower than 300 mg/kg, this being the most numerous group, then a group with a polyphenolic content between 300 and 400 mg/kg and finally another group with varieties that exceed 400 mg/kg. Given that the latter group is more interesting due to its polyphenols composition, they are identified in Figure 1 with the corresponding number assigned to the varieties in Table 1. The varieties are identified in the graphics with the number that accompanies these varieties in Table 1. Figure 2 shows the average weight of the olives, it also represents the polyphenols content, which is represented by the area of the circles therein. The phenolic compounds present in EVOO have proven ability to inhibit leukocytic 5-lipoxygenase [9]. On the other hand, have established the ability of EVOO polyphenols to neutralize free radicals [40]. With diets rich in oleic acid and therefore with a regular intake of EVOO, cardiovascular pathologies improve since a lower oxidation of lipoproteins, LDL (with a high atherogenic potential) can be observed, as well as an improvement in the blood lipid profile that leads to a lower elevation of triglycerides and a more rapid disappearance of the triglyceride-rich lipoproteins in blood plasma, which does not seem to solely depend on the oleic acid content but also on its relations with linoleic acid.

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Figure 2. Polyphenol content and olive weight according to oil variety origin.

It has been found [24] that regular consumption of olive oil and onion lead to a clear decrease in systolic pressure, a tendency to decrease diastolic pressure and an improvement of the blood flow due to a drop of hematocrit. Olive oil, which is rich in monounsaturated fatty acids, produces favorable changes in plasma lipoproteins and achieves lower oxidation. In recent years, olive oil, always Extra Virgin, has increased its role in light of certain chronic diseases, especially cancer, and breast cancer in particular due to its protective effect in response to the aberrant cell proliferation problem, which increasingly affects women. After two decades of research on disease prevention and research with natural polyphenols applied as bioactive components, it has been possible to verify their effectiveness in prevention as antiradical in degenerative diseases such as atherosclerosis, coronary heart disease, rheumatism and arthritis [8]. Recent research [3, 35] has also verified the traditional olive oil potential as antimicrobial, in plant disease control, the advantage of the human use of the olive tree, both its oil and leaves as antimicrobial and antioxidant [20]. The specific role of the promising expectations that olive oil shows linked to health should certainly be investigated in detail and depth before drawing definitive conclusions.

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[33] Regulation (EC) nº 109/2008 of the European Parliament and the Council of 15 January 2008 by which it was modified the Regulation (EC) nº 1924/2006. [34] Rodis P. S., Karathanos V. T., Mantzavionu A. (2012). Partitioning of olive oil antioxidants berween oil and wáter phases. J. Agric. Food Chem. 50, 596-601. [35] Rounds L., Havens C. M., Feinstein Y., Friedman M., Ravishankar S. (2013). Concentration-dependent inhibition of Escherichia coli O157:H7 and heterocyclic amines in heated ground beef patties by apple and olive extracts, onion powder and clove bud oil. Meat Science. 94, 461-467. [36] Ruiz-Gutierrez, V., Muriana F. J. G., Villar J. (1998). El aceite de olive virgen y las enfermedades cardiovascualres. Perfil lipídico en plasma y composición lipídica de la membrana de eritrocito humano. Grasas y aceites. 49 (1), 9-29. [Olive oil and cardiovascular disease. Lipidic profile in plasma and lipidic composition of the human erythrocyte membrane. Oils and fats. 49(1), 9-29]. [37] Sáez-Tormo G., Puig-Parellada P. (1999). Radicales libres y estrés oxidativo en biomedicina, importancia y utilidad de los antioxidantes en la prevención de procesos fisiopatológicos relacionados. FVFA Generalitat Valencia. Ed. Valencia. 177 pp. [Free radicals and oxitative stress in biomedicine, importance and usefulness of antioxidant in prevetion of related pathophysiological processes. FVFA Generalitat Valencia. Ed. Valencia. 177 pp]. [38] Trichopoulou A., Georgiou E., Bassiakos Y., Lip-Worth L., Lagion P., Proukakis C., Trichopoulos D. (1997). Energy intake and monounsaturated fat in relation to bone mmineral density among women and men in Greece. Prev. Med. 26, 395-400. [39] Valenzuela (1940). Curso de olivicultura e industrias derivadas. Hays Bell Ed. Buenos Aires. Argentina. 160 pp. [Olive growing and derived industries course. Hays Bell Ed. Buenos Aires. Argenitna. 160 pp]. [40] Visoli F., Bellomo G., Galli C. (1998). Free radical scavenging properties of olive oil polyphenols. Biochem. Biophys. Res. Commun. 247 (1), 60-4.

In: Handbook of Olive Oil Editor: Jozef Miloš

ISBN: 978-1-53612-356-2 © 2017 Nova Science Publishers, Inc.

Chapter 2

OLIVE VARIETIES AND PHENOLIC COMPOUNDS IN OLIVE OILS Gassan Hodaifa* and Santiago Rodriguez-Perez Molecular Biology and Biochemical Engineering Department, Universidad Pablo de Olavide, Seville, Spain

ABSTRACT The olive stage of ripeness influences in the olive composition i.e., phenolic compounds and concentration vary along ripeness process. Phenolic compounds are key in the oil quality as they are natural antioxidants that contribute to protecting the oil stability against oxidation. Phenols are present in the olive oil in a percentage from 0.1% to 0.3% depending on the olives state of ripeness from which olive oil was extracted. Olives and olive oil contain polyphenols such as oleuropein, hydroxy-tyrosol, tyrosol, rutin, and quercetin, as well as caffeic, vanillic, and o- and p-coumaric acids. Hydroxytyrosol significantly inhibits the lipid oxidation of olive oil and has a positive effect on human health. Previous studies have suggested that polyphenols present in the olive oil could improve the oxidative stability of canned products and reduce the concentration of carcinogenic compounds such as heterocyclic amines in fry processes. In fact, diets rich in natural foods and food-derived components such as phenolic compounds receive a great deal of attention because they are perceived as ‘safe’ and ‘non-medicinal’ which some are known to function as chemopreventive agents against oxidative damage, cerebrovascular disease, and aging. The phenolic composition and concentration are different for each olive variety, being the variety “Picual” the one of the highest phenolic concentration. The most abundant polyphenol among the varieties studied was hydroxytyrosol, with concentrations of 1.4–12 mg L−1. In general, reduction in polyphenols content along ripeness process was observed. This reduction has been noted in the hydroxy-tyrosol concentration over the ripeness process and varied according to the olive variety, by 10, 2.8, and 1.6 mg L−1 for the varieties ‘Hojiblanca’, ‘Picual’, and *

Corresponding author: [email protected].

20

Gassan Hodaifa and Santiago Rodriguez-Perez ‘Arbequina’, respectively. The evaluation of the influence of the degree of ripeness on oxidative stability of olive oils is important for decisions on producing oils with a certain overall quality. The practice of harvesting ripe olives as soon as possible to produce oils of high phenolic contents and thus high oxidative stability implies bitter and piquant oils, this being admissible for oils of some varieties but detrimental to others appreciated in the market for being more fruity and sweeter.

Keywords: olive variety, olive oil, ripeness, polyphenols, stability

1. OLIVES AND OLIVE OIL COMPOSITION The origin of olive cultivation, Olea europaea L., is probably located in Syria, Iran and, Palestine. The olive cultivation was extended through Cyprus and Egypt to the Mediterranean area. In the fifteenth century, the olive cultivation was carried to the New World, and nowadays, it is spread across all continents. The olives production mostly concentrated (the olive-growing zone) in the Mediterranean basin (Borges et al., 2017). The olives are composed of three different parts: endocarp or pit, mesocarp or pulp, and epicarp or peel. The fruit begins to develop after fertilization following these three phases. In phase I, the endocarp reaches its definitive size that represents 80% of the fruit volume. In phase II, the fruit growth slows down, keeping the fruit size constant and endocarp is hardening. Finally, in phase III, mesocarp and epicarp grow intensely until the physiological ripeness of the fruit. In this phase, the accumulation of oil occurs at the same time as the fruit ripening. The virgin olive oil was defined in the Regulation EEC of 1966 on the common organization of the market in oils and fats. The virgin olive oil is obtained from the olive fruit, only by mechanical or other physical methods, under conditions that do not cause the alteration of the oil, and that have not undergone any treatment other than washing, decantation, centrifugation, and filtration. It is express excluded of oils obtained with solvents, re-esterification processes or any mixture of oils of another nature. The current legal framework defining the categories of olive oils is based on Regulation of the Commission of the European Communities nº 2568/1991 and its amendments (EC Regulation 2568, 1991). Virgin olive oil is composed of a glyceride fraction consisting mainly of triglycerides and a small proportion of minor compounds of a heterogeneous nature. The compounds present in the glyceride fraction i.e., triglycerides, monoglycerides, diglycerides, phosphatides and free fatty acids, make up to 98% (Bengana et al., 2013). Among the minority compounds that can make up to 2% are present sterols and terpenes; aliphatic alcohols; esters in glycerides; hydrocarbons; volatile compounds; waxes; tocopherols; pigments and phenols (Sánchez de Medina et al., 2015; Bengana et al., 2013). Most of the fatty acids in olive oil are esterified to glycerol molecules, forming triglycerides. One of the characteristics of olive oil is the composition and abundance of

Olive Varieties and Phenolic Compounds in Olive Oils

21

fatty acids. Oleic acid is the most abundant and can have an abundance up to 84% of fatty acids (Reboredo-Rodríguez et al., 2015). In addition, linoleic (up to 21%) and linolenic (up to 1.5%) acids are significantly abundant. Other fatty acids that can be reached significant abundances are palmitic acid (up to 21%), palmitoleic acid (up to 3.5%) and stearic acid (up to 5.3%). The fatty acids composition is very varied and depends on multiple factors. Linoleic and linolenic acids are essential fatty acids that should be included in every diet. However, its presence in vegetable oils can vary because these fatty acids are highly susceptible to oxidized. Olive oil has a great advantage compared to other vegetable oils because it is more resistant to oxidation than other vegetable oils (Franco et al., 2014). This higher resistance is partly due to the greater abundance of antioxidants and oleic acid, which is more resistant to oxidation than other more polyunsaturated seed oils. The minor compounds fraction is composed of compounds with very different chemical characteristics. These compounds influence significantly on the olive oil quality and conservation against oxidation, despite their low concentration. Moreover, the nutritional value and health benefits of these compounds is very important and worldwide topic of many studies (Sánchez de Medina et al., 2015). Legislations are strictly controlled olive oil quality; in fact, it is very exigent with the organoleptic characteristics control. Compounds such as alcohols, esters, ketones, and aldehydes, among others are formed aromatic fraction of olive oil. Volatile compounds are the main responsible of olive oil odor, which significantly influences in the determination of olive oil quality. The composition and concentration of aromatic fraction in olive oil extracted into a large extent on the enzymatic activity that occurs during the milling and whipping process of the olives. Therefore, olive variety, conditions of extraction process of olive oil and olives ripeness are influence on the quality of the olive oil produced. Tocopherols contribute significantly to the stability of the olive oil. The α-tocopherol is the most abundant tocopherol in the olive oil, up to 95% of the total tocopherols compounds. α-tocopherol shows a nutritional value because it is the main homolog of the forms of vitamin E present in olive oil despite of its important role in the olive oil conservation. Tocopherol concentrations in olive oil depends heavily on the olives variety, the climatology of the olive growing area, environmental factors, olive oil extraction process used and the olives ripeness (Debbabi et al., 2016). The color of olive oil is associated to pigment concentrations. Color intensity mainly related to the olive variety and ripeness. Legislations did not considering the color of olive oil within the olive oil commercial categorization. However, color of olive oil is an important indicator of olive oil quality for consumers (Criado et al., 2008). The composition and concentration of pigments in the olives determine the concentration and composition of pigments transferred to the olive oil. Olives contain chlorophylls, carotenes, xanthophylls and anthocyanins, which change its concentrations throughout

Gassan Hodaifa and Santiago Rodriguez-Perez

22

olives ripeness. In the process of olive oil extraction, these pigments are distributed between solid and liquid phases according to the distribution constant. Furthermore, some chemical and enzymatic activity are occurs in olive paste during milling and whipping which change the pigments composition of olive oil. Pigments contribute to nutritional and commercial values of olive oil considering that some carotenes are vitamin A precursors (Criado et al., 2008). Squalene is the hydrocarbon with the greatest abundance in olive oil, which is one of the main compounds of the minority compounds. Olive oil is the vegetable oil that has the highest concentration of squalene. This fact is important because squalene is a biochemical precursor to the synthesis of sterols such as beta-sitosterol that interferes with the intestinal absorption of cholesterol.

2. PHENOLIC COMPOUNDS Phenols are metabolites derived from the shikimic acid pathway and from the phenylpropanoid metabolism, which have in their structure an aromatic group with one or more -OH groups. Phenols are not essential for plant growth, development or reproduction. However, phenols have a significant influence on the relationship between the plant and its environment. The plant synthesizes phenols in response to biotic or abiotic stress. The main functions attributed to phenols are: 





 

Protection against ultraviolet rays: Phenols protect plants from ultraviolet-B (UV-B) radiation (280-320 nm). This function is very important because the UVB radiation causes the alteration of plant metabolism through the generation of oxygen reactive species. The phenols act by adjusting the antioxidant systems of the plant. Chemical signaling: The release of phenols into the medium can affect the nutrients absorption, the growth of other plants and even infection by mycorrhizal fungi. Pigmentation of flowers and fruits: some phenols such as anthocyanins, chalcones or flavones, act as pigments in flowers and fruits, which favors the pollination of the plant. Protection against pathogens: The phenols concentration in the plant is associated with its resistance to fungal, bacterial and viral pathogens. Protection against herbivores: Phenols reduce and even destroy the pleasant taste of the plant.

In general, phenolic compounds are present in foods and therefore in any diet. Despite their low concentration, they are considered important components from both

Olive Varieties and Phenolic Compounds in Olive Oils

23

organoleptic and nutritional points of view. Phenols are mainly responsible for the bitter taste and astringency, properties appreciated in some foods and despised in others. Many authors because of their nutritional value study phenols and their health benefits attributed to them (Gutierrez-Rosales et al., 2012). The main benefit is the antioxidant function, which it is developed, even at low concentrations. This antioxidant capacity is important for the food itself, but also for the organisms that eat the food (Franco et al., 2014). Low concentrations of phenols in foods will protect them from oxidation and small concentrations of ingested phenols will protect the body’s cells against oxidation. This property is the main cause that relates to phenols as antiarteriosclerotic, cardio-protective, neuroprotective, anti-inflammatory, anti-mutagenic, anti-carcinogenic, hormonal modulators, endothelial protectors, immune system protectors, antiallergenic and antidiabetic agents (Sánchez de Medina et al., 2015; Reboredo-Rodríguez et al., 2015; Talhaoui et al., 2014; Allalout et al., 2009). Phenolic compounds are found in significantly quantities in olives, especially in glucoside form, i.e., structurally water-soluble compounds. The oleuropein, a bitter glucoside of the olive, is the most abundance of the total phenols. Glucosides can transform into less complex molecules, called aglucons (esters) and simple phenols, soluble both in water as well as in oil due to the enzymatic action. The phenols concentration in olive oil ranging from 0.1% to 0.3%, depending mainly on the olive oil extraction methods, olive variety, and ripeness (Nieto et al., 2010). This presence in olive oil is key for its quality due to the antioxidant function mentioned before proving its stability against oxidation. In addition, polyphenols are the main contributors to olive oil bitterness, astringency, and pungency. Attempts have made to correlate concentrations of individual phenols to panel test scores. The most representative phenolic compounds presents in olives and olive oil are phenolic acids, phenolic alcohols, flavonoids and secoiridoids. The phenolic profile of olives and olive oil shows phenols as oleuropein, ligostroside, hydroxytyrosol, tyrosol, rutin, and quercetin, as well as caffeic, vanillic, and o- and p-coumaric acids, all them with excellent antioxidant properties. These phenol compounds are effective radical scavengers with the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) test, and it has been demonstrated that the antioxidant activity of phenolic compounds in linoleic acid was tyrosol < caffeic acid < oleuropein < hydroxy-tyrosol (Nieto et al., 2010). Hydroxy-tyrosol significantly inhibits the lipid oxidation of olive oil and has a positive effect on human health. It has reported that oleuropein and hydroxy-tyrosol enhance the oxidative stability of oil-in-water emulsions, while quercetin inhibits lipid oxidation in raw fish and beef. Several authos have concluded that the remarkable olive oil resistance to oxidation is closely linked to its total phenol concentration (Allalout et al., 2009; Nieto et al., 2010; Fuentes de Mendoza et al., 2013). Only olive oils, extracted exclusively by physical methods, possess these substances. In this sense, is important to indicate that phenols are not present in refined oils, i.e., oils

24

Gassan Hodaifa and Santiago Rodriguez-Perez

from seeds, refined olive oil, or olive pomace oil since the physicochemical refinement destroy most of these components or disperse them in the neutralization and washing processes. Nieto et al., 2010 observed an improvement in oxidative stability of canned fish when olive oil was used instead of refined oils. The improvement in the oxidative stability was explained by the presence and concentration of phenols in olive oil, usually removed in the refinery process of refined oils. The same authors observed that frying beef patties in virgin olive oil reduced the content of heterocyclic amines, a well-known carcinogenic compound. The spoiling of the olive oil can be delayed but could not permanently prevented. Olive oil has a powerful natural antioxidant that protects it from oxidation during a relatively long period. The phenolic fraction has this property, as it can inhibit the formation of hydrogen peroxides and unsaturated fatty acids for a certain length of time.

3. OLIVE OIL STABILITY AND RIPENESS INDEX Oxidative stability, although not considered a standard parameter of quality, is useful to provide information on the oil’s hypothetical shelf life. It usually evaluated by the induction time (time period until a critical point of oxidation is reached) with a corresponding sensorial degradation of the oil as consequence of a sudden acceleration of the oxidative process. The oxidative stability, usually evaluated by the Rancimat method, reveals the resistance of the product to begin the oxidation process characterized by free radical reactions. The resistance to oxidative deterioration usually attributed to two main factors: (a) The fatty acid composition of olive oil that characterized by a high monounsaturated-to-polyunsaturated fatty acid ratio. (b) The pool of minor compounds of powerful antioxidant activity that constituted mainly by tocopherols and polyphenols, In addition, to chlorophylls and carotenoids. Antioxidants scavenge free radicals and chelate metal ions that trigger free radical reactions. Previous studies have established the kinetics of the oxidative reactions which shows that it could be determined a proportional relationship between the induction time and the antioxidants concentration (Nieto et al., 2010). The olive oil composition results of the interaction between genetic, environmental and technological factors that are determinants in the development stage, olives ripeness and olive oil extraction. There is much variation in the composition and concentration of phenolic compounds between the different olive oils. The concentration range in phenols in olive oils is very wide, which it makes the comparison between studies difficult.

Olive Varieties and Phenolic Compounds in Olive Oils

25

There are multiple factors that have the ability to modify the phenolic composition in olive oil. It has observed that the concentration and phenolic composition of olive oils vary depending on both intrinsic and extrinsic factors of the olives. The content in phenolic substances of the olive oils influenced by the following factors: 







Olive variety: The olives characteristics such as color, size, and yield in oil have determined for centuries the selection between different varieties of olives. These differences significantly influence the composition of olive oil, both in the glyceride fraction and in the minority components such as phenols. The olives variety influences qualitatively and quantitatively the phenolic composition of the extracted oil. Many authors have carried out studies to describe the phenolic profiles of different olives varieties. These studies showed that oleuropein is the most abundant compound in almost olives varieties, with concentrations between 35 and 2400 mg kg-1 olives. However, some compounds have proposed as varietal identifiers because they have identified only in a limited group of olives varieties such as “demetiloleuropeína” (Coratina, Leccino, Cailletier, L11 and Arbequina). There are many different phenolic concentrations in the olives varieties. However, the highest total phenolic concentration are show in Arbequina, Cornicabra, Picolimón y Picual. Environmental factors: These factors condition the olives growth and their ripeness that influences considerably in its phenols composition and concentration. According to Dag et al., (2011) there is certain evidence that relates some environmental factors to the phenolic concentration. The influence of temperature has given contradictory results. However, it has observed that low rainfall rates related to an increase in phenolic concentration (Borges et al., 2017). Similarly, at lower altitudes and latitudes, higher concentrations of phenols have observed for the same varieties (Borges et al., 2017). As for the soil type, higher concentrations of phenols are differentiated according to the water retention capacity of the soil, so that from the highest to the lowest phenolic concentration observed, calcareous soils, clay, and loam are classified (Dag et al., 2011). Technological factors: The oil extraction process composed by different stages such as the olives milling, whipping and oil separation. During the process, tissue breakdown activates the enzymatic activity that decreases the phenols concentration in the oil. In recent years, some modifications investigated to reduce this enzymatic activity, such as the boning of olives before milling or the use of nitrogen in the pulp whipping. Ripeness: The degree of ripeness of the olive has great importance in the concentration of phenols in the olive oil. In general, it can deduced that in the olive oil independently of the olive variety used, the total phenol content declines

26

Gassan Hodaifa and Santiago Rodriguez-Perez as the ripeness progressed (Franco et al., 2014). In fact, there are phenols that could not detected at the end of the ripeness according to the olive variety. Hydroxytyrosol is the most abundant phenol in the olive oil independently of the olive variety. Although the hydroxyltyrosol reduced as ripeness progress, this phenol is the most abundant in all the ripeness stages.

During the olives ripening, the phenolic compounds concentration decreases. In the early stages of ripeness, oleuropein is the most abundant compound (Talhaoui et al., 2014). However, its concentration begins to decrease, in general terms, with the olives ripening. This fact may related to an increased activity of the hydrolytic enzymes. Ortega-García and Peragon, (2009) studied Picual, Arbequina, Verdial and Frantoio varieties suggested the possible relationship between the enzymatic activity of the phenylalanine ammonium lyase (PAL) and polyphenol oxidase (PPO) enzymes with the phenols concentration. A decrease in the activity of the PAL enzyme observed as the ripening of the olives advanced. This enzyme regulates the biosynthesis of the phenylpropanoid compounds. However, the activity of the enzyme PPO, which is the enzyme that carries out the oxidative degradation of the phenolic compounds, increases as the ripening of the olives advances. It could asserted that oils from olives harvested in early ripening stages will have a higher concentration of phenolic compounds than oils from mature olives. However, this assertion does not give an accurate picture of the fluctuation of the phenolic compounds concentration in the ripening process. Some authors have observed that during the ripening process, after an initial reduction of the phenolic compounds. A second synthesis of these is given in some varieties such as Picual, Arbequina, Arbosana, Chetoui, Chemlale, Gemlik, Moraiolo, Frantoio and Morisca (Bacourri et al., 2008; Camposeo et al., 2013; Dagdelen et al., 2013; Cecchi et al., 2013; Franco et al., 2014), (Table 1). Table 1 shows the profile of phenolic fraction in olive oil corresponding a different olive varieties, geographical zones and ripeness stages (Bacourri et al., 2008; Allalout et al., 2009; Nieto et al., 2010; Gutierrez Rosales et al., 2010; Dag et al., 2011; Bengana et al., 2013; Camposeo et al., 2013; Dagdelen et al., 2013; Cecchi et al., 2013; Franco et al., 2014; Reboredo Rodríguez et al., 2015; Sánchez de Medina et al., 2015). The determination of the relationship between olive variety and the most phenolic production is quite difficult because of there are too many external conditions which influence in each study such as geographic location, irrigation regimen, climatology, and harvesting technology. In general, the results show that compounds such as hydroxyltyrosol, rutin and luteolin-7-glucoside increased its concentration as the olives ripening advanced in most varieties. This increase could be due to the degradation of oleuropein by enzymatic activity. In the end stage of the ripening process, the reduction of the oleuropein concentration is very fast, reaching even zero in some varieties like Leccino.

Table 1. Phenolic compounds profiles on function of olive variety and olive ripeness stage (B: Beginning, M: Middle, E: End)

Phenolic compound Tyrosol Vanillic acid Decarboxymethyl oleuropein aglycon (+)-1-acetoxypinoresinol Decarboxymethyl ligstroside aglycon p-Coumaric acid Oleuropein aglycon Ligstroside aglycon Secoiridoids Simple phenols Hydroxy-tyrosol Total1

B 2.99 0.33 91.6 n.d. 80.3 1.25 172.3 10.9 263.9 8.86 4.3 363.9 B

Hydroxytyrosol Tyrosol Vanillic acid Vanillin p-Coumaric acid

Chétoui M 17.8 1.19 86.0 n.d. 35.1 1.29 302.3 7.04 395.4 39.9 19.6 470.4 Arbequina M 3.38 7.72 0.86 0.15 0.33

E 35.6 1.71 109.5

Olive variety /Olive ripeness stage Chemlali B M E 3.13 9.54 7.5 0.36 1.29 1.84 12.6 9.93 1.78

n.d. 60.9

n.d. 24.8

n.d. 31.3

n.d. 15.1

2.54 216.6 6.54 332.3 97.4 57.6 490.9

2.37 17.8 7.32 37.8 7.72 2.86 70.4

3.67 11.1 3.6 16.5 14.7 1.65 46.2

E

B

3.27 32.8 19.8 62.5 18.3 4.21 112.0 Arbosana M 28.5 15.1 0.85 0.23 0.14

E

Reference B

M

E Bacourri et al., 2008 (Tunisia)

B

Koroneiki M 11.4 14.6 0.82 0.21 0.17

E Allalout et al., 2009 (Tunisia)

Table 1. (Continued)

Phenolic compound B 4-(acetoxyethyl)-1,2dihydroxybenzene p-Coumaric acid 4-(acetoxyethyl)-1,2dihydroxybenzene Ferrulic acid dialdehydic form of elenolic acid linked to tyrosol 4-(acetoxyethyl)-1Hydroxybenzene dialdehydic form of elenolic acid linked to tyrosol Pinoresinol Acetoxypinerosol 3,4-DHPEA-EA p-HPEA-EA Tyrosol secoridoids Hydroxytyrosol secoridoids Total2

Protocatechuic acid Vanillic acid Caffeic acid

B 0.02 0.78 0.10

Arbequina M 0.41

E

Olive variety /Olive ripeness stage Arbosana B M E 0.45

B

Koroneiki M 0.62

0.33 0.41

0.14 0.45

0.17 0.62

2.71 9.31

3.04 30.1

16.8 69.8

0.5

1.50

1.76

21.0

23.3

62.6

13.8 0.84 8.71 2.65 31.4 21.4

8.05 0.86 32.0 6.83 45.2 90.6

13.4 0.85 37.1 3.43 80.6 118.3

108.3 Hojiblanca M 0.01 0.50 n.d.

E n.d. 0.23 n.d.

B n.d. 0.15 n.d.

137.8 Picual M n.d. 0.52 n.d.

E n.d. 1.7 n.d.

B n.d. 0.29 n.d.

236.5 Arbequina M 0.01 0.43 n.d.

E

E 0.07 n.d. n.d.

Reference

Nieto et al., 2010 (Spain)

Phenolic compound Vanillin Syringic acid p-Coumaric acid Ferulic acid t-Cinnamic acid Rutin Quercetin Hydroxy-tyrosol Oleuropein Total3

B 0.85 0.16 0.04 0.08 0.36 0.04 0.05 11.9 0.03 14.41

3,4-DHPEA-EA p-DHPEA-EA Total4

B 201.1 45.0 753.7

Total5

B 268.7

Hojiblanca M 0.36 n.d. n.d. n.d. n.d. n.d. 0.04 6.03 0.02 6.96 Hojiblanca M 9.31 14.4 n.d. Barnea M 169

Demethyloleuropein Nuzhenide

B 51.2 183.8

Frantoio M 128.8 252.9

E 0.35 n.d. n.d. n.d. n.d. n.d. 0.01 2.00 0.01 2.6 E 4.03 2.40 112.2 E 32.29

E 55.4 199.8

Olive variety /Olive ripeness stage Picual B M E 0.19 0.22 1.03 n.d. 0.06 n.d. n.d. n.d. n.d. 0.03 0.02 0.01 n.d. n.d. n.d. n.d. n.d. n.d. 0.01 0.01 0.01 7.13 5.44 4.5 0.01 0.01 0.01 7.52 6.28 7.26 Arbequina B M E 196.4 5.41 0.07 18.2 10.4 0.02 511.6 n.d. 126.2 Souri B M E 108.3 94.09 75.27

B n.d. 312.2

Moraiolo M n.d. 446.8

E n.d. 264.2

B 0.36 0.06 n.d. n.d. n.d. 0.11 0.64 3.03 n.d. 4.49 B

Arbequina M 0.33 n.d. n.d. 0.11 n.d. n.d. 0.16 1.97 n.d. 3.01 M

Reference E 0.12 n.d. n.d. n.d. n.d. n.d. 0.11 1.48 0.03 1.81 E GutierrezRosales et al., 2010 (Spain)

B

M

E Dag et al., 2011 (Isreal)

B n.d. 332.5

Leccino M 107.9 371.4

E 59.1 168.8

Cecchi et al., 2013 (Italy)

Table 1. (Continued)

Phenolic compound Hydroxytyrosyl acyclodihydroelenolate Caffeoyl-6′secologanoside Oleuropein aglycones Oleuropein Comselogoside Ligstroside Oleoside Rutin Hydroxytyrosol glucoside Hydroxytyrosol Tyrosol Total6

B 162.7 130.4 478.6 375.7 167.0 25.4 68.7 437.6 86.4 158.2 103.3 11,698.9

Frantoio M 39.4 90

Total7

B 341

990.3 1159.5 137.8 41.5 301.8 485.3 65.6 180 30.4 11,600.9 Arbequina M 395

Vanillic Syringic p-cumaric Chlorogenic

B 0.66 0.01 0.2 n.d.

Ayvalık M 1.04 0.02 0.09 n.d.

E 28.7

Olive variety /Olive ripeness stage Moraiolo B M E 58.7 10.2 31.9

B 20.2

Leccino M 6.4

E 15.8

192.7

157.4

86.9

78.6

78.5

49

186

1005.8 139.4 164.1 10.7 132.7 357.7 87.8 90.8 32.6 7,905.5

2,127.8 1,038.6 199.5 57.3 328.1 647.2 92.8 168.8 107 18,332.6

409.4 353.8 144 24.1 280.4 755.2 239.7 104.5 29.8 12,102.7

1,439.3 1,685 202.7 27.8 400 566 347.7 332 207.1 17,468.7

E 261

B 530

1,211.8 1,783.5 161.2 37.5 460.7 1,198.1 171.5 107.7 36.5 19,416.3 Arbosana M 578

E 298

B 970

1,312.1 478.2 230.2 7.4 262.3 493.9 192.1 137.2 35.1 10,568.9 Coratina M 850

E 1.09 0.01 0.4 0.02

B 0.08 0.01 n.d. n.d.

Domat M 1.49 0.01 0.02 n.d.

E 1.78 0.01 0.24 0.02

B 0.98 0.02 0.05 n.d.

Gemlik M 1.65 0.01 0.04 n.d.

Reference

274.7 55 226 8.9 106.3 394.1 190.5 201.1 73.9 9117.8 E 640

E 1.15 0.01 0.07 0.03

Camposeo et al., 2013 (Italy)

Dagdelen et al., 2013 (Turkey)

Phenolic compound Ferulic Hiydroxytyrosol Tyrozol Vanillin Apigenin Luteolin Rutin Total8

B n.d. 0.09 0.99 0.7 0.36 0.27 5.98 9.26

Ayvalık M 0.07 0.8 0.68 0.18 0.81 1.67 n.d. 5.36

B Total9

3,4-DHPEA-AC 3,4-DHPEA-EDA p-HPEA-EDA 3,4-DHPEA-EA p-HPEA-EA 3,4-DHPEA p-HPEA Vanillic acid

126

B 42.3 75.5 59.9 33.7 16.5 1.34 1.95 2.00

E 0.12 0.52 1.13 0.16 0.93 2.28 n.d. 6.66

M 94 Arbequina M 98.2 68.2 86.9 122.2 14.6 0.75 1.3 0.43

E

Olive variety /Olive ripeness stage Domat B M E n.d. 0.05 0.12 n.d. 1.15 0.78 0.18 0.66 0.8 4.09 0.13 0.12 0.23 0.06 0.23 n.d. 0.23 1.42 1.35 5.5 n.d. 5.94 9.3 5.52 Chemlal B M E

B n.d. 0.16 1.57 0.65 0.06 0.28 3.64 7.41 B

Gemlik M 0.04 0.63 1.19 0.19 0.38 0.53 n.d. 4.66 M

Reference E 0.03 0.35 0.53 0.08 0.9 1.74 n.d. 4.89 E Bengana et al., 2013 (Algeria)

85

E 36.8 44.7 36.8 104.4 9.68 0.39 0.64 0.16

B 7.33 141.1 73.5 68.4 82.9 2.84 3.49 1.16

Carrasqueña M 6.89 148.4 84.1 75.7 40.0 3.77 6.43 0.47

E 6.99 88.7 36.1 95.2 14.3 0.65 3.44 0.44

B 6.20 70.6 41.4 30.4 51.0 1.28 3.00 0.49

Morisca M 33.5 171.0 91.5 133.4 151.2 1.04 1.97 0.32

E 43.3 42.5 40.3 19.0 23.9 1.38 3.17 0.26

Franco et al., 2014 (Spain)

Table 1. (Continued)

Phenolic compound Vanillin p-cumaric acid Ferulic acid Luteolin Apigenin Total10

B 0.47 0.81 n.d. 2.68 0.91 238.0 B

Σ Hydroxytyrosol derivatives11 Σ Tyrosol derivatives11 Σ Phenolics11 B Hydroxytyrosol Tyrosol Caffeic acid p-Coumaric acid Ferulic acid Oleuropein Luteolin Apigenin Vanillic acid Vanillin Quercetin

Arbequina M 0.41 0.22 n.d. 6.21 1.87 401.3 Arbequina M 85 77 162 Arbosana M 2.07 18.7 0.026 0.24 0.152 0.22 4.14 2.91 0.8 0.1 0.045

E 0.17 0.24 n.d. 2.76 n.d. 260.2 E

E

Olive variety /Olive ripeness stage Carrasqueña B M E 0.36 0.23 0.17 1.37 0.18 0.53 n.d. n.d. 0.16 0.91 1.55 1.6 0.34 0.47 n.d. 383.7 355.0 246.2 Picual B M E 522

B

177 699 Cornicabra M 1.43 4.28 0.03 0.22 0.105 0.022 0.84 0.77 0.16 0.42 n.d.

Morisca B M E 0.32 0.19 0.19 0.84 0.41 0.17 n.d. n.d. 0.15 2.96 1.8 3.78 0.67 0.44 n.d. 209.2 588.95 178.1 Mansa (60%) and Brava (40%) B M E 114 232 346

E

B

FS-17 M 33.9 23.6 0.105 0.98 0.283 0.023 0.17 0.51 0.51 0.1 0.043

Reference

ReboredoRodríguez et al., 2015 (Spain)

E Sanchez de Medina et al., 2015 (Spain)

Phenolic compound B Rutin 3,4-DHPEA-AC 3,4-DHPEA-EDA p-HPEA-EDA 3,4-DHPEA-EA p-HPEA-EA Total12 B Hydroxytyrosol p-hydroxybenzoic acid Caffeic acid Epicatechin p-coumaric acid Ferulic acid Rutin Quercetin Total13 1

Arbosana M 0.04 1.09 6.96 10.4 8.55 2.23 59.6 Barnea M 1.38 1.55 n.d. n.d. n.d. n.d. 18.8 0.42 149.3

E

E

Olive variety /Olive ripeness stage Cornicabra B M E 0.04 7.66 10.0 15.7 51.1 39.1 132.6 Coratina B M E 2.39 2.48 n.d. n.d. n.d. n.d. 16.97 0.49 58.9

B

B

FS-17 M 0.037 9.6 9.94 9.22 60.4 16.9 166.6 Koreniki M 1.58 3.51 0.16 n.d. 0.12 0.45 21.7 0.71 180.2

Reference E

E

Phenolic compounds calculated as mg of 3,4-dihydroxyphenylacetic acid kg-1 of oil. 3 Phenolic compounds calculated as mg kg-1 as synergic acid. Phenolic compounds calculated as mg of phenol L-1. 4 -1 5, 8, 9, 10, 11, 13 Phenolic compounds calculated as mol of phenol g . Phenolic compounds calculated as mg of phenol kg-1. 6 Phenolic compounds calculated as mg of tyrosol kg-1on dry matter. 7 Phenolic compounds calculated as mg gallic acid per 100 g dry weight. 12 Phenolic compounds calculated as g of phenol kg-1. 2

Xiang et al., 2017 (China)

34

Gassan Hodaifa and Santiago Rodriguez-Perez

The biosynthesis of oleuropein is very active during the ripening process of the olives. This fact produces an increase in the concentration of oleuropein in the olives. However, this activity decreases conform increases the activity of the enzyme βglucosidase. The enzymatic action of this enzyme produces a decrease in the concentration of oleuropein, producing its derivatives. The biosynthesis of oleuropein presents some differences according to the olives variety. In the Arbequina variety, the biosynthetic routes are active for a longer time than in the Hojiblanca variety. Therefore, the phenolic concentration of olives and olive oil of this variety usually presents high values throughout the ripening process of the olives, in comparison with other varieties. The highest phenolic concentrations have detected in Picual, Hojiblanca, Arbequina, Chetoui, Morisca and Coratina. The enzyme β-glucosidase acts on the glycosylated phenolic compounds, influencing its concentrations and those of its aglycone forms during the olives ripening (GutierrezRosales et al., 2010). The main compounds resulting from the action of this enzyme are the ellolic acid and the aglycone forms of oleuropein and ligustroside (Gutierrez-Rosales et al., 2010). The location of this enzyme varies during the ripening process of the olives. It has observed that in the early stages of olives ripening when pit formed, the βglucosidase enzyme is located in the central part of the olive (Gutierrez-Rosales et al., 2012). This fact can indicate that the enzyme participates in the transport of the monolignols that give rise to the lignin that forms the pit. In the middle stages of the olives ripening, with the already hardened pit the enzyme has distributed in the cells of the olives mesocarp (Gutierrez-Rosales et al., 2012). This distribution can explain by the enzymatic action of defense against external agents. Some studies have determined that the activity of this enzyme is greater in the Arbequina variety than in the Hojiblanca variety. The lignans present a maximum of concentration before the hardening of the pit, after which its concentration decreases (Gutierrez-Rosales et al., 2012). A second maximum has observed in intermediate ripening stages in some varieties of olives. The synthesis of flavonoids begins when the pit is formed, which could be related to the decrease of lignans synthesis. The evaluation of the degree of ripeness seems an important decision to obtain olive oils with a high oxidative stability with a certain overall quality. The control of the harvesting ripe olives stage can maximize the phenolic concentration in the olive oil (Franco et al., 2014; Dag et al., 2011). Therefore, a ripening index determination seems a key parameter to optimize the high-quality olive oil production. A good ripening index has to be objective, easily quantifiable and preferably not destructive (Camposeo et al., 2013). In order to achieve the objective classification, the index must to base on the physiological, physic and biochemical significant variations that produced along the ripening process.

Olive Varieties and Phenolic Compounds in Olive Oils

35

The olive ripeness index (RI) based on the changes in the color of both epicarp and mesocarp of olives. The determination of the RI requires a representative sample of the agricultural area studied and from this representative sample, it take 100 olives. These olives classified according to their pigmentation in a scale of eight levels (8 categories) of pigmentation related to the degree of ripening. In this sense, it have been considered ripeness period the time from the appearance of violaceous spots until the final coloration of the skin (Humanes-Guillen and Civantos-López, 1992). The color of olives classified in different categories: 1. Category A (with coefficient value equal to 0) corresponding to olives with intense green epicarp. 2. Category B (with coefficient value equal to 1) corresponding to olives with green-yellow epicarp. 3. Category C (with coefficient value equal to 2) corresponding to olives with reddish males epicarp in less than half the fruit. 4. Category D (with coefficient value equal to 3) corresponding to olives epicarp with reddish males or light violet throughout the fruit (more than half the fruit). 5. Category E (with coefficient value equal to 4) corresponding to olives with black epicarp and white mesocarp. 6. Category F (with coefficient value equal to 5) corresponding to olives with black epicarp with less than half of the purple mesocarp. 7. Category G (with coefficient value equal to 6) corresponding to black olives with purple mesocarp without reaching the bone. 8. Category H (with coefficient value equal to 7) corresponding to olives with black epicarp and total purple mesocarp. Once the olives have been classified by category (maturity/color levels), the number of olives is counted for each category and multiplied by the coefficient of each category. The calculation of RI do by applying the following equation (1): RI =

A×0 + B×1 + C×2 +D×3 + E×4 + F×5 + G×6 + H×7 100

(1)

where A, B, C, D, E, F, G and H are the number of olives count for each category. According to general information of studies published, the IR value equal to 3 a 4 shows the best results in both quantity and quality of olive oil (Baccouri et al., 2008). Due to IR is a laborious method, it usually used in combination with the flesh firmness index or detachment index. These methods correlated with the olives resistance to damage and the harvesting efficiency, respectively. The damage produced in the harvesting or manipulation can influence in the olive oil chemical composition as it has mentioned before. The main disadvantage of these indexes is that are destructive indexes.

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Gassan Hodaifa and Santiago Rodriguez-Perez

Recently, the colorimetric index was introduced in the olive culture. This is a not destructive index already used for other fruit tree species successfully. Colorimetric index could control the olives color changes up to the selected level. This method could represent a simple and quick method which it may replace all other. As it has mentioned before, external damage can influence significantly in the enzymatic activity along the ripening process. The ripening process actives the enzymatic activity which it makes olives sensitive to external damage which can increase the enzymatic activity which influences in the phenolic biosynthesis. Therefore, the criteria for choosing the harvesting time have also take into account the external factors that can influence in its concentration i.e., the mechanical technics to harvesting. Thus, some authors note that the criteria have to be first the maximum harvesting mechanical efficiency and then the maximum oil quality (Camposeo et al., 2013). This priority of criteria has its sense because the final olive oil quality is influenced also by the harvesting mechanism. Which is better ripening index and priority criteria to determine the optimal harvesting time? The speed of the ripening process that depends on the olive variety could determine the answer. This factor is determined by the start of the ripening process and its duration. Table 2 shows the usual ripening development for some olive varieties. A non-destructive ripening index could selected for varieties with a medium to short ripening process (Late ripening) such as Hojiblanca (Camposeo et al., 2013). While varieties with a long ripening process (early ripening) as Arbequina, a traditional index could be used (Camposeo et al., 2013). According to published information, olive oils with lower phenolic concentration usually are fruitier and less bitter than olive oils with higher phenolic concentration (Allalout et al., 2009). However, oils with phenolic concentration higher will show notable oxidative stability (Nieto et al., 2010; Allalout et al., 2009; Fuentes de Mendoza et al., 2013). The evaluation of the influence of the degree of ripeness on oxidative stability of olive oils is important for decisions on producing oils with a certain overall quality (Franco et al., 2014; Dag et al., 2011). The practice of harvesting ripe olives as soon as possible to produce oils of high phenolic contents and thus high oxidative stability implies bitter and piquant oils, this being admissible for oils of some varieties but detrimental to others appreciated in the market for being more fruity and sweeter (Nieto et al., 2010). Therefore, according to the variety or varieties of olives from which the olive oil extracted, the optimal harvesting time must be determined (Dag et al., 2011). The duration of the ripening process should taken into account to select the ripening index. The harvesting, extraction and store process must to looking for minimizing the external agents’ effect that might modify the organoleptic profile of the olive oil, achieving high-quality olive oils.

Olive Varieties and Phenolic Compounds in Olive Oils

37

Table 2. Usual ripening development (early, middle and late) for olive varieties mentioned in this study Olive variety Picual Hojiblanca Verdial Manzanilla/Carrasqueña Morisca Cornicabra Arbequina Shikitita Arbosana Chétoui Chemlali Koroneiki Barnea Souri Frantoio Moraiolo Leccino Coratina Urano® Domat Gemlik

Ripening development Early Late Late Early Early Late Early Early Late Late Late Early Early Middle Late Early Early Late Early Late Early

Finally, the increase of the acidity value is observed as ripening process advanced, with significant differences between the early and late stage of ripeness. These results have observed from many authors. This acidification might be due to a progressive activation of the lipolytic activity along the ripening process. This change in the physicochemical characteristics makes olives especially sensitive to the pathogenic infections and mechanical damage, which can produce oils with high acidity values (Mendoza et al., 2013).

4. ORGANOLEPTIC CHARACTERISTICS OF OLIVE OIL Sensory characteristics of olives influence in the final olive oil quality. Olive’s organoleptic characteristics undergo changes related to the tree age and external agents of deterioration that increase with the olives ripeness process. While the ripening process advances, olives organoleptic profile changes due to metabolic processes that affect to compounds such as triglycerides, fatty acids, phenols, tocopherols, chlorophylls, and

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carotenoids. These changes highly influence in the aroma, nutritional value and oxidative stability i.e., olive oil quality. The methods to determine the ripeness stage described above can facilitate the olive oil production with the highest possible phenolic concentration according to the range determined by the olive variety, environmental factors, and harvesting technology. However, the phenolic compounds not only have a positive influence on olive oil and are beneficial to health but also influence the sensory characteristics of the olive oil. As mentioned above, the phenolic compounds influence the bitterness, astringency, and pungency of the olive oil. So, how are the sensory characteristics of olive oil evaluated? A trained panel, according to Appendix XII of the European Community (EC) Regulation no. 796/2002, must carry out the sensory analysis. The panelists should ideally be experts in the olive oil sensory evaluation and, recognized by the International Olive Council. In the sensory analysis, each sample is tasted for its bitterness, pungency and fruitiness, which are positive attributes, and any negative attributes, according to the official procedure. Results are usually expressed as the mean intensity of the sensory perceptions of the tasters. Nieto et al., 2010, Reboredo-Rodriguez et al., 2016 and Fernandez-Silva et al., 2013 have studied the intensity or the Pearson correlation of the components in the olive oil with the sensorial attributes of olive oil. It is well accepted that phenol compounds and the bitterness are correlated. It is well known that bitterness and pungency are mainly related to secoiridoid compounds (Allalout et al., 2009). The olive oil usually has a high content of a bitter glucoside called oleuropein, which is formed by glucose, elenolic acid, and the o-diphenol hydroxytyrosol. The concentration of this secoiridoid glucoside in fruits depends on many factors such as variety, irrigation, and degree of ripeness. Indeed, the level of oleuropein in olives decreases with ripening as it has been mentioned. Some studies have suggested the heating of raw olives to reduce bitterness in virgin olive oil (Garcia et al., 2005). This method would inactivate the enzyme β-glucosidase, and the bitter secoiridoid aglycons would not be formed. According to Garcia et al., (2005) which have studied the negative attributes in the different olive oils varieties over the harvest season, it seems clear that these values increased with the advance of the ripening process. Olive varieties with long ripening process showed that the mean value of negative attributes takes longer to vary than the varieties with early ripening process. Sensory defects are detected due to the high concentrations of some volatile oils versus the profile of high-quality olive oils. Some studies have demonstrated that the highest sensory significance, evaluated by odor values, corresponds to 1-octen-3-ol for mustiness–humidity; ethyl butanoate, propanoic, and butanoic acids for fusty sensory defects; acetic acid, 3-methyl butanol and ethyl acetate for winy-vinegary defects; and

Olive Varieties and Phenolic Compounds in Olive Oils

39

several saturated and unsaturated aldehydes and acids for rancid sensory defects (Morales et al., 2005). According to Nieto et al., (2010) and Reboredo-Rodríguez et al., (2016) the variation of the mean of the fruity attribute could have a negative correlation with the increase phenolic compounds. It has been observed that varieties with a long ripening process as Arbequina showed lower values of the fruity attribute than varieties with a short ripening process at the beginning of the season. However, varieties with long ripening processes not only showed an increase of the fruity attribute at the middle of the season but also kept values higher than varieties with an early ripening process. Table 3 shows the sensory intensity of some olive oil varieties at the end of the harvest. Table 3. Intensity of sensory attributes from some olive oils studied at the end of harvest

Olive variety Hojiblanca Picual Arbequina 90% Brava/10% Mansa 70% Brava/30% Mansa

fruitiness 2.5 2.2 2.6 2.5 3.3

Attributes defects bitterness 1.7 2.7 1.9 3.4 1.5 2.0 2.5 2.3 4.7 4.1

pungency 3.4 3.7 3.0

Reference Nieto et al., 2010 ReboredoRodríguez et al., 2016

Mendoza et al., 2013 have observed that olive oils extracted from olive growing zone under stressed conditions showed pungency and bitterness more pronounced. At the same time, in these olive oils had high phenolic concentration. Moreover, previous studies that have studied Chétoui and Cornicabra varieties oils observed high phenolic compounds levels along the ripening process (Table 2). This fact may be attributed to its markedly late ripening compared to other varieties with early ripening process such as Gemlik variety (Table 2). The sensory characteristics of bitterness and pungency are due to the activation of taste receptors and trigeminal nerve endings associated with taste buds in fungiform papillae, sensitive to chemical stimuli. In olive oils these sensations are related to the presence of phenolic compounds and can persist for rather long times after deglutition, showing a clear after-effect that can greatly vary among olive oils in intensity and duration and might affect consumer acceptance. Phenolic compounds in olive oil are mainly aglycones and other secoiridoid derivatives. For example, oleuropein and ligstroside have an antioxidant effect and are the main compounds responsible for the shelf life of virgin olive oils. During storage, the oils undergo qualitative and quantitative changes due to decomposition and oxidation. The total phenols contents decrease and, consequently, the typical bitter taste and pungent note of fresh olive oil decrease in intensity.

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Many authors have studied the relationship between the individual phenolic compounds and bitter and pungent sensations in olive oils (Nieto et al., 2010; FernandesSilva et al. 2013). Some authors have indicated the dialdehydic form of decarboxymethyl elenolic acid linked to hydroxy-tyrosol (3,4-DHPEA-EDA) and oleuropein aglycone (3,4-DHPEA-EA) as the main compounds responsible for bitter taste (Garcia et al., 2005). Meanwhile, other authors have attributed bitter and pungent notes to ligstroside derivatives such as p-HPEA-EDA (Tovar et al., 2005), even identifying as the main compound responsible for the pungent sensation on the back of the tongue (Andrewes et al., 2003). Also, some studies have reported a linear correlation between bitter taste and derivatives of oleuropein and ligstroside aglycones (Gutierrez-Rosales et al., 2003), while others have registered a highly significant correlation of bitter olive oil taste with the aldehydic form of oleuropein aglycone in a concentration range between 0.03 and 0.5 mmol kg-1 (Mateos et al., 2004).

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Gutierrez, F., Albi, M. A., Palma, R., Ríos, J. J. and Olías, J. M. (1989). Bitter tasting of virgin olive oil: correlation of sensory evaluation and instrumental HPLC analysis. J. Food Sci., 54: 68–70. Gutierrez-Rosales, F., Rios, J. J. and Gomez-Rey, M. L. (2003). Main polyphenols in the bitter taste of virgin olive oil: structural confirmation by on-line high performance liquid chromatography electrospray ionization mass spectrometry. J. Agric. Food Chem., 51: 6021–6025. Gutierrez-Rosales, F., Romero, M. P., Casanovas, M., Motilva, M. J. and MinguezMosquera, M. I. (2010). Metabolites Involved in Oleuropein Accumulation and Degradation in Fruits of Olea europaea L.: Hojiblanca and Arbequina Varieties. J. Agric. Food Chem., 58: 12924–12933. Gutierrez-Rosales, F., Romero, M. P., Casanovas, M., Motilva, M. J. and MinguezMosquera, M. I. (2012). β-Glucosidase involvement in the Formation and Transformation of Oleuropein during the Growth and Development of Olive Fruits (Olea europaea L. cv. Arbequina) Grown under Different Farming. J. Agric. Food Chem., 60: 4348−4358. Hrncirik, K. and Fritsche, S. (2005). Relation between the endogenous antioxidant system and the quality of extra virgin olive oil under accelerated storage conditions. J. Agric. Food Chem., 5: 2103–2110. Humanes-Guillen, J. and Civantos-López, M. (1992). Production of high quality olive oil. Influence of the culture. Andalusian Ministry of Agriculture and Fisheries, Seville, Spain. Jadhav, S. J., Nimbalkar, S. S., Kulkarni, A. D. and Madhavi, D. L. (1995). Lipid oxidation in biological and food systems, in: Food Antioxidants, Technological, Toxicological and Health Perspectives (Ed.) by Madhavi, D. L., Deshpande, S. S., Salunkhe, D. K., Marcel Dekker Inc, New York, pp. 5–63. Jolayemi, O. S., Tokatli, F. and Izmir, B. O. (2016). Effects of malaxation temperature and harvest time on the chemical characteristics of olive oils. Food Chem., 211: 776– 783. Kiritsakis, A. K. (1998). Flavor components of olive oil: a review. J. Am. Oil Chem. Soc., 75: 673–681. Lavelli, V. (2002). Comparison of the antioxidant activities of extra virgin olive oils. J. Agric. Food Chem., 50: 7704–7708. Leonardis, A. and Macciola, V. (2002). Catalytic effect of the Cu(II)- and Fe(III)cyclohexanebutyrates on olive oil oxidation measured by Rancimat. Eur J. Lipid Sci. Technol., 104: 156–160. Manna, C., Galleti, P., Cucciolla, V., Montedoro, A. and Zappia, V. (1999). Olive oil hydroxyl-tyrosol protects human erythrocytes against oxidative damage. J. Nutr. Biochem., 10: 159–165.

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Mateos, R., Dominguez, M. M., Espartero, J. L. and Cert, A. (2003). Antioxidant effect of phenolic compounds, α-tocopherol, and other minor components in virgin olive oil. J. Agric. Food Chem., 51: 7170–7175. Mateos, R., Cert, A., Perez-Camino, M. C. and Garcia, J. M. (2004). Evaluation of virgin olive oil bitterness by quantification of secoiridoid derivatives. J. Am. Oil Chem. Soc., 81: 71–75. Matos, L. C., Pereira, J. A., Andrade, P. B., Scabra, R. M. and Oliveira, M. B. (2007). Evaluation of a numerical method to predict the polyphenol content in monovarietal oils. Food Chem., 102: 976–983. Medina, I., Sacchi, R., Biondi, L., Aubourg, S. and Paolillo, L. (1998). Effect of packing media on the oxidation of canned tuna lipids, antioxidant effectiveness of extra virgin olive oil. J. Agric. Food Chem., 46: 1150–1157. Mendoza, M. F., Gordillo, C. M., Expóxito, J. M., Casas, J. S., Cano, M. M., Vertedor, D. M. and Baltasar, Mª. N. F. (2013). Chemical composition of virgin olive oils according to the ripening in olives. Food Chem., 141: 2575–2581. Morales, M. T., Luna, G. and Aparicio, R. (2005). Comparative study of virgin olive oil sensory defects. Food Chem., 91: 293–301. Morello, J. R., Vuorela, S., Romero, M. P., Morilva, M. J. and Heinonen, M. (2005). Antioxidant activity of olive pulp and olive oils phenoliccompounds of the ‘Arbequina’ cultivars. J. Agric. Food Chem., 53: 2002–2008. Morellò, J. R., Motilva, M. J., Tovar, M. J. and Romero, M. P. (2004). Changes in commercial virgin olive oil (cv ‘Arbequina’) during storage, with special emphasis on the phenolic fraction. Food Chem., 85: 357–364. Nieto, L. M., Hodaifa, G. and Peña, J. L. L. (2010). Changes in phenolic compounds and Rancimat stability of olive oils from varieties of olives at different stages of ripeness. J. Sci. Food Agric., 90: 2393–2398. Obied, H., Bedgood, D. R., Prenzler, P. D. and Robards, K. (2007). Bioscreening of Australian olive mill waste extract: biophenol content, antioxidant, antimicrobial and molluscidal activities. Food Chem. Toxicol., 45: 1238–1248. Ortega-García, F. and Peragón, J. (2009). Phenylalanine ammonia-lyase, polyphenol oxidase, and phenol concentration in fruits of Olea europaea L. cv. Picual, Verdial, Arbequina, and Frantoio during ripening. J. Agric. Food Chem., 57 (21): 10331‐ 1034. Paiva-Martins, F. and Gordon, M. H. (2002). Effects of pH and ferric ions on the antioxidant activity of olive polyphenols in oil-in-water emulsions. J. Am. Oil Chem. Soc., 79: 571–576. Papadopoulos, G. and Boskou, D. (1991). Antioxidant effects of natural phenols on olive oil. J. Am. Oil Chem. Soc. 68: 669–671.

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Persson, E., Graziani, G., Ferracane, R., Fogliano, V. and Skog, K. (2003). Olive oil phenols reduce the formation of heterocyclic amines during frying of beef burgers. Food Chem. Toxicol., 41: 1587–1597. Ramanathan, L. and Das, N. P. (1992). Studies on the control of lipid oxidation in ground fish by some polyphenolic natural products. J. Agric. Food Chem., 40: 17–21. Reboredo-Rodríguez, P., González-Barreiro, C., Cancho-Grande, B., Fregapane, G., Salvador, M. D. and Simal-Gándara, J. (2015). Characterisation of extra virgin olive oils from Galician autochthonous varieties and their co-crushings with Arbequina and Picual cv. Food Chem., 176: 493–503. Reboredo-Rodríguez, P., González-Barreiro, C., Cancho-Grande, B., Valli, E., Bendini, A., Toschi, T. G. and Simal-Gándara, J. (2016). Characterization of virgin olive oils produced with autochthonous Galician varieties. Food Chem., 212: 162–171. Salvador, M. D., Aranda, F. and Fregapane, G. (1999). Contribution of chemical components of Cornicabra virgin oils to oxidative stability: a study of three successive crop seasons. J. Am. Oil Chem. Soc., 76: 427–432. Sánchez de Medina, V., Priego-Capote, F. and Luque de Castro, M. D. (2015). Characterization of monovarietal virgin olive oils by phenols profiling. Talanta, 132: 424–432. Servili, M. and Montedoro, G. (2002). Contribution of phenolic compounds to virgin olive oil quality. Eur J. Lipid Sci. Technol., 104: 602–613. Shahidi, F., Zheng, Y. and Saleemi, Z. O. (1993). Stabilization of meat lipids with flavonoids and flavonoid-related compounds, J. Food Lipids 1: 69–78. Sousa, A., Malheiro, R., Casal, S., Bento, A. and Pereira, J. A. (2015). Optimal harvesting period for cvs. Madural and Verdeal Transmontana based on antioxidant potential and phenolic composition of olives. Food Sci. Technol., 62: 1120-1126. Talhaoui, N., Gómez-Caravaca, A. M., León, L., De la Rosa, R., Segura-Carretero, A. and Fernández-Gutiérrez, A. (2014). Determination of phenolic compounds of ‘Sikitita’ olive leaves by HPLC-DAD-TOF-MS. Comparison with its parents ‘Arbequina’ and ‘Picual’ olive leaves. Food Sci. Technol., 58: 28-34. Toschi, G., Biguzzi, B., Cerretani, L., Bendini, A., Rotondi, A. and Lerker, G. (2004). Effect of crushing time and temperature of malaxation on the oxidative stability of a monovarietal extra-virgin olive oil, obtained by different industrial processing systems. Prog. Nutr., 6: 132–138. Tovar, M. J., Motilva, M. J. and Romero, M. P. (2001). Changes in the phenolic composition of virgin olive oil from young trees (Olea europaea L. cv. ‘Arbequina’) grown under linear irrigation strategies. J. Agric. Food Chem. 49: 5502–5508.

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Whitehead, M. C., Breeman, C. S. and Linsella, B. A. (1985). Distribution of taste and general sensory nerve endings in fungiform papillae of the hamster. Am. J. Anat., 173: 185–201. Xiang, C., Xu, Z., Liu, J., Li, T., Yang, Z. and Ding, C. (2017). Quality, composition, and antioxidant activity of virgin olive oil from introduced varieties at Liangshan. Food Sci. Technol., 78: 226-234.

In: Handbook of Olive Oil Editor: Jozef Miloš

ISBN: 978-1-53612-356-2 © 2017 Nova Science Publishers, Inc.

Chapter 3

BIOMEDICINAL ASPECTS AND ACTIVITIES OF OLIVE OIL PHENOLIC COMPOUNDS Cvijeta Jakobušić Brala*, Monika Barbarić, Ana Karković Marković and Stanko Uršić Faculty of Pharmacy and Biochemistry, University of Zagreb, Zagreb, Croatia

ABSTRACT Olive oil is a basic component of the Mediterranean diet. Mediterranean populations experience reduced incidence of cardiovascular disease, atherosclerosis, diabetes mellitus, metabolic syndrome, neurodegenerative diseases, certain types of cancer and higher life expectancy. Following impressive number of various biomedicinal studies related to the phenomena that accumulate for decades, these health benefits could be at least partially attributed to the olive oil, and more specifically the phenolic compounds naturally present in olive oil. While a number of reports have linked the health benefits of olive oil with its phenolic content, there is a great number of in vitro and in vivo studies that have demonstrated positive effects of olive oil phenolic compounds following analysis of certain physiological parameters. Thus, the phenolic compounds are deemed to be of central importance for beneficial antioxidant, antiatherogenic and antiinflammatory, antimicrobial, cardiovascular, anticancer, and neuroprotective effects that can be ascribed to the consumption of extra virgin olive oils. The aim of this chapter is to review the biomedicinal activities of olive oil phenolic compounds, including antioxidant, anti-inflammatory, antimicrobial, cardiovascular, endocrine, anticancer and central nervous system effects. Also, some insights related to bioavailability and synergistic activities are presented.

*

Corresponding Author: [email protected].

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Keywords: olive oil, phenolic compounds, biomedicinal, hydroxytyrosol, tyrosol, oleuropein, oleochantal, bioavailability

INTRODUCTION The Mediterranean diet (MD), traditional dietary model in countries of the Mediterranean basin, is characterized by a high intake of olive oil, fruits, vegetables, nuts, legumes, cereals; moderate intake of fish and poultry; a low intake of dairy products, red meat, processed meats, and sweets; and wine in moderation, consumed with meals. It was recommended by UNESCO as an Intangible Cultural Heritage since 2013. High adherence to MD is associated with improved ageing and reduced incidence of agerelated diseases, including cardiovascular diseases (CVDs), diabetes mellitus, metabolic syndrome, certain types of cancer, neurodegenerative disease, cognitive decline and increased longevity. Olive oil (OO) as the main source of fat is one of the main properties of the MD (Sofi 2010, Estruch 2013, Knight 2016, Fernandez del Rio 2016, Muros 2015). Throughout history, OO has been very appreciated, as potent pharmacological agent. Olive cultivation persist from olden times. Thus, for example, at Lun (island of Pag, Croatian Adriatic) there are hundreds of olive trees 1000-1600 years old (Jakobušić Brala 2015). Homer called OO “liquid gold” and Hippocrates, who mentions approximately 60 health conditions where OO use can be beneficial, called it “great healer.” Traditionally, many beneficial properties associated with OO have been ascribed to its high oleic acid content. Today, according to great number of in vitro, in vivo, epidemiological and clinical studies, it is clear that many of OO health effects are due to its minor components, phenolic compounds (Visioli 2002, Tripoli 2005, Cicerale 2010). Natural phenolic compounds are secondary plant metabolites, chemically characterized by the presence of one or more aromatic rings with one or more hydroxyl substituents. Major olive oil phenolic compounds (OOP, see also Figure 1 below) include the phenolic alcohols, hydroxytyrosol (HT, 3,4-dihydroxyphenylethanol, DHPEA) and tyrosol (TY, phydroxyphenylethanol, p-HPEA) and their secoiridoid precursors: oleuropein (OLE, the HT ester of elenolic acid glucoside), oleuropein aglycon (3,4-DHPEA-EA), the dialdehydic derivative of decarboxymethyl elenolic acid bound to HT, known as oleacin (3,4-DHPEA-EDA) or to tyrosol, known as oleochantal (p-HPEA-EDA) (Bendini 2007).

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Figure 1. Major olive oil phenolic compounds (OOP). HT: hydroxytyrosol; TY: tyrosol; OLE: oleuropein; 3,4-DHPEA-EA: oleuropein aglycon; 3,4-DHPEA-EDA: oleacin; p-HPEA-EDA: oleochantal.

This chapter presents OOP beneficial health effects, including antioxidant, antiinflammatory, antimicrobial activity and particularly recent findings concerning their possible use for prevention/treatment of CVDs, atherosclerosis, daibetes mellitus, metabolic syndrome, neurodegenerative diseases, cancer, as well as their bioavailability, metabolism, and synergistic activity.

ANTIOXIDANT ACTIVITY During normal physiological functions, significant concentrations of reactive free radicals and nonradical species, commonly referred to as reactive oxygen species, ROS, are formed. Under conditions of physiological disorders or excessive stress, the production of ROS can overcome the capacity of the endogenous antioxidant system, causing oxidative stress. Oxidative stress can cause oxidative damage to biomolecules, lipids, DNA, proteins, what is considered to be a critical step in the pathophysiology of many chronic diseases such as cardiovascular disease, atherosclerosis, diabetes mellitus, cancer, neurodegenerative diseases and other degenerative diseases (Conti 2016). Olive oil has been shown to be effective against oxidative stress associated diseases as well as with aging (Fito 2007, Fernandez del Rio 2016). This can be mainly attributed to OO phenolic compounds, since they have been proved, in vitro and in vivo, to have strong antioxidant activity (Visioli 2002, Bendini 2007). Particularly strong antioxidant activity exert hydroxytyrosol and oleuropein due to its catecholic structure. The presence of second hydroxyl group at the ortho-position enhances antioxidant capacity (GranadosPrincipal 2010, Rodriguez-Morató 2016, Hassen 2015). It has been shown that also tyrosol is the effective cellular antioxidant, in spite of its weak antioxidant activity,

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probably by intracellular accumulation (Di Benedetto 2007). Hydroxytyrosol and tyrosol metabolites (for example HT and TY sulphates) also show significant antioxidant activity (Atzeri 2016). OOP can exert their antioxidant activity directly, by scavenging ROS generated during oxidative stress (Visioli 1998) or metal chelation (Kitsati 2016) and indirectly by increasing the organism endogenous defense systems against an oxidative stress (Rodriguez-Morató 2016). OOP decrease oxidative damage of biological macromolecules, which have been shown to have a subsequent positive effect on disease risk. Low-density lipoprotein (LDL) oxidation is considered to be a major risk factor for the development of atherosclerosis and CVD. The protective effect of OOP on LDL oxidation has been reported in a series of papers (Visioli 1995, Covas 2006a, de la Torre-Carbot 2010). Oxidative damage to DNA could precede human carcinogenesis. Research concerning DNA damage shows the intake of phenol-rich olive oil decreases oxidative DNA damage in vivo in humans by up to 30% (Fabiani 2008). Further, randomized cross-over study found the OOP had significantly lowered F2-isoprostane levels; the F2-isoprostanes formed as a result of the free radical induced peroxidation of arachidonic acid, a common membrane bound fatty acid (Ruano 2005). In vitro research has shown that OOP reduce detrimental oxidative damage to red blood, renal and intestinal cells (Paiva-Martins 2013, Martinez Lara 2016).

ANTI-INFLAMMATORY ACTIVITY It is known that the pathophysiology of common disease states such as CVD, atherosclerosis, neurodegenerative disease, rheumatoid arthritis, cancer etc. could take root in chronic inflammation. Olive oil phenolic compounds have been reported to possess significant antiinflammatory properties (Parkinson 2016, Schwingshackl 2015a, Tomé-Carneiro 2016). Current evidence for a role of OOP in reducing inflammatory processes is, in the main, related to atherosclerosis. The release of pro-inflammatory cytokines, like interleukins and tumor necrosis factor-α (TNF-α), within the vascular wall affect endothelial function. Excessive inflammatory processes of the endothelium are predictors of future cardiovascular events. Phenol-rich OO is effective in modulating inflammatory mediators derived from arachidonic acid, as well as other inflammatory markers, which have been proposed to play an important role in the development of atherosclerosis (Perona 2006, Bogani 2007, Fito 2008, Moreno-Luna 2012, Rigacci 2016). It has been demonstrated that OO improves inflammation and atherosclerosis biomarkers in HIVinfected patients receiving antiretroviral treatment (Dokmanović 2015). Further, due to its anti-inflammatory properties, OOP have a beneficial effect in the prevention of neurodegenerative diseases (Rodríguez-Morató 2015). There is some

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evidence that OOP may be effective in the treatment of some immune-inflammatory diseases such as rheumatoid arthritis, systemic lupus erythematosus and inflammatory bowel disease (Aparicio-Soto 2016, Angeles Rosillo 2016, Kaulmann 2016). It has been demonstrated that HT has the potential as a chondroprotective compound against osteoarthritis, as an autophagy and sirtuin-1 inducer (Cetrullo 2016). Olive oil decreased inflammatory response and oxidative damage in pressure ulcers, and in that way promotes wound healing (Donato-Trancoso 2016). It could be proposed that phonophoresis with virgin olive oil is as effective as piroxicam gel for the treatment of female athletes’ anterior knee pain (Nakhostin-Roohi 2016). It has been observed that OOP affect several inflammative pathways (Rigacci 2016, Parkinson 2016, Bonura 2016). The anti-inflammatory mechanisms of OOP are suggested to include: inhibition of pro-inflammatory enzymes, such as cyclooxygenase-1 (COX-1) and COX-2, lipoxygenase, inducible nitric oxide synthase (iNOS), phosphoinositide 3-kinase, tyrosine kinases, NF-κB, and downregulation of various proinflammatory cytokines such as chemokines, TNF-α, interleukins and monocyte chemotactic protein-1 (MCP-1). In vitro, oleocanthal (dialdehydic derivative of decarboxymethyl elenolic acid bound to tyrosol, p-HPEA-EDA) has been shown to inhibit both COX-1 c and COX-2, inflammatory enzymes involved in the biosynthesis of inflammatory prostaglandins, in a dose-dependent manner, and is more effective than ibuprofen in inhibiting these enzymes at equimolar concentrations (Beauchamp 2005). It has also been shown that oleocanthal attenuates inflammatory mediators such as iNOS which plays a role in the pathogenesis of joint degenerative disease (Iacono 2010). Further evidence suggests that oleocanthal may be a potent pharmacological agent in the treatment of neurodegenerative disease. In addition to neuroprotective properties, this compound attenuates markers of inflammation implicated in Alzheimer’s disease (Abuznait 2013, Qosa 2015). Oleuropein inhibits TNFα induced matrix metalloproteinase 9 (MMP-9) in a monocyte cell line. Monocytes, and the molecules they secrete play a significant role in inflammatory disease development (Dell’Agli 2010). Hydroxytyrosol exerts a vascular protective effect since it decreases MMP-9 release and reduces COX-2 and NF-κB activation (Scoditti 2014). Oleacein enhances the anti-inflammatory activity of human macrophages by increasing CD163 receptor expression (Filipek 2015).

ANTIMICROBIAL ACTIVITY Olive oil phenolic compounds have a wide spectrum of antibacterial, antiviral, and antiprotozoal activities. OO has shown a broad spectrum of antibacterial activity against a large number of Gram positive and Gram negative, aerobic and anaerobic, intracellular and extracellular

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bacteria (Karaosmanoglu 2010, Medina 2006). Antibacterial activity was correlated with OOP content, and refined olive oil, without OOP, showed no antibacterial activity. This indicates that OOP may be useful therapeutic agents in the treatment of some infectious diseases (Tripoli 2005). OOP, including oleocanthal, hydroxytyrosol, and tyrosol have been shown to possess potent activity against several strains of bacteria responsible for intestinal and respiratory infections in vitro (Bisignano 1999, Cicerale 2010). Contrary, recent study indicates that HT did not show significant antimicrobial activity (MedinaMartinez 2016). Oleocanthal has been found to aid in inhibiting the growth of Helicobacter pylori; the bacterium associated with peptic ulcer and gastric cancer development (Romero 2007). OOP have also been found to exhibit antibacterial activity against the beneficial bacteria, Lactobacillus acidophilus and Bifidobacterium bifidum (Tripoli 2005, Medina 2006). The inhibition of these health-benefiting bacteria may cause a detrimental effect on wellbeing and therefore requires further investigation. The antibacterial mechanism of action of OOP has not been thoroughly investigated though they are known to penetrate cell membranes of both Gram negative and Gram positive bacteria causing damage to the peptidoglycans and cell membrane structure. Oleuropein and hydroxytyrosol have shown antiviral activities against HIV-1 and influenza virus, in vitro (Lee-Huang 2007, Bedoya 2016, Yamada 2009). The mechanism of action of the antiviral activity of OOP is suggested to include interference with viral amino acid production, prevention of virus shedding, inhibition of viral replication, neutralization of reverse transcriptase and protease in retroviruses, prevention of virus entry to cells, disruption of virus structure and stimulation of phagocytosis. It has been demonstrated that tyrosol and hydroxytyrosol have antitrypanosomal and antileishmanial activity. Trypanosomiasis and leishmaniasis, infectious diseases caused by protozoan parasites, affect millions of people around the world, especially in tropical and subtropical and present one of the major causes of hunger and poverty in sub-Sahara Africa (Belmonte-Reche 2016).

CARDIOVASCULAR EFFECTS Cardiovascular diseases are the world’s leading cause of death (Mozaffarian 2016). Numerous risk factors, like hypertension and dyslipidemia, are known to promote the pathogenesis of CVD. Although considerable progress has been made in the treatment and prevention of CVD with pharmacological therapy, diet therapy and lifestyle remains the base of clinical intervention. The lower incidence of CVD observed in Southern Europe might, at least in part, be explained by the adherence to Mediterranean diet (MD) rich in extra virgin olive oil. A number of studies and meta-analyses have reported the cardioprotective effect of the MD (Huedo-Medina 2016, Bloomfield 2016, Martinez-Gonzalez 2016, Delgado-Lista 2014,

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Sofi 2010, Fung 2009). In particular, the PREDIMED (PREvencion con DIetaMEDiterranea, Prevention with Mediterranean diet) trial, a multicentre randomized intervention study conducted in Spain, evaluated the impact of MD, supplemented with extra-virgin olive oil, on the rate of major CV events (myocardial infarction, stroke or death from CV causes). It has been concluded that the MD supplemented with extravirgin olive oil reduced the incidence of major CV events by almost 30% (Estruch 2013, Martinez-Gonzalez 2015). Although it is difficult to isolate individual dietary factors, cumulative evidence suggests that OO may play a key role in the observed cardiovascular benefit (Covas 2015, Delgado-Lista 2016, López-Miranda 2010). This is probably due to the high amount of monounsaturated fatty acids, but it is also the result of the presence of phenolic compounds. There are a great number of reviews about the effect of OOP on the incidence of CVD (Rigacci 2016, Tomé-Carneiro 2016, Hohmann 2015). There are multiple mechanisms by which OOP might impact the development of CVD: reducing oxidative damage, decreasing inflammation, improving endothelial function and influencing platelet aggregation, increasing high-density lipoprotein (HDL) cholesterol and decreasing blood pressure.

Antioxidant Activity Oxidative stress is involved in the complex pathology of CVD. ROS degrade NO, which plays an important role in artery endothelium relaxation. Diminished NO level leads to platelet aggregation, thrombosis, and vascular inflammation. As discussed previously, OOP are strong antioxidants capable of neutralizing ROS and these antioxidant properties are associated with beneficial cardiovascular effects (Bogani 2007, Siti 2015). Scavenging activity of hydroxytyrosol has been demonstrated with respect to hypochlorous acid (HOCl), a potent oxidant produced in vivo at the site of inflammation (Visioli 1998). This activity may be critical for the protection from atherosclerosis since HOCl can oxidize the apoproteic component of LDL. It has been reported in a great number of papers that consumption of OOP protects LDL particles from oxidative damage (Marrugat 2004, Covas 2006b). The suppressed formation of oxidized LDL (oxLDL) depends upon the phenolic content applied. Postprandial LDL phenolic content and LDL oxidation are modulated by OOP intake. In a randomized controlled trial (RCT) after 3 weeks of sustained daily OO (629 mg total OOP/L) consumption, LDL phenolic content increased, oxLDL decreased relative to refined OO (with no OOP) (de la TorreCarbot 2010). This was confirmed in another RCT study; a 2-month consumption of a phenol-rich OO (∼30 mg/day) diet led to a significant decrease in oxLDL compared to baseline values in pre-hypertensive women (Moreno-Luna 2012). The European Food Safety Authority (EFSA) released a health claim about the role of OOP in protecting LDL from oxidation in vivo: “A daily intake of 20 g of olive oil, which contains at least 5

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mg of hydroxytyrosol and its derivatives (e.g., oleuropein and tyrosol) provides the expected beneficial effects” (EFSA NDA Panel 2010, Martín-Peláez 2013).

Anti-Inflammatory Activity, Endothelial Dysfunction, and Platelet Aggregation The cardiovascular endothelium is the main target of all major risk factors for CVD (hypertension, hyperglycemia, hyperlipidemia, inflammation, aging), and its damage is one of the first steps in the development of CVD. Endothelial dysfunction is a hallmark of vascular aging and is considered to be the first pathological symptom of atherosclerosis. Low-grade, chronic synthesis and release of pro-inflammatory cytokines within the vascular wall affect endothelium and may lead to endothelial dysfunction. Beneficial effects of MD on markers of both inflammation and endothelial function have been reported in a great number of papers (Schwingshackl 2015a, Cicerale 2010, Esposito 2004). Within the PREDIMED cohort sub-study, it has been shown that the increase in OOP intake, which was unequivocally identified as increased total urinary phenolic excretion, was associated with decreased inflammatory biomarkers (MedinaRemón 2016). A general improvement in inflammation markers following the consumption of phenol-rich olive oil has been seen in a number of RCTs (Fito 2008, Bogani 2007). OOP are able to restore endothelial function and to reduce lipid accumulation within the atherosclerotic lesions (Claro 2015). Intervention trial investigated the influence of OOP (51 mg oleuropein, 10 mg HT) on vascular function and inflammation, in a postprandial setting, and provided the evidence of a positive modulation of vascular function in healthy volunteers (Lockyer 2015). The administration of phenol-rich OO has been demonstrated to improve endothelial function in hypercholesterolemic patients and in hypertensive women (Ruano 2005, Moreno-Luna 2012). In vitro, OLE and HT inhibited endothelial adhesion molecule expression (Carluccio 2003). OOP may stimulate the production of endothelial vasodilator factors and have a direct, endothelium-independent relaxant effect on vascular smooth muscle, an effect that usually occurs at higher concentrations. Oleuropein aglycon and oleacin have an endothelium-dependent vasorelaxant effect mediated by an enhanced NO production, probably through a redox mechanism within endothelial cells and, at higher concentrations, an endothelium-independent vasorelaxant effect. Taking into account the plasmatic concentrations of these phenols, it is clear that OOP could modulate vascular tone in vivo (Segade 2016). HT and secoiridoids downregulated proteins related to proliferation and migration of endothelial cells and occlusion of blood vessels in aorta and proteins related to heart failure in heart tissue (Catalan 2016). OOP have been shown to influence platelet aggregation, which is a key factor in the development of thrombus and myocardial infarction or angina (Covas 2015, Delgado-

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Lista 2011). The acute intake of phenol-rich OO has been associated with a smaller postprandial increase of tissue factor (TF), activated coagulation factor VII and fibrinogen, as well as a greater decrease in the fibrinolysis pathway factors such as tissue plasminogen activator and plasminogen activator inhibitor type 1 in both healthy and hypercholesterolemic subjects compared with the intake of a low-phenol OO (Ruano 2007). HT, HT acetate, TY, and OLE have been proved to prevent platelet aggregation (Granados-Principal 2010). HT and its acetate ester exerted anti-platelet effects similar to acetylsalicylic acid (Aspirin) both in vitro and in rats (Gonzalez Correa 2009).

Dyslipidemia Dyslipidemia is important CVD risk factor and refers to a variety of lipid abnormalities, including elevated total cholesterol, LDL cholesterol and triglyceride levels and low high-density lipoprotein (HDL) cholesterol levels, which have been associated with the development and progression of atherosclerosis and CVD. Olive oil has a beneficial effect on LDL and HDL particles (Catapano 2016). OOP provide benefits on lipoprotein particle atherogenic ratios and subclasses profile distribution. Lipoprotein particle measures and associated ratios may be better markers for atherosclerosis risk than conventional lipid measures (Fernandez-Castillejo 2016). In the EUROLIVE study, 200 healthy, nonsmoking male volunteers were given 25 mL per day of olive oil with high (366 mg/kg), medium (164 mg/kg) and low (3 mg/kg) phenolic content in a randomized, cross-over, double-blind and controlled trial. It has been observed a decrease in in vivo lipid oxidative damage and also an increase in HDL cholesterol, in a direct, dose-dependent manner with the phenolic content of the olive oil (Covas 2006b). HDLs play a central role in reverse cholesterol transport. They remove excess cholesterol from peripheral cells (the cholesterol efflux capacity, CEC) and transport it to the liver for further metabolism and excretion. CEC has been shown to predict coronary event incidence and to be inversely related to the development of early atherosclerosis. HDL function reflects the physiological role of the lipoprotein better than the quantity of HDL cholesterol. The consumption of OOP increased the cholesterol efflux capacity (Hernaez 2016, Berrougui 2015). It has been observed an increase in the biological metabolites of OOP bound to the HDLs (hydroxytyrosol sulphate, and homovanillic acid sulphate and glucuronate). These compounds could exert a local antioxidant protection in HDLs that may prevent oxidative modifications of the apolipoprotein A-I, the main HDL protein involved in CEC, and of other HDL proteins. Such protection would also avoid oxidative modifications of HDL lipids, making the lipoprotein more fluid and thus more functional (Atzeri 2016). HDLs could act as transporters of several derivatives of OOP to the endothelial cells where they may prevent oxidative damage. OOP have a significant

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role in the up-regulation of genes involved in the cholesterol efflux from cells to HDL, in vivo in humans (Meza-Miranda 2016). Functional, phenol-enriched OOs are a promising therapy to improve CEC (Farràs 2015).

Atherosclerosis Atherosclerosis is a chronic inflammatory disease affecting large and medium arteries and is considered to be a major underlying cause of cardiovascular disease. Several epidemiological studies have reported a correlation between increased levels of olive oil in the diet and a lower risk of developing atherosclerosis (Moss 2016, GranadosPrincipal 2010, Tripoli 2005). There is increasing evidence that oxidative modification of LDL plays a key role in the development of atherosclerosis, as it induces plaque formation within the arterial wall. OOP reduce oxidative stress and protect LDL from oxidation, and may also interfere with the inflammatory response within the atherosclerotic lesion, thus improving vascular stability. Also, OOP positively modulate arterial stiffness (Bogani 2007, Rigacci 2016). OO rich in phenolic compounds modulates the expression of atherosclerosis-related genes in vascular endothelium (Meza-Miranda 2016). The antiatherogenic activity of TY, HT, and OLE has been extensively studied in vitro and in vivo (Granados-Principal 2010, Rodríguez-Morató 2016). OLE appears to possess the highest anti-atherosclerotic power among all OOP, mostly resulting from cholesterol regulation (Rigacci 2016). Also, oleacin might play a special role in decreasing the progression of atherosclerosis (Naruszewicz 2015).

Hypertension High blood pressure (BP) and hypertension are another important risk factors for CVD. Hypertension was found to be inversely proportional to consumption of the MD and particularly to consumption of OO (Lopeza 2016, Psaltopoulou 2004). Within the PREDIMED cohort, the increase in OOP intake was associated with a decrease in systolic and diastolic BP (Medina-Remón 2015). Further, in RCT study, mildly hypertensive young women received phenol-rich olive oil (30 mg/day) and OOP-free olive oil. Only phenol-rich olive oil significantly decreased both systolic and diastolic BP (Moreno-Luna 2012). In another RCT study, hypertensive men with stable coronary heart disease administered virgin OO and refined OO for 3 weeks. The phenolic content was about 10 times higher in virgin OO (7.4 mg TP/day) than in the refined oil (0.67 mg TP/day). There was a significant reduction in systolic BP after the intake of virgin OO, compared to refined OO (Fito 2005). However, a meta-analysis investigating the effects of phenol-rich olive oils on risk factors of the cardiovascular system, using data from

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RCTs performed with healthy or CVD patients, where high phenolic OOs were compared with low phenolic ones, found mild effects for lowering systolic BP, but no effects in diastolic BP (Hohmann 2015). Nevertheless, the authors acknowledged that there was the small number of studies/participants to get firm conclusions. OOP may control hypertension through their antihypertensive and potent antioxidant activities. Oxidative stress is a significant factor leading to accelerated structural and functional damage in hypertension. Also, it has been demonstrated that OOP enhance NO concentrations and may help dilate arteries, stimulate ROS synthase system, and increase plasma nitrites/nitrates, which reduces BP (Moreno-Luna 2012).

ENDOCRINE EFFECTS Diabetes Mellitus Consumption of olive oil exerts beneficial health effects towards type 2 diabetes mellitus (T2DM). T2DM is a condition closely associated with obesity and CVD, together known as metabolic syndrome, which also includes non-alcoholic fatty liver disease (NAFLD), a condition whose severity spans from simple triglyceride accumulation in the liver parenchyma (steatosis) to non-alcoholic steatohepatitis (NASH) (Bonaccio 2016, Rigacci 2016, Parkinson 2016, Salas-Salvado 2008). The clearest evidence that olive oil may prevent diabetes comes from PREDIMED study including patients at high CVD risk. The participants following a Mediterranean diet rich in extra virgin olive oil had a 40 % reduced incidence of T2DM than the control group (Martínez-González 2015, Salas-Salvado 2014). Consumption of phenol-rich olive oil improved metabolic control in patients with T2DM (Santangelo 2016). Further, it has been observed that higher OO intake is associated with modestly lower risk of T2DM in women (Guasch-Ferre 2015). Olive oil phenolic compounds can positively modulate T2DM and other pathological states associated with deregulation of carbohydrate and lipid metabolism (Rigacci 2016). There is a great number of in vitro and in vivo studies demonstrating the different mechanism of action. Oleuropein and hydroxytyrosol demonstrated hypoglycemic activity in various diabetic animal models. HT (0.5 to 10 mg/kg/day orally) influenced the major biochemical processes leading to diabetic vasculopathy and reduced cell proliferation in the vascular wall in rats with experimentally-induced diabetes mellitus (Antonio Lopez-Villodres 2016). Tyrosol may also play an important role in the treatment of diabetes mellitus, as it exerts antiinflammatory effects on the liver and pancreas of streptozotocin-induced diabetic rats via its antioxidant activity (Chandramohan 2016), as well as it inhibits endoplasmatic reticulum stress-induced apoptosis in pancreatic β-cell (Lee 2016). Dysfunction of pancreatic β-cells is a major determinant for the development of T2DM.

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Obesity Obesity is an increasingly widespread condition affecting millions of people mainly in developed countries. The PREDIMED study has also shown that an olive oil-rich diet was effective in the prevention of weight gain (Buckland 2015). Observational and dietary intervention trials consistently show that Mediterranean diet rich in olive oil does not contribute to obesity and according to a systematic review including twenty-one epidemiological studies may actually help decrease it (Buckland 2008). The EPICPANACEA cohort study showed that individuals with high adherence to MD, including olive oil, were significantly less likely to develop overweight or obesity (Romaguera 2010). OOP have been shown to influence the expression of genes related to obesity, to reduce the size of fat cells leading to the decreased risk of obesity. Oleuropein, hydroxytyrosol, and others have been shown to be effective against obesity by suppressing dose-dependently intracellular triglyceride accumulation and the expression of adipogenesis-stimulating factors during adipocyte differentiation. HT, at nutritionally relevant concentrations, exerts adiponectin downregulation in inflammed adipocytes (Scoditti 2015), as well as reduces triglyceride accumulation and promotes lipolysis in human primary visceral adipocytes during differentiation (Stefanon 2016). OLE demonstrated TGR5 agonist activity. TGR5 is a G-protein coupled receptor in adipose tissue that increases energy expenditure, glucose metabolism and hence can combat obesity. These data indicate that OOP may have a protective role against excessive fat accumulation associated with systemic oxidative stress.

Liver Disease OOP display significant protection against liver disease resulting from altered lipid metabolism. The anti-steatosis effects of OOP have been associated with increased lipid metabolism and modulation of glucose homeostasis. OOP have been reported to downregulate lipid synthesis in primary cultured rat hepatocytes, suggesting that a decrease in hepatic lipid metabolism, particularly lipid synthesis, may represent a possible mechanism underlying the reported hypolipidemic effect of these substances (Priore 2015). HT exerts a beneficial effect in the prevention of early inflammatory events responsible for the onset of insulin resistance and steatosis, reducing hepatic inflammation and nitrosative/oxidative stress and restoring glucose homeostasis and intestinal barrier integrity (Pirozzi 2016). Tyrosol significantly increased hepatic cystathionine β-synthase (CBS) and cystathionine gamma-lyase (CSE) expression and H2S synthesis in high fat diet-fed mice. CBS and CSE are major enzymes responsible for endogenous H2S, which has emerged as a potential therapeutic target in NAFLD (Sarna 2016).

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Osteoprotective Effects Hormonal changes during the menopausal transition ultimately result in a decline in estrogen levels which lead to the development of osteoporosis. Considerable evidence has demonstrated that pro-inflammatory cytokines such as IL-1, IL-6, and TNF-α are involved in the regulation of bone turnover, by increasing bone resorption. Lower incidence of osteoporosis in the Mediterranean area has been attributed mainly to MD (Puel 2008). Olive oil can effectively reduce bone loss in ovariectomized rats, and with no or only mild effects on the uterine endothelium (Zheng 2016). OO and OLE were able to prevent osteopenia most probably via antagonizing inflammatory mediators. In vitro, OLE increased osteoblast differentiation at the gene expression level. OOP hydroxytyrosol and few others, stimulate human osteoblastic cell proliferation and further research should be carried out to develop new interventions and adjuvant therapies using OO for bone health in osteoporosis (Garcia-Martinez 2016).

CENTRAL NERVOUS SYSTEM EFFECTS Neurodegenerative disorders encompass a large number of chronic diseases characterized by progressive and irreversible injury of the neurons that results in loss of function and/or structure and cell death. Currently, only symptomatic and palliative medications are available to treat neurodegenerative diseases, such as Alzheimer’s (AD) and Parkinson’s disease (PD). A number of theories have been proposed to explain the neurodegenerative process. The brain is the largest consumer of oxygen in the human body. The abundance of readily oxidizable polyunsaturated fatty acids and catalytic metal ions, along with inefficient antioxidant defense make the brain a major site for developing oxidative stress (Markesbery 1997, Rodríguez-Morató 2015). Adherence to the MD has been associated with a reduced incidence of neurodegenerative diseases, decreased risk of cognitive impairment and dementia and better cognitive performance. Virgin olive oil, the main source of fat in the MD, rich in minor phenolic components, may contribute to neuroprotective effects credited to MD (Sofi 2010, Singh 2014, Knight 2016, Petersson 2016, Wu 2017). Neuropsychological tests in a PREDIMED subcohort of cognitively healthy individuals demonstrated that adherence to the MD supplemented with OO (1 L/week) was associated with improved cognitive functions at 4-year follow-up (Valls-Pedret 2015). A growing body of evidence from animal models to clinical studies indicates that OOP compounds may have neuroprotective effects in several pathologies of the nervous system, such as Alzheimer’s disease, Parkinson’s disease, stroke and also in better cognitive performance, through the control of oxidative stress, inflammation, apoptosis and mitochondrial dysfunction (Davinelli 2016, Rodríguez-Morató 2015). Recent findings in animal models and humans

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show that OOPs may have a role in regulating neurotrophins levels, in particular nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), suggesting that OOPs may also induce their protective effects through the potentiation of neurotrophins action (Carito 2016). Alzheimer’s disease (AD) is one of the most studied neurodegenerative amyloid diseases, a significant cause of dementia in the elderly. It is characterized by the deposition of typically aggregated proteins/peptides in tissues, associated with degeneration and progressive functional impairment. Neuropathological hallmarks of AD include the accumulation of extracellular neuritic plaques (which are formed of amyloidβ peptides) and intracellular neurofibrillary tangles (whose major component is hyperphosphorylated tau protein). Amyloid-β peptides induce oxidative stress that is manifested by lipid peroxidation, protein and DNA oxidation, free radical formation and neurotoxicity. Inflammatory processes are also involved in the pathology of AD. Numerous clinical and preclinical studies have suggested several health promoting effects of the consumption of olive oil that could protect and decrease the risk of developing Alzheimer’s disease (Martorell 2016, Qosa 2015). Studies conducted both in vivo and in vitro have revealed the great potential of the OOP, mainly oleocanthal and oleuropein aglycone, in counteracting amyloid aggregation and toxicity. Protective effect of oleocanthal against AD has been related to its ability to prevent amyloid-β (A-β) and tau aggregation in vitro, and enhance A-β clearance from the brain of wild-type mice in vivo (Qosa 2015, Abuznait 2013). Oleuropein aglycone counteracts amyloid aggregation and toxicity affecting different pathways: amyloid precursor protein processing, amyloid-β peptide and tau aggregation, autophagy impairment and neuroinflammation (Martorell 2016). It has been demonstrated in vitro that also hydroxytyrosol and oleuropein may be effective as tau aggregation inhibitors at low concentrations (10 μM) (Daccache 2011). Also, OO has a neuroprotective effect, attributable to its hydroxycinnamic acids component, against A-β-induced cytotoxicity and oxidative stress (Villareal 2016). Parkinson’s disease (PD) is a progressive neurodegenerative disorder, primarily affecting dopaminergic neurons in the substantia nigra. According to the catecholaldehyde hypothesis, monoamine oxidase (MAO) inhibition should slow the progression of Parkinson’s disease, by decreasing production of the autotoxic dopamine (DA) metabolite 3,4-dihydroxyphenylacetaldehyde. However, hydroxytyrosol inhibits both enzymatic and spontaneous oxidation of endogenous DA and mitigates the increase in spontaneous oxidation during MAO inhibition (Goldstein 2016). HT has a protective effect against DA and 6-hydroxydopamine induced dopaminergic cell death, supporting the beneficial effect of olive oil in preventing DA-metabolism related dopaminergic neuron dysfunction (Yu 2016a). OLE may prevent neuronal degeneration in a cellular dopaminergic model of PD, due to neuroprotective, anti-oxidative and autophagyregulating activity (Achour 2016).

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OOP can improve some age-related dysfunctions by differentially affecting different brain areas. It has been described that the maternal administration of hydroxytyrosol to rats is able to restore the impaired neurogenesis and cognitive function (learning capacity and memory) caused by prenatal stress (Zheng 2015a). HT improves neuronal survival, mitochondrial function and reduces oxidative stress in the brain cortex of mice (Zheng 2015b). Oleuropein pretreatment attenuates cognitive dysfunction and oxidative stress induced by some anesthetic drugs in the hippocampal area of rats (Alirezaei 2017). OLE has a neuroprotective effect on cerebral ischemia and reperfusion injury in a middle cerebral artery in mice, what may indicate that it is a potential therapeutic for stroke (Yu 2016b). Also, it has been suggested that tyrosol is an appropriate candidate to be used in stroke therapy since it exhibited protective effects against the sensory motor dysfunction in rats (Bu 2007). A study in a mouse model of ethanol addiction has demonstrated that OOP may confer protection against ethanol-induced oxidative stress by reducing serum free oxygen radicals but not affecting the free oxygen radicals defense. Occasional exposure to alcohol is sufficient to induce an imbalance in the intracellular redox state and produce tissue damage, particularly brain and liver (Carito 2017). Study on rats has demonstrated that OLE administration can protect the hypothalamic paraventricular nucleus from oxidative stress, what is associated with occurrence of hypertension (Sun 2017).

ANTICANCER EFFECTS Cancer is a complex disease characterized by the uncontrolled growth and spread of abnormal cells. Alongside tobacco and infectious organisms, an unhealthy diet is deemed to be the most important external cancer-causing factor. Cancers figure among the leading causes of morbidity and mortality on a global scale. According to the statistics in 2012, there were an estimated 14.1 million cases of cancer diagnosed around the world and 8.2 million cancer deaths. Due to the growth and aging of population, those numbers are expected to increase further, reaching about 21.7 million of new cancer cases and 13.0 million cancer deaths in 2030 alone (American Cancer Society 2016). Few recent meta-analyses of observational studies have demonstrated that adherence to Mediterranean diet has a protective effect against various cancers in terms of risk reduction for overall cancer incidence and/or mortality (Sofi 2013, Schwingshackl 2014, Schwingshackl 2015b) and with lower incidence of several cancer types, especially colorectal, breast, gastric, prostate, liver, and head and neck cancer (Schwingshackl 2015b). Olive oil, the main dietary fat in Mediterranean population, is already recognized as very important health-promoting component with effects that include, among many others, a reduced risk of cancer (Pirrodi 2017, Parkinson 2016, Buckland 2015, Kwan

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2017). A contemporary meta-analysis of observational studies revealed that olive oil consumption is inversely related to cancer prevalence, with most prominent results observed for breast cancer and cancers of the digestive system (Psaltopoulou 2011). Over the last few years, many in vitro and in vivo studies have demonstrated that olive oil phenolic alcohols and their secoiridoid derivatives are able to interfere with proliferation, apoptosis and differentiation of different tumor cells by acting on the expression of genes controlling these processes (Casaburi 2013, Cárdeno 2013a, Hashim 2014, Rosignoli 2016, Fabiani 2016, Pirrodi 2017). Hydroxytyrosol acts as potent antioxidant that induces apoptotic cell death and mitochondrial dysfunction by generating ROS in colon cancer DLD1 cells (Sun 2014). Some of the newest research results confirmed the antiproliferative and apoptotic activities of hydroxytyrosol, as well as its main microbial metabolites, in colon Caco-2 and HT-29 cancer cell lines (López de las Hazas 2017). A purified extracts from olive mill waste water, rich in hydroxytyrosol, have shown similar cancer chemoprotective activity as determined by inhibition of proliferation, migration, invasion, adhesion, sprouting of colon cancer cells and release of angiogenic, pro-inflammatory cytokines VEGF and IL-8 both in vitro and in vivo (Bassani 2016). Furthermore, anticarcinogenic effects of hydroxytyrosol are demonstrated in human hepatocarcinoma cells (Giordano 2014, Zhao 2014) and in papillary and follicular thyroid cancer cells (Toteda 2017). Oleuropein induces antimetastatic, antiproliferative and apoptotic effects in human breast cancer cells by modulation (up- or down-regulation) of expression of certain genes (Hassan 2012, Elamin 2012, Hassan 2013). In vivo study on animal model with MCF-7 human breast tumor xenografts confirmed that oleuropein possesses a potent anti-cancer activity inhibiting both the MCF-7 cells xenograft growth and their invasiveness into the lung (Sepporta 2014). Apart from breast cancer, anticarcinogenic effects of oleuropein and its effective mechanisms were demonstrated in a variety of cancer cells such as cervical cancer (Yao 2014), prostate cancer (Acquaviva 2012), thyroid cancer (Bulotta 2013), colon cancer (Cárdeno 2013b) and neuroblastoma (Seçme 2016) cells. Anticarcinogenic effects of oleocanthal are well documented throughout literature, showing the diversity of its way of actions. Oleocanthal induces cell death via induction of lysosomal membrane permeabilization in cancerous cells, which tend to have fragile lysosomal membranes compared to non-cancerous cells (LeGendre 2015). Study on human breast cancer cells has demonstrated reduced c-Met kinase activity, cell growth, migration, and invasion of breast cancer cells in presence of oleocanthal (Akl 2014). Disruption of c-Met related pathways by oleocanthal led to design of a novel semisynthetic oleocanthal-based c-Met inhibitor named homovanillyl sinapate with excellent therapeutic potential to control c-Met-dependent malignancies (Mohyeldin 2016). Additionally, some anticancer mechanisms of oleocanthal are investigated against human malignant melanoma cells with promising results (Fogli 2016).

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BIOAVAILABILITY AND METABOLISM An important topic among recent research of olive oil phenolic compounds presents the analysis of bioavailability and ADME (absorption, distribution, metabolism and excretion) after ingestion to find out the mechanisms through the compounds exert their activity in the human body. However, such beneficial effects depend on the proportion of active substances that are absorbed from the gastrointestinal tract (Fantini 2015). The quantity of a compound that is absorbed and metabolized in the human body after it is ingested is called bioavailability, and it is commonly measured in terms of maximum plasma concentration (Cmax). In order to produce effects in vivo, a compound must enter the circulation and reach the tissues, in its native or metabolized form, in a sufficient quantity to exert biological activity (Marzocchella 2011). Among others, hydroxytyrosol, tyrosol, oleuropein aglycone and their glycosides are of central interest with regard to bioavailability and the formation and activities of its metabolites. The first report about the bioavailability of hydroxytyrosol and tyrosol and their metabolites in urine (involving also the glucuronide conjugates) was provided by Visioli (2000). Further results of ADME studies report about extremely poor bioavailability of hydroxytyrosol and tyrosol due to an extensive first-pass intestinal/hepatic metabolism, leading to the formation of sulphate and glucuronide conjugates (Miró-Casas 2003, Khymenets 2010). Also, the concentrations of these phenolic compounds in their free forms in urine were extremely low (about 5-10% recovered, respectively) (Rodríguez-Morató 2016). On the other hand, studies showed that hydroxytyrosol and tyrosol are well absorbed in the gastrointestinal tract in a dose-dependent manner (Visoli 2000, Weinbrenner 2004, Covas 2006, Pastor 2016). Once absorbed, these phenolic compounds are widely distributed throughout the body. In a study in rats, the oral administration of increasing doses of hydroxytyrosol (given in a refined olive oil matrix), demonstrated that hydroxytyrosol accumulated in a dose-dependent manner not only in plasma and urine, but also in the liver, kidney and brain (López de las Hazas 2015). The ability of hydroxytyrosol to reach the brain after crossing the blood-brain barrier in vivo demonstrated that it fulfils this essential requirement to be used as a neuroprotective agent (Rodríguez-Morato 2016). Comparing the bioavailability of hydroxytyrosol in humans, it was observed that recoveries were much higher after its administration as a natural component of olive oil (44.2% of hydroxytyrosol administered) than after its addition to refined olive oil (23% of hydroxytyrosol administered) or yogurt (5.8% of dose or approximately 13% of that recorded after virgin olive oil intake) (Visioli 2003). The human absorption of high amounts (2.5 mg/kg) of pure hydroxytyrosol, administered as an aqueous solution, was very low and it produced a great variety of metabolites, leading to poor bioavailability (1,000-fold sensitization) when co-cultured in the presence of oleuropein aglycone. Indeed, the nature of the interaction between oleuropein aglycone and trastuzumab was found to be strongly synergistic in Tzb-resistant SKBR3/Tzb100 cells. Mechanistically, the oleuropein aglycone treatment significantly reduced the HER2 (human epidermal growth factor receptor 2) Extracellular Domain cleavage and subsequent HER2 auto-phosphorylation, while it dramatically enhanced the Tzb-induced down-regulation of HER2 expression. The study of Fabiani (2011) demonstrated that, when tested in a complex mixture, the olive oil phenols exerted a more potent chemopreventive effect compared to single compounds. This phenomenon could be due to a synergistic interaction among the different compounds present in the extracts. However, it cannot be excluded that the complex extracts contain some other unidentified component(s) having potent chemopreventive activity. Other results showed that extra virgin olive oil extract significantly inhibited the proliferation and clonogenic ability of T24 and 5637 bladder cancer cells in a dosedependent manner (Coccia 2016). The researchers also evaluated the ability of low doses of extra virgin olive oil extract to modulate the in vitro activity of paclitaxel or mitomycin, two antineoplastic drugs used in the management of different types of cancer.

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In: Handbook of Olive Oil Editor: Jozef Miloš

ISBN: 978-1-53612-356-2 © 2017 Nova Science Publishers, Inc.

Chapter 4

AN OVERVIEW OF SAMPLE TREATMENT AND ANALYTICAL METHODOLOGIES FOR THE DETERMINATION OF PHENOLIC COMPOUNDS IN OLIVE OILS L. Molina-García, M. L. Fernández-de Córdova and E. J. Llorent-Martínez* Department of Physical and Analytical Chemistry, Faculty of Experimental Sciences, University of Jaén, Jaén, Spain

ABSTRACT The increasing popularity of olive oil is mainly attributed to its high content in phenolic compounds, which corresponds with the minor components fraction. Some polar olive oil phenols are not generally present in other fats, and this is one of the reasons that make this product unique. Phenolic compounds comprise a large family of secondary metabolites of plants and present a wide variety of health benefits. These compounds act as natural antioxidants and play an important role in the prevention of human diseases. Due to the chemical diversity of phenolic compounds, and to the fact that some of them are found at very low concentrations, their analysis is relatively complex. Briefly, assays for phenols in olive oil can be classified as those determining the total content of polyphenols, and those allowing the determination of the individual phenolic profile. Most of the analytical methods used for the quantitative determination of total phenols in olive oil are based on colorimetric assays. The analysis of o-diphenolic compounds is also carried out by this type of determination. Qualitative and quantitative composition of phenols in olive oil varies because of the strong dependence on variety and ripeness of the fruit, agronomic factors, and the processing system used for its *

Corresponding Author address, Email: [email protected].

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L. Molina-García, M. L. Fernández-de Córdova and E. J. Llorent-Martinez extraction. Therefore, the identification and quantification of the individual components of olive oil are of great interest. Extraction, chromatographic separation, and characterization are the three basic steps involved in the analysis of the phenolic profile of olive oil. The extraction procedures are mainly based on liquid-liquid extraction (LLE) and solid-phase extraction (SPE) using, in most cases, methanol as solvent. Regarding the analytical separation, it can be accomplished by capillary gas chromatography (GC) and, mainly, reverse phase high-performance liquid chromatography (RP-HPLC) with different detectors. Capillary electrophoresis (CE) has also been used for this purpose, achieving the same aims than HPLC but higher resolution, and reducing sample volume and analysis time. Nevertheless, nowadays liquid chromatography coupled to mass spectrometry (HPLC-MS) is widely accepted as the main tool in identification, structural characterization, and quantitative analysis of phenolic compounds in olive oil. Nuclear magnetic resonance (NMR) spectroscopy is also a powerful complementary technique for structural assignment in the cases where mass spectral data are insufficient to establish a definitive structure for phenolic compounds. In recent years, the hyphenation of HPLC with the most information-rich spectroscopic technique NMR has been proposed for structure elucidation of phenolic compounds in olive oil. Several studies have been carried out regarding the development of efficient and accurate analytical methods for the qualitative and quantitative analysis of phenolic compounds in olive oil. This chapter pretends to show an overview of different analytical approaches, including the most recent advances, and the difficulties regarding phenolic compounds determination in olive oil.

Keywords: virgin olive oil, polyphenols, analytical methods

INTRODUCTION The main antioxidants of virgin olive oil (VOO) are phenolic compounds, including lipophilic and hydrophilic phenols, and pigments (carotenoids, chlorophylls). While the lipophilic phenols, among which are tocopherols, can be found in other vegetable oils, some hydrophilic phenols of VOO are not generally present in other oils and fats. The health effects attributed to the consumption of VOO are mainly due to the olive hydrophilic phenols (Servili et al., 2009; Omar, 2010), commonly named as ‘phenolic compounds’ or ‘polyphenols’. Different groups of polyphenols can be found in VOO, such as phenolic acids, phenolic alcohols, hydroxy-isochromans, flavonoids, secoiridoids, and lignans. While tocopherols, phenolic alcohols, phenolic acids, and flavonoids are present in many fruits and vegetables belonging to several botanical families, secoiridoids are found only in plants belonging to the Olearaceae family, being the main class of phenols present in both olive fruit and VOO (Bendini et al., 2007a). They come from the secondary metabolism of terpenes and are characterized by the presence in their molecular structure of either elenolic acid or elenolic acid derivatives, in their glycosidic form or as aglycone (Garrido et al., 1997). The two main secoiridoids present in olive fruits are oleuropein and ligstroside, which are esters of elenolic acid (EA) with hydroxytyrosol (3,4-DHPEA) and tyrosol (p-HPEA), respectively; they are found in

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glycoside form. The crushing of olive fruits and the posterior malaxation of the milled olives, which are carried out during the production of VOO, gives rise to the formation of the aglycone derivatives from oleuropein and ligstroside through either a hydrolytic mechanism or -glucosidase activity (Cerretani et al., 2009); the dialdehydic form of decarboxymethyl EA linked to hydroxytyrosol or tyrosol termed 3,4-DHPEA-EDA and p-HPEA-EDA, the ligstroside aglycone mono-aldehyde (p-HPEA-EA) and oleuropein aglycone mono-aldehyde (3,4-DHPEA-EA) are the most abundant derivatives found in fresh VOO (Bendini et al., 2007b; Gómez-Alonso et al., 2007; Montedoro et al., 1993). Both agronomical (cultivar, geographic origin of olives, ripening stage, and trees irrigation) and technological factors influence the concentration of secoiridoids in VOO (Servili et al., 2004; Cerretani et al., 2006). According to European Food Safety Authority (EFSA), polyphenols in olive (olive fruit, olive mill waste waters or olive oil, Olea europaea L. extract and leaf), standardized by their content of hydroxytyrosol and its derivatives, contribute to the maintenance of normal blood HDL-cholesterol concentrations (without increasing LDL-cholesterol concentrations), which is a beneficial physiological effect (EFSA, 2012). Many studies have also demonstrated the anti-oxidant, anti-microbial, cardioprotective, hypoglycemic, anticarcinogenic and anti-inflammatory properties of VOO polyphenols (Bogani et al., 2007; Tripoli et al., 2005; Moreno-Luna et al., 2012). All these compounds also contribute to the stability and long shelf life typically observed in VOO in contrast with other vegetable oils. Besides olive oil stability, the nature and amount of polyphenols transferred from the olive fruit to the VOO have an important impact on their organoleptic properties. Thus, the contribution of some polyphenols - in particular secoiridoid derivatives of hydroxytyrosol - to the bitterness of VOO has been demonstrated by different authors (Gutiérrez-Rosales et al., 2003). The chemesthetic perceptions of pungency and astringency in some VOO are originated by other phenolic molecules such as p-HPEA-EDA, which can stimulate the free endings of the trigeminal nerve located in the palate and gustative buds (Andrewes et al., 2003). Identification as well as overall and/or individual quantitation of phenolic compounds in VOO are of great interest due to their effect on olive oil quality and beneficial health effects. To date, many analytical methods have been proposed for determining polyphenols in VOO, using different extractions, separations and quantification techniques. Polyphenols are present at trace levels in the oil matrix; therefore, they have to be extracted, cleaned up and concentrated before their analytical separation and detection. The extraction procedure is usually considered the most crucial step in analytical methods, having a significant impact on the results accuracy. The analysis of polyphenols in VOO is a challenging analytical problem due to the great variety of compounds that can be present, which differ in physicochemical behavior and size (from simple phenolic acids to tannins). Moreover, extreme care must be taken to ensure correct extraction, devoid of chemical modification, since a lot of polyphenols are easily

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hydrolyzed and all of them are relatively easily oxidized, which will invariably result in artefacts (Ryan et al., 1998). The approaches commonly used for the extraction of polyphenols from VOO are traditional liquid-liquid extraction (LLE) and direct phase (diol) solid phase extraction (SPE) (Capriotti et al., 2014). During the last few years there has also been a consistent increase in the development of environmentally friendly miniaturized extraction techniques for polyphenols (Godoy-Caballero et al., 2013), such as liquid-liquid microextraction (LLME), dispersive liquid-liquid microextraction (DLLME) and reversed-phase dispersive liquid-liquid microextraction (RP-DLLME), aimed to overcome common drawbacks of traditional methods. The methods so far adopted for the analysis of polyphenols in VOO are mainly based on spectrophotometric approaches or separation techniques. Spectrophotometric approaches, such as the Folin Ciocalteu (F-C) method, are used for overall determination. Nevertheless, the great diversity of phenolic compounds, the limited selectivity of the traditional methods, and the present and growing importance of VOO polyphenols require the replacement of overall quantitation methods by others providing individual information about each phenol. Chromatographic and electrophoretic methods are employed to accomplish individual separation and quantification of these compounds after their extraction. The results obtained by gas chromatography (GC) are very reliable, but a derivatization step is commonly necessary, so this technique is not commonly used. Electrophoretic techniques, such as capillary electrophoresis (CE) coupled with UV, mass spectrometry (MS) and MS/MS or capillary electrochromatography (CEC), are very attractive for the determination of polyphenols because of their high efficiency and short analysis time, but their drawback is the low sensitivity. Liquid chromatography (HPLC) with UV detection or coupled to MS (HPLC-MS) or MS/MS (HPLC-MS/MS) is the most effective and widely employed technique for the structural characterization and determination of both low and high molecular weight polyphenols in VOO (Motilva et al., 2013). For years NMR spectroscopy has been successfully applied to the analysis of olive oil (Vlahov, 1999) and its usefulness has been increasing steadily due to its noninvasiveness, rapidity, and sensitivity to a wide range of compounds in a single measurement (Bendini et al., 2007a). Currently there are high-resolution NMR techniques, such as two dimensional (2D) NMR spectroscopy, which combined with different strategies for treatment of data allows the analysis of complex mixtures of polyphenols without the necessity of coupling it with a separative technique (Christophoridou e Dais, 2009). On the other hand, problems such as strong signal overlap, diversity of intensities due to various concentrations of the phenolic compounds, or dynamic range problems have been resolved by coupling a high-resolution spectroscopic technique such as nuclear magnetic resonance (NMR) to HPLC (HPLCNMR or HPLC-NMR/MS) (Christophoridou et al., 2005). As a consequence of the great antioxidant activity and health benefits of o-diphenols, there is an increasing interest in the determination of their concentration in VOO. For this

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purpose, spectrophotometric methods have been commonly used. Recently, the potential of using electroanalytical techniques, such as voltammetry and amperometry, for the quantification of o-diphenols has been demonstrated due to their good sensitivity. Additional advantages include the use of low-cost and portable instrumentation, as long as the potential miniaturization of the devices (Dossi et al., 2017). These methods usually take advantage of the ability of o-diphenols to form complexes with molybdenum (VI) (Duhmer-Klair et al., 2003) or the fact that oxidation of mono-phenols is an irreversible process, occurring in one step at a relatively high potential, whereas o-diphenol oxidation takes place at a low potential in a two-electron-proton reversible mechanism (Enache et al., 2013). Discrepancies in the content of polyphenols in VOO samples are frequently found, probably due to the wide variety of analytical methods used (particularly methods for overall determination, drastically influenced by the standard selected) and/or by the expression of the results in different formats (El Riachy et al., 2011). This chapter is focused on the revision and assessment of the analytical methods proposed for qualitative and/or quantitative determination of polyphenols in olive oils.

SAMPLE PREPARATION Sample Treatment Prior to Isolation VOO is a viscous matrix, which makes weighing the oil rather than measuring its volume the most accurate choice (El Riachy et al., 2011). Then, it is common to dissolve samples in a non-polar organic solvent and to homogenize by stirring or vortexing. In this way, the lipid fraction is removed and it is easier to extract the phenolic compounds with a polar solvent. Although petroleum ether and chloroform have been used, hexane is the most widely reported solvent (Gutfinger, 1981; Tasioula-Margari and Okogeri, 2001b; Hrncirik and Fritsche, 2004). Other authors carry out an extraction with methanolic solvents, followed by a clean-up procedure with hexane prior to analysis (Gosetti et al., 2015). Due to the presence of conjugated phenolics, a hydrolysis step has been included by some authors before their extraction from VOO. This step minimizes interferences, especially when appropriate standards are not available. Acid hydrolysis is the most common approach to measure aglycones from flavonoid glycosides and phenolic acids from their respective esters (Carrasco-Pancorbo et al., 2005). Alkaline hydrolysis has been used more recently considering the stability of plant phenols under these conditions. Alkaline hydrolysis has been utilized, for instance, for secoiridoids (Carrasco-Pancorbo et al., 2005). Therefore, in this context, acid or basic hydrolysis can be used (Litridou et al., 1997; Oliveras-Lopez et al., 2007).

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Extraction of Phenolic Compounds from VOO The polar phenolic fraction is present at trace levels in the VOO matrix, so it has to be extracted, cleaned and concentrated before the analytical separation and detection (Capriotti et al., 2014). This isolation step is a prerequisite in order to obtain a sample extract that contains all the compounds of interest. In addition, this extract needs to be free from interfering matrix components, so a high selectivity can be obtained in the subsequent quantification (Tura and Robards, 2002). LLE (Bonoli et al., 2004; CarrascoPancorbo et al., 2005; Aturki et al., 2008; Suarez et al., 2008; Lerma-Garcia et al., 2009) and SPE (Mateos et al., 2001; Guttierrez-Rosales et al., 2003; De La Torre-Carbot et al., 2005; Carrasco-Pancorbo et al., 2006b; Garcia-Villalba et al., 2009) have been the main techniques traditionally utilized for the VOO simple and complex polar phenols isolation. The diverse extraction procedures differ in further aspects such as the amount of sample extracted, the kind and volume of solvents, and/or the solid phase cartridges (Hrncirik and Fritsche, 2004; El Riachy et al., 2011).

Liquid-Liquid Extraction (LLE) Methanol or methanol/water mixtures have been the most used extracting solvents for phenolic compounds. However, the selection of the best percentage of this solvent mixture for the complete recovery of phenolic compounds from VOO is subjected to controversy. Initially, a high percentage of methanol in water seemed to provide the best extraction yields. However, pure methanol was later mentioned as the best extraction solvent, due to the incomplete extraction observed for some phenolic compounds when a mixture of methanol/water was used, probably due to the emulsion formed between water and oil (Angerosa et al., 1995). However, other studies demonstrated that reducing the percentage of methanol to 60% increased the recovery efficiency of phenolic compounds (Pirisi et al., 2000; Ballus et al., 2014). Other organic solvents, such as ethanol, acetonitrile or N,N dimethylformamide (DMF) have also been proposed, observing interesting results in terms of recovery efficiency and sample manipulation (Brenes et al., 2000). Some improvements in the LLE, such as shortened extraction time, reduced reagent and sample volume and higher extraction efficiency, can be obtained by using ultrasound and microwaves as auxiliary extraction energies (Ruiz-Jimenez and Luque De Castro, 2003). Extraction of the polar fraction of olive oil using tetrahydrofuran/water (80:20, v/v) instead of methanol/water (60:40, v/v), provided 5 and 2 times higher recoveries for hydroxytyrosol and tyrosol, respectively (Cortesi, et al., 1995b). Moreover, tensiaoactive substances (2% v/w Tween-20) have also been added in order to liberate the phenolic compounds from the lipoprotein membranes (Montedoro and Cantarelli, 1969). Regarding the amount of sample and solvent used in LLE, the conventional systems usually used large amounts, making it time consuming, expensive and laborious (Gómez-

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Caravaca et al., 2005). To overcome these disadvantages, LLME (Becerra-Herrera et al., 2014) has been used, being faster, requiring a smaller amount of sample, and generating smaller volumes of residues. A comparison study demonstrated that both methods presented good repeatability and reproducibility, but lower amounts of total phenols extracted from VOO were observed when using LLE (Pizarro et al., 2013). DLLME and RP-DLLME have been other LLE techniques used with the same purpose (GodoyCaballero et al., 2013). An innovative and recent study has used “green solvents,” namely deep eutectic solvents (DESs), for the VOO phenolic extraction. In this work, a comparison of the yields with those obtained with conventional extraction methods was made. DES provided good solubility of phenolic compounds with different polarities and important enhancements in the recoveries of two secoiridoid derivatives (Garcia et al., 2016). A clean-up step can be introduced after the extraction in order to remove potential interferences, hence improving selectivity and sensitivity. On the one hand, one strategy consists in the overnight storage at sub-ambient temperature of the extracts, followed by filtration and centrifugation (Angerosa et al., 1995). On the other hand, some authors have cleaned-up the extracts with hexane, petroleum ether and chloroform, both pure or solvent mixtures (Lavelli and Bondesan, 2005). Sorbent columns such as Sephadex and Policlar AT/Celite 560 have also been used for extracts cleaning (Solinas and Cichelli, 1981). Evaporation of the extractant under vacuum or nitrogen stream and subsequent reconstitution with an appropriate solvent is usually carried out for pre-concentration purposes (Angerosa et al., 1995). Ambient or moderate temperature is required to avoid degradation.

Solid Phase Extraction (SPE) During the last decade, SPE for phenolic compound isolation from VOO has been reported in the literature. Several types of sorbents with different characteristics, such as C18, diol, amino or C8, and diverse eluents, namely methanol, ethanol and methanolwater mixtures, have been applied for both clean-up and pre-concentration purposes. (Gutierrez et al., 1989; Mannino et al., 1993; Servili et al., 1999; Pirisi et al., 2000; Mateos et al., 2001; Hrncirik and Fritsche, 2004; Gomez-Rico et al., 2008). The extraction of polar phenolic compounds by SPE was carried out for the first time with C18 cartridges and methanol as eluent (Mannino et al., 1993). C8 and C18 have been utilized for further experiments and the results have shown that normal-phase SPE is more suitable than reversed-phase due to the incomplete extraction of polar phenolic compounds and the partial oil separation provided by the last one (Cortesi et al., 1995a; Pirisi et al., 2000). In other study, C8 cartridges showed to be fast and simple for phenolic compound isolation (Pirisi et al., 2000). Two commercially available cartridges, octadecyl C18 (2g, 6 mL) and octadecyl C18E (end capped; 2g, 6 mL) have also been compared by other authors, who concluded that unsatisfactory recoveries were observed

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for C18 EC, but full quantitative recoveries for C18 (Liberatore et al., 2001). Amino and diol-bonded phase cartridges were also compared and the first one showed to be problematic because of the formation of interference artifacts during extraction, whereas recoveries > 90% were obtained for all major olive phenolic compounds when diol-bond phase SPE cartridges were used (Mateos et al., 2001). Polar compounds were retained on diol-bond by passing a hexane solution of the oil. Then, hydrocarbons waxes, tocopherols and triacylglycerols were eliminated by washing with hexane, and the polar fraction was eluted with methanol. Diol cartridges also provided higher recovery efficiency in other studies, where a comparison was made with other bonded-phases such as C8, C18 and Sax-SPE (Bendini et al., 2003; Gómez-Caravaca et al., 2005). Two SPE schemes have commonly been utilized for phenols extraction. On the one hand, a solution of the oil in hexane can be extracted in a reversed-phase cartridge; rising steps with hexane-ethoxyethane or hexane-cyclohexane solvents were used to remove the non-polar lipid fraction previously to the polar phenols elution with methanol or acetonitrile (Mannino et al., 1993). On the other hand, the polar fraction extracted with aqueous methanol solution can be fractionated by SPE; simple phenols and phenolic acids are eluted with methanol/water (50:50, v/v) and a more complex nature extract is obtained with a methanol/chloroform mixture (Litridou et al., 1997). Several comparative studies lead to controversial data regarding the most suitable extraction technique for phenols extraction from VOO. Some authors have concluded that LLE using methanol/water (60:40, v/v) as extractant led to higher extraction efficiencies than SPE-diol or SPE-C18 (Hrncirik and Fritsche, 2004). Another comparative study revealed that C18-SPE was more efficient than LLE using methanol/water (80:20, v/v) for the separation of simple phenols, but secoiridoids derivatives were extracted more efficiently with LLE. Due to the ease in handling and the short time needed, SPE has been considered by some authors the best choice for the extraction of phenolic compounds from VOO (Gómez-Caravaca et al., 2005). However, in other studies, SPE has been reported to be only effective for fresh VOO due to the potential stationary phase interaction with oxidized phenols (Armaforte et al., 2007). Sometimes, LLE and SPE have also been combined in the same extraction procedure (Litridou et al., 1997; Buiarelli et al., 2004).

Super Critical Fluid Extraction (SFE) Procedures using less organic solvents, such as SFE, have been used in recent years for the extraction of polyphenols from plant sources (Espinosa-Pardo et al., 2017; Fernandes et al., 2017; Garcia-Mendoza et al., 2017) due to their interesting advantages regarding extraction process, being more effective because of the high capacity for diffusion and high solvation power (Santos-Buelga. e Williamson, 2003). SFE has also been used to concentrate phenols from olive leaves (Le Floch et al., 1998; Xynos et al., 2012) although recoveries higher than 45% could not be achieved. In addition, SFE was

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not suitable for phenols extraction from VOO due to the co-extraction of simple lipids, mainly triacylglycerols, which are miscible with carbon dioxide and may cause interferences (Le Floch et al., 1998).

PHENOLICS DETERMINATION Assays for Total Phenolic Content Determination Basically, there are two general approaches for the determination of the phenolic content in olive oil. The first option, discussed in this section, is the determination of the total phenolic content (TPC), without any separation of the individual phenolics. The second approach – discussed in a following section – consists in the determination of the individual phenolic compounds after their proper separation. It is worth mention that a suitable extraction of the phenolics is required, no matter the methodology used for their determination. Obviously, the second approach provides more valuable information than the TPC determination, as each phenolic compound presents different bio(chemical) properties, hence the importance of as detailed the information as possible. However, the TPC is also widely used, due to its simplicity and the ease to compare the obtained results with other authors (as TPC procedures are usually similar between different laboratories). The F-C assay is the most commonly used procedure to determine TPC in food extracts. This assay is a colorimetric method based on electron transfer reactions between the F-C reagent and phenolic compounds. However, this method presents an important handicap for several food samples: its lack of specificity for TPC determinations. Compounds such as ascorbic acid, reducing sugars, organic acids or other reducing compounds can also reduce the F-C reagent, interfering in the determination of the TPC (Sánchez-Rangel et al., 2013). In this regards, the absence of ascorbic acid and sugars in olive oils favours the application of F-C assay in these samples. Total phenolics determined by the F-C assay are frequently expressed in gallic acid equivalents, although other standards may be used for olive oil samples. For instance, this assay has been applied to the determination of TPC in olive oil, expressing the results in gallic acid equivalents (Houshia et al., 2014), oleuropein equivalents (Cioffi et al., 2010), and caffeic acid equivalents (García-Villalba et al., 2010). Other authors proposed alternative methods to the F-C assay for olive oil samples. Some examples follow: a) an enzymatic assay (Mosca et al., 2000) was reported to compare favorably with the F-C assay, presenting higher sensitivity; b) the use of a chemometric analysis of UV-Vis spectral data of olive oil samples, with no prior clean-up or extraction of the sample (Fuentes et al., 2012); the results obtained presented good correlation with an HPLC reference method; c) the use of near-infrared spectroscopy for

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the determination of phenolic compounds (among others), although the results showed that higher validation was still required (Mailer, 2004); d) use of fluorescence spectroscopy, obtaining good correlation with F-C assay, as long as higher sensitivity (Papoti and Tsimidou, 2009). However, although each method presented some advantages, such as better sensitivity or no sample treatment, they still provide the analyst only with TPC, and not individual amounts of each phenolic. Therefore, the use of separation techniques to improve the quality and quantity of the results obtained is still required.

Assays for o-Diphenols Determination VOO contains a large number of phenolic compounds including simple phenols, lignans, and secoiridoids, which exhibit antioxidant properties (Owen et al., 2000). The highest antioxidant activity is generally attributed to those having o-diphenolic structures (González-Quijano et al., 1977; Baldioli et al., 1996). Phenols having an orthodihydroxyl structure have also been reported to play important protective role against oxidative stress damage in biological systems. The ability of o-diphenols, in particular an isomer of decarboxymethyloleuropein aglycon (3,4-DHPEA-EDA), to reduce the oxidized forms of tocopherols was also hypothesized (Bendini et al., 2006). Later, this hypothesis was substantiated by other authors (Pazos et al., 2007) who demonstrated that o-diphenols exhibit a synergistic effect when they are in association with α-tocopherol, and can be potentially active in the regeneration of this latter via reduction of the αtocopheroxyl. On the other hand, important polyphenols such as oleoeuropein have odiphenolic structure and are important markers of product correct storage and preservation since they are oxidized during long term or improper olive oil storage. Therefore, the development of analytical methods for the selective detection of the antioxidants fraction bearing o-diphenol functionalities is important for the valorization of VOO. In 2009, the Council of Members of the International Olive Council, in its Decision No DEC-17/97-V/2009, recommended the provisional application of the colorimetric method of A. Cert (Cert et al., 2007) for the determination of o-diphenolic compounds in olive oils. This method provides a quantitative determination of phenolic compounds more active as antioxidants, such as hydroxytyrosol, its oleosidic forms and luteolin. For the application of the assay, a previous separation and purification of the phenolic fraction by SPE is required. The SPE step is carried out by using a polar phase, specifically diol-bonded cartridges, which is more appropriate for the extraction of polar fractions from nonpolar matrices than nonpolar phases. A solution of the oil in n-hexane is passed through the diol-bonded cartridge (previously conditions with methanol and nhexane) and the polar compounds are retained on the solid phase. After this, nonpolar

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compounds such as triacylglycerols, hydrocarbons, tocopherols and waxes, which interfere in the colorimetric analysis, are eliminated by washing with hexane. A subsequent washing with hexane/ethyl acetate (90:10, v/v) allows removing the major part of oxidized sterols, diacylglycerols and triacylglycerols. Finally, the very polar fraction is eluted with methanol. After solvent evaporation, the residue is extracted with water/methanol (1:1, v/v) at 40 ºC, obtaining a colorless extract that contains all the phenolic compounds (Mateos et al., 2001). The colorimetric assay is carried out on the methanolic extract obtained from the olive oil by SPE by reaction with a 5% solution of sodium molybdate dihydrate in ethanol/water (1:1, v/v) as a reactive and spectrophotometric measurement at 370 nm after 15 min. A blank is obtained by measuring a mixture of phenolic solution with 1 mL of ethanol/water (1:1, v/v). The calibration curve is constructed with pyrocatechol solutions in the range 0.02-0.07 mg/mL and the result is expressed as millimol of o-diphenols per kg of oil. Other standards such as hydroxytyrosol (Mateos et al., 2001) and gallic acid (Douzane et al., 2013) are also used for calibration purposes. Some of the contents in o-diphenols found by different authors are 0.16 mmol/kg for Arbequina olive oil, 1.01 mmol/kg for Manzanilla olive oil and 0.52 mmol/kg for Picual olive oil (Mateos et al., 2001); 0.310.56 mmol/kg for monovarietal VOO from twenty-one Algeria cultivars (Douzane et al., 2013) and 0.085-0.62 mmol/kg for 13 samples of VOO from Italy. When comparing to FC method, the o-diphenol assay is more time-saving and selective for compounds having a catecholic structure (Cerretani et al., 2009). The ability of o-diphenols to form complexes with molybdenum (VI) (Duhmer-Klair et al., 2003) has also been exploited for their selective electrochemical determination in olive oil (Del Carlo et al., 2012). The proposed flow injection method takes advantage of the formation of such complexes in order to provide selectivity in the determination of odiphenols by amperometry at fixed potential using molybdate immobilized onto the surface of a carbon paste electrode as electrochemical mediator. The calibration range obtained with this amperometric system (0.2-50 mg/L) was wider than that provided by the spectrophotometric assay and covered two orders of magnitude. In the analysis of 13 olive oils samples by this method, o-diphenol concentrations ranging from 0.04 to 0.33 mmol/kg were found. The comparison of these results with those found by applying the colorimetric method (Cert et al., 2007) showed a general overestimation of this latter over the amperometric method, particularly for the samples containing low amounts of odiphenols. According to the authors, the overestimation of the colorimetric assay can be attributed to the lower sensitivity of the analysis. Taken into account the intrinsic sensitivity of the electrochemical measurement and, in addition, the rapidity, selectivity and ease of operation of the assay, it seems to be a promising approach for the development of a rapid assay for analysis of o-diphenols in olive oils. A new spectrophotometric approach (García et al., 2013) for determining total odiphenolic compounds in VOO, exploiting their oxidation to the corresponding o-

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quinones in a basic medium through the consumption of molecular oxygen, has also been proposed and compared with the traditional method of SPE followed by colorimetric determination using sodium molybdate. In this method a solution of the sample in hexane is extracted with a 0.5 mol/L NaOH solution and, after 25 min, the absorbance of the aqueous phase is measured at 410 or 530 nm. The autopolimerisation of the resulting oquinones originates coloured derivatives (Hotta et al., 2002), the only chromophores that are expected to be soluble in aqueous media. In this case, the calibration curve is constructed with standard solutions of oleuropein, and the results are expressed as mmol oleuropein/kg oil. The o-diphenol content found in the analysis of 50 virgin olives oils ranged from 0.013 to 2.35 mmol/kg. It is well established that the electrochemical oxidation of o-diphenols occurs at low potentials through a two electron two-proton reversible process, while an irreversible one-electron one-proton process at higher potentials is involved in the electrochemical oxidation of mono-phenols (Lin et al., 2015). This different mechanism in the oxidation of mono-phenols and o-diphenols has been exploited for the development of several electroanalytical methods for the analysis of the latter. Enache et al. proposed a rapid and sensitive method using square wave voltammetry at screen printed electrodes, with a detection limit for hydroxytyrosol of 0.40 mM (Enache et al., 2013). Recently, a simple, sensitive device consisting of a dual electrode detector pencil-drawn at the end of a paper microfluidic channel, defined by hydrophobic barriers, has been proposed for the fast determination of o-diphenols (Dossi et al., 2017). The discrimination between monophenols and o-diphenols of extra virgin olive oil (EVOO) is possible due to the use of two different working electrodes; o-diphenols are the sole species displaying a reversible behavior in their oxidation process. The approach also allows easily distinguishing EVOO from other vegetable oils.

Separation Analytical Techniques for Analysis of Phenolic Compounds It is widely known that phenolic compounds present several beneficial health effects on human health. However, not all the compounds contribute in the same way. Therefore, although traditional methods for the analysis of TPC in olive oil are still used, it is important to make use of separative techniques to obtain a profile of the individual phenolics that are present, together with their individual concentrations. HPLC is usually the selected technique for the analysis of phenolic compounds, although GC and CE have also been used.

Gas Chromatography (GC) Several research groups have developed analytical methods using GC, although their use is limited in this field, mainly due to the derivatization step required before the

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analysis. Moreover, the high temperatures may damage the phenolics. Although the flame ionization detector (FID) is common in GC, and it has been used for the analysis of phenolics (Farajzadeh et al., 2014), its main handicap is the small number of compounds that can be analyzed. In addition, the use of analytical standards is required, and a complete accurate identification of the compound cannot be achieved. Hence, the best option is the coupling of GC and MS. MS allows the identification of a higher number of compounds, even without using analytical standards. GC-ion trap-MS (Rios et al., 2005) and GC-time of flight MS (GCTOF MS) (García-Villalba et al., 2011) have been satisfactorily applied to the identification of more than 20 compounds in virgin olive oil. In addition, a recent paper analyzed several virgin olive oils by both LC-TOF MS and GC-TOF MS to obtain the complete phenolic profile (Bajoub et al., 2016). Using chemometrics, both profiles were compared, and the data obtained allowed the authors to classify the olive oils into different olive varieties. However, the authors also concluded that the results obtained by LC-MS were superior to those of GC-MS.

Capillary Electrophoresis (CE) CE is usually reported as a fast and efficient separation technique. Although this is true, several handicaps appear when analyzing large numbers of compounds, especially in complex samples, such as olive oil. However, different approaches have been recently described to solve some of the hurdles inherent to CE. The analysis of phenolic compounds by CE has been usually carried out by UV detection, although the coupling with MS has increased the range of applications. Using UV detection, the analysis of selected phenolic compounds has been reported in several works (Bonoli et al., 2003; Carrasco-Pancorbo et al. 2006b; Carrasco-Pancorbo et al., 2006c). In most cases, only qualitative and/or quantitative analyses were carried out, although the relation between phenolic profiles and sensorial properties has also been evaluated (Carrasco-Pancorbo et al., 2009). Considering that the most critical aspect of CE-UV is the separation of the analytes, the most important CE parameters have to be carefully optimized. In this sense, multivariate statistical techniques have proven useful (Ballus et al., 2011). UV detection lacks of sensitivity and selectivity. Selectivity can be improved by proper sample preparation and high-resolution separation techniques. However, UV signal is intrinsic to the analyzed compounds. To improve sensitivity, online preconcentration can be carried out in CE instrumentation (Monasterio et al., 2013). The use of fluorescence along with UV-visible detection has also been reported to provide better and more complete results (Godoy-Caballero et al., 2012). However, methods using fluorescence and/or UV detection need to use commercial standards, which are not always available. In fact, some authors used HPLC-MS isolated standards (Carrasco-Pancorbo et al., 2006b) in the absence of commercial ones. Although this is an

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interesting approach, it is obvious that conventional detection techniques present important obstacles, hence the need of MS. The coupling of CE with MS has been recently reported for the analysis of phenolic compounds in olive oil. However, this coupling is not as straightforward as in GC and HPLC, which is an important limitation to develop reliable CE-MS methodologies. Using electrospray ionization, seven phenolic acids were analyzed in virgin olive oil (Nevado et al., 2009), although the analysis of a higher number of phenolics was also reported (Carrasco-Pancorbo et al., 2006a). Aiming to improve the sensitivity of CE-MS, a coupling configuration originally designed for HPLC-MS has been adapted to CE-MS instrumentation (Nevado et al., 2010); in addition to higher sensitivity, the repeatability of the method was also improved in comparison with commercial CE-MS configurations. Finally, it is worth mentioning the development of analytical methods which used both CE and HPLC with MS detection (Carrasco-Pancorbo et al., 2007; García-Villalba et al., 2009) to increase the number of phenolic compounds that could be analyzed. Considering the low number of analytical methodologies that use CE-MS, and the necessity to use HPLC-MS in some cases, it is clear that further research is still needed in this field.

Liquid Chromatography (HPLC) HPLC is by far the most versatile separation technique for the analysis of a large number of compounds in complex samples. For instance, CE usually presents repeatability problems, which are commonly solved in HPLC. The scientific literature regarding the analysis of phenolic compounds in food – including olive oil - is huge, so only selected papers will be cited. The most common approach consists in the use of HPLC-MS. However, other detection techniques have been reported. HPLC-UV methods (Tasioula-Margari and Okogeri, 2001a; 2001b) are simple and cheap, but they lack of sensitivity and selectivity. The use of fluorescence (Selvaggini et al., 2006) improves the analytical performance of the methods, although limited information is still obtained. The use of chemometrics to analyze HPLC data has increased considerably in the last decade, no matter the detection technique. An interesting approach consists in the combination of HPLC with UV and fluorescence detection with chemometrics (Rueda et al., 2016; Bajoub et al., 2017). In this way, the fingerprints of the phenolic fraction of several oils were recorded and, after proper data treatment, good recognition and prediction abilities were obtained to differentiate between several varieties of olive oil, and between olive oil and other edible vegetable oils. However, HPLC-MS is by far the best option, usually working in negative electrospray ionization mode. The most common procedure for the analysis of phenolic compounds consists in the use of HPLC-UV-MS (usually MS/MS) (De La Torre-Carbot et al., 2005). Although most of the information is extracted from MS data, the use of UV provides additional information for the most abundant compounds. In addition to the use of HPLC, ultra high

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performance liquid chromatography (UHPLC) is gaining ground due to its inherent advantages, mainly its high sample throughput. As an example, the determination of eight polyphenols by UHPLC-MS-MS in extra-virgin olive oil samples was achieved in less than 5 min, obtaining low detection limits (Gosetti et al., 2015). MS detection presents important advantages, such as its sensitivity, selectivity, multicomponent analysis, and potential identification of compounds without the need of analytical standards – provided that the MS spectra are already known. However, the identification of unknowns directly from the MS spectra is not an easy task. Therefore, other techniques are needed. NMR spectroscopy is a valuable research technique for the identification of unknown compounds, and it has been used for the analysis of phenolics in olive oil samples. The phenolic fraction of an extra virgin olive oil was studied by hyphenated HPLC-DAD-NMR/MS techniques. In addition to the identification of 25 compounds by MS, a new diastereoisomer of oleuropein was characterized by NMR (Pérez-Trujillo et al., 2010). Therefore, the use of several detection techniques is highly encouraged to increase the amount and quality of information that can be collected.

Analytical Difficulties and Challenges Phenols are a complex family of compounds presenting diverse chemical and physical properties such as differences in polarity, stability, molecular size, UV absorption or extractability by solvents. Agronomical conditions such as cultivar, fruit ripening, pedoclimatic conditions, conservation and irrigation can modify the qualitative and quantitative composition of hydrophilic phenols in VOO (Esti et al., 1998; Motilva et al., 2000; Ouni et al., 2011). It has also been shown that the mechanical extraction process, being crushing and malaxation the most critical technological points, strongly affects the concentration of hydrophilic phenols in VOO (Servili et al., 1994; Di Giovacchino et al., 2002). This lack of phenols homogeneity in VOO implies problems related to their determination. It has been difficult to find universally accepted extraction procedures and analytical methods for quantification of single components. As a consequence, it is difficult to make a direct comparison among the concentration of olive oil phenols reported in the literature because of the discrepancies found in the results (Pirisi et al., 2000). Another important difficulty regarding quantification of phenolic compounds in VOO is that the most naturally occurring phenolic compounds, such as secoiridoids, are not commercially available. Therefore, the total recovery of phenolic compounds is difficult to evaluate, which has been one of the major concerns and challenges of researchers over decades. Different strategies have been carried out in order to evaluate the VOO phenols efficiency recovery. For example, peanut oil was fortified with a commercial standards

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mixture and subjected to different extraction systems, analyzed and compared (Bendini et al., 2003). Other studies utilized compounds presenting similar structures to calculate the response factors relative to internal standards (Mateos et al., 2001). For example, three commercially available standards, namely tyrosol, oleuropein and pinoresinol, have been suggested for quantification of tyrosol derivatives, oleuropein derivatives and lignans, respectively, considering the similarities found in their response factors and by simple multiplication of the corresponding molecular weight ratios (Daskalaki et al., 2009). However, the behavior and response of the analytes contained in the oil could be different of those corresponding to the standards utilized and consequently the recovery results can only be estimates in such case. To overcome this problem, a SPE method was proposed in which LLE-prepared phenolic extract of VOO were used to fortify phenolic-free refined oil, and the recovery efficiency was calculated (Gómez-Caravaca et al., 2005). Nevertheless, an important factor to be considered is the interaction between target compounds and matrix sample. The VOO water content has shown to be influential on phenolic recovery extraction in both LLE (Gomez-Caravaca et al., 2007) and SPE (Lozano-Sanchez et al., 2012) systems since these compounds are located in the water/oil interface (Huang et al., 1996; Ambrosone et al., 2006). The elimination of water content by filtration makes more phenolic compounds available for subsequent extraction. It has been demonstrated, in various studies, that those belonging to secoiridoids group are some of the phenols affected in terms of better recoveries when water is eliminated previously (Gomez-Caravaca et al., 2007; Lozano-Sanchez et al., 2012; Bakhouche et al., 2014). This fact raises doubts about the accuracy of data reported in different studies where phenolic profiles have been used as fingerprint but the variation in VOO water content during phenolic extraction was not considered (Rotondi et al., 2004; GarciaVillalba et al., 2010; Ouni et al., 2011; Karkoula et al., 2012). This fact should be taken into account because fruit ripening stage, olive variety and geographical area have been shown to be influential in the VOO water content (Motilva et al., 2000; Taamalli et al., 2010). All the difficulties above mentioned imply the need for new investigations in the development of extraction methods for all kinds of VOO, taking into account phenolic fraction and water content, as well as the other important factors already mentioned. The analysis of the phenol fraction extracted also presents some problematic aspects when chromatographic methods are applied. Although analytical techniques and equipment available are powerful and characterized by very low detection limits, chromatographic data are not homogenous and, due to the high number of subclasses and the complexity of secoiridoids, the total structural characterization of the phenolic fraction is sometimes impossible. The major concern of researchers is related to the high number of isomers that could be originally present or could be artificially formed during chromatographic analysis. Oleuropein aglycone, ligstroside aglycone and EA have been demonstrated to have an elevated number of isomers coming from hydrolysis during

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olive ripening and olive oil processing (Fu et al., 2009; Vichi et al., 2013). In addition, isomers of oleuropein and ligstroside aglycone, among others, have been found to be formed during chromatographic analysis when water or methanol were used as mobile phase (Karkoula et al., 2012; Karkoula et al., 2014). Especially problematic is the use of water as mobile phase, which is the case in the official and validated method proposed until date. For this reason, and one more time, data available over the estimation of this fraction could be questionable and future research should be directed to clarify the best conditions for the analytical parameters in order to provide comparable results regarding recovery and quantification of this family of antioxidants in VOO. Finally, the combination of different detection techniques in HPLC, such as NMR and MS, is a very powerful tool that should not be ignored. In addition, the use of bi- or multi- dimensional chromatography along with high-resolution MS is another important approach that should be taken into account to improve the analytical results in future research.

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Gutierrez-Rosales, F., Rios, J.J., Gomez-Rey, M.L., (2003). Main polyphenols in the bitter taste of virgin olive oil. Structural confirmation by on-line high-performance liquid chromatography electrospray ionization mass spectrometry. Journal of Agricultural and Food Chemistry 51, 6021-6025. Hotta, H., Ueda, M., Nagano, S., Tsujino, Y., Koyama, J., Osakai, T., (2002). Mechanistic study of the oxidation of caffeic acid by digital simulation of cyclic voltammograms. Analytical Biochemistry 303, 66-72. Houshia, O.J., Qutit, A., Zaid, O., Shqair, H., Zaid, M., (2014). Determination of total polyphenolic antioxidants contents in West-Bank olive oil. Journal of Natural Sciences Research 4, 71-76. Hrncirik, K., Fritsche, S., (2004). Comparability and reliability of different techniques for the determination of phenolic compounds in virgin olive. European Journal of Lipid Science and Technology 106, 540-549. Huang, S.W., Hopia, A., Schwarz, K., Frankel, E.N., German, J.B., (1996). Antioxidant activity of alpha-tocopherol and Trolox in different lipid substrates: Bulk oils vs oilin-water emulsions. Journal of Agricultural and Food Chemistry 44, 444-452. Karkoula, E., Skantzari, A., Meliou, E., Magiatis, P., (2014). Quantitative measurement of major secoiridoid derivatives in olive oil using qNMR. Proof of the artificial formation of aldehydic oleuropein and ligstroside aglycon isomers. Journal of Agricultural and Food Chemistry 62, 600-607. Karkoula, E., Skantzari, A., Melliou, E., Magiatis, P., (2012). Direct measurement of oleocanthal and oleacein levels in olive oil by quantitative H-1 NMR. Establishment of a new index for the characterization of extra virgin olive oils. Journal of Agricultural and Food Chemistry 60, 11696-11703. Lavelli, V., Bondesan, L., (2005). Secoiridoids, tocopherols, and antioxidant activity of monovarietal extra virgin olive oils extracted from destoned fruits. Journal of Agricultural and Food Chemistry 53, 1102-1107. Le Floch, F., Tena, M.T., Rios, A., Valcarcel, M., (1998). Supercritical fluid extraction of phenol compounds from olive leaves. Talanta 46, 1123-1130. Lerma-Garcia, M.J., Lantano, C., Chiavaro, E., Cerretani, L., Herrero-Martinez, J.M., Simo-Alfonso, E.F., (2009). Classification of extra virgin olive oils according to their geographical origin using phenolic compound profiles obtained by capillary electrochromatography. Food Research International 42, 1446-1452. Liberatore, L., Procida, G., d'Alessandro, N., Cichelli, A., (2001). Solid-phase extraction and gas chromatographic analysis of phenolic compounds in virgin olive oil. Food Chemistry 73, 119-124. Lin, Q., Li, Q., Batchelor-McAuley, C., Compton, R.G., (2015). Two-electron, twoproton oxidation of catechol: kinetics and apparent catalysis. The Journal of Physical Chemistry C 119, 1489-1495.

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Litridou, M., Linssen, J., Schols, H., Bergmans, M., Posthumus, M., Tsimidou, M., Boskou, D., (1997). Phenolic compounds in virgin olive oils: Fractionation by solid phase extraction and antioxidant activity assessment. Journal of the Science of Food and Agriculture 74, 169-174. Lozano-Sanchez, J., Cerretani, L., Bendini, A., Gallina-Toschi, T., Segura-Carretero, A., Fernandez-Gutierrez, A., (2012). New filtration systems for extra-virgin olive oil: Effect on antioxidant compounds, oxidative stability, and physicochemical and sensory properties. Journal of Agricultural and Food Chemistry 60, 3754-3762. Mailer, R.J., (2004). Rapid evaluation of olive oil quality by NIR reflectance spectroscopy. Journal of the American Oil Chemists Society 81, 823-827. Mannino, S., Cosio, M.S., Bertuccioli, M., (1993). High performance liquid chromatography of phenolic compounds in virgin olive oils using amperometric detection. Italian Journal of Food Science 4, 363–370. Mateos, R., Espartero, J.L., Trujillo, M., Rios, J.J., Leon-Camacho, M., Alcudia, F., Cert, A., (2001). Determination of phenols, flavones, and lignans in virgin olive oils by solid-phase extraction and high-performance liquid chromatography with diode array ultraviolet detection. Journal of Agricultural and Food Chemistry 49, 2185-2192. Monasterio, R.P., Fernandez, M.D., Silva, M.F., (2013). High-throughput determination of phenolic compounds in virgin olive oil using dispersive liquid-liquid microextraction- capillary zone electrophoresis. Electrophoresis 34, 1836-1843. Montedoro, G., Cantarelli, C., (1969). Investigation on olive oil. Phenolic compounds. Rivista Italiana Sostanze Grasse 46, 115-124. Montedoro, G.F., Servili, M., Baldioli, M., Miniati, E., (1993). Simple and hydrolyzable phenolic compounds in virgin olive oil. 3. Spectroscopic characterizations of the secoiridoid derivatives. Journal of Agricultural and Food Chemistry 41, 2228-2234. Moreno-Luna R., Muñoz-Hernandez, R., Miranda, M.L., Costa, A.F., Jimenez-Jimenez, L., Vallejo-Vaz, A.J., Muriana, F.J.G., Villar, J., Stiefel, P., (2012). Olive oil polyphenols decrease blood pressure and improve endothelial function in young women with mild hypertension. American Journal of Hypertension 25, 1299-304. Mosca, L., De Marco, C., Visioli, F., Cannella, C., (2000). Enzymatic assay for the determination of olive oil polyphenol content: Assay conditions and validation of the method. Journal of Agricultural and Food Chemistry 48, 297-301. Motilva, M.J., Serra, A., Macia, A., (2013). Analysis of food polyphenols by ultra-high performance liquid chromatography coupled to mass spectrometry: an overview. Journal of Chromatography A 1292, 66-82. Motilva, M.J., Tovar, M.J., Romero, M.P., Alegre, S., Girona, J., (2000). Influence of regulated deficit irrigation strategies applied to olive trees (Arbequina cultivar) on oil yield and oil composition during the fruit ripening period. Journal of the Science of Food and Agriculture 80, 2037-2043.

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Nevado, J.J.B., Penalvo, G.C., Robledo, V.R., (2010). Advantages of using a modified orthogonal sampling configuration originally designed for LC-ESI-MS to couple CE and MS for the determination of antioxidant phenolic compounds found in virgin olive oil. Talanta 82, 548-554. Nevado, J.J.B., Penalvo, G.C., Robledo, V.R., Martinez, G.V., (2009). New CE-ESI-MS analytical method for the separation, identification and quantification of seven phenolic acids including three isomer compounds in virgin olive oil. Talanta 79, 1238-1246. Oliveras-Lopez, M.J., Innocenti, M., Giaccherini, C., Ieri, F., Romani, A., Mulinacci, N., (2007). Study of the phenolic composition of Spanish and Italian monocultivar extra virgin olive oils: Distribution of lignans, secoiridoidic, simple phenols and flavonoids. Talanta 73, 726-732. Omar, S.H., (2010). Oleuropein in olive and its pharmacological effects. Scientia Pharmaceutica 78, 133-154. Ouni, Y., Taamalli, A., Gomez-Caravaca, A.M., Segura-Carretero, A., FernandezGutierrez, A., Zarrouk, M., (2011). Characterisation and quantification of phenolic compounds of extra-virgin olive oils according to their geographical origin by a rapid and resolutive LC-ESI-TOF MS method. Food Chemistry 127, 1263-1267. Owen, R.W., Mier, W., Giacosa, A., Hull, W.E., Spielgelhalder, B., Bartsch, H., (2000). Phenolic compounds and squalene in olive oils: The concentration and antioxidant potential of total phenols, simple phenols, secoiridoids, lignans and squalene. Food Chemistry Toxicology 38, 647-659. Papoti, V.T., Tsimidou, M.Z., (2009). Looking through the qualities of a fluorimetric assay for the total phenol content estimation in virgin olive oil, olive fruit or leaf polar extract. Food Chemistry 112, 246-252. Pazos, M., Andersen, M.L., Medina, I., Skibsted, L.H., (2007). Efficiency of natural phenolic compounds regenerating α-tocopherol from α-tocopheroxyl radical. Journal of Agricultural and Food Chemistry 55, 3661-3666. Perez-Trujillo, M., Gomez-Caravaca, A.M., Segura-Carretero, A., Fernandez-Gutierrez, A., Parella, T., (2010). Separation and identification of phenolic compounds of extra virgin olive oil from Olea europaea L. by HPLC-DAD-SPE-NMR/MS. Identification of a new diastereoisomer of the aldehydic form of oleuropein aglycone. Journal of Agricultural and Food Chemistry 58, 9129-9136. Pirisi, F.M., Cabras, P., Cao, C.F., Migliorini, M., Muggelli, M., (2000). Phenolic compounds in virgin olive oil. 2. Reappraisal of the extraction, HPLC separation, and quantification procedures. Journal of Agricultural and Food Chemistry 48, 11911196. Pizarro, M. L., Becerra, M., Sayago, A., Beltran, M., Beltran, R., (2013). Comparison of different extraction methods to determine phenolic compounds in virgin olive oil. Food Analytical Methods 6, 123-132.

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Rios, J.J., Gil, M.J., Gutierrez-Rosales, F., (2005). Solid-phase extraction gas chromatography-ion trap-mass spectrometry qualitative method for evaluation of phenolic compounds in virgin olive oil and structural confirmation of oleuropein and ligstroside aglycons and their oxidation products. Journal of Chromatography A 1093, 167-176. Rotondi, A., Bendini, A., Cerretani, L., Mari, M., Lercker, G., Toschi, T.G., (2004). Effect of olive ripening degree on the oxidative stability and organoleptic properties of cv. Nostrana di Brisighella extra virgin olive oil. Journal of Agricultural and Food Chemistry 52, 3649-3654. Rueda, A., Samaniego-Sanchez, C., Olalla, M., Gimenez, R., Cabrera-Vique, C., Seiquer, I., Lara, L., (2016). Combination of analytical and chemometric methods as a useful tool for the characterization of extra virgin argan oil and other edible virgin oils. Role of polyphenols and tocopherols. Journal of AOAC International 99, 489-494. Ruiz-Jimenez, J., Luque de Castro, M.D., (2003). Flow injection manifolds for liquidliquid extraction without phase separation assisted by ultrasound. Analytica Chimica Acta 489, 1-11. Ryan, D., Robards, K., (1998). Phenolic compounds in olives. Analyst 123, 31R-44R. Sánchez-Rangel, J.C., Benavides, J., Basilio Heredia, J., Cisneros-Zevallos, L., JacoboVelázquez, D.A., (2013). The Folin-Ciocalteu assay revisited: improvement of its specificity for total phenolic content determination. Analytical Methods 5, 59905999. Santos-Buelga, C., Williamson, G., (2003) in: Methods in Polyphenols Analysis. Cambridge, UK., The Royal Society of Chemistry, pp. 11. Selvaggini, R., Servili, M., Urbani, S., Esposto, S., Taticchi, A., Montedoro, G., (2006). Evaluation of phenolic compounds in virgin olive oil by direct injection in highperformance liquid chromatography with fluorometric detection. Journal of Agricultural and Food Chemistry 54, 2832-2838. Servili, M., Baldioli, M., Montedoro, G.F., (1994). Phenolic composition of virgin olive oil in relationship to some chemical and physical aspects of malaxation. Acta Horticulturae 356, 331–336. Servili, M., Baldioli, M., Selvaggini, R., Miniati, E., Macchioni, A., Montedoro, G., (1999). High-performance liquid chromatography evaluation of phenols in olive fruit, virgin olive oil, vegetation waters, and pomace and 1D-and 2D-nuclear magnetic resonance characterization. Journal of the American Oil Chemists Society 76, 873882. Servili, M., Esposto, S., Fabiani, R., Urbani, S., Taticchi, A., Mariucci, F., Selvaggini, R., Montedoro, G.F., (2009). Phenolic compounds in olive oil: Antioxidant, health and organoleptic activities according to their chemical structure. Inflammopharmacology 17, 1-9.

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Servili, M., Selvaggini, R., Esposto, S., Taticchi, A., Montedoro, G., Morozzi, G., (2004). Health and sensory properties of virgin olive oil hydrophilic phenols: Agronomic and technological aspect of production that affect their occurrence in the oil. Journal of Chromatography A 1054, 113-127. Solinas, M., Cichelli, A., (1981). Sulla determinazione delle sostanze fenoliche dell’olio di oliva Rivista Italiana Sostanze Grasse 58, 159–164 [On determination of phenolic substances of olive oil. Italian Journal of Fats Substances 58, 159-164]. Suarez, M., Macia, A., Romero, M.P., Motilva, M.J., (2008). Improved liquid chromatography tandem mass spectrometry method for the determination of phenolic compounds in virgin olive oil. Journal of Chromatography A 1214, 90-99. Taamalli, A., Gomez-Caravaca, A.M., Zarrouk, M., Segura-Carretero, A., FernandezGutierrez, A., (2010). Determination of apolar and minor polar compounds and other chemical parameters for the discrimination of six different varieties of Tunisian extra-virgin olive oil cultivated in their traditional growing area. European Food Research and Technology 231, 965-975. Tasioula-Margari, M., Okogeri, O., (2001a). Isolation and characterization of virgin olive oil phenolic compounds by HPLC/UV and GC-MS. Journal of Food Science 66, 530-534. Tasioula-Margari, M., Okogeri, O., (2001b). Simultaneous determination of phenolic compounds and tocopherols in virgin olive oil using HPLC and UV detection. Food Chemistry 74, 377-383. Tripoli, E., Giammanco, M., Tabacchi, G., Di Majo, D., Giammanco, S., De La Guardia, M., (2005) The phenolic compounds of olive oil: structure, biological activity and beneficial effects on human health. Nutrition Research Reviews 18, 98-112. Tura, D., Robards, K., (2002). Sample handling strategies for the determination of biophenols in food and plants. Journal of Chromatography A 975, 71-93. Vichi, S., Cortes-Francisco, N., Caixach, J., (2013). Insight into virgin olive oil secoiridoids characterization by high-resolution mass spectrometry and accurate mass measurements. Journal of Chromatography A 1301, 48-59. Vlahov, G. (1999) Application of NMR to the study of olive oils. Prog. Nucl. Magn. Reson. Spectrosc., 35, 341-357. Xynos, N., Papaefstathiou, G., Psychis, M., Argyropoulou, A., Aligiannis, N., Skaltsounis, A.L., (2012). Development of a green extraction procedure with super/subcritical fluids to produce extracts enriched in oleuropein from olive leaves. Journal of Supercritical Fluids 67, 89-93.

In: Handbook of Olive Oil Editor: Jozef Miloš

ISBN: 978-1-53612-356-2 © 2017 Nova Science Publishers, Inc.

Chapter 5

THE NATURAL VARIATION OF PHENOLIC COMPOUNDS IN THE FRUITS AND OILS OF OLIVE (OLEA EUROPAEA L.) Ana G. Pérez1, Angjelina Belaj2, Mar Pascual1 and Carlos Sanz1,* 1

Department of Biochemistry and Molecular Biology of Plant Products, Instituto de la Grasa, CSIC, Seville, Spain 2 IFAPA, Centro Alameda del Obispo, Cordoba, Spain

ABSTRACT Different scientific evidences suggest that the long term dietary consumption of virgin olive oil (VOO) seems to be related to an attenuation of the inflammatory response and reduction of the associated risk of chronic inflammatory disease states. VOO phenolic compounds are claimed to be the main responsible for these positive health benefits. They are mainly synthetized from phenolic glucosides present in the olive fruit by the action of glucosidases occurring when they come together once the olive fruit is crushed during olive oil extraction. The genetic variability of the major phenolic compounds was studied in a representative sample of olive cultivars (Olea europaea L.) from the World Olive Germplasm Collection established at IFAPA Centre “Alameda del Obispo” in Cordoba, Spain. The most abundant phenolic components found in VOO are the secoiridoid derivatives resulting from the enzymatic hydrolysis of oleuropein, ligstroside and demethyloleuropein present in the fruit, which showed to be on average the main phenolic glucosides. The mean content of phenolic compounds in the oils was 494.51 µg/g oil, displaying a variability range of 63.74-1432.04 µg/g oil. The mean *

Corresponding autor: Carlos Sanz. Department of Biochemistry and Molecular Biology of Plant Products, Instituto de la Grasa, CSIC, Campus UPO, Building 46, Ctra. Utrera km 1, 41013-Seville, Spain. Tel: +34 954611550, Fax: +34 954616790. E-mail: [email protected].

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Ana G. Pérez, Angjelina Belaj, Mar Pascual et al. content of phenolic compounds in the fruits was 12384.23 µg/g fruit with a range of 3754.13-30696.39 µg/g fruit. Total phenolic compounds in the fruits and the oils were significantly correlated (r = 0.66). Thus, the composition and biochemical status of the olive fruit seem to be the most important variables determining the synthesis of the VOO phenolic compounds during the oil extraction process. On the other hand, the content of oleuropein and derivatives in the oils and fruits showed a correlation coefficient (r = 0.64) lower than that observed for ligstroside and derivatives (r = 0.73). These findings might be related to the higher oxidation rates of the former due to the action of oxidative enzymes during the oil extraction process as a consequence of the orthodiphenolic structure they possess. Data on phenolic composition would be of interest for the selection of optimal parents in olive breeding programs with the aim of obtaining new cultivars with improved nutritional quality.

Keywords: Olea europaea L., fruit, virgin olive oil, phenolic compounds, variability

INTRODUCTION The need for diversification of olive (Olea europaea L.) cultivars is based mainly on the new intensive industry and the new production areas outside the Mediterranean basin. Olive breeding has traditionally focused on the agronomic traits but as a consequence of the growing number of scientific proofs supporting the positive impact of virgin olive oil (VOO) consumption on human health, the nutritional quality of VOO has become an important olive breeding target (Konstantinidou et al., 2010; León et al., 2011; Estruch et al., 2013). VOO represents the primary lipid source and one of the main features that distinguish the Mediterranean diet. It is one of the oldest known plant oils and it is unique among them since it can be consumed as a fruit juice. Recent attention has been given to the phenolic fraction of VOO because of their benefits for human health (Lucas et al. 2011; Visioli and Bernardini, 2011; Konstantinidou et al., 2010). However, phenolics are important not only from a nutritional point of view but also in terms of sensory quality. VOO phenolics are responsible for the bitter and pungent sensory notes of this oil (Inarejos-García et al. 2009; Mateos et al., 2004; Andrewes et al., 2003). Bitterness and pungency are common and desirable attributes in VOOs when present at low to moderate intensity, but they are rejected by consumers when present at high intensity. Thus, due to their organoleptic and health promoting properties, phenolic compounds are currently being used as quality markers for VOO (León et al., 2011). The synthesis of phenolic compounds responsible for the nutritional and sensory quality of VOO occurs when enzymes and substrates meet as olive fruit is crushed during the olive oil extraction process. Among the phenolic compounds so far identified in VOO, secoiridoids, lignans, flavonoids and hydrophilic phenols such as phenolic alcohols and phenolic acids are the most important classes. There are many differences in the phenolic profiles among cultivars (Cicerale et al., 2009; García-González et al., 2010) due to genetics, the edafo-climatic conditions, and the industrial procedures used during olive

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oil extraction. Although the latter may influence the VOO phenolic profile (Servili et al., 2004), the composition and biochemical status of the olive fruit seems to be the most important variables responsible for the synthesis of the VOO phenolic compounds during the oil extraction process. In this sense, the presence of phenolic compounds in VOO might be directly related to the content of phenolic glucosides initially present in the olive fruit tissues and the activity of hydrolytic and oxidative enzymes acting on these glucosides (García-Rodríguez et al., 2011; Romero-Segura et al., 2012). The main phenolic glucosides present in olive fruit are oleuropein, ligstroside and demethyloleuropein, although many others such as verbascoside, luteolin glucoside and apigenin glucoside have also been identified (Obied et al., 2008; Sevarese et al., 2007). The secoiridoid derivatives resulting from the enzymatic hydrolysis of oleuropein, ligstroside and demethyloleuropein, are the dialdehydic forms of decarboxymethyloleuropein and decarboxymethylligstroside aglucones (3,4-DHPEAEDA and p-HPEA-EDA, respectively) and the aldehydic forms of oleuropein and ligstroside aglucones (3,4-DHPEA-EA and p-HPEA-EA, respectively), which are in turn the most abundant phenolic components found in olive oils (Montedoro et al., 2002; Pérez et al. 2014). These secoiridoids contain in their molecules the phenolic alcohol tyrosol (p-HPEA) or its hydroxyl derivative hydroxytyrosol (3,4-DHPEA), the latter providing the strongest antioxidant activity to the oil (Artajo et al., 2006).

HEALTH AND SENSORY PROPERTIES As mentioned before, the beneficial effects of the traditional Mediterranean diet on human health have been widely reported. This diet reduces the risk of a number of diseases, mainly those associated to inflammation such as cardiovascular disease, diabetes, metabolic syndrome, arthritis, Alzheimer’s disease and certain types of cancer (Lucas et al. 2011; Estruch et al., 2013). Particularly, the long term dietary consumption of VOO phenolics seems to be linked to an attenuation of the inflammatory response and reduction of the associated risk of chronic inflammatory disease states (Visioli, and Bernardini, 2011; Lucas et al., 2011). Among these VOO phenolics, secoiridoid compounds have been described as being responsible for most of the nutritional quality of VOO because they are the most abundant class of phenolics in the oils and because most of them have an orthodiphenol chemical structure with strong in vitro antioxidant properties. Particularly, p-HPEA-EDA and 3,4-DHPEA-EA have demonstrated important nutritional and organoleptic properties (Visioli and Bernardini, 2011). p-HPEA-EDA, also known as oleocanthal, possesses similar anti-inflammatory properties to ibuprofen so that it is considered as one of the main factors within the Mediterranean diet reducing the risk of a number of diseases containing an inflammatory component (Lucas et al., 2011). More recently, Scotece et al. (2013) also demonstrated that p-HPEA-EDA inhibits

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proliferation of myeloma cells. Additionally, this compound seems to be the main phenolic responsible for the VOO pungency, producing a strong burning pungent sensation at the back of the throat, which is very important for VOO acceptation by consumers (Andrewes et al., 2013). On the other hand, beyond its potent antioxidant properties, 3,4-DHPEA-EA seems to be the main compound responsible for the bitterness of VOO, which is also very important from the consumer point of view (Mateos et al., 2004). Partial hydrolysis of VOO secoiridoid compounds during gastric and intestinal digestion has been widely described (Pinto et al., 2011; Pereira-Caro et al., 2012). This process increases the level of p-HPEA and, especially, 3,4-DHPEA in the colon. Thus, extensive investigation has focused on 3,4-DHPEA as a chronic disease preventive agent. Its acetylated derivative, 3,4-DHPEA acetate, has been reported to possess anticancer activity against human adenocarcinoma (Mateos et al., 2011) and to protect against oxidative stress in human cervical cells (Bouallagui et al., 2011) and human hepatoma cells (Mateos et al., 2005), probably through the proven protection against oxidative DNA damage that they provide (Grasso et al., 2007). Due to the lower polarity of this compound, it seems to be better absorbed in differentiated Caco-2 cell monolayers than free 3,4-DHPEA (Mateos et al., 2011). However, there are no data of its presence in plasma after sustained and moderate doses of VOO consumption as it has been demonstrated for 3,4-DHPEA and p-HPEA (Miró-Casas et al., 2003). Although most studies relate the beneficial effect of VOO consumption with the level of 3,4-DHPEA in plasma (Visioli and Bernardini, 2011) different beneficial effects of p-HPEA have been also widely demonstrated despite the lack of an orthodiphenolic structure and the consequent lower in vitro antioxidant activity compared to 3,4-DHPEA (Caruso et al., 1999; De la Puerta et al., 1999; Giovannini et al., 1999). This antioxidant activity of the minor phenols (or simple phenols) depends on the number of hydroxyl groups in the molecule that would be strengthened by steric interference. In this sense, the electronwithdrawing properties of the carboxylate group in benzoic acids (vanillic acid) have a negative influence on the H-donating abilities of the hydroxy benzoates. On the other hand, hydroxylated cinnamates (cinnamic, p-coumaric and ferulic acids) seem to be more effective for electron-withdrawing than the benzoate structural equivalents (Dziedzic and Hudson, 1983). Structurally similar to estradiol, which is the primary estrogen hormone in humans, lignans are one of the major classes of chemical compounds referred to collectively as phytoestrogens. Different scientific researches indicate that lignans, among other olive oil chemicals, might play an active role in protecting against breast cancer (Menéndez et al., 2008; 2009). Finally, flavonoids, such as luteolin and apigenin, are important for human health because of their high pharmacological activities as radical scavengers and high antioxidant capacity in both in vivo and in vitro systems (Cook and Samman, 1996; RiceEvans et al., 1995).

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NATURAL VARIATION OF PHENOLIC COMPOUNDS The wide genetic heritage of the olive tree is one of the main attributes of this crop and it is represented by thousands of local and old cultivars, restricted to specific areas where they originally grew. Conservation of olive genetic resources has led to the establishment of different germplasm banks. In this sense, the World Olive Germplasm Collection (WOGC), located at IFAPA Centre “Alameda del Obispo” in Cordoba, Spain, is an international reference on olive germplasm due to the high number of accessions included and their high degree of identification, which makes the collection unique for breeding purposes (Belaj et al., 2012; 2016). As mentioned above, olive breeding programs have focused traditionally on improving agronomic traits. However the major breeding targets are recently including also the sensory and nutritional qualities of VOO (León et al., 2011; El Riachy et al., 2012). In this sense, taking into account the proven relationship between the phenolic composition of VOO and its benefits for human health, the phenolic fractions of olive fruits and the corresponding oils produced by a subset of 64 cultivars from the WOGC were assessed. This cultivar subset is highly diverse in terms of phenotypic characteristics and geographical origin along the Mediterranean basin. For this purpose, phenolic compounds were extracted from olive fruits according to Romero-Segura et al. (2012), whereas olive oils phenolics were isolated by solid-phase extraction. Both phenolic extracts were analyzed by reversed-phase HPLC following a previously described procedure by Pérez et al. (2014). Olive fruit Picholine-70 Cipresino Coratina Zalmati Torcio de Cabra Caninese Patronet Argudell Bosana Picholine marroqui Lastovka Cornicabra Ouslati Figueretes Arbequina Blanqueta Chorreao de Montefrio Ulliri i Kuq Vaneta Zaity Canetera Galega Vulgar Haouzia Pecoso Shami-1041 Azapa Wardan Borriolenca Vallesa Kaesi Sabatera Pequeña de Casas Ibañez Vera Verdial Velez-Malaga Curivell Vinyols Amygdalolia Arbosana Majhol-152 Klon-14-1081 Elmacik Lechin de Sevilla Agouromanakolia Aggezi Shami-1 Verde Verdelho Abbadi Abou Gabra Levantinka Palomar Kolybada Rapasayo Corbella Blanqueta-48 Kan Celebi Yun Gelebi Kalokerida Verdial de Badajoz Istarska Bjelica Joanenca Maurino Jaropo Lentisca Majhol-1013 Negrinha Merhavia

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Figure 1. Main phenolic compound groups in the olive fruits and oils from the WOGC cultivar subset.

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A high degree of variability for the content of phenolic compounds has been found in olive (Figure 1). The mean content of phenolic compounds in the olive fruits was 12384.23 µg/g fruit with a range of 3754.13-30696.39 µg/g fruit. Olive fruit phenolics are mainly composed of oleuropein and ligstroside and their demethyl derivatives, as well as flavonoid compounds in a minor proportion. However, just a small fraction of those phenolics remains in the oil after extraction. Thus, the mean content of phenolic compounds in the oils was 494.51 µg/g oil, displaying a variability range of 63.741432.04 µg/g oil. Olive oil phenolics mostly belong to four different groups, compounds derived from tyrosol, which contain this phenolic alcohol (p-HPEA) or its hydroxyl derivative hydroxytyrosol (3,4-DHPEA) in their molecules, lignans, flavonoids, and an array of phenolic acids and alcohols (simple phenols). The tyrosol derivatives are the most abundant phenolics in the oils, especially those with a secoiridoid chemical structure such as the dialdehydic forms of decarboxymethyloleuropein (3,4-DHPEAEDA) and decarboxymethyl-ligstroside (p-HPEA-EDA) aglucones and the aldehydic forms of oleuropein and ligstroside aglucones (3,4-DHPEA-EA and p-HPEA-EA, respectively) (Figure 2). Their contents were on average up to 70 times higher than those of the rest of phenolic groups in the oils. Among them, the most abundant on average were those secoiridoids derived from hydroxytyrosol, 3,4-DHPEA-EA and 3,4-DHPEAEDA, whose mean contents in the oils were quite similar (168.91and 158.90 µg/g oil, respectively), displaying a variability range of 7.17-742.05 for 3,4-DHPEA-EA and 4.29475.87µg/g oil for 3,4-DHPEA-EDA. Thus, these secoiridoids turn out to be the main antioxidants in the oil due to their high contents and their orthodiphenolic structure. The secoiridoids derived from tyrosol (p-HPEA-EDA and p-HPEA-EA) represent on average around a quarter of the total secoiridoids in the oils of the cultivar subset. However, whereas p-HPEA-EDA displayed a mean value of 100.69 µg/g oil and a content range of 6.58-435.97 µg/g oil, p-HPEA-EA showed lower mean value (16.79 µg/g oil) and variability range (1.84-90.09 µg/g oil). This high content of secoiridoids in the oils reflects the levels of oleuropein, ligstroside and their derivatives in the olive fruits (Figure 3). Their contents were on average 37 times higher than those of the flavonoids in the fruits. Among them, the most abundant on average were those derived from hydroxytyrosol, oleuropein and its demethylated derivative demethyloleuropein, whose mean contents in the fruits were 7638.29 and 2061.41µg/g fruit, respectively, displaying a variability range of 0-23358.09 and 0-11633.15 µg/g fruit. Among the tyrosol and hydroxytyrosol derivatives not displaying a secoiridoid structure, 3,4-DHPEA acetate showed the highest mean content in the oils (6.41 µg/g oil) (Figure 2). The cultivar subset also showed a high content variability for 3,4-DHPEA acetate with a range of 0.25-22.39 µg/g oil. Contents of 3,4-DHPEA and p-HPEA in the oils were the lowest among the tyrosol and hydroxytyrosol derivative group of phenolic compounds. The mean contents for 3,4-DHPEA and p-HPEA were 1.56 and 4.99 µg/g oil, respectively.

The Natural Variation of Phenolic Compounds in the Fruits …

Secoiridoids

80

600

Lignans

70 Content (µg/g oil)

60 50

300

40

Flavonoids

12

500 400

14

121

10 8 6

30

200

4

20 100 0

3,4-DHPEA-EDA

3,4-DHPEA-EA p-HPEA-EDA

p-HPEA-EA

27

10

2

0

0 pinoresinol 1-acetoxypinoresinol

luteolin

apigenin

Simple phenols

24 Content (µg/g oil)

21 18 15 12 9 6 3 0 3,4-DHPEA

vanillic acid p-HPEA

vanillin

p-coumaric acid cinnamic acid 3,4-DHPEA acetate

ferulic acid

Figure 2. Ranges and distributions of the contents (µg/g oil) of the main phenolic compounds in the oils. Horizontal lines in the interior of the boxes are median values. The height in a box is equal to the interquartile distance, indicating the distribution for 50% of the data. The outliers (open dots) and extreme data (solid dots) are indicated outside the whiskers (the lines extending from the top and bottom of the box).

Lignans represented on average the second major group of phenolics in the oils although they are at a concentration 17 times lower than those of the tyrosol and hydroxytyrosol derivatives (Figure 2). As displayed in Figure 2, the most abundant lignan was 1-acetoxypinoresinol. The mean value in the oils was 22.75 µg/g oil and the contents ranged in the interval 1.44-85.08 µg/g oil. The mean value found for pinoresinol was 4.53 µg/g oil, and the content range was 0.70-16.44 µg/g oil.

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24000

Content (µg/g fruit)

20000

Oleuropein and derivatives

2500

Ligstroside and derivatives

2000 16000 1500 12000 1000 8000 500 4000 0 0 3,4-DHPEA glucoside Oleuropein Demethyloleuropein

Verbascoside

p-HPEA glucoside Ligstroside Demethylligstroside

1400

Content (µg/g fruit)

1200

Flavonoids FLAVONOIDS

1000 800 600 400 200 0 -200 Luteolin glucoside Luteolin Apigenin glucoside

Apigenin

Figure 3. Ranges and distributions of the contents (µg/g fruit) of the main phenolic compounds in the olive fruits. Horizontal lines in the interior of the boxes are median values. The height in a box is equal to the interquartile distance, indicating the distribution for 50% of the data. The outliers (open dots) and extreme data (solid dots) are indicated outside the whiskers (the lines extending from the top and bottom of the box).

Considering that simple phenols are mainly composed of tyrosol derivatives, the third group of importance in the oils would be the flavonoids. Luteolin was on average the major flavonoid quantified in the oils (Figure 2), displaying a mean value of 4.85 µg/g oil and the contents ranged in the interval 0.88-14.36 µg/g oil. The mean value found for apigenin was 1.86 µg/g oil and the content range was 0.08-10.00 µg/g oil. The content of flavonoids in the oils also reflects the relatively high levels of conjugated flavonoids in the olive fruits (Figure 3). Among them, the most abundant on average was luteolinglucoside, whose mean content in the fruits was 300.98 µg/g fruit, displaying a variability range of 45.78-1247.26 µg/g fruit. The relationships among the main groups of phenolic compounds in the fruits and oils of the WOGC cultivar subset are shown in Table 1. Total phenolic compounds in the fruits and oils were significantly correlated (r = 0.66), which suggests that fruit phenolics are one of the main factors responsible for the phenolics content in the oil. The level of

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oleuropein derivatives in the oils showed a correlation coefficient with the oleuropein and derivatives in fruits (r = 0.64) lower than that observed for ligstroside derivatives (r = 0.73). These findings might be related to the higher oxidation rates of the former due to the action of oxidative enzymes on their orthodiphenolic structure during the oil extraction process. On the other hand, the content of flavonoids in the oils and fruits displayed the highest correlation coefficient (r = 0.74). Table 1. Pearson’s correlation coefficients among the main groups of phenolic compounds found in the cultivar subset of the WOGC Olive fruit

Olive oil

Total phenols

Oleuropein and

Ligstroside and

derivatives

derivatives

Flavonoids

Tyrosol derivatives

Total phenols

*** 0.66

*** 0.63

*** 0.65

0.15

*** 0.66

Secoiridoids

*** 0.66

*** 0.63

*** 0.64

0.14

*** 0.67

Oleuropein derivatives

*** 0.66

*** 0.64

*** 0.53

0.16

*** 0.66

Ligstroside derivatives

*** 0.48

** 0.42

*** 0.73

0.05

*** 0.48

Flavonoids

0.05

0.02

0.01

*** 0.74

0.02

Tyrosol derivatives

*** 0.66

*** 0.63

*** 0.65

0.14

*** 0.67

Lignans

0.18

0.15

** 0.34

-0.03

0.18

Simple phenols

-0.22

-0.24

-0.06

0.03

-0.23

Marked correlations are significant at: ** p < 0.01, *** p < 0.001. 1,0 .

1

p-HPEA

0,8 .

p-HPEA glucoside

1 0,6 .

1 Factor 2

0,4 .

1

p-HPEA-EDA

Apigenin glucoside

0,2 .

Ligstroside

3,4-DHPEA glucoside

3,4-DHPEA

1

3,4-DHPEA acetate p-HPEA-EA

0,0 .

1

3,4-DHPEA-EA

Luteolin

-0,2 .

Apigenin

Demethyloleuropein

1

Luteolin glucoside

Demethylligstroside

Oleuropein 3,4-DHPEA-EDA

-0,4 .

1 -1,0 .

-0,8 .

-0,6 .

-0,4 .

-0,2 .

0,0 .

0,2 .

0,4 .

0,6 .

0,8 .

1,0 .

1

1

1

1

1

Factor 1

1

1

1

1

1

Figure 4. Factor analysis. Position of the main phenolic compounds in the oils and in the fruits (boxed) from the WOGC cultivar subset on the first two factors using the normalized Varimax method.

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Factor analysis allowed explaining the pattern of correlations within the main groups of phenols in the olive fruits and oils, those which contain tyrosol or hydroxytyrosol moieties in their molecules. Figure 4 shows the factor analysis bi-plots using the normalized Varimax method and considering factors with eigenvalues higher than 1. Those factors explained 79.04% of the total variance. First factor explained 21,92% of the variance and second factor 17,93%. As shown, most of the components distributed along Factor 1 axis except for oil p-HPEA and its glucoside in the fruit, and different groupings can be noticed that might have a metabolic sense. Thus, major fruit phenolics oleuropein and ligstroside group together with their aglucones in the oils at the far end of the Factor 1 axis. Interestingly, their demethyl derivatives are located at the opposite place in the axis, which suggest that both might have parallel but separated synthetic ways. It is worth mentioning also that 3,4-DHPEA acetate is located in the vicinity of demethyloleuropein. Supposedly, 3,4-DHPEA acetate would not be simply formed by an acetylation of the 3,4-DHPEA molecule but from a cleaving process, occurring during the oil extraction, of an unstable secoiridoid structure derived from the deglucosylation of oleuropein. In this sense, no significant increases in the content of 3,4-DHPEA acetate was observed in in vitro deglucosylation of demethoxyoleuropein by olive β-glucosidase activity (Romero-Segura et al., 2012). On the other hand, Figure 4 also shows grouping of the fruits and oils flavonoids and of the oil tyrosol and hydroxytyrosol with their corresponding glucosides present in the fruit. The latter along the positive Factor 2 axis but separated, which might suggest that although they are chemically related their synthesis could occur in a parallel rather than in a sequential way through a simple hydroxylation. Moreover, compound location in the plot of Figure 4 would suggest that these phenolic alcohols present in the oil apparently would come from the hydrolysis of their glucosides present in the fruit more than from successive cleavages from oleuropein and ligstroside secoiridoid derivatives. Principal component analysis (PCA) was very useful for analyzing the data from the main groups of phenolic compounds (Figure 5). The first two principal components carried a high amount of important information and accounted for 66.44% of the total variance. PCA bi-plots showed strong associations between the secoiridoid compounds (Figure 5-A) and a number of cultivars present mainly along the positive Factor 1 axis and the first quadrant (Figure 5-B). Also cultivars displaying high contents of lignans in their oils are located in the first quadrant. Lignans and the oleuropein and ligstroside derivatives are close in the plot, so that it is possible to select cultivars for breeding programs whose oils have a potential high estrogenic activity (lignans) as well as a high level of antioxidants (secoiridoids derived from hydroxytyrosol). Meanwhile, cultivars closely associated to high level of flavonoids are located mainly along the negative Factor 2 axis and the fourth quadrant. Thus, it is possible to pinpoint specific cultivars

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whose oils possess potent radical scavenger and antioxidant properties due to their high content in flavonoids (Cook and Samman, 1996; Rice-Evans et al., 1995).

Figure 5. Principal component analysis of the main groups of phenolic compounds in the oils and in the fruits (boxed) from the WOGC cultivar subset. A: vector distribution of the phenolic compounds, B: distribution of the cultivars.

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Figure 6. Principal component analysis of the tyrosol and hydroxytyrosol derived phenolic compounds in the oils and in the fruits (boxed) from the WOGC cultivar subset. A: vector distribution of the phenolic compounds, B: distribution of the cultivars.

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Based on their nutritional and sensory implications and their importance from a quantitative point of view, PCA was performed separately for each of the tyrosol and hydroxytyrosol derivatives (Figure 6). Vectors displayed associations quite similar to those shown in the Factor Analysis (Figure 4). However, it is more clear the relationship that exists between the oil simple alcohols (3,4-DHPEA and p-HPEA) and their glucosides in the fruit. Also, it is more evident the relationship between the demethyl derivatives of oleuropein and ligstroside and the demethoxy derivatives of their aglucones (3,4-DHPEA-EDA and p-HPEA-EDA). This distribution of the vectors allows identifying in the fourth quadrant cultivars which presumably give rise to oils with remarkable health-promoting properties. These properties would be consequence of their high content of the antioxidant 3,4-DHPEA-EDA and the anti-inflammatory potential due to their elevated content of oleocanthal (p-HPEA-EDA) (Lucas et al., 2011; Visioli and Bernardini, 2011). However, the sensory aspects related to these oils should be also considered because of the importance from the perspective of the consumer acceptability. Despite most of these cultivars would give rise to oils with a mild bitter taste, which is mainly due to 3,4-DHPEA-EA (Mateos et al., 2004), they would be characterized by a high level of pungency due to the high level of p-HPEA-EDA. In this sense, Visioli and Bernardini (2011) recommended consumers to be trained and informed on how to choose high-quality olive oils based on their organoleptic attributes. Oils rich in polyphenols are characterized by a bitter and pungent taste. Moreover, it would be possible to select genotypes whose oils would have a high levels of 3,4-DHPEA-EDA and oleocanthal (pHPEA-EDA) in combination with a high content of 3,4-DHPEA acetate located along the Factor 1 positive axis. The vector distribution displayed in Figure 6-A permits also to select cultivars in the third quadrant (Figure 6-B) whose oils would be characterized by a high health-promoting capacity and low pungency, but highly bitter.

CONCLUSION Taking into account the proven benefits for human health of the VOO phenolic fraction, which is main responsible for the sensorial and nutritional quality of this key element of the Mediterranean diet, the assessment of the phenolic profile might be very useful to identify olive cultivars producing oils with an improved nutritional quality. The analytical data demonstrated that a high degree of variability for the content of phenolic compounds is found in olive. The use of multivariate analysis allowed to identify cultivars particularly interesting in terms of phenolic composition and deduced organoleptic and nutritional quality. This information can be used both to identify old olive cultivars which give rise to oils with a high nutritional quality and in breeding programs for parent selection. As mentioned above, the major targets in olive breeding

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are shifting recently more towards the sensory and nutritional quality of VOO in contraposition to the traditionally focus on the agronomic trait improvement.

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Estruch, R., Ros, E., Salas-Salvadó, J., Covas, M.I., Corella, D., et al. (2013). Primary prevention of cardiovascular disease with a Mediterranean diet. N. Engl. J. Med., 368, 1279-1290. García-González, D.L., Tena, N. & Aparicio, R. (2010). Quality characterization of the new virgin olive oil var. Sikitita by phenols and volatile compounds. J. Agric. Food Chem., 58, 8357-8364. García-Rodríguez, R., Romero-Segura, C., Sanz, C., Sánchez-Ortiz, A. & Pérez, A. (2011). Role of polyphenol oxidase and peroxidase in shaping the phenolic profile of virgin olive oil. Food Res. Int., 44, 265-635. Giovannini, C., Straface, E., Modesti, D., Coni, E., Cantafora, A., et al. (1999). Tyrosol, the major olive oil biophenol, protects against oxidized-LDL induced injury in Caco2 cells. J Nutr, 129, 1269-1277. Grasso, S., Siracusa, L., Spatafora, C., Renis, M. & Tringali, C. (2007). Hydroxytyrosol lipophilic analogues: Enzymatic synthesis, radical scavenging activity and DNA oxidative damage protection. Bioorganic. Chem., 35, 137-152. Inarejos-García, A.M., Androulaki, A., Salvador, M.D., Fregapane, G. & Tsimidou, M. (2009). Discussion on the objective evaluation of virgin olive oil bitterness. Food Res. Int., 42, 279-284. Konstantinidou, V., Covas, M.I., Muñoz-Aguayo, D., Khymenets, O., de La Torre, R., et al. (2010). In vivo nutrigenomic effects of VOO polyphenols within the frame of the Mediterranean diet: a randomized trial. Faseb J, 24, 2546-2557. León, L., Beltrán, G., Aguilera, M. P., Rallo, L., Barranco, D., et al. (2011). Oil composition of advanced selections from an olive breeding program. Eur. J. Lipid Sci. Technol., 113, 870-875. Lucas, L., Russell, A. & Keast, R. (2011). Molecular mechanisms of inflammation. Antiinflammatory benefits of virgin olive oil and the phenolic compound oleocanthal. Curr. Pharm. Design, 17, 754-768. Mateos, R., Cert, A., Pérez-Camino, M.C. & García, J.M. (2004). Evaluation of virgin olive oil bitterness by quantification of secoiridoid derivatives. J. Am. Oil Chem. Soc., 81, 71-75. Mateos, R., Goya, L. & Bravo, L. (2005). Metabolism of the olive oil phenols hydroxytyrosol, tyrosol and hydroxytyrosyl acetate by human hepatoma HepG2 cells. J. Agric. Food Chem., 53, 9897–9905. Mateos, R., Pereira-Caro, G., Saha, S., Cert, R., Redondo-Horcajo, M., et al. (2011). Acetylation of hydroxytyrosol enhances its transport across differentiated Caco-2 cell monolayers. Food Chem., 125, 865- 872.

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Menéndez, J.A., Vazquez-Martín, A., García-Villalba, R., Carrasco-Pancorbo, A., Oliveras-Ferraros, C., et al. (2008). tabAnti-HER2 (erbB-2) oncogene effects of phenolic compounds directly isolated from commercial Extra-Virgin Olive Oil (EVOO). BMC Cancer, 8, 1-23. Menéndez, J.A., Vazquez-Martín, A., Oliveras-Ferraros, C., García-Villalba, R., Carrasco-Pancorbo, A., et al. (2009). Extra-virgin olive oil polyphenols inhibit HER2 (erbB-2)-induced malignant transformation in human breast epithelial cells: relationship between the chemical structures of extra-virgin olive oil secoiridoids and lignans and their inhibitory activities on the tyrosine kinase activity of HER2. Int. J. Oncol., 34, 43-51. Miró-Casas, E., Covas, M.I., Fito, M., Farré-Albadalejo, M., Marrugat, J., et al. (2003). Tyrosol and hydroxytyrosol are absorbed from moderate and sustained doses of virgin olive oil in humans. Eur. J. Clin. Nutr., 57, 186-190. Montedoro, G., Baldioli, M., Selvaggini, R., Begliomini, A.L. & Taticchi, A. (2002). Relationship between phenolic compounds of olive fruit and olive oil: Importance of the endogenous enzymes. Acta Hort., 586, 551-556. Obied, H.K., Prenzler, P.D., Ryan, D., Servilli, M., Taticchi, A., et al. (2008). Biosynthesis and biotransformations of phenol-conjugated oleosidic secoiridoids from Olea europaea L. Nat Prod Rep, 25, 1167-1179. Pereira-Caro, G., Sarria, B., Madrona, A., Espartero, J.L., Escuderos, M.E., et al. (2012). Digestive stability of hydroxytyrosol, hydroxytyrosyl acetate and alkyl hydroxytyrosyl ethers. Int. J. Food Sci. Nutr., 63, 703-707.32. Pérez, A.G., León, L., Pascual, M., Romero-Segura, C., Sánchez-Ortiz, A., de la Rosa, R.& Sanz, C. (2014). Variability of virgin olive oil phenolic compounds in a segregating progeny from a single cross in Olea europaea L. and sensory and nutritional quality implications. PLOS ONE 9, e92898. Pinto, J., Paiva-Martins, F., Corona, G., Debnam, E.S., Oruna-Concha, M.J., et al. (2011). Absorption and metabolism of olive oil secoiridoids in the small intestine. British J. Nutr., 105, 1607-1618. Rice-Evans, C.A., Miller, N.J., Bolwell, P.G., Broamley, P.M. & Pridham, J.B. (1995). The relative antioxidant activities of plant-derived polyphenolic flavonoids. Free Radic. Res., 22: 375-383. Romero-Segura, C., García-Rodríguez, R., Sánchez-Ortiz, A., Sanz, C. & Pérez, A.G. (2012). The role of olive β-glucosidase in shaping the phenolic profile of virgin olive oil. Food Res. Int. 45: 191-196. Savarese, M., De Marco, E. & Sacchi, R. (2007). Characterization of phenolic extracts from olives (Olea europaea cv. Pisciottana) by electrospray ionization mass spectrometry. Food Chem., 105, 761-770.

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Scotece, M., Gomez, R., Conde, J., López, V., Gomez-Reino, J.J., et al. (2013). Oleocanthal inhibits proliferation and MIP-1 alpha expression in human multiple myeloma cells. Curr. Med. Chem., 20, 2467-2475. Servili, M., Selvaggini, R., Esposto, S., Taticchi, A., Montedoro, G., et al. (2004). Health and sensory properties of virgin olive oil hydrophilic phenols: Agronomic and technological aspects of production that affect their occurrence in the oil. J. Chromatogr. A, 1054: 113-117. Visioli, F. & Bernardini, E. (2011). Extra virgin olive oil’s polyphenols: Biological activities. Curr. Pharm. Design, 17, 786-804.

In: Handbook of Olive Oil Editor: Jozef Miloš

ISBN: 978-1-53612-356-2 © 2017 Nova Science Publishers, Inc.

Chapter 6

HYDROXYTYROSOL AND TYROSOL, PHENOLIC COMPOUNDS OF VIRGIN OLIVE OIL, COULD ACT LIKE ANTI-INFLAMMATORIES IN CHRONIC INFLAMMATION Cristina Sánchez-Quesada, PhD1,2,3 and José J. Gaforio, MD, PhD1,2,3,4, 1

Center for Advanced Studies in Olive Grove and Olive Oils, University of Jaén, Spain 2 Immunology Division. Department of Health Sciences, Faculty of Experimental Sciences, University of Jaén, Jaén, Spain 3 Agrifood Campus of International Excellence, ceiA3, Spain 4 CIBER-ESP, Instituto de Salud Carlos III, Madrid, Spain

ABSTRACT Hydroxytyrosol (HT) and tyrosol (TY) are two phenols present in virgin olive oil (VOO), the principal fat used in Mediterranean countries. It is well known the benefits of Mediterranean diet in the prevention and development of certain illness as cancer or cardiovascular diseases as well as the natural protection exerted by VOO in these same diseases. When there is an injury in the body, inflammation is one of the early response to the damage; immune cells drive the immunologic response to clean and heal the damage. An uncontrolled response of the immune system could derive in the appearance of several illness. Macrophages are the main cells that control this process and their appearance and activity is the key of the development of cancer or Crohn’s diseases, among others. In this chapter, the effects in macrophages of two natural compounds (HT 

Correspondence: [email protected]; Tel.: +34-953-212-002.

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Cristina Sánchez-Quesada and José J Gaforio and TY) present in VOO are studied. Results showed that both compounds are able to manage M1 macrophage response into an anti-inflammatory state, which could be very useful in the treatment of several diseases as Crohn’s or inflammatory bowel diseases. Even more, there could be able to prevent chronic inflammation, which in turn is one of the reason of certain disease appearances.

INTRODUCTION Mediterranean countries have been differentiated from others because of their low prevalence of several diseases, such as cancer, cardiovascular diseases, etc [1, 2]. Recently, many studies demonstrate that diet plays a central role in the appearance and development of chronic inflammation, and some studies describe the “inflammatory potential” of certain diets [3]. Olive oil, the main fat of the Mediterranean diet, has been shown to possess anti-inflammatory effects in several diseases [4, 5]. Many of its minor compounds have been described to prevent or treat several different diseases [6-8], but there is no description of which olive oil compounds are responsible of these antiinflammatory properties and whether they can be used in several inflammatory diseases (such as Crohn’s disease, inflammatory bowel syndrome, cancer, among others) or not. Hydroxytyrosol and tyrosol are two of the main phenols present in virgin olive oils [9]. These phenols are described to possess several anti-inflammatories activities and to prevent several diseases as cardiovascular and cancer diseases, both related directly with chronic inflammation [10, 11]. Nevertheless, any study describing the action that they have in inflammation process or in macrophage behavior. Inflammation is a complex set of interactions among soluble factors and cells and can arise in any tissue in response to traumatic, infectious, post-ischemic, toxic or autoimmune injury. Macrophages are the main and first cells that appear at the injured area and are capable of managing and controlling the inflammatory response. M1 phenotype macrophages are usually cytotoxic effectors that mediate the Th1 cytotoxic and pro-inflammatory response, whereas M2 phenotype macrophages possess antiinflammatory properties and drive the Th2 inflammatory response [12, 13]. Following the interaction of both types of macrophages, the healing process normally leads from infection to recovery. However, if targeted destruction and assisted repair are not properly phased, inflammation can lead to persistent tissue damage from leukocytes, lymphocytes and collagen. Inflammation can be considered in terms of its checkpoints, where binary or higher-order signals drive each commitment to escalate, ‘go signals’ trigger ‘stop signals’, and molecules responsible for mediating the inflammatory response can also suppress inflammation depending on the timing and context. The noninflammatory state does not passively exist because of the absence of inflammatory stimuli; rather, the maintenance of health requires the positive actions of specific gene products to suppress reactions to potentially inflammatory stimuli that do not warrant a

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full response [14]. Therefore, persistent chronic inflammation could lead to several diseases, such as inflammatory bowel syndrome or cancer. Many studies shows that chronic inflammation occurs prior to the appearance and development of several cancers [15]. In our study, the effects of hydroxytyrosol (HT) and tyrosol (TY) on the proinflammatory responses of M1 macrophages were studied. The cytokines and molecules involved in inflammatory responses were studied after treatments with both compounds in a human monocyte cell line (THP-1), which was differentiated into M1 macrophages. To our knowledge, this is the first time that HT and TY have been studied in a proinflammatory human cellular model.

MATERIALS AND METHODS 1. Chemicals The following were purchased from Sigma-Aldrich Co. (St Louis, MO, USA): 2hydroxyphenyl ethanol (Tyrosol, CAS 501-94-0 (TY)) purity 98%; Hepes solution; Sodium Pyruvate solution; Non-Essential Amino Acids mixture 100× (NEAA); Lipopolysaccharides from Escherichia coli 055:B5 (LPS); 2,3-Bis(2-methoxy-4-nitro-5sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt (XTT sodium salt) (purity ≥90%); N-Methylphenazonium methyl sulfate (PMS) (purity ≥98%); Phorbol 12-myristate 13acetate (PMA) (purity ≥99%); phosphate buffer saline (PBS); sodium chloride (NaCl) (purity ≥99,5%); L-Arginine (L-Arg) (purity 98.5-101.0%) suitable for cell culture and Triton X-100. Fetal Bovine Serum (FBS) was obtained from PAA Laboratories GmbH (Pasching, Austria). Minimum essential medium with Eagle’s salts (MEM) and PhenolRed-free Roswell Park Memorial Institute 1640 medium (RPMI) were obtained from Gibco® Life Technologies Ltd (Paisley, UK). Methanol dry (max 0,005%), Magnesium Chloride (50% MgCl2 powder QP) (MgCl2) and ethanol absolute PRS was purchased from Panreac Quimica S.L.U. (Barcelona, SPAIN). TrypLE Express was obtained from Invitrogen (Eugene, OR, USA). β-Mercaptoethanol was purchased from Applichem GmbH (Darmstadt, GERMANY). PIPES (98,5 + %) was obtained from Acrōs Organics (Geel, BELGIUM). Culture plates were obtained from Starlab (Hamburg, GERMANY). NFκβ p65 (F-6) antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). NFκβ p65 Sandwich ELISA kit from Cell Signaling Technology (CST, Danvers, MA, USA). RayBio® Human Cytokine Antibody Array (Human Inflammation Array I) was purchased from RayBiotech, Inc. (Norcross, GA, USA). TNF-α Enzyme Immunometric Assay Kit were pursached from (Stressgen) Enzo Life Science, Inc. (Farmingdale, NY, USA). 2-(3,4-dihydroxyphenyl) ethanol (Hydroxytyrosol, CAS

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10597-60-1 (HT)) purity ≥98% was obtained from Cayman Chemical (Ann Arbor, MI, USA).

2. Cell Culture and Treatment The THP-1 (human acute monocytic leukemia) cell line was obtained from American Type Culture Collection (ATCC, Rockville, MD, USA). The THP-1 monocytes were maintained at 37 oC in a humidified atmosphere under 5% CO2 in MEM supplemented with 10% FBS, 1% hepes buffer, 1% sodium pyruvate, 1% NEAA and 0,05 mM 2mercaptoethanol. THP-1 cells were sub-cultured at least twice per week, and discarded and replaced by frozen stocks after 25 passages for achieving an optimal condition of growth. Macrophages differentiation was induced according to Sánchez-Quesada et al., [16] and it was followed by hydroxytyrosol (HT) or tyrosol (TY) treatment at 1, 10 and 100 µM along 24h. All the assays were conducted under these conditions except for those specified below.

3. Cytotoxicity Assay THP-1 cells survival, measured as the cellular growth of treated cells versus untreated controls, was carried out using an XTT-based assay according to SánchezQuesada et al., [16]. Absorbance of cell survival was measured at 450nm wavelength (620nm as reference) in a plate reader (TECAN GENios Plus, Tecan Trading AG, Switzerland). Viability was calculated using the formula: % viable cells = [(A treated cells) / (A control)] x 100 where A is the difference in absorbance between optical density units (A = OD450 – OD620). All measurements were performed in quadruplicate and each experiment was repeated at least three times.

4. Raybio® Human Cytokine Antibody Array in M1 State THP-1 Macrophages Differentiated THP-1 cells were stimulated with LPS (1 µg/mL) at 24 h. After that, HT or TY treatments were realized. Then, supernatants were isolated and processed according to manufacturer instructions. Arrays membranes were directly detected using a

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chemiluminescence imaging system (FluorChem E System, ProteinSimple) for achieve production levels of the following cytokines/proteins: eotaxin, eotaxin-2, interleukin 1 alfa (IL-1 α), interleukin 1 beta (IL-1β), interleukin 2 (IL-2), interleukin 3 (IL-3), interleukin 4 (IL-4),interleukin 6 (IL-6), interleukin 7 (IL-7), interleukin 8 (IL8),interleukin 10 (IL-10), interleukin 11 (IL-11), interleukin 12 p40 (IL-12 p40), interleukin 12 p70 (IL-12p70), interleukin 13 (IL-13), interferon-gamma (IFN-γ), granulocyte colony-stimulating factor (GCSF), granulocyte macrophage colonystimulating factor (GMCSF), chemokine CCL-1 (I-309), metallopeptidase inhibitor 2 (TIMP-2). Data were analyzed with the RayBio® Human Inflammation Antibody Array 1 Analysis Tool (Cat # SO2-AAH-INF-1). Data are expressed as chemiluminescents arbitrary units acquired by the chemiluminescence imaging system (FluorChem E, Protein Simple, CA, USA) after normalization (positive control) and background subtraction.

5. TNFα Production After results obtained in cytokine array, TNFα molecule was measured for corroborate the production of anti-inflammatory cytokines in M1 macrophages. After treatments with HT and TY for 24h, TNFα production were measured with TNF-α Enzyme Immunometric Assay Kit (Stressgen) according to manufacturer protocol in a microplate reader (TECAN GENios Plus, Tecan Trading AG, Switzerland). Data are expressed as the main (of three independent assays) of total produced protein (pg/mL).

6. NFκβ Detection in M1 State THP-1 Macrophages After stimulation of differentiated THP-1 cells with LPS (1µg/mL) and HT or TY treatments, NFκβ production was measured according to manufacturer protocol (PathScan Total NFκβ p65 Sandwich ELISA kit (Cell Signaling Technology)). Cells were analysed in a microplate reader (TECAN GENios Plus, Tecan Trading AG, Switzerland). Data expressed as the main (of three replicates) respect to control, set as 100%.

7. NO Production in M1 Type THP-1 Macrophages Nitric oxide (NO) production was measured according to Sánchez-Quesada et al. [16]. NO production was analysed by a NO analyser (NOA 280i de SIEVERS, GE Water

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and Process Technologies, Pennsylvania, USA). Data are expressed as the main of three independent experiments relative to untreated control, which was set as 100%.

8. Statistical Analysis For all the assays except for cytokine antibody array, data are displayed as the mean of at least three independent experiments (± SEM) run in triplicate; for cytotoxicity assay, results are expressed as a percentage relative to the untreated control cells (which was defined as 100 %). A general variance analysis (ANOVA) was carried out and Student ttest. A p value 5% of total liver weight [30] and involves a broad spectrum of pathologies including steatosis, steatohepatitis (involving inflammation) and cirrhosis [8]. About 43% of subjects with steatosis develop to more severe stages such as steatohepatitis and 7 to 16% of them develop hepatic cirrhosis [31]. There are several molecular events that can result in accumulation of lipids in hepatocytes, most of them are: i) increased lipid uptake in the liver due to increased lipolysis in adipose tissue; ii) increased hepatic lipogenesis; iii) deficient synthesis and/or secretion of lipoproteins, sequestering TG and cholesterol into the liver; iv) reduction of mitochondrial -oxidation; v) deficiency of peripheral lipid storage due to pathologies such as IR, leading to TG accumulation in the liver [7]. One of the most common triggers of hepatic steatosis in subjects with obesity and poor eating habits is IR, both peripheral and hepatic [31]. The hepatocyte, by not adapting to the IR, loses its functionality leading even to cellular necrosis or apoptosis and finally liver failure. In contrast, if the

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hepatocyte adapts and retains its functionality, it remains vulnerable to stimuli that may trigger an inflammatory response. Eventually, it may be triggered: i) OS, due to the increase in the production of ROS; ii) lipid peroxidation of membranes; iii) abnormal production of cytokines; iv) hepatic mitochondrial dysfunction; v) metabolic disorders of fatty acid metabolism, mainly altered levels of enzymes and nuclear transcription factors such as PPARα and vi) metabolic endotoxemia [31]. Chronic inflammation, together with OS, can lead to an increase in proinflammatory mediators that activate fibrogenesis, promoting the evolution from simple steatosis to steatohepatitis and cirrhosis [32].

Figure 1. Progression of liver damage: Dysfunctional adipose tissue releases high levels of FFA and proinflammatory cytokines that pass into the blood. Hepatic IR, common pathology in obese subjects, generates an increase in de novo synthesis of fatty acids in liver, greater accumulation of TG in hepatocytes and lower release of VLDL by the hepatocytes, promoting a greater accumulation of FFA and eventually TG, leading to the development of steatosis. Proinflammatory cytokines that reach the liver from dysfunctional adipose tissue generate a state of inflammation, which in turn generates mitochondrial dysfunction, condition exacerbated by the large amount of accumulated FFA, which leads to OS, allowing the progression of steatosis to steatohepatitis. Steatohepatitis is capable of activate stellate cells of the liver, which increase the production of various components of the extracelular matriz generating fibrosis and subsequently cirrhosis.

EVOO, rich in MUFAs, has been used for preventive and treatment purposes in liver steatosis, both alone [33, 34] or in conjunction with other compounds, such as n-3 PUFAs (especially EPA (C20:5 n-3) and DHA (C22:6 n-3)) [35]. EVOO, which is in particular rich in polyphenols, decreases the FFA-induced steatosis in HepG2 cells, reducing the

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number and size of fat globules and the accumulation of TG [36]. A study in rats with hepatic steatosis induced by high-fat diet showed that HT supplementation (10 mg/kg/day) was able to restore PPARα and carnitine palmitoyltransferase 1 (CPT1) levels, promoting -oxidation. In addition, HT was able to decrease the production of proinflammatory cytokines such as IL-6 and TNF-, which resulted in a histological decrease of steatosis and inflammation [11].

3.2. EVOO and Non-Alcoholic Steatohepatitis Non-alcoholic steatohepatitis is a condition characterized by the presence of predominantly macrovesicular hepatic steatosis and lobular and portal inflammation with hepatocyte injury (ballooning) with or without fibrosis [37, 38]. Steatohepatitis may progress to cirrhosis in approximately 20% of subjects over a 10-year period [39], to liver failure and rarely to hepatocarcinoma [37]. Once steatohepatitis is established, stellate cells in the liver activate various fibrogenic mechanisms [40], beginning to secrete large amounts of collagen, among other substances, transforming the liver into a fibrotic and dysfunctional tissue. Non-alcoholic steatohepatitis is frequently associated with obesity and diabetes mellitus type 2 (DM2) [32]. In addition, its appearance since the progression of simple steatosis may be due, among other factors, to a deficiency of mitochondrial FA oxidation [7], as well as to an excessive accumulation of lipids in the hepatocytes, which leads to inflammation. A study in humans reported that subjects with steatohepatitis present a decrease in apoB-100 related to IR, compromise that retains or block the secretion of VLDL from the liver promoting hepatic steatosis [40] due to lipid accumulation in the hepatocytes. Eventually inflammation may occur, due to the increase of proinflammatory cytokines and the production of ROS by dysfunctional mitochondrias, thus establishing steatohepatitis [7]. HT, one of the most effective antioxidants of natural origin and present in EVOO, has shown important effects on steatohepatitis, reducing inflammation and OS, thus preventing the progression of liver damage to steatohepatitis, or even by preventing the further progressing of the disease. Within the anti-inflammatory mechanisms of HT, characterized in a human cell line of THP-1 monocytes, was identified the inhibition of nitric oxide synthase, inhibition of cyclooxygenase-2 enzyme expression, increased transcription of TNF-, and a suppression of the increase in the formation of nitric oxide stimulated by lipopolysaccharides [41]. In addition, HT is able to eliminate ROS and activate the endogenous defense system mediated by Nrf2 [17], thus decreasing OS that allows the progression to steatohepatitis and improving the clinical picture.

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3.3. EVOO and Cirrhosis Hepatic cirrhosis is a chronic disease considered irreversible that alters the structure and function of the liver. It is defined histopathologically as a triad of cellular necrosis, fibrosis and regeneration nodules, being hepatic dysfunction it is most important clinical manifestation [42]. Cirrhosis is the end stage of various chronic liver diseases, such as excessive alcohol consumption, indiscriminate consumption of drugs and some pharmaceuticals, obesity, environmental toxicants, heavy metals or autoimmune diseases [43]. It is a terminal stage of liver damage that can progress to hepatocarcinoma, but previously produces a serie of alterations and complications, such as gastrointestinal hemorrhages and hepatic encephalopathy, among others [43]. Cirrhosis is a stage of hepatic tissue damage, characterized by fibrosis, which involves an increased production of the extracellular matrix by the stellate cells and myofibroblasts [44]. Fibrosis resulting from hepatic tissue damage correlates with the extracellular deposition of type 1 collagen, smooth muscle, actin, elastin and other proteins involved in the remodeling of the extracellular matrix by macrophages and stellate cells; is the abundance of these proteins which affects the progression and severity of fibrosis evolving to cirrhosis [45]. Several studies have evaluated the effect of EVOO on fibrosis by improving the histological and clinical picture. In this sense, experimental tissue damage has been induced with various agents, including CCl4, a substance that causes fibrosis. Wang et al., showed that rats fed with EVOO had less damage in the hepatic architecture and less formation of fibrous tissue compared to animals fed corn oil [15]. They also demonstrated that EVOO was able to induce lower lipid peroxidation and decreased smooth muscle actin alpha (αSMA) expression, a protein involved in the cells structure, thus decreasing fibrosis [15]. Lee et al. demonstrated that EVOO is able to decrease both adipose vesicles (steatosis) and fibrosis produced by CCl4 [16].

3.4. EVOO and Hepatocellular Carcinoma Inflammation and tissue damage generated by liver cirrhosis may also generate dysplastic lesions in hepatocytes involving DNA damage. This damage may eventually trigger liver cancer [46]. Hepatocellular carcinoma is the most common primary liver cancer in adults, accounting for approximately 80% of cases [47], being the fifth most common cause of cancer worldwide and the second leading cause of cancer death [48, 49]. Hepatic cirrhosis is the most important predisposing factor for the development of hepatocellular carcinoma [30]. It is estimated that between 1 and 8% of subjects with liver cirrhosis will develop hepatocellular carcinoma every year [30]. Among the possible treatments, curative therapies such as surgical resection and liver transplantation are the most common; their choice depends mainly on the number and size of the lesions, the

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patient’s general condition and the hepatic functions preserved at the undamaged tissue [30]. Several studies have tested the effect of polyphenols of EVOO on different types of cancer [50]. Referred to hepatocellular carcinoma, HT has shown to be able to inhibit the enzyme xanthine oxidase reducing the production of superoxide anion, thus protecting DNA damage [14]. A study conducted in cell lines of human hepatocellular carcinoma showed that HT is able to induce apoptosis and inhibit the proliferation of cancer cells by suppressing the activation of nuclear factor kappa B (NF-kB) (a transcription factor involved in the proinflamatory signal pathway), via AKT and the consequent decrease in the transcription of regulated genes by NF-kB [14]. Since AKT, once activated, is able to accelerate the degradation of IKB (an inhibitor of NF-kB) and phosphorylate the NFkB/p65 complex, promoting translocation to the nucleus of NF-kB [51]. In this sense HT suppresses the activation of AKT and therefore of NF-kB [14]. Also, a study in human hepatoma cell lines showed that oleuropein (10-80 uM), a polyphenol of EVOO and precursor of HT, is capable of inhibiting, in a dose-dependent manner, cell growth, increasing cell death and decreasing colony formation by inactivating the PI3K/AKT pathway, promoting cellular apoptosis [52].

3.5. EVOO and Ischemia-Reperfusion Injury Ischemia is a condition in which there is a transient or permanent decrease of the blood flow to some organ or tissue [53]; reperfusion is the restoration of blood flow. During ischemia, there is a lack of oxygen and nutrients [54], which may cause cell damage and the production of proinflammatory cytokines. Once the blood flow has been restored, all these mediators are returned to the tissue producing greater damage, known as ischemia-reperfusion injury (I/R) [54]. Hepatic I/R damage is a consequence of liver transplantation, hepatic resection or vascular reconstruction and trauma, hypovolemic shock, and tumor excision [55, 56, 57, 58]. The lack of oxygen in the tissue and the conversion of hepatocellular metabolism to anaerobic pathways due ischemia [56], induces a proinflammatory state that leaves the tissue vulnerable to reperfusion [59]. Rapid restoration of blood flow is necessary to restore the cellular functions lost during ischemia, but reperfusion is also capable of initiating a cascade response of OS causing deep injury in hepatocytes [56]. I/R hepatic injury is characterized by OS, inflammation and cellular apoptosis [60]. I/R damage has two phases: i) early phase, associated with the activation of kupffer cells causing the formation of ROS, TNF-α and IL-1 production, among other proinflammatory factors [61, 62, 63, 64, 65] and; ii) late phase, at this stage, high levels of proinflammatory factors promote the recruitment of polymorphonuclear lymphocytes, perpetuating and amplifying liver damage due to the release of ROS, TNFα and the activation of proteases [61, 62, 66, 67].

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There are few studies evaluating the effect of EVOO or some of its components on I/R damage. However, Pan et al., evaluated the effect of HT on an in vivo mouse model, where I/R was induced by surgery. HT (10 mg/kg) administered 4 hours before ischemia achieved reduced AST and ALT levels compared to controls, as well as cell reduced apoptosis and accumulation of ROS and malondialdehyde (MDA) as indicators of oxidative damage. HT administration also attenuated the increase in inflammatory mediators (TNF-α, IL-6 and MIP-2) [60]. In an in vitro model of anoxia/reoxygenation in an anaerobic chamber, HT (100 uM) was able to decrease the number of apoptotic cells and the levels of ROS [60].

4. MOLECULAR MECHANISMS INVOLVING THE PROTECTIVE EFFECT OF EVOO Alteration of lipid metabolism is frequently observed among subjects with some degree of liver damage, generating: i) increased hepatic lipogenesis; ii) decreased lipoprotein secretion; iii) lipid peroxidation and iv) hepatic depletion of n-3 PUFA [7, 34, 68, 69], due to the decrease in the activity of the enzymes Δ5 and Δ6 desaturases and OS. In this context, EVOO has been shown to exert significant hepatoprotective effects. EVOO in liver is involved in the activation of various metabolic pathways in order to prevent inflammation, OS, endoplasmic reticulum stress (overload of poorly folded proteins in the lumen), mitochondrial dysfunction and IR, key situations in the onset and progression of hepatic tissue damage. Among the most important molecular effects of EVOO in the prevention of liver damage are:

(i) Activation of Nuclear Transcription Factor Nrf2,Inducing a Cellular Antioxidant Response, by the Gene Expression of Antioxidant Enzymes or of Enzymes Involved in Cell Detoxification OS and the damage it produces is a relevant factor in the progression of liver damage, in this context the role of antioxidant enzymes or cellular detoxifiers molecules are of vital importance. Among the antioxidant enzymes are glutathione peroxidase (GPx) and glutathione S-transferase (GST) involved in the removal of peroxides, being the primary mechanism of defense against oxidative damage; on the other hand enzyme glutathione reductase (GR) is responsible for the regeneration of oxidized glutathione a strong nonenzymatic antioxidant molecule [70, 71, 72]. HT, known for its antioxidant activity, is able to increase the levels of phosphorylated AKT and ERK in hepatocytes, by regulating these pathways (AKT and ERK), the polyphenol increases the phosphorylation of Nrf2 in

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its serine or tyrosine residues, achieving the dissociation of the Nrf2/Keap1 complex which manintains Nrf2 in the cytosol, increasing the proteasomal degradation of Keap1 and the translocation to the nucleus of Nrf2; in the nucleus, Nrf2 is able to bind to the antioxidant responsive element (ARE) and increase the expression of GPx, GST and GR, which results in an increase of the respective coded enzymes and in a decrease of the OS and the damage produced by this process (see Figure 2) [17]. However, in a randomized, double-blind study in healthy humans, where the subjects where their own control, the activation of phase II enzymes mediated by Nrf2 were tested after doses of HT of 5 and 25 mg/day. No increase in the activity of phase II enzymes was obtained, which could be due to the dose or the short time of the treatment. However, more studies in humans are needed to verify the possible protective action of this polyphenol [73]

(ii) Inactivation of Nuclear Transcription Factor NF-kB, Preventing the Cellular Inflammatory Response NF-kB is a transcription factor that plays a regulatory role in the cytokine gene expression; within the genes that are regulated by this factor are those that code for proteins involved in inflammation, cell proliferation, apoptosis, angiogenesis, metastasis, among others [74, 75]. NF-kB can be activated by the activation of the AKT pathway, i.e., when AKT is phosphorylated. EVOO is able to decrease the levels of phosphorylated AKT, inactivating NF-kB. A study conducted in hepatocellular carcinoma cells revealed that HT is able dose-dependently to decrease phosphorylated AKT levels, inducing apoptosis in the cells; HT was able to decrease DNA binding activity, and in a dosedependent manner, it decreased NF-kB and the genes regulated by this transcription factor [14].

(iii) Inhibition of the PERK Pathway, Preventing the Reticulum Stress, through the UPR System, and Autophagy In various conditions, such as during increased protein demand, or conditions that alter protein folding in the endoplasmic reticulum, such as OS, there is an overload of poorly folded proteins in the lumen of the endoplasmic reticulum, which is known as reticule stress. Reticular stress generates a series of complications that can aggravate hepatic tissue damage. Faced to reticulum stress cells activate an adaptive response known as UPR (Unfolded Protein Response). UPR is a signaling pathway that carries information about the state of protein folding between the reticulum and the nucleus, in order to inhibit the protein synthesis [76] and decrease the load of unfolded proteins; to this purpose three transduction signals that count the presence of unfolded proteins in the

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lumen of the organelle are activated: i) Activation of IRE-1α by oligomerization and autophosphorylation, leading to the cut in two positions of the X-box binding protein 1 (XBP1) mRNA, the resulting exons are assembled generating an mRNA that encodes a transcription factor that promotes the expression of chaperones and other genes of the UPR [77]. ii) Migration of ATF 6α, in response to the reticulum stress, this factor migrates to the Golgi apparatus, where its transmembrane domain is cut off and the nterminal fragment migrates to the nucleus, where it acts as a transcription factor to increase protein folding capacity [78, 79]. iii) Activation of PERK, that phosphorylates the alpha subunit of eIF2, leading to the inactivation of this translational initiation factor, leading to a decrease in protein synthesis and reducing reticulum stress [80, 81, 82, 83]. When these cellular pathways are not sufficient to prevent damage and accumulation of protein complexes, cellular autophagy is triggered.

Figure 2. Effect of HT on Nrf2. EVOO and especially its component HT, is able to increase the phosphorylation of AKT and ERK in the hepatocytes. By activating these metabolic pathways, is phosphorylated Nrf2 in its residues of serine and tyrosine, which allows the dissociation of the complex Nrf2/Keap1, which maintains Nrf2 in the cytosol. This dissociation allows the proteasomal degradation of Keap1 and the translocation of Nrf2 to the nucleus. In the nucleus Nrf2 binds to ARE and increases the transcription of genes coding for GPx, GST and GR, resulting in an increase in the translation of the respective enzymes, which allows the reduction of OS and the damage produced by this condition.

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The role of EVOO, especially through HT, is to activate the cellular antioxidant enzymatic machinery, by increasing the activity and translocation to the nucleus of Nrf2. In this way, OS decreases, avoiding or decreasing the reticulum stress, therefore there is no activation of the PERK pathway described above, thus preventing autophagy and the continuous tissue damage generated by the accumulation of unfolded proteins. The effects of EVOO and of its components on the liver have been widely evaluated by several researchers. In this regard, Priore et al., evaluated the effect of EVOO on healthy hepatocytes from Wistar rats, comparing to refined olive oil lacking polyphenols, but with the same fatty acid profile; Authors highlighted the synergistic effect of polyphenols in this model expressed on the inhibition of FA and cholesterol synthesis [33]. In addition, HT and oleuropein in particular (25 uM) were able to inhibit the synthesis of FA and cholesterol [84], which is related to the decrease in the enzymatic activity of acetyl-CoA carboxylase (ACC), promoting β-oxidation [84, 85]. Lama et al., using a high-fat diet-induced NALFD model, showed that EVOO is able to reduce glycemia and insulin, due to a greater phosphorylation of AKT and greater expression of GLUT2 in hepatocytes, improving the uptake of glucose and the approach of the insulin receptor to the membrane [86, 87]. On the other hand, EVOO is able to improve lipid metabolism by increasing AMPK phosphorylation and PPARα expression, also the oil decreases inflammation (decreased TNF-α, IL-1 and IL-10), decreases the expression of cyclooxygenase-2 enzyme and lipid peroxidation, improving the integrity of the hepatocyte [86]. EVOO, on the other hand, activates PPARα and reduces the activation of SREBP-1C, inhibiting hepatic lipogenesis and decreasing the accumulation of TG [87]. HT accumulates in a dose-dependent manner in a large number of tissues, including hepatic tissue [88, 89, 90]. This polyphenol has shown to be able to mediate lipid metabolism, thus reducing the size of the lipid globules and the accumulation of TG in the hepatocytes [11]. HT is also able to bind to the cytoplasmic domain of PPARα, thus PPARα heterodimerizes with 9-cis-RXR [88, 89, 90], producing the translocation of this dimer to the nucleus and its binding to the PPAR response zone in the promoter region of the target genes that regulates this transcription factor [91, 92, 93]. Among genes regulated are the gen that codes CPT-1α, which is involved in the transport of FA to the interior of the mitochondria, through the carnitine-dependent system, promoting βoxidation. On the other hand HT also increases the translation of ACOX-1, key protein of the peroxisome, indispensable for the oxidation of FA of high hydrocarbon chain [91, 92, 93]. HT also increases ACC phosphorylation, induced by activation of the AMPK pathway, this modification inactivates the enzyme by decreasing malonyl CoA levels, thereby reducing hepatic CPT-1α inhibition, favoring β-oxidation and reducing fatty acid synthesis [85]; in this way EVOO is able to reduce liver steatosis. By suppressing NF-kB, HT is able to reduce inflammation and OS [14]; added to the activation and greater translocation to the nucleus of Nrf2, HT activates the endogenous antioxidant defense

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system, reducing lipid peroxidation and ballooning, avoiding the progression of liver damage [11, 14, 17, 19] to steatohepatitis or cirrhosis. Likewise, polyphenols from EVOO (10-80 uM oleuropein) has been shown to inhibit cell growth and increase cell apoptosis in human hepatoma cell lines by decreasing the levels of phosphorylated AKT, with maintenance of AKT levels, inducing the excision of caspase-3 and anti-poly ADP ribose polymerase (PARP), leading to apoptosis by suppression of the PI3K/AKT pathway [52]. Another important effect of HT has been seen in I/R damage, subsequent to liver transplantation or surgical resection. In this condition it has been shown that HT is able to increase the activity of SOD 1 and SOD 2, which eliminate the superoxide anion, protecting the hepatocyte from oxidative damage; and the activity of CAT, which catalyzes the conversion of hydrogen peroxide to oxygen and water, both in vivo and in vitro, thus protecting the hepatocyte from the damage caused by ROS that are generated during ischemia and returned to tissue during reperfusion [60].

CONCLUSION EVOO is a food characteristic of the Mediterranean diet to which are attributed various benefits to human health, especially as a protector of cardiovascular health. Among the most remarkable components of EVOO for its benefits to human health are OA and polyphenols, especially HT, which is able to modulate various metabolic pathways and act at the molecular level to exert their effects on the prevention of hepatic tissue damage. Among the most remarkable effects of HT is the decrease of NF-kB and of its regulated genes, as well as the increase in the activity and translocation to the nucleus of Nrf2, reducing OS, reticular stress and thus preventing liver damage progression. Scientific investigations regarding EVOO and liver damage are focused especially on the role of HT, which has been extensively tested in cellular and animal models. However, more research is needed in humans to find not only an adequate dosage for HT, but to verify that the effects seen in these models are replicable to humans. It is also necessary to elucidate that a healthy diet that include EVOO, the main source of HT and other polyphenols, which is recommended for preventive purposes to healthy subjects, may also have therapeutic effects in subjects who already have obesity, IR or some degree of hepatic injury.

ABBREVIATIONS ACC ALT

Acetyl-CoA carboxylase Alanine transaminase

Extra Virgin Olive Oil and Hepatoprotective Effects ARE AST CAT CCl4 CPT1 DM2 EVOO FA FFA GPx GR GST HT IR I/R MDA MUFA NAFLD NF-kB OA OS PARP PPAR PUFAs ROS SFA -SMA SOD SREBP TG UPR XBP1

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Antioxidant responsive element Aspartate transaminase Catalase Carbon tetrachloride Carnitine palmitoyltransderase 1 Diabetes mellitus type 2 Extra virgin olive oil Fatty acid Free fatty acid Glutathione peroxidase Glutathione reductase Glutathione S-transferase Hydroxytyrosol Insulin resistence Ischemia-Reperfusion Malondialdehyde Monounsaturated fatty acids Non-alcoholic fatty liver disease; Nuclear factor kappa B Oleic acid Oxidative stress Anti-poly ADP ribose polymerase Peroxisome proliferator activated receptor alpha Polyunsaturated fatty acids Reactive oxygen species Saturated fatty acids Smooth muscle actin alpha Superoxide dismutase Sterol regulatory element binding protein triglycerides Unfolded protein response X-box binding protein 1

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[30] Poniachik, J. Enfermedad por hígado graso no alcohólico. En: Diagnóstico y tratamiento de las enfermedades digestivas, Sociedad Chilena de Gastroenterología ed. Santiago de Chile, 2013: 321-25. (Non-alcoholic fatty liver disease. In: Diagnosis and treatment of digestive diseases, Chilean Society of Gastroenterology ed. Santiago of Chile, 2013, 321-25). [31] Folch, J; Lees, M; Sloane-Stanley, GH. A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem, 1957, 226, 497–509. [32] Buque, X; Aspichueta, P; Ochoa, B. Fundamento molecular de la esteatosis hepática asociada a la obesidad. Rev Esp Enferm Dig, 2008, 100(9), 565-578. (Moleculas basis of hepatic steatosis associated with obesity. Rev Esp Enferm Dig, 2008, 100(9), 565-578). [33] Priore, P; Caruso, D; Siculella, L; Gnoni, GV. Rapid down-regulation of hepatic lipid metabolism by phenolic fraction from extra virgin olive oil. Eur J Nutr, 2015, 54, 823-833. [34] Baraldi, FG; Vicentini, TM; Teodoro, BG; Dalalio, FM; Dechandt, CRP; Prado, IMR; et al. The combination of conjugated linoleic acid (CLA) and extra virgin olive oil increases mitocondrial and body metabolism and prevents CLA-associated insulin resistance and liver hypertrophy in C57BL/6 mice. J Nutr Biochem, 2016, 28, 147-54. [35] Valenzuela, R; Espinosa, A; Llanos, P; Hernandez-Rodas, M; Barrera, C; Vergara, D; et al. Anti-steatotic effects of an n-3 LCPUFA and extra virgin olive oil mixture in the liver of mice subjected to high-fat diet. Food Funct, 2016, 7, 140. [36] Hur, W; Kim, SW; Lee, YK; Choi, JE; Hong, SW; Song, MJ; et al. Oleuropein reduces free fatty acid-induced lipogenesis via lowered extracellular signalregulated kinase activation in hepatocytes. Nutr Res, 2012, 32(10), 778–86. [37] Chalasani, N; Younossi, Z; Lavine, JE; Diehl, AM; Brunt, EM; Cusi, K; et al. The Diagnosis and Management of Non-Alcoholic Fatty Liver Disease: Practice Guideline by the American Association for the Study of Liver Diseases, American College of Gastroenterology, and the American Gastroenterological Association Gastroenterology, 2012, 142(7), 1592-609. [38] Ludwig, J; Viggiano, TR; McGill, DB; Oh, BJ. Nonalcoholic steatohepatitis: Mayo Clinic experiences with a hitherto unnamed disease. Mayo Clin Proc, 1980, 55, 434-438. [39] McCullough, AJ. The clinical features, diagnosis and natural history of nonalcoholic fatty liver disease. Clin Liver Dis, 2004, 8, 521–533. [40] Charlton, M; Sreekumar, R; Rasmussen, D; Lindor, K; Nair, KS. Apolipoprotein synthesis in nonalcoholic steatohepatitis. Hepatology, 2002, 35(4), 898-904. [41] Zhang, X; Cao, J; Zhong, L. Hydroxytyrosol inhibits pro-inflammatory cytokines, iNOS, and COX-2 expression in human monocytic cells. Naunyn Schmiedeberg’s Arch Pharmacol, 2009, 379(6), 581–6.

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[42] Esteller Pérez, A; González Gallego, J. Nutrición en las enfermedades hepatobiliares. En: Gil A Ed. Tratado de nutrición. Tomo IV. Acción Médica, Madrid, 2005, pp. 907-57. (Nutrition in hepatobiliary diseases. In: Gil a Ed. Nutrition Treaty., Volume IV. Medical Action, Madrid, 2005, pp. 907-57). [43] Mesejo, A; Juan, M; Serrano, A. Cirrosis y encefalopatía hepaticas: consecuencias clínico-metabólicas y soporte nutricional. Nutr Hosp, 2008, 23, 8-18. (Hepatic Cirrhosis and encephalopathy: clinical-metabolic consequences and nutritional support. Nutr Hosp, 2008, 23, 8-18). [44] Duarte, S; Baber, J; Fujii, T; Coito, AJ. Matrix metalloproteases in liver injury, repair and fibrosis. Matrix Biol, 2015, 44, 147–159. [45] Lytle, KA; Depner, CM; Wong, CP; Jump, DB. Docosahexaenoic acid attenuates Western diet-induced hepatic fibrosis in Ldlr-/- mice by targeting the TGFβ-Smad3 pathway. J Lipid Res, 2015, 56(10), 1936-46. [46] Zender, L; Spector, MS; Xue, W; Flemming, P; Cordon-Cardo, C; Silke, J; et al. Identification and validation of oncogenes in liver cancer using an integrative oncogenomic approach. Cell, 2006, 125(7), 1253–1267. [47] cancer.net (Internet). USA: American Society of Clinical Oncology (ASCO): 2005 (cited abril 2017). Available from: http:// www. cancer.net/es/tipos-decáncer/cáncer-de-h%C3%ADgado/ introducción. [48] globocan.iarc.fr (Internet). France: International Agency for Research on Cancer; 2012 (cited april 2017). Available from: http://globocan.iarc.fr/Pages/fact_sheets _cancer.aspx. [49] Ferlay, J; Shin, HR; Bray, F; Forman, D; Mathers, C; Parkin, DM. Estimates of worldwide burden of cáncer in 2008, GLOBOCAN 2008. Int J Cancer, 2010, 127, 2893-2917. [50] Vilaplana-Perez, C; Auñon, D; Garcia-Flores, LA; Gil-Izquierdo, A. Hydroxytyrosol and potential uses in cardiovascular diseases, cancer, and AIDS. Front Nutr, 2014, 27, 1, 18. [51] Vivanco, I; Sawyers, CL. The phosphatidylinositol 3-Kinase AKT pathway in human cáncer. Nat Rev Cancer, 2002, 2(7), 489-501. [52] Yan, CM; Chai, EQ; Cai, HY; Miao, GY; Ma, W. Oleuropein induces apoptosis via activation of caspases and suppression of phosphatidylinositol 3kinase/protein kinase B pathway in HepG2 human hepatoma cell line. Mol Med Rep, 2015, 11(6), 4617-24. [53] rae.es (Internet). Spain: Real Academia Española (Royal Spanish Academy), 1993 (cited 10 april 2017). Available from: http://dle.rae.es/?id=MCri5PV. [54] Teoh, N; Dela Pena, A; Farrell, G. Hepatic ischemic preconditioning in mice is associated with activation of NF-κB, p38 kinase, and cell cycle entry. Hepatology, 2002, 36, 94-102.

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[55] Ramírez, P; Marín, JM; Piñero, A; Chávez-Cartaya; Parrilla, P. Investigación experimental aplicada a la clínica: isquemia-reperfusión hepática. Cir Esp, 2000,67,281-91. (Experimental investigation applied to the clinic: hepatic ischemiareperfusion. Cir Esp, 2000, 67, 281-91). [56] Visioli, F; Borsani, L; Galli, C. Diet and prevention of coronary heart disease: the potential role of phytochemicals. Cardiovasc Res, 2000, 47, 419–425. [57] Lemasters, J; Thurman, RG. Reperfusion injury after liver preservation for transplantation. Annu. Rev. Pharmacol Toxicol, 1997, 37, 327–338. [58] Olthoff, KM. Can reperfusion injury of the liver be prevented? Trying to improve on a good thing. Pediatr Transplant, 2001, 5, 390–393. [59] Jaeschke, H. Molecular mechanism of hepatic ischemia-reperfusion injury and preconditioning. Am J Physiol Gastrointest Liver Physiol, 2003, 284, G15-G26. [60] Pan, S; Liu, L; Pan, H; Ma, Y; Wang, D; Kang, K; et al. Protective effects of hydroxytyrosol on liver ischemia/reperfusion injury in mice. Mol Nutr Fodd Res, 2013, 57, 1218-1227. [61] Cutrín, JC; Perrelli, MG; Cavalieri, B; Peralta, C; Roselló-Catafau, J; Poli, G. Microvascular disfunction induced by reperfusion injury and protective effect of ischemic preconditioning. Free Radical Biol Med, 2002, 33, 1200-8. [62] Lichtman, SN; Lemasters, JJ. Role of cytokines and cytokine-producing cells in reperfusion injury to the liver. Sem Liver Dis, 1999, 19, 171-204. [63] McCord, JM. Oxygen-derived free radicals in postischemic tissue injury. N Engl J Med, 1985, 312(3), 159-63. [64] Adkison, D; Höllwarth, ME; Benoit, JN; Parks, DA; McCord, JM; Granger, DN. Role of free radicals in ischemia-reperfusion injury to the liver. Acta Physiol Scand Suppl, 1986, 548, 101-7. [65] Carden, DL; Granger, DN. Pathophysiology of ischaemia-reperfusion injury. J Pathol, 2000, 190(3), 255-66. [66] Colletti, LM; Kunkel, SL; Walz, A; Burdick, MD; Kunkel, RG; Wilke, CA; Strieter, RM. The role of cytokine networks in the local liver injury following hepatic ischemia/reperfusion in the rat. Hepatology, 1996, 23(3), 506–514. [67] Jaeschke, H; Farhood, A; Smith, CW. Neutrophils contribute to ischemia/reperfusion injury in rat liver in vivo. FASEB J, 1990, 4(15)3355–3359. [68] Valenzuela, R; Videla, L. The importance of the long-chain polyunsaturated fatty acid n- 6/n-3 ratio in development of non-alcoholic fatty liver associated with obesity. Food Funct, 2011, 2, 644. [69] Delarue, J; Lallés, JP. Nonalcoholic fatty liver disease, Roles of the gut and the liver and metabolic modulation by some dietary factors and especially long-chain n3 PUFA. Mol Nutr Food Res, 2016, 60(1), 147-59. [70] Lei, XG; Cheng, WH; McClung, JP. Metabolic Regulation and Function of Glutathione Peroxidase-1. Annu Rev Nutr, 2007, 27, 41–61.

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[71] Hayes, JD; Flanagan, JU; Jowsey, IR. Glutathione transferases. Annu. Rev. Pharmacol Toxicol, 2005, 45, 51–88. [72] Argyrou, A; Blanchard, JS. Flavoprotein disulfide reductases: advances in chemistry and function. Prog. Nucleic Acid Res Mol Biol, 2004, 78, 89–142. [73] Crespo, MC; Tomé-Carneiro, J; Burgos-Ramos, E; Loria Kohen, V; Espinosa, MI; Herranz, J; Visioli, F. One-week administration of hydroxytyrosol to humans does not activate Phase II enzymes. Pharmacol Res, 2015, 95-96, 132-7. [74] Aggarwal, BB. Nuclear factor-kappaB: the enemy within, Cancer Cell, 2004, 6, 203–208. [75] Karin, M. Nuclear factor-kappaB in cancer development and progression. Nature, 2006, 441, 431–436. [76] Kaufman, R. Stress signaling from the lumen of the endoplasmic reticulum: coordination of gene transcriptional and translational controls. Genes Dev, 1999, 13, 1211-1233. [77] Lee, A; Iwakoshi, N; Glimcher, L. XBP-1 regulates a subset of endoplasmic reticulum resident chaperone genes in the unfolded protein response. Mol Cell Biol, 2003, 23, 7448-7459. [78] Haze, K; Yoshida, H; Yanagi, H; Yura, T; Mori, K. Mammalian transcription factor ATF6 is synthesized as a transmembrane protein and activated by proteolysis in response to endoplasmic reticulum stress. Mol Biol Cell, 1999, 10, 3787-99. [79] Ye, J; Rawson, R; Komuro, R; Chen, X; Dave, U. ER stress induces cleavage of membrane-bound ATF6 by the same proteases that process SREBPs. Mol Cell, 2000, 6, 1355–1364. [80] Giordano, E; Davalos, A; Nicod, N; Visioli, F. Hydroxytyrosol attenuated tunicamycin-induced endoplasmic reticulum stress in human hepatocarcinoma cells. Mol Nutr Food Res, 2014, 58, 954-962. [81] ncbi.nlm.nih.gov (Internet). USA: NCBI, 1988 (cited 2 May 2017). Available from: https://www.ncbi.nlm.nih.gov/gene/9451. [82] Harding, H; Zhang, Y; Ron, D. Protein translation and folding are coupled by an endoplasmic-reticulum-resident kinase. Nature, 1999, 397, 271–74. [83] Lin, J; Walter, P; Yen, T. Endoplasmic Reticulum Stress in Disease Pathogenesis. Annu Rev Pathol, 2008, 3, 399-425. [84] Priore, P; Siculella, L; Gnano, GV. Extra virgin olive oil phenols down-regulate lipid synthesis in primary-cultured rat-hepatocytes. J Nutr Biochem, 2014, 25(7), 683-91. [85] Aguilera, CM; Gil-Campos, M; Canete, R; Gil, A. Alterations in plasma and tissue lipids associated with obesity and metabolic syndrome. Clin Sci (Lond), 2008, 114(3), 183–93.

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[86] Lama, A; Pirozzi, C; Mollica, MP; Trinchese, G; Di Guida, F; Cavaliere, G; et al. Polyphenol-rich virgin olive oil reduces insulin resistance and liver inflammation and improves mitocondrial dysfunction in high-fat diet fed rats. Mol Nutr Food Res, 2017, 61(3), 1600418. [87] Assy, N; Nassar, F; Nasser, G; Grovoski, M. Olive oil consumption and nonalcoholic fatty liver disease. World J Gastroenterol, 2009, 15(15), 1809-1815. [88] MacLaren, L; Guzeloglu, A; Michel, F; Thatcher, WW. Peroxisome proliferatoractivated receptor (PPAR) expression in cultured bovine endometrial cells and response to omega-3 fatty acid, growth hormone and agonist stimulation in relation to series 2 prostaglandin production. Domest Anim Endocrinol, 2006, 30(3), 15569. [89] Desvergne, B; Wahli, W. Peroxisome proliferator-activated receptors: nuclear control of metabolism. J Clin Endocrinol Metab, 1999, 20, 649–688. [90] Escher, P; Braissant, O; Basu-Modak, S; Michalik, L; Wahli, W; Desvergne, B. Rat PPARs: Quantitative Analysis in Adult Rat Tissues and Regulation in Fasting and Refeeding. Endocrinology, 2001, 142(10), 4195-202. [91] Gross, B; Pawlak, M; Lefebvre, P; Staels, B. PPARs in obesity-induced T2DM; dyslipidaemia and NAFLD. Nat Rev Endocrinol, 2017, 13(1), 36-49. [92] ncbi.nlm.nih.gov (Internet). USA: NCBI; 1988 (cited 9 Dec. 2016). Available from: https://www.ncbi.nlm.nih.gov/gene/11430. [93] ncbi.nlm.nih.gov (Internet). USA: NCBI; 1988 (cited 9 Dec. 2016). Available from: https://www.ncbi.nlm.nih.gov/gene/12894.

In: Handbook of Olive Oil Editor: Jozef Miloš

ISBN: 978-1-53612-356-2 © 2017 Nova Science Publishers, Inc.

Chapter 10

THE HEALTH BENEFITS OF OLEOCANTHAL AND OTHER OLIVE OIL PHENOLS Roberto Ambra*, Sabrina Lucchetti† and Gianni Pastore‡ Food and Nutrition Research Centre Council for Agricultural Research and Economics, Rome, Italy

ABSTRACT Different phenolic compounds are present in extra-virgin olive oil (EVOO). The biological effects of the more characterizing for EVOO, i.e., secoiridoids, have been studied since their discovery and their health beneficial properties, that go beyond merely antioxidant activities, are becoming more and more recognized. Oleocanthal is the secoridoid responsible for the oral irritation induced by EVOO and perceptually it gives an oropharyngeal sensation similar to that produced by ibuprofene. Oleocanthal similarity with ibuprofene is also at the pharmacological level since it induces a dose dependent inhibition of COX enzymes even greater than the drug. Furthermore, oleocanthal is able to inhibit in vivo the formation of neurofibrillary aggregates responsible for Alzheimer’s disease. This review summarizes the knowledge of the in vitro and in vivo effects of oleocanthal comparing, where available, data with related EVOO phenols, and focusing on its anti-inflammatory, chemotherapeutic, neuroprotective and antimicrobial activities, discussing also bioavailability and experimental concentration issues.

Keywords: extra-virgin olive oil (EVOO), oleocanthal, olive phenols, bioavailability

*

[email protected] (Corresponding author). [email protected]. ‡ [email protected]. †

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INTRODUCTION Health properties of extra virgin olive oil (EVOO) have been attributed both to its high levels of oleic acid and to several minor components, including EVOO phenolic compounds. Phenolic compounds possess one or more aromatic rings with one or more hydroxyl groups. They are the most widely distributed secondary metabolites of plants, with more than 8000 chemical structures, and are involved in several functions, from defense against UV radiation and pathogens to decomposition of organic substances. The amount of phenolic compounds in EVOO can exceed one gram per kg of oil, depending on many factors including the cultivar, growth conditions and oil processing. EVOO contains at least 36 structurally different phenolic compounds, which can be divided into 6 main groups based on similarity of structure: secoiridoids, phenylalcohols, phenylacids, hydroxyisocromans, flavonoids and lignans. Secoiridoids are the most caractherizing molecules of the Oleaceae family and can represent 80-90% of total oil phenols. The most abundant secoiridoids in EVOO are: 



 

3,4-DHPEA-EDA; the dialdehydic form of the decarboxymethyl elenolic acid linked to hydroxytyrosol; oleuropein aglycone dialdehyde; oleacein; DOA (deacetoxy oleuropein aglycone) – up to 600 mg/kg p-HPEA-EDA; the dialdehydic form of the decarboxymethyl-elenolic acid linked to tyrosol; ligstroside aglycone dialdehyde; oleocanthal; DLA (deacetoxy ligstroside aglycone) - up to 400 mg/kg 3,4-DHPEA-EA; the elenolic acid linked to hydroxytyrosol; oleuropein aglycone monoaldehyde; OA (oleuropein aglycone) - up to 300 mg/kg p-HPEA-EA; the elenolic acid linked to tyrosol; ligstride aglycone monoaldehyde - LA (ligstroside aglycone) up to 50 mg/kg

Then, in order of abundance, the phenyl alcohols hydroxytyrosol (3,4-di hydroxyphenyl ethanol; HT) and tyrosol (4-hydroxyphenyl ethanol; TY), respectively up to 200 and 180 mg/kg. Oleocanthal and oleacein differ from the molecules from which they derive (ligstroside and oleuropein) for the lack of a glycosidic group and a carboxymethyl group in position 4 (Figure 1). Phenols are responsible of the two main features of EVOO, i.e., its stability, protecting it from oxidation, and its sensory properties (bitterness, pungency, astringency). Then, they have several activities, from antioxidant, anti-inflammatory, antitumor, antimicrobial, and cardiovascular protective and neuroprotective effects. This review focusses mainly on the properties of EVOO phenyl alcohols and secoiridoids (from here on in named EVOOLS), with a special attention to oleocanthal.

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Oleocanthal was firstly reported in 1993 by Montedoro and collaborators [1] that found the molecule in the secoiridoid fraction. Subsequently oleocanthal was identified as the molecule responsible for oral irritation induced by EVOO [2], perceptually similar to that produced oropharyngeal by ibuprofene [3]. Oleocanthal was shown to direct interact with the transient receptor potential cation channel, subfamily A, member 1 (TRPA1), localized within the oral cavity into the pharynx. However, differently from other TRPA1 agonists, TRPA1 activation by oleocanthal does not involve the classical cysteine covalent modification [4].

Figure 1. Structures of oleuropein (1), oleacein (2), ligstroside (3), oleocanthal (4), hydroxytyrosol (5), and tyrosol (6) (from Vougogiannopoulou et al. (5).

ANTIOXIDANT AND ANTI-INFLAMMATORY EFFECTS Inflammatory processes are important since their role in the elimination of pathogens and the activation of repair processes at damaged tissues. However, if inflammation becomes chronic, for example because of bad dietary habits, the sustained release of chemokines induces macrophages and lymphocytes T attraction, and the release of toxic molecules such reactive oxygen or nitrogen species, and prostaglandins. Several natural anti-inflammatory compounds exist. Such molecules have analgesic and antipyretic properties and are known in traditional medicine since centuries. Some of them are known as natural nonsteroidal anti-inflammatory drugs (NSAIDs), and their antiinflammatory properties are attributed to the inhibition of arachidonic acid metabolites,

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i.e., COX enzymes. One important example is willow bark, that was mentioned in texts of ancient Mesopotamia and was currently used by Hippocrates. In 1763, its properties were clinically demonstrated and in 1971 Vane and coworkers reported that inhibition of COX by that salicylic acid happens in a dose-dependent manner [6]. The similarity with ibuprofene pushed Beauchamps and colleagues to isolate, extract and synthesize oleocanthal and establish that similarity was also at the pharmacological level [7]. Oleocanthal was shown to induce a dose dependent inhibition of COX1 and COX2 greater than ibuprofen (41-57% vs 13-18%) at the 25mM equimolar concentrations. Although oleocanthal constitutes only 10% of the total EVOOLS [8], Beauchamp and collaborators hypothesized that it could be sufficient to support EVOOLS ability to modify some physiological functions and potentially reduce the risk of inflammatory diseases [7]. Starting from ten years ago, near all EVOOLS were shown to reduce the expression of COX2. Firstly in human colon adenocarcinoma cells (an EVOOLS extract was used), through p38 phosphorylation inhibition [9], in HepG2 hepatocytes (HT) [10], then in THP-1 monocytes (TY and HT) [11] and in intestinal Caco2 cells (HT) [12]. In addition, EVOOLS induced NF-B inhibition in cultured THP-1 monocytes (an EVOOLS extract or OA) [13]. Finally, NF-B inhibition was observed in circulating monocytes from healthy volunteers, using both an EVOOLS extract [14] and EVOO [15], and from metabolic syndrome volunteers (EVOO) [16]. More recently Rosignoli et al. showed that in peripheral blood mononuclear cells (PBMC), oleocanthal reduces not only COX2 (gene and protein expression), compared to other phenols, but also reduces the production of the superoxide radical and PGE2 prostaglandin [17]. The stronger effect of oleocanthal resides in its catechol group and depends on TNF-α production [17, 18], inhibition of IL-6 expression and secretion and inhibition of macrophage inflammatory protein 1α expression [19, 20]. The antiinflammatory activity of oleocanthal has been associated also with 5-lipoxygenase inhibition (even if higher for oleacein) [5], an enzyme catalyzing the initial biosynthesis steps of leukotrienes and therefore considered a potential target for the treatment of inflammatory diseases. EVOOLS have been shown to protect intact membranous systems like red blood cells (EVOOLS tested were HT, oleacein and OA) [21], liver cells (HT) [22] and epithelial pigment cells retinal (HT) [23] from oxidative stress. The membrane localization of phenols is at least in part responsible for the antioxidant activity, by quenching peroxyl radicals from the aqueous phase and preventing lipid peroxidation and the propagation of oxidation from the interface to the inner membrane [24]. Previous studies using reconstituted membranes have shown in fact that EVOOLS stick inside the lipid membrane and remain on its surface without penetrating it, which is consistent with conformational motility of non-planar structures and phenols hydrophilic properties [25].

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Recent data indicate that oleocanthal can induce rapid cell death due to permeabilization of lysosomal membrane in several cancer cell lines [26]. Antioxidants capabilities of EVOOLS have been linked to other properties, from antidiabetic to hypoglycemic, observed in chemically-induced diabetic rats (HT) [27], and increase of longevity in C. elegans (TY) [28].

ANTITUMOR EFFECTS One of the first acknowledgment that EVOO consumption could interfere with cancer was reviewed in year 2000 by Trichopoulou and coworkers that estimated that a shift to the EVOO-containing traditional healthy Mediterranean diet could induce a reduction of the incidence for several types of cancer i.e., colorectal, breast, prostate, pancreas, and endometrial [29]. Chemo-preventive abilities of EVOOLS were initially associated to their antioxidant properties. Accordingly, the ability to stop cell proliferation and induce apoptosis in HL60 promyelocytic cells were observed only in presence of the two phenolic hydroxyl groups of HT ring, since TY did not have such effect [30]. Following studies performed on cell lines and animal models demonstrated that EVOOLS can also directly inhibit proliferation and promote apoptosis of several tumor cell lines, and different mechanisms were identified. Accordingly, both ROS-dependent and independent mechanisms of action of HT-induced apoptosis have been proposed [31], even if the ROS one showed higher efficacy, since it was observed in more cell lines and irrespectively of the tissue of origin, eg. breast (MCF-7 and MDA), prostate (PC3 and LNCaP) and colon (SW480 and HCT116). Downstream, HT-induced apoptosis was shown to involve cytochrome c and activation of caspase 3, but not by the activation of the FAS (TNF receptor superfamily member 6) and the death receptor pathways. Subsequently authors have reported that the apoptosis pathway activated by HT in HL-60 cells involves activation of c-jun through the c-jun NH2-terminal kinase [30]. The activation of caspase-3 and a clear pro-apoptotic effect was demonstrated also for oleocanthal, together with PARP activation, phosphorylation of p53 and DNA fragmentation [32]. Upstream the apoptosis cascade, Morrozzi and coworkers demonstrated that the effects of EVOOLS (a mixture, or oleocanthal or oleacein alone) on HL-60 cells depend on a specific accumulation in G1 and S-phase and involve p21Waf/Cip1 and p27Kip1 cyclin kinases inhibitors [33, 34]. On the other hand, Margarucci and colleagues showed that the pro-apoptotic effects of oleocanthal on HeLa and U937 cell lysates were associated to a G2/M accumulation [35] and were mediated by direct binding on HSP90 (subsequently corroborated by means of in-living-cell mass-spectrometry [36]), an already known target

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in cancer therapy. In particular, oleocanthal specifically downregulated the cellular protein levels of HSP90 targets Cdk4 and AKT [35]. Recently Cardeno and the Casaburri groups reviewed that the protective effect of EVOOLS is extended to several other kinds of cancer, and the involvement of both antioxidant or not or even yet unidentified mechanisms, possibly with synergetic effects were suggested [37, 38]. Akl and colleagues recently demonstrated that oleocanthal anti-proliferative effects, specific for breast cancer cells (with IC50 of 16.2 and 40.8 µM for MDA-MB-231 and MCF-7 respectively) and absent at the same doses in non-tumors cells (MCF10A), are mediated by the inhibition of HGF-induced activation of the tyrosine kinase c-Met and mitogenic downstream pathways [39]. The same authors previously predicted that oleocanthal could target the c-Met kinase domain, inhibiting the growth, the migration and the invasive properties in cancerous mammary cells and prostate cancer cells (IC50 10-20 μM). Oleocanthal was also shown to possess anti-angiogenic properties through the downregulation of CD31 in endothelial cells [40]. In this last work, the authors reported that oleocanthal induces a dose-dependent inhibition of the G1/S progression. In this regard, the authors reported that the effect on the cell cycle was associated with a dose-dependent reduction (5-15 µM) of cyclin D1 and CDK6 levels and with an increase in p27. Cyclin D1 downregulation by oleocanthal was observed also in human hepatocellular carcinoma cells, and the effect was accompanied by Bcl-2 downregulation and mediated upstream by STAT3 [41]. Treatment with higher doses of oleocanthal (25 μM, 72h) were associated with induction of apoptosis in the MDA-MB-231 cell line, detected by cytofluorometry for V and PI and by WB of caspases 3 and 8, PARP and RIP. Downstream of c-Met, oleocanthal effect occured with a dose-dependent block of AKT and MAPK phosphorylation. Inhibition of AKT phosphorylation by oleocanthal was recently reported also in human malignant melanoma cells, at the micromolar range of concentration [42]. By means of wound healing and transwell chamber assays and nude athymic mice, oleocanthal was shown to suppress respectively HGF-induced or MDA-MB-231-induced tumor migration and invasion [40]. Similar results were obtained recently by Gu and coworkers, that demonstrated that oleocanthal inhibited proliferation, migration, and invasion in human umbilical vascular endothelial cells and induced potent anti-tumor growth in a subcutaneous xenograft model [43]. Finally, recent data on MDAMB-231 cells indicates that oleocanthal is also able to strongly inhibit the enzymatic activity of mammalian target of rapamycin (mTOR), establishing a link with the neuroprotective effects of the phenol [44]. Another property of EVOOLS, reported in HL-60 cells but also monocytes, involves DNA protection against oxidative damage (HT only was tested) [45]. The effect was demonstrated also in vivo, in circulating cells of both rats [46] and of postmenopausal women (50g for 8 weeks) [47], using EVOO rich in phenols. A recent study found that HT is capable of preventing the genotoxic damage also in cancerous mammary epithelial

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cells (MCF-7 and MDA-MB-231) [48] and similar results were obtained on HeLa cells treated with total phenols [49]. Importantly, like flavonoids and lignans, EVOOLS strongly inhibited the overexpression in mammary tumor cells of the fat acid synthase (FASN) [50], a key enzyme involved in the anabolic conversion of carbohydrates into fat. Using cancer cells, authors found that a mixture of secoiridoids, rather than single ring phenols (TY, HT and elenolic acid) or single phenols [51], compete with the binding of ATP to the kinase domain of HER2 inhibiting the activity of the tyrosine kinase receptor and promoting its degradation, rather than its transcriptional activity [52].

CARDIOVASCULAR PROTECTIVE EFFECTS Both long and short intervention studies demonstrated that the intake of EVOO rich in phenols, protects against cardiovascular disease [53, 54], improving the antioxidant status also in the elderly [55] and the endothelial dysfunction in patients with early atherosclerosis [56]. In particular, increased consumption of EVOO was associated with lowered conjugated dienes [57] and plasma levels of oxLDL, well known pro-oxidants that cause tissue damage and are considered a risk factor for developing cardiovascular disease [58]. A direct, protective, binding of EVOOLS to LDL was suggested by some studies [57, 59] indicating that the interaction originates glucuronated and sulphated phenol-LDL, complexes that are actually hypothesized able to prevent atherosclerosis, or even considered potential markers of cardiovascular disease [57, 60]. More specifically, EVOO consumption was associated with the appearance of specific phenolic LDLs, depending on the administration protocol: acute intake was found accompanied by a rapid but transient appearance of HT and TY glucuronated forms [60], while chronic intake (25 mL for 3 weeks, at fasting) was characterized mostly by sulphated forms [57]. MiroCasas and coworkers measured that HT and TY half-lives after and acute intake are respectively 2.5 and 5-8 hours [61], which is consistent with the observation that HT or TY glucuronated forms are removed faster from LDL than sulphated forms.

NEUROPROTECTIVE PROTECTIVE EFFECTS The formation of neurofibrillary tangles, has a main role in the neurological devastation of Alzheimer’s disease (AD). Another protective property of EVOOLS, and more specifically oleocanthal, involves inhibition of the formation of neurofibrillary aggregates, through direct interaction with the fibrillogenic group [62] of the TAU microtubule associated proteins [63]. In particular, through circular dichroism and surface

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plasma resonance studies, it was observed that oleocanthal is able to induce a conformational rearrangement of TAU from random coil to alpha helix [62]. Data in the literature indicate that AD develops mainly due to an excessive accumulation of amyloid beta plaques (A) in the brain as a result of insufficient clearance across the blood brain barrier (BBB) [64]. Another potential mechanism induced by oleocanthal involves a direct interaction with A and alteration of the aggregation state [65]. In particular, Abuznait and colleagues observed that oleocanthal, both in vitro (murine endothelial brain cells) and in vivo (mice), increased levels of factors involved in A clearance from the BBB, namely the P-glycoprotein (P-gp) and the LDL lipoprotein receptor-related protein 1 (LRP1) and of the degradating enzymes neprilysin (NEP) and the insulin degrading enzyme (IDE) [66]. Later a similar effect was demonstrated in an invertebrate model of AD fed with oleacein, that was shown able to inhibit Aβ plaque deposition, and reduce toxic Aβ oligomers [67]. Recently, Batarseh and coworkers reported that oleocanthal reduction of Aβ toxicity involves downregulation of glutamine and glucose transporters in astrocytes [68]. With respect to other EVOOLS, HT was shown in an in vitro model of differentiated sympathetic neurons to interact with integral membrane proteins, accelerating the in-out shift of the norepinephrine transporter [69]. On the other hand, TY was shown to possess a protective effect against death by chemically induced mitochondrial dysfunction in catecholaminergic CATH.a neuronal cells, another in vitro model of AD [70].

ANTIMICROBIAL EFFECTS Due to their low intestinal absorption and predominant excretion as metabolites (see next section), Garcia-Villalba and coworkers suggested that EVOOLS may have antimicrobial action, always in the gastrointestinal tract, as reported for tea phenols and certain fruits [71]. In agreement, oleacein, oleocanthal, HT and TY showed a potent in vitro activity against bacterial strains responsible for intestinal and respiratory infections, but unfortunately also against beneficial bacteria such as Lactobacillus acidophilus and Bifidobacterium bifidum [72]. Using an in vitro system that mimics gastric conditions, Romero and colleagues found that oleocanthal may escape acid hydrolysis and help the growth inhibition of Helicobacter pylori, responsible for peptic ulcer and gastric cancer development [73]. Actually oleocanthal is stable at high temperatures (240°C, 90min), only degrading by 16% [3]. A study of antimicrobial capacity of food-derived bacteria also demonstrates that these properties are greater if different phenol mixtures are used than individual phenols, suggesting a synergistic action between the different EVOOLS [74].

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BIOAVAILABILITY AND EVOOLS METABOLISM In 2004 Vissers and colleagues reported the literature on EVOO bioavailability since 1966 and concluded that despite being well absorbed, with a 55-66% efficiency and an excretion rate of only 5-16% of the total amount ingested [75], dietary EVOOLS are not sufficient to give a biologically significant effect, because of their non-significant reduction of LDL oxidisability or other oxidation markers in humans [76]. In the attempt to explain low bioavailability, Visioli and collaborators assumed that the achievable plasma concentration of EVOOLS is strictly affected by the matrix through which the compounds are administered [77, 78]. Anyway, in vivo data has been available almost for a decade only for simple TY and HT phenols: in 2000, HT was shown to be transported to small intestinal cells by passive diffusion [79]. Tuck and collaborators demonstrated using oral (or venous) administration of radioactively labeled TY and HT in laboratory animals, that the phenols are bioavailable [80]. Similarly, Visioli and co-workers reported that 98% of TY and HT are absorbed in humans, proportionally to the concentration present in the oil [81] and that they are found in plasma and urine as conjugated forms mostly glucuronates [78]. Later, as mentioned in the previous section, Garcia-Villalba and coworkers reported that almost all phenols contained in an EVOO oral administration appeared in urine as metabolites [82], possibly due to hepatic metabolism [83], suggesting that they are not absorbed but could have local antioxidant action in the gastrointestinal tract, in accordance with the free radical scavenging capabilities that have been reported both in the fecal matrix and intestinal cells [84]. Finally, it was demonstrated from one hand, that after a single ingestion of EVOOLS a multitude of phenolic compounds can be detected in various animal tissues [85], and from the other, that not only HT and TY but also complex phenols like OA enter the tenuous intestine [37, 86]. EVOOLS metabolism has been also analyzed using cell models, i.e., Caco2/TC7 intestinal cells and an in vitro system that simulates gastric and intestinal digestion phases with the aim of assessing the resistance of phenols to digestion, metabolism and transport [83]. In particular, the authors found a good stability of secoiridoids to gastric digestion, which is not unexpected since these molecules have already undergone, during the oil production process, an acidic and enzymatic beta-glucosidase treatment that converts the glucosidic forms, present in raw olives, in the aglycone forms typical of EVOO [87]. On the other hand, lower resistance to the small intestine conditions was found (mimicked by means of incubation with pancreatin and bile salts at 37°C for 2 hours at pH 6.5), as demonstrated by low recovery and presence of metabolites, mainly methylated and to a lesser extent sulphated and glucuronated, and consistently with in vivo data [82]. Consistently with Soler’s hypothesis of EVOOLS being processed in the liver [83], Mateos and colleagues observed low absorption but high metabolism within HepG2

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hepatic cells, mostly in glucuronated and methylated forms [88]. The lack of sulphated metabolites is consistent with data from Visioli et al. [78] and Garcia-Villalba et al. [82]. Other cell models have been used, i.e., JIMT-1 breast cancer cells, in order to study the cellular fate of EVOOLS, including oleocanthal [89]. EVOOLS were undetectable in breast cancer cells, confirming their low intracellular accumulation, as previously reported in HepG2 cells [88]. Nonetheless, metabolites were found, mainly methylated, suggesting the presence of a catechol-O-methyltransferase activity in tumor cells. Analyzing for 24h the fate of phenols, authors observed a greater and faster (already at 15 min) disappearance of secoiridoids containing HT, already in the first few minutes for oleacein and more slowly, between 30 min and 2 h, for OA. TY-related secoiridoids showed instead a delayed disappearance reaching the maximum absorption between 1-2h for LA and after 6h for oleocanthal. According to the authors, this slower absorption depends on the lower degree of hydroxylation. In any case, at 24h all secoiridoids disappeared, probably due to spontaneous degradation, according to the fact that it was observed even in the absence of cells. The simpler phenols HT and TY showed unreliable amounts, most likely because their concentration is subjected to variations due to the spontaneous degradation of the secoiridoids. With respect to the metabolites recoverable in the culture medium at different incubation times, authors found that TY is poorly metabolized, while HT is metabolized in methyl-HT, similarly to what previously reported on Caco-2 [79, 83] and HepG2 cells [88]. Regarding secoiridoids transformations, authors argued that HT-related ones are rapidly methylated (with a maximum concentration of up to 2 hours), while the TY-ones do not form methylated intermediates, in accordance with the fact that the enzyme responsible for the transformation (catechol-O-methyl transferase) requires an ortho-diphenolic group [79].

EXPERIMENTAL CONCENTRATION CONSIDERATIONS As stated in the previous section, after EVOO intake, phenols are extensively modified, giving rise to quantifications issues in tissues. For example, after a single administration of 25-40mL of EVOO, plasma concentrations of phenols were found ranging from lower than micromolar [61] to close to 20µM [90], possibly because of individual metabolic differences rather than merely methodological aspects linked to oil production. Anyway, such question is particularly relevant when setting up in vitro experiments, due to the appropriateness of the concentration used for cell treatment, compared with that achievable physiologically in vivo. According to a daily intake of 50g of EVOO [91] containing an average of between 230 to 510 mg/kg of phenols [92] and considering that about 45% of the phenols are not absorbed [75], Sacchi and collaborators estimated that the concentration of phenols which reaches the colon is in the order of 11 mg [93]. Since

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the average volume of the large intestine is about 220ml [94], the resulting concentration of phenols should be around 50g/ml of phenols, which is consistent with doses used normally in in vitro experiments. The matter is different when only one phenol is considered. For example, Fogliano questioned the conclusions of the work of Beauchamp [7], i.e., the idea that protective effects of the MD diet can be attributed to a daily consumption of oleocanthal uniquely, in a dose (9 mg) corresponding to about 10% of the standard dose ibuprofen, taken daily with 50g of EVOO. According to Fogliano, the daily consumption of oleocanthal is 10 times less, for several reasons. Firstly, even considering the richest EVOOs (up to 1000mg/kg of total phenolic compounds) produced from unripe olives, since oleocanthal never exceeds 10% of total phenolic compounds [95], 50g of EVOO may not contain more than 5mg of oleocanthal. In addition, because of their high content of phenolic compounds, such EVOOs are too sharp and bitter and would be discarded in favor of other EVOOs with a lower content in phenols, between 100 and 300mg/kg, corresponding, for 50g of EVOO, to a oleocanthal content between 0.5 and 1.5mg, that is 10 times lower than that reported by Beauchamps. Finally, consumption of EVOO is very far from 50g daily [96]. Nonetheless, recent data support the direct involvement of oleocanthal in the beneficial properties of EVOO against rheumatoid arthritis [97]: the phenol was found able to directly inhibit the production of iNOS induced by LPS in murine chondrocytes [98], in a dose-dependent way and at non-toxic μM concentration corresponding to the amount of oleocanthal introduced with a daily intake of 25-50 mL [99].

FUTURE RESEARCH Even if several experimental indications support the idea that oleocanthal is relevant for the nervous system in the amounts achievable by dietary EVOO (see [100] for a recent review), actually, very little is known of oleocanthal absorption and metabolism. Moreover, scientific proofs that oleocanthal is sufficient, in the amounts available in EVOO, for anti-inflammatory or antitumoral health effects of EVOO are still very scarce. More research and especially intervention studies are needed to increase the knowledge on oleocanthal (and other EVOOLS) metabolism, in order to draw conclusions on its biological properties. In particular, the study of oleocanthal metabolism appears particularly relevant for its anti-inflammatory properties, since its hydrolysis is expected to produce the dialdehydic elenolic acid, which is molecularly more similar to ibubrofen than oleocanthal itself [8]. Moreover, since high anti-inflammatory doses of ibuprofen have serious side effects, for example for arthritis (> 2400mg), a prospective nutraceutical use of oleocanthal is highly desirable.

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Also, an increase of the knowledge on anti-tumor activities of oleocanthal, through different cell models, is mandatory, in order to elucidate its molecular mechanisms of action and envisage possible medical applications. In fact, oleocanthal was shown to induce an opposite and cell-dependent effect on p38 phosphorylation, positive on human cultured chondrocytes [98] and inhibitory in human colon adenocarcinoma cells [9]. A similar opposite and cell line-dependent effect was reported more recently in a study aimed to verify the possible estrogenic properties of oleocanthal: using gene reporter systems, binding of oleocanthal to estrogen receptors was demonstrated, with both estradiol-agonistic or antiagonistic effects [101], possibly because of cell-specific differences in EVOOLS metabolism. As long as a single component of a food item should not be considered as an active ingredient of a drug, both in vitro and in vivo studies are mandatory, in order to compare the effects of the entire food, of all the components and their content and also the absorption and the interaction with other components of the food itself. In fact, especially for the cardiovascular level, results (probably exacerbated by at least the antiinflammatory and the LDL-binding activities of EVOOLS) strongly support the idea that the activities of a food item do not reside in individual molecules that compose it but in the complex mixture of phenols, that act synergistically for beneficial effects on human health. In this context, an olive oil containing a specific combination of EVOOLS, with a proper concentration of active compounds or/and natural excipients, possibly modulating ad hoc the activity of endogenous olive enzymes [102], could work usefully as a nutraceutical in cardiovascular disease or cancer prevention. With this respect, we have recently shown that oleocanthal is, among nutritionally relevant EVOOLS, one whose concentration is more influenced by the technological preparation procedures and by the genetic background and environmental plant growing conditions [103, 104], allowing to easily obtain samples with different ratios of phenols vs squalene or tocopherols, useful for the in vitro testing of the synergistical actions of EVOOLS.

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[40] Elnagar AY, Sylvester PW, El Sayed KA. (-)-Oleocanthal as a c-Met inhibitor for the control of metastatic breast and prostate cancers. Planta Med. 2011;77(10):1013-9. [41] Pei T, Meng Q, Han J, Sun H, Li L, Song R, et al. (-)-Oleocanthal inhibits growth and metastasis by blocking activation of STAT3 in human hepatocellular carcinoma. Oncotarget. 2016;7(28):43475-91. [42] Fogli S, Arena C, Carpi S, Polini B, Bertini S, Digiacomo M, et al. Cytotoxic Activity of Oleocanthal Isolated from Virgin Olive Oil on Human Melanoma Cells. Nutr Cancer. 2016;68(5):873-7. [43] Gu Y, Wang J, Peng L. (-)-Oleocanthal exerts anti-melanoma activities and inhibits STAT3 signaling pathway. Oncol Rep. 2017;37(1):483-91. [44] Khanfar MA, Bardaweel SK, Akl MR, El Sayed KA. Olive Oil-derived Oleocanthal as Potent Inhibitor of Mammalian Target of Rapamycin: Biological Evaluation and Molecular Modeling Studies. Phytother Res. 2015;29(11):1776-82. [45] Fabiani R, Rosignoli P, De Bartolomeo A, Fuccelli R, Servili M, Montedoro GF, et al. Oxidative DNA damage is prevented by extracts of olive oil, hydroxytyrosol, and other olive phenolic compounds in human blood mononuclear cells and HL60 cells. J Nutr. 2008;138(8):1411-6. [46] Jacomelli M, Pitozzi V, Zaid M, Larrosa M, Tonini G, Martini A, et al. Dietary extra-virgin olive oil rich in phenolic antioxidants and the aging process: long-term effects in the rat. J Nutr Biochem. 2010;21(4):290-6. [47] Salvini S, Sera F, Caruso D, Giovannelli L, Visioli F, Saieva C, et al. Daily consumption of a high-phenol extra-virgin olive oil reduces oxidative DNA damage in postmenopausal women. Br J Nutr. 2006;95(4):742-51. [48] Warleta F, Quesada CS, Campos M, Allouche Y, Beltran G, Gaforio JJ. Hydroxytyrosol protects against oxidative DNA damage in human breast cells. Nutrients. 2011;3(10):839-57. [49] Erol O, Arda N, Erdem G. Phenols of virgin olive oil protects nuclear DNA against oxidative damage in HeLa cells. Food Chem Toxicol. 2012;50(10):3475-9. [50] Menendez JA, Vazquez-Martin A, Oliveras-Ferraros C, Garcia-Villalba R, Carrasco-Pancorbo A, Fernandez-Gutierrez A, et al. Analyzing effects of extravirgin olive oil polyphenols on breast cancer-associated fatty acid synthase protein expression using reverse-phase protein microarrays. Int J Mol Med. 2008;22(4):433-9. [51] Menendez JA, Vazquez-Martin A, Oliveras-Ferraros C, Garcia-Villalba R, Carrasco-Pancorbo A, Fernandez-Gutierrez A, et al. Extra-virgin olive oil polyphenols inhibit HER2 (erbB-2)-induced malignant transformation in human breast epithelial cells: relationship between the chemical structures of extra-virgin olive oil secoiridoids and lignans and their inhibitory activities on the tyrosine kinase activity of HER2. Int J Oncol. 2009;34(1):43-51.

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[52] Menendez J, Vazquez-Martin A, Garcia-Villalba R, Carrasco-Pancorbo A, Oliveras-Ferraros C, Fernandez-Gutierrez A, et al. tabAnti-HER2 (erbB-2) oncogene effects of phenolic compounds directly isolated from commercial ExtraVirgin Olive Oil (EVOOO). BMC Cancer. 2008;8(1):377. [53] Martin-Pelaez S, Covas MI, Fito M, Kusar A, Pravst I. Health effects of olive oil polyphenols: recent advances and possibilities for the use of health claims. Mol Nutr Food Res. 2013;57(5):760-71. [54] Ross SM. Effects of extra virgin olive oil phenolic compounds and the Mediterranean diet on cardiovascular health. Holist Nurs Pract. 2013;27(5):303-7. [55] Oliveras-Lopez MJ, Molina JJ, Mir MV, Rey EF, Martin F, de la Serrana HL. Extra virgin olive oil (EVOOO) consumption and antioxidant status in healthy institutionalized elderly humans. Arch Gerontol Geriatr. 2013;57(2):234-42. [56] Widmer RJ, Freund MA, Flammer AJ, Sexton J, Lennon R, Romani A, et al. Beneficial effects of polyphenol-rich olive oil in patients with early atherosclerosis. Eur J Nutr. 2013;52(3):1223-31. [57] de la Torre-Carbot K, Chavez-Servin JL, Jauregui O, Castellote AI, LamuelaRaventos RM, Nurmi T, et al. Elevated circulating LDL phenol levels in men who consumed virgin rather than refined olive oil are associated with less oxidation of plasma LDL. J Nutr. 2010;140(3):501-8. [58] Meisinger C, Baumert J, Khuseyinova N, Loewel H, Koenig W. Plasma oxidized low-density lipoprotein, a strong predictor for acute coronary heart disease events in apparently healthy, middle-aged men from the general population. Circulation. 2005;112(5):651-7. [59] Gonzalez-Santiago M, Fonolla J, Lopez-Huertas E. Human absorption of a supplement containing purified hydroxytyrosol, a natural antioxidant from olive oil, and evidence for its transient association with low-density lipoproteins. Pharmacol Res. 2010;61(4):364-70. [60] de la Torre-Carbot K, Chavez-Servin JL, Jauregui O, Castellote AI, LamuelaRaventos RM, Fito M, et al. Presence of virgin olive oil phenolic metabolites in human low density lipoprotein fraction: determination by high-performance liquid chromatography-electrospray ionization tandem mass spectrometry. Anal Chim Acta. 2007;583(2):402-10. [61] Miro-Casas E, Covas MI, Farre M, Fito M, Ortuno J, Weinbrenner T, et al. Hydroxytyrosol disposition in humans. Clin Chem. 2003;49(6 Pt 1):945-52. [62] Monti MC, Margarucci L, Tosco A, Riccio R, Casapullo A. New insights on the interaction mechanism between tau protein and oleocanthal, an extra-virgin oliveoil bioactive component. Food Funct. 2011;2(7):423-8. [63] Li W, Sperry JB, Crowe A, Trojanowski JQ, Smith AB, 3rd, Lee VM. Inhibition of tau fibrillization by oleocanthal via reaction with the amino groups of tau. J Neurochem. 2009;110(4):1339-51.

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[64] Bates KA, Verdile G, Li QX, Ames D, Hudson P, Masters CL, et al. Clearance mechanisms of Alzheimer’s amyloid-beta peptide: implications for therapeutic design and diagnostic tests. Mol Psychiatry. 2009;14(5):469-86. [65] Pitt J, Roth W, Lacor P, Smith AB, 3rd, Blankenship M, Velasco P, et al. Alzheimer’s-associated Abeta oligomers show altered structure, immunoreactivity and synaptotoxicity with low doses of oleocanthal. Toxicol Appl Pharmacol. 2009;240(2):189-97. [66] Abuznait AH, Qosa H, Busnena BA, El Sayed KA, Kaddoumi A. Olive-oil-derived oleocanthal enhances beta-amyloid clearance as a potential neuroprotective mechanism against Alzheimer’s disease: in vitro and in vivo studies. ACS Chem Neurosci. 2013;4(6):973-82. [67] Diomede L, Rigacci S, Romeo M, Stefani M, Salmona M. Oleuropein Aglycone Protects Transgenic C. elegans Strains Expressing Aβ42 by Reducing Plaque Load and Motor Deficit. PLoS ONE. 2013;8(3):e58893. [68] Batarseh YS, Mohamed LA, Al Rihani SB, Mousa YM, Siddique AB, El Sayed KA, et al. Oleocanthal ameliorates amyloid-beta oligomers’ toxicity on astrocytes and neuronal cells: In vitro studies. Neuroscience. 2017;352:204-15. [69] Luzon-Toro B, Geerlings A, Hilfiker S. Hydroxytyrosol increases norepinephrine transporter function in pheochromocytoma cells. Nucl Med Biol. 2008;35(7):801-4. [70] Dewapriya P, Himaya SW, Li YX, Kim SK. Tyrosol exerts a protective effect against dopaminergic neuronal cell death in in vitro model of Parkinson’s disease. Food Chem. 2013;141(2):1147-57. [71] Selma MV, Espin JC, Tomas-Barberan FA. Interaction between phenolics and gut microbiota: role in human health. J Agric Food Chem. 2009;57(15):6485-501. [72] Medina E, de Castro A, Romero C, Brenes M. Comparison of the concentrations of phenolic compounds in olive oils and other plant oils: correlation with antimicrobial activity. J Agric Food Chem. 2006;54(14):4954-61. [73] Romero C, Medina E, Vargas J, Brenes M, De Castro A. In vitro activity of olive oil polyphenols against Helicobacter pylori. J Agric Food Chem. 2007;55(3):680-6. [74] Karaosmanoglu H, Soyer F, Ozen B, Tokatli F. Antimicrobial and antioxidant activities of Turkish extra virgin olive oils. J Agric Food Chem. 2010;58(14):823845. [75] Vissers MN, Zock PL, Roodenburg AJ, Leenen R, Katan MB. Olive oil phenols are absorbed in humans. J Nutr. 2002;132(3):409-17. [76] Vissers MN, Zock PL, Katan MB. Bioavailability and antioxidant effects of olive oil phenols in humans: a review. Eur J Clin Nutr. 2004;58(6):955-65. [77] Visioli F, Galli C. Olive oil: more than just oleic acid. Am J Clin Nutr. 2000;72:853.

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[78] Visioli F, Galli C, Grande S, Colonnelli K, Patelli C, Galli G, et al. Hydroxytyrosol excretion differs between rats and humans and depends on the vehicle of administration. J Nutr. 2003;133(8):2612-5. [79] Manna C, Galletti P, Maisto G, Cucciolla V, D’Angelo S, Zappia V. Transport mechanism and metabolism of olive oil hydroxytyrosol in Caco-2 cells. FEBS Lett. 2000;470(3):341-4. [80] Tuck KL, Freeman MP, Hayball PJ, Stretch GL, Stupans I. The in vivo fate of hydroxytyrosol and tyrosol, antioxidant phenolic constituents of olive oil, after intravenous and oral dosing of labeled compounds to rats. J Nutr. 2001;131(7):1993-6. [81] Visioli F, Galli C, Bornet F, Mattei A, Patelli R, Galli G, et al. Olive oil phenolics are dose-dependently absorbed in humans. FEBS Lett. 2000;468(2-3):159-60. [82] Garcia-Villalba R, Carrasco-Pancorbo A, Nevedomskaya E, Mayboroda OA, Deelder AM, Segura-Carretero A, et al. Exploratory analysis of human urine by LC-ESI-TOF MS after high intake of olive oil: understanding the metabolism of polyphenols. Anal Bioanal Chem. 2010;398(1):463-75. [83] Soler A, Romero MP, Macià A, Saha S, Furniss CSM, Kroon PA, et al. Digestion stability and evaluation of the metabolism and transport of olive oil phenols in the human small-intestinal epithelial Caco-2/TC7 cell line. Food Chemistry. 2010;119(2):703-14. [84] de la Torre R. Bioavailability of olive oil phenolic compounds in humans. Inflammopharmacology. 2008;16(5):245-7. [85] Serra A, Rubio L, Borras X, Macia A, Romero MP, Motilva MJ. Distribution of olive oil phenolic compounds in rat tissues after administration of a phenolic extract from olive cake. Mol Nutr Food Res. 2012;56(3):486-96. [86] de Bock M, Thorstensen EB, Derraik JG, Henderson HV, Hofman PL, Cutfield WS. Human absorption and metabolism of oleuropein and hydroxytyrosol ingested as olive (Olea europaea L.) leaf extract. Mol Nutr Food Res. 2013;57(11):2079-85. [87] Bendini A, Cerretani L, Carrasco-Pancorbo A, Gomez-Caravaca AM, SeguraCarretero A, Fernandez-Gutierrez A, et al. Phenolic molecules in virgin olive oils: a survey of their sensory properties, health effects, antioxidant activity and analytical methods. An overview of the last decade. Molecules. 2007;12(8):1679719. [88] Mateos R, Goya L, Bravo L. Metabolism of the olive oil phenols hydroxytyrosol, tyrosol, and hydroxytyrosyl acetate by human hepatoma HepG2 cells. J Agric Food Chem. 2005;53(26):9897-905.

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[89] Garcia-Villalba R, Carrasco-Pancorbo A, Oliveras-Ferraros C, Menendez JA, Segura-Carretero A, Fernandez-Gutierrez A. Uptake and metabolism of olive oil polyphenols in human breast cancer cells using nano-liquid chromatography coupled to electrospray ionization-time of flight-mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci. 2012;898:69-77. [90] Covas MI, de la Torre K, Farre-Albaladejo M, Kaikkonen J, Fito M, Lopez-Sabater C, et al. Postprandial LDL phenolic content and LDL oxidation are modulated by olive oil phenolic compounds in humans. Free Radic Biol Med. 2006;40(4):608-16. [91] Ferro-Luzzi A, Sette S. The Mediterranean Diet: an attempt to define its present and past composition. Eur J Clin Nutr. 1989;2:13-29. [92] Owen RW, Mier W, Giacosa A, Hull WE, Spiegelhalder B, Bartsch H. Phenolic compounds and squalene in olive oils: the concentration and antioxidant potential of total phenols, simple phenols, secoiridoids, lignansand squalene. Food and Chemical Toxicology. 2000;38(8):647-59. [93] Sacchi R, Paduano A, Savarese M, Vitaglione P, Fogliano V. Extra virgin olive oil: from composition to “molecular gastronomy”. Cancer Treat Res. 2014;159:325-38. [94] Cummings JH, Banwell JG, Segal I, Coleman N, Englyst HN, Macfarlane GT. The amount and composition of large bowel contents in man. Gastroenterology. 1990;98:A408. [95] Monti SM, Ritieni A, Sacchi R, Skog K, Borgen E, Fogliano V. Characterization of phenolic compounds in virgin olive oil and their effect on the formation of carcinogenic/mutagenic heterocyclic amines in a model system. J Agric Food Chem. 2001;49(8):3969-75. [96] De Lorenzo A, Alberti A, Andreoli A, Iacopino L, Serrano P, Perriello G. Food habits in a southern Italian town (Nicotera) in 1960 and 1996: still a reference Italian Mediterranean diet? Diabetes Nutr Metab. 2001;14(3):121-5. [97] Skoldstam L, Hagfors L, Johansson G. An experimental study of a Mediterranean diet intervention for patients with rheumatoid arthritis. Ann Rheum Dis. 2003;62(3):208-14. [98] Iacono A, Gomez R, Sperry J, Conde J, Bianco G, Meli R, et al. Effect of oleocanthal and its derivatives on inflammatory response induced by lipopolysaccharide in a murine chondrocyte cell line. Arthritis Rheum. 2010;62(6):1675-82. [99] Corona G, Spencer JP, Dessi MA. Extra virgin olive oil phenolics: absorption, metabolism, and biological activities in the GI tract. Toxicol Ind Health. 2009;25(4-5):285-93. [100] Rigacci S. Olive Oil Phenols as Promising Multi-targeting Agents Against Alzheimer’s Disease. Adv Exp Med Biol. 2015;863:1-20.

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[101] Keiler AM, Djiogue S, Ehrhardt T, Zierau O, Skaltsounis L, Halabalaki M, et al. Oleocanthal Modulates Estradiol-Induced Gene Expression Involving Estrogen Receptor alpha. Planta Med. 2015;81(14):1263-9. [102] Ramírez E, Brenes M, García P, Medina E, Romero C. Oleuropein hydrolysis in natural green olives: Importance of the endogenous enzymes. Food Chemistry. 2016;206:204-9. [103] Ambra R, Natella F, Lucchetti S, Forte V, Pastore G. alpha-Tocopherol, betacarotene, lutein, squalene and secoiridoids in seven monocultivar Italian extravirgin olive oils. Int J Food Sci Nutr. 2016;8:1-8. [104] Raffo A, Bucci R, D’Aloise A, Pastore G. Combined effects of reduced malaxation oxygen levels and storage time on extra-virgin olive oil volatiles investigated by a novel chemometric approach. Food Chemistry. 2015;182:257-67.

In: Handbook of Olive Oil Editor: Jozef Miloš

ISBN: 978-1-53612-356-2 © 2017 Nova Science Publishers, Inc.

Chapter 11

HEALTH AND ECONOMIC IMPACT: ORGANIC OLIVE OIL Marilene Lorizio Department of Law, University of Foggia, Italy

ABSTRACT Agriculture not only responds to meet food needs, but it must also respond to the needs and institutional context. The agricultural sector supports and creates employment opportunities and also the economic viability of rural areas with low settlement. In this scenario, organic olive oil area is one of the most important of the agricultural sector because olive oil is the link between health and nutrition. The aim of the paper is to investigate organic olive oil sector, thus to demonstrate it’s importance in terms of wealth and economic impact.

INTRODUCTION Today, agriculture is called to respond to new and different needs that affect the institutional conditions that form the context in which it operates. In fact, the role of agriculture within the economic system of a country is somewhat different compared to the common belief according to which the agricultural sector performs almost exclusively to the primary function of the fulfillment of the food requirements. Today, agriculture is called to respond to new and different needs that affect the institutional conditions that form the context in which it operates. In fact, the role of agriculture within the economic 

[email protected].

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system of a country is somewhat different compared to the common belief according to which the agricultural sector performs almost exclusively to the primary function of the fulfillment of the food requirements. The agricultural sector is examined as a whole as a supplier of many secondary products and services, such as the support and creation of employment opportunities, the economic survival of the low settlement rural areas and rural development. It also plays a multifunctional role that, recently, the company attributes to ensure maximum compliance with food safety standards, environmental regulations and those on the wellbeing of the community while preserving the beauty of the landscape and contributing to the vitality of rural areas. The result is a vision and a consideration of the broader agricultural and multifaceted. The agricultural sector is facing many challenges and difficulties. They range from recent addresses in food consumption with related research of the “quality” of agricultural products, the increasing specialization of production phases, which translates into a reformulation of the vertical chain, to the central agricultural policies. Especially the quality is strictly subjective requirement is becoming more and more the structure of a connotation objective guarantee and reliability.

AGRICULTURE: A NEW VISION The new and different needs that agriculture today is called to respond affect the strategic importance of farming and research planning. The stringent objective, traditionally attributed to the sector of maximization of production for the purpose of meeting the food needs, while still claiming and especially its extraordinary relevance, comes from one side to compete with new production ideologies, which enhance the variety and peculiarities of input, emphasizing in particular the local dimension, the origin and history. On the other hand, the same primary function - producing food prices - is to combine with other needs, now reputed equally fundamental environmental issues and food safety issues. This accumulation of circumstances leads to a reformulation of agricultural activities. The new role of agriculture in different economic and social systems affects the sector configuration and farmer's decisions and strategies, attributing more and more attention to the production of positive externalities. This is a relatively new requirement for the sector, given the inadequacy - welcome shift over time – of the traditional model of agriculture in producing changes and improvements which ensure greater environmental and social sustainability. It would seem therefore taking place in the agricultural sector a paradigm shift based on new needs:

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Reorganization of the agricultural activity in multifunctional terms; Reformulation of priority orders related to the different functions attributed to the sector; Identifying new and different trajectories of technical and technological progress development that will characterize the new paradigm.

The multifunctionality of agriculture and integrated rural development programming are therefore of extreme importance and relevance. With the notion of multifunctionality you are essentially wishes understanding of agriculture's ability to respond to the community needs not only in productive and social terms, but also environmental. And because of the nature of the needs that meets, agriculture has always been, and continues to be, a politically strategic sector on which to intervene. Relevant, latest requirements emerged related to the sector. In fact, the recent need by the community for a better environment, and more particularly to a more attentive care of the land, has given new motivation to support agriculture. The agricultural activity is implemented by businesses throughout the territory, and it also takes side tasks producer of public goods and the protection and guarantee of public health (Tripoli et al. 2005). In recent years, the development of consumption, in a global negative trend, is characterized by the good performance of sectors like organic food or well-being. The greatest transformation has taken place in the agro-food industry. This sector, is driven by a growing criticism over its impact on the environment and on health, and pressured by stringent demands from public opinion and the political world, has increasingly based its own choices on the criteria of social responsibility. The agricultural sector today provides considerable non-monetary assets, such as job opportunities and the economic survival of some rural areas with low settlement and rural development. These values are priceless because they relate to the quality of life of present and future generations. Finally, the agricultural sector has the multifunctional role of ensuring compliance with food and environmental safety standard. Therefore, it now faces new challenges and difficulties affecting the strategic importance of agricultural planning. Indeed, on the one hand the primary objective of satisfying food demand is faced with new productive philosophies which highlight the variety and the particularity of the inputs, their local feature, their source and their history; on the other, the same primary objective is linked to other strategic objectives, such as environmental and food safety issues. All this involves a different characterization of agricultural activities and of the role of agriculture itself in the various economic and social systems. The configuration of the sector is changing to meet new demands of increased environmental and social sustainability, which were not covered by the traditional production model. Growing consideration of the ‘‘social function’’ of agriculture has increased over the years, not only in Italy, but in many European countries. It is seen as a way of life, as heritage, as a cultural identity, as a safeguard of the ecosystem. From this point of view, agriculture produces an added value,

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which is not directly connected to economic features, but rather is linked to issues of inclusion and social cohesion. In this scenario, organic olive oil area is one of the most important of the agricultural sector because olive oil is the link between health and nutrition.

LITERATURE There is an increasing focus of the modern consumer to the diet-health relationship (Belletti and Marescotti, 1996). The main responses to these evolutionary signals have been developing foods dedicated to the maintenance of health and the introduction into the market of a large quantity of products bearing the label nutrition and functionality: functional and health claims (Katan and De Roos, 2004). From a nutritional standpoint, olive oil exploitation begins from studies conducted in the '50s by American nutritionist Dr. Ancel Keys who, in carrying out research on cardiovascular disease, had observed how the Mediterranean populations were less exposed to cardiac problems because their diet was, at first sight, low in fat. On this basis, the US Department of Agriculture (USDA) in 1992 drew up a guide designed to orient the people to make dietary choices that maintain good health and reduce the risk of chronic diseases. It was the so-called food pyramid, subsequently revised and completed that, in addition to emphasize the need to carry out a regular and daily exercise, is very close to the Mediterranean Diet. This type of diet is reconnected to secular eating habits of the peoples of the Mediterranean basin and it is characterized by abundance of plant foods from cereals, legumes, fruits, vegetables, as well as marine-derived foods such as fish and a very common type of fat, such as olive oil. This diet contains little saturated fatty acids, is rich in complex carbohydrates and fiber, and has a high content of monounsaturated fatty acids that are derived primarily from the assumption of olive oil. Modern nutritional science on nutrition claims that the main part of the lipid portion should come from vegetable oils and preferably olive oil. As is known, the olive oil is for food tradition and connection with the territory, one of the food and agriculture undisputed protagonist of the Mediterranean Diet (Keys, 1995). Most nutritional guidelines (Food Based Dietary Guidelines) of Western countries recommend replacing saturated fats with monounsaturated fats, polyunsaturated. These guidelines encourage above all the consumption of extra virgin olive oil through the enhancement of its nutritional properties (Hite et al., 2010). This would bring the socalled model of the Mediterranean diet (less cardiovascular disease, obesity, and reducing inflammatory markers, including the commonly accepted positive effects). The European Food Safety Authority (EFSA), the highest scientific body in the field of food safety in Europe, has determined that for normal operation/good health of an organ/body function,

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can be used to boast nutritional oil extra virgin olive oil in the following Table (EFSA, 2011). Although only the presence of polyphenols is exclusive oil (extra virgin) olive, the other two features (presence of vitamin E and monounsaturated fats) determine the healthiness and attractiveness of the product to consumers. Health authorities national base their communications/suggestions about what and how much fat to consume on all three of these health aspects (Garcia et al. 2002). Studies on the relationship between diet and the incidence of cardiovascular disease and cancer have highlighted the crucial role of diet as a possible contributing factor in the onset of these processes. Until recently, fats and vegetable oils were considered irrelevant but recent analyzes suggest the possibility that olive oil produces a protective effect against some types of cancer and, in particular, of breast cancer (Trichopoulou, 1997). The nutritional properties of olive oil are determined by its very balanced composition characteristics and its use as the main source of dietary fat, within the recommended limits of intake of lipid level, plays an important role in providing protection to a diet health of consumers of all ages. Table 1. Nutritional Features and Recommended doses NUTRITIONAL ELEMENT

Vitamin E

REQUIRED PORTION FOR A BENEFICIAL EFFECT At least 15% of 20 mg, equal to 3 mg

Monounsaturated and Polyunsaturated Fats

--------

Olive Oil Polyphenols

5 mg of hydroxytyrosol and derivatives (oleoeuropeina, tyrosol) per day by eating a balanced diet

HEALTHY EFFECTS RECOGNIZED Extra virgin olive oil is a food rich in vitamin E, which protects the body cells from oxidative damage Replacing saturated fats with monounsaturated fats and polyunsaturated content in oil extra virgin oil can help maintain normal levels of LDL cholesterol in the blood The olive oil polyphenols can prevent oxidative stress, they have antioxidant effects, improve fat metabolism, and protect the fraction LDL from oxidative damage.

Extra virgin olive oil is the seasoning with the best balance of fat. It is particularly rich in monounsaturated fatty acids, which among fatty substances are the most active in the prevention of cardiovascular disorders, and poor instead of saturated fats, responsible for the increase of cholesterol levels in the blood and directly linked to issues such as the occlusion of the arteries, atherosclerosis, and myocardial infarction. Extra virgin olive oil contains very few, to the benefit of health. Particularly abundant in oleic acid, a monounsaturated fat that can regulate cholesterol levels (reduces the level of LDL cholesterol, “bad,” compared to the level of HDL cholesterol, the “good”).

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Olive oil is a precious nourishment for children, thanks to the great contribution of oleic acid, also present in breast milk; it is appropriate also for sportsmen, because it represents a unique source of energy readily digestible. Extra virgin olive oil is the only condiment that plugs into the weaning of infants because it contains essential polyunsaturated fatty w6 and w3 in proper relationship to each other, as is the case in human milk. In the old age it is equally important, since it limits the loss of calcium in the bones. The oil-renowned oil as a Mediterranean product with potential health benefits has now crossed the borders of cardiovascular and oncological to get to be studied even as a remedy or preventive for many other conditions. More and more studies showing favorable effects from the consumption of olive oil in respect of hypertension, diabetes, obesity, gastroduodenal ulcer, gall stones, rheumatoid arthritis, up to brain cognitive deficits. Olive oil also exerts a positive effect on the tone and gallbladder activity, due to its cholagogue properties; in fact exists an inverse relationship between consumption of vegetable fats and incidence of gallstones. While saturated fatty acids stimulate the formation of gallstones, unsaturated fatty acids oppose it. In addition, the regular use of olive oil would seem to exert a positive action in reducing - beyond, of course, the genetic predisposition - the risk of onset of diabetes mellitus (insulin-dependent). Biochemical and clinical studies conducted in the US and Europe on different populations show that a diet with high content of saturated fats, common in many countries of Western Europe and North America, raises the LDL cholesterol, favoring the onset of hypertension, a tendency to thrombosis, a cell multiplication which favors arterial lesions. The olive oil may also exert, thanks to a high content of oleic acid and the simultaneous presence of antioxidant substances, a protective role against neurodegenerative diseases such as Alzheimer and Parkinson. Extra virgin olive oil is, finally, a kind of elixir of life because thanks to the important heritage of substances with high antioxidant, prevent and combat many chronic diseases that occur with advancing years. In addition to oleic acid and its benefits, including linoleic acid, contained in olive oil, it would be beneficial to health, especially with regard to autoimmune diseases. These acids are called essential fatty acids because they cannot be synthesized by the body and must necessarily come from the diet, otherwise the occurrence of deficiencies. The beneficial effects of olive oil would therefore attributable, at least as regards its main components, the balance between saturated fatty acids, monounsaturated and polyunsaturated, and between those agents and antioxidants. Olive oil is the only oil obtained by cold extraction from a fruit with only mechanical means and can be consumed unrefined. Define the olive oil simply a “seasoning” certainly seems an understatement given that its nutritional properties and its beneficial effects go far beyond those attributable to the individual components therein. It can therefore rightly say that olive oil represents a natural “functional food” or a nutraceutical offered by nature to fortify the human organism.

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LINKS BETWEEN HEALTH AND NUTRITION The link between diet and health has been recognized since ancient times and it is always more to the modern consumer attention, for which the power must not only meet the nutritional requirements but also be a tool for the prevention and even the treatment of diseases and disorders. From this point of view the so-called functional foods are an important tool available to the consumer, in conjunction with a good lifestyle, to achieve their objectives healthful. The success of this food category depends, among other reasons, the progressive aging of the population (Cox et al. 2004), the negative impact on the health of lifestyles inadequate (Zou and Hobbs 2006), as well as the development of scientific knowledge (Murase et al. 2008) and the agro-food technology that makes possible the development of innovative foods (Syrian et al. 2008). For these reasons, functional foods is an area in continuous development and not just in rich countries, despite the often high information asymmetry that characterizes them and characterizing food chains in general. The term functional food was introduced in Japan and has been applied in numerous definitions classified according to four main dimensions (Doyon and Labrecque 2008): to the characteristics, the health benefits, the level of functional attributes in and aptitude for consumption, namely the fact that the food is in itself part of the normal diet. A functional food is, therefore, a common food 'modified' or 'innovated' in order to provide the consumer with additional nutritional benefits beyond those. There are different types of functional foods: Siro et al. (2008) distinguish between fortified products (products in which it is increased the content of a component/nutrient already present), enriched products (in which instead was added a nutrient/component normally absent), altered products (for which has been reduced or removed a 'harmful' component), and enhanced commodities (foods in which the agricultural raw material has been improved by means of appropriate cultivation or breeding techniques, genetic manipulation, different composition of feed or other factors of production). This classification also distinguishes between foods that improve the lives foods that reduce health-risks and foods that make life easier and feeding. For all its nutritional and healthful qualities, extra virgin olive oil has obtained one of the most important international awards directly from the Food and Drug Administration (FDA), the “Qualified Health Claim.” From millennia protagonist in the Mediterranean table, olive oil is a useful coverage for the containment of various types of diseases for which it is necessary correct information. Correct information should cover producers, in order to improve the quality of their product, and consumers, and should never stop to consider it a food of great importance and a valuable ally for their own health. In extra virgin olive important components for health are polyphenols, which give the characteristic flavor to the oil: the more there are the more spicy and fruity. Polyphenols are a family of chemical compounds strongly revalued because they are antioxidants;

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then, they fight the free radicals of organism, which are capable of activating forms of cancer and other diseases. They are able to reduce bad cholesterol (LDL) circulating in the blood, which thus remains more smoothly and with less risk of heart attacks. The Italian olives, especially the South-Central ones, generally contain more polyphenols. In fact, in Apulia, the region that alone provides almost half the National oil, the type of olives most widely is the so-called Coratina, which has a high content of polyphenols, and therefore the Apulian oil is spicy and fruity. Among other things, the polyphenols, with their antioxidant action, longer oil life, whose alteration is due, in fact, mainly to the action of oxygen. The other benefits of extra virgin oil components are unsaturated fatty acids. In particular, oleic acid, which is monounsaturated, is present for about 75%, and can be termed the “keeper” of the arteries as it binds to cholesterol in the blood and drags it away. Cholesterol is of two types: the “good” (HDL), which protects the arteries, and the “bad” (LDL), which tends to clog arteries with clot formation. it should be mentioned that olive oil contains absolutely no cholesterol and that oleic acid has the ability to not only reduce the level of bad cholesterol, but also raise the good cholesterol (Rhee et al. 1991). In addition, it makes the extra virgin olive oil more assimilable also facilitating the transport of the vitamins contained in it. A study carried out by the Faculty of Pharmacy at the University of Milan, has shown the antioxidant properties of phenols present in extra virgin olive oil. It has shown that in vitro phenols have antioxidant properties against the LDL, inhibit thrombus formation, fight inflammation and cause the increase of nitric oxide syntax, with vasodilating action, as well as reduction of free radicals. All the benefits of Oil Extra Virgin Olive Oil. The extra virgin olive oil, raw or heated, is the most suitable fat for feeding, not only for its aroma and its flavor, but also for the whole of its properties, including in particular its acid composition, with a predominance of monounsaturated fatty acids, a perfect balance of polyunsaturated fats, the content of vitamin E, provitamin a and antioxidants. The products made from olive: 

They help the circulation, they regulate blood pressure and are good for your heart: oleic acid prevents arterial injury, lowers blood pressure and reduces by about 30% the risk of heart attack, stroke, thrombosis, and coronary heart disease in general. The polyphenols and vitamin E in the oil extra virgin olive oil, thanks to their antioxidant, helping to prevent atherosclerosis and slow down the aging of cells; the diet rich in animal fat instead increases the amount of blood cholesterol, a major risk factor in cardiovascular diseases, while vegetable oils are rather protective action. The extra virgin olive oil, compared to other vegetable oils has further beneficial effects as oil produced by simply pressing and crushing of the fruit, without further chemical-physical manipulations. The

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seed oil is instead produced through the use of special chemicals and equipment, such as butane, propane, hexane They are the most powerful antioxidants in existence in nature. Extra virgin olive oil has antioxidant and anti-radical. The presence of tocopherols and polyphenols give extra virgin olive oil an important role in antioxidant and anti radical limiting cellular aging. Percentage of the levels of arthritis and osteoporosis. There are not yet clear the mechanism by which it works. Recent studies have shown that people who adopt a diet rich in extra virgin olive oil, are less likely to develop rheumatoid arthritis. They were also shown benefits for bone status with regard to the prevention of osteoporosis. In fact it seems that the regular intake of olive oil enhances the absorption of calcium by the body. It has anti-cancer effects. Many studies relate the use of extra virgin olive oil with a reduction of certain types of cancer.(Braga et al. 1998) In particular the breast. Moreover, the specific mortality is highest in northern European countries than in countries of the Mediterranean area. Metabolize lipids and carbohydrates and reduce sugar and fat in the blood. Food prepared with extra virgin olive oil have excellent gastric and intestinal tolerance (Serrano, 1997). In fact, olive oil protects the mucous membranes and avoids the effects, thus reducing the risk of gastric and duodenal ulcers. It exerts a laxative, more effective on an empty stomach and helps to correct the chronic constipation. It stimulates the gall bladder and inhibits the secretion of bile. He also protective effect against the formation of gallstones, due to activation of bile flow and an increase in high density lipoprotein (HDL). It is effective against diabetes. An oil-rich diet can reduce the risk of type 2 diabetes by nearly 50%. It was always thought that a high-fat diet could increase the risk of heart disease, cancer and diabetes. We now know that is not the amount of fat to affect our health but rather the quality. A diet rich in monounsaturated fatty acid diet such as extra virgin olive oil content and dried fruit, is able to protect the body from many of these diseases. Sharpen depression. A study all Spanish claims that a diet rich in unsaturated fatty acids that are present in virgin olive oil, fish and some vegetables, have properties that can be beneficial in the prevention of depression. They are diuretics and detoxifies the body. An appreciable characteristics of olive oil is definitely the fragrance that gives the dishes and this increases the palatability of food by promoting the digestive secretory stimuli, thus inducing a better digestibility. In fact it is also used as a medicinal oil to cleanse the digestive system. This improves bowel movement with preventive effects against constipation.

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The olive oil is effective against cancers of the skin, due to its high content of squalene. This is a substance that is abundant in olive oil (even more in shark liver, hence the name) would be effective in fighting cancer of the skin; in Italy and Greece, where the olive oil consumption is particularly high, life expectancy is higher than in Northern European countries, where it tends to consume a higher proportion of animal fat. All this despite the intense sun exposure of the two Mediterranean countries and a greater presence of smokers compared to Northern Europe. Sharpen the evolution of Alzheimer's. A study published in Chemical Neuroscience, showed how the oleocantale, the substance responsible for its itching in the throat, have properties useful in reducing the risk of developing Alzheimer's disease. Moreover, the Mediterranean diet is also known for its beneficial properties with regard to cognitive functions, particularly of older people and adults. A study conducted in France in adult and elderly patients has shown that regular use of extra virgin olive oil is able to improve visual memory and verbal ability.

Monounsaturated fats also make the extra virgin olive oil is particularly resistant to alterations. This means that, if properly stored, not rancid but especially stands up well to cooking, reaching high temperatures also (e.g., frying) without giving rise to the formation of harmful substances, which instead happens when using polyunsaturated fatrich oils (such as most seed oils). To distinguish extra virgin olive oil from other oils is also the content of polyphenols, very specific antioxidants. Normally, in nature, the oil is accumulated inside of a seed: it happens for the hazelnut, soy, and sunflower and so on. But the olive nature has thought of something new and very unusual: the fat concentrate mainly on the outside of the seed, in the pulp. This, however, provides an unstable fat and decidedly uncomfortable environment, due to the presence of water and enzymes that, in the moment in which the oil is removed from the tree, can continue their degrading action of the fruit. The olive fats thus arise in a disadvantageous situation compared to those of the seeds. It is at this point that come into the picture polyphenols, powerful natural antioxidants that can protect their fat when they are in an unfavorable environment, rich in water, such as in the olive pulp but also the fats found in ' aqueous environment of our cells. And so the polyphenols also become important for our health: protect the integrity of cell membranes, providing a defense against the formation and development of many types of cancer, and it seems they are also able to counteract the loss of memory and l ' alteration of other cognitive functions associated with aging. Polyphenols have a bitter flavor and spicy, so if an oil has these two notes of flavor, it means that polyphenols are present. Only high-quality oils containing at least 250 mg per kilogram of polyphenols can boast the label the presence of polyphenols. However, there are oils that contain many more, and they are real elixir of health.

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Italy has a well-established international reputation as a producer of olive oil. The Italian extra virgin olive oil has a strong penetration in the markets of the United Kingdom and Germany. One area with considerable potential is also represented by the production of biological oil. Through time, consumers have become more alert to not only environmental issues but also those related to food safety. This led to a simultaneous increase in both supply and demand for organic food products, driven by the need for more natural and healthy food. In the context of organic agriculture, the organic olive oil production is one of the most dynamic areas of agro-food Italian, which is characterized by a sudden, substantial and continuous growth and the number of organic farms and their size presents a significant and widespread increase in the national agricultural system. In addition to the production of organic olive oil, Italy is particularly competitive at the international level in other sectors which, together with olive oil, is generally attributed largely to the “made in Italy” (pasta, dairy, fresh fruit and vegetables, fruit and vegetables transformed, wines; meat preparations). Looking at the factors that drive consumers to choose organic foods, some have a subjective dimension, in the sense that directly affect the well-being of the person who consumes the product. Others have a public value, because they increase the welfare of the community and only indirectly the individual welfare. The first category includes features like the taste and food security (although this has actually also a public dimension), while the latter includes for example the benefits of using environmentally friendly practices or the promotion of local development. Positive externalities of organic production: the least environmental impact, the development of local agriculture, the practice of agriculture even in less productive areas, such as hills and mountains. With regard to the structural properties of the olive oil sector, it should be noted that the dynamics that characterize it are undergoing an evolution with important changes linked mainly to its international dimension. Almost all of the world's production is concentrated in the Mediterranean with 72% located in the European Union and 25% in the countries of North Africa and the Middle East; the remaining portion, of approximately 3%, is made in the new olive-growing areas, in a manner particular Australia, Chile and Argentina. For operators of the challenges the sector are increasing and being present on the market often means making choices segmentation up and decide to produce quality, also to meet the increasingly complex needs of consumers and evolved. Although the olive oil market can be considered mature and stable in Europe little under 2 million tonnes per year over the past five years - they are found some important trends about the rise in consumption, both on a global scale, both with no reference to the countries producing olive oil. In fact, slowly, the consumption and the production of olive oil have gone hand in hand, arriving in fact doubled in volume. At the same time, there has been the entry of new consumer countries, which has led to decrease the weight of the first 4 global consumers from 71% to 52% (Table 2).

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1991/92 2009/10

Total world First 4-producing countries Total world First 4-producing countries

PRODUCTION (tons) 2.206 86% 3.204 77%

CONSUMPTION (tons) 1.857 71% 2.873 52%

Often there is a conflict cases between dominant national culture (“use of the most widespread” fat) and perception of health, and this could lead the authorities responsible not to give hard enough indications “improvement” about the most significant fat from one point of view qualitative. From this point of view, olive oil and even more extra virgin olive oil leaving penalized and not fully recognized at a basic level of “evidence based policy making.” Thus, a timely initiative would bring to light the olive oil perceived in non-Latin cultures, to see if the positive aspect prevails (“monounsaturated fat and polyphenols”) or negative (“so fat”), and in what segments of the population. A better understanding of these mechanisms would lead to a better work of information and targeting of related messages. Considering the recent progress in the knowledge of its health properties, the initiatives in question could consist of targeted promotional campaigns in European countries and third countries, which seem more receptive to the consumption of olive oil, or have seen in recent years, increasing more imports and consumption (World Health Organization European Region, 2003) These campaigns could have an interesting public-private partnership, because of the synergies that would be created and the broader win-win situation between producers, consumers and national health actors (which could reduce disease and health care costs through increased dissemination of fat monounsaturated and generally extra-virgin olive oil). In all promotional campaigns nutritional aspects of extra virgin olive oils should be the main elements to be included in the programs, to enable consumers to choose the product, not only for its real characteristics, but also for the positive effects of some elements such vitamin E, monounsaturated/polyunsaturated fats, polyphenols. Aiming at better and better transparency to the consumer, which can allow him to make purchases more and more conscious about the price/quality ratio, and the promotion intended as training are key conditions to introduce this product.

REFERENCES Braga, C., La Vecchia C., Franceschi S. (1998), Olive oil, other seasoning fats and the risk of colorectal carcinoma. Cancer 82, 448-53.

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EFSA (2011) Scientific Opinion on the substantiation of health claims related to polyphenols in olive oil. EFSA Journal 2011;9(4):2033 [25 pp.]. doi:10.2903/j.efsa.2011.2033. Garcia, A., Brenes, M., Romero, C., Garcia, P., Garrido, A. (2002) Study of phenolic compounds in virgin olive oils of the Picual variety. European Food Research Technology 215:407-412. Hite A. H., Feinman R. D., Guzman G. E., Satin M., Schoenfeld P. A., Wood R. J. (2010). In the face of contradictory evidence: Report of the Dietary Guidelines for Americans Committee. Nutrition 26, 915–924. Keys A., (1995), Mediterranean diet and public health: personal reflections, Am. J. Clin. Nutr. 61: S 1321-3. Rhee J.C., Chang T.M., Lee Ky, Jo Yh, Chey Wy (1991), Mechanism of oleic acid induced inhibition of gastric acid secretion in rats, Am. J. Physiol. 260: G564-G570. Serrano P. (1997), Influence of type of dietary fat (olive and sunflower oil) upon gastric acid secretion release of gastrin, somatostatin, peptide y y in man, Dig. Dis. Sci.42: 626-633. Trichopoulou A., (1997), Olive oil and breast cancer, Cancer Causes Control, 6: 475-6. Tripoli, E., Gianmarco, M., Tabacchi, G., Di Majom D., Gianmarco, S., La Guardia, M. (2005). The phenolic compounds of olive oil: structure, biological activity and beneficial effects on human health. Nutrition Research Reviews 18, 98-112. World Health Organisation European Region. Food based dietary guidelines in the WHO European Region. Copenhagen: WHO, Europe, 2003.

In: Handbook of Olive Oil Editor: Jozef Miloš

ISBN: 978-1-53612-356-2 © 2017 Nova Science Publishers, Inc.

Chapter 12

POTENTIAL OF VIRGIN OLIVE OIL AGAINST CANCER: AN OVERVIEW OF IN VITRO AND IN VIVO STUDIES Houda Nsir, Amani Taamalli* and Mokhtar Zarrouk Laboratoire de Biotechnologie de l’Olivier, Centre de Biotechnologie de Borj-Cérdia, Tunisie

ABSTRACT Mediterranean diet (MD) represents the gold standard in preventive medicine due to its association with lower overall mortality patterns. Its role in human nutrition is one of the most important areas of investigation, therefore a wide range of epidemiological studies found an inverse correlation between the MD consumption and the incidence of certain cancers in populations living in the Mediterranean area, compared with populations living in Northern Europe or the USA, probably because of the harmonic combination of many elements with antioxidant and anti-inflammatory properties. Extra virgin olive oil (EVOO) stands for the main source of fat in MD, characterized by bioactive components particularly phenolic compounds, it has a potential preventive and functional action on Cancer disease, liver, breast, colon which present the second cause of death after cardiovascular diseases worldwide. The beneficial effects of EVOO have been attributed mostly to its phenolic fraction and its anti-proliferative properties causing apoptosis of human cancer cells. In this regard, this chapter presents an overview of benefits of olive oil against cancer, assessing and discussing the mechanism of action undersigning these effects as well as case studies.

Keywords: Mediterranean diet, olive oil, cancer, prevention, nutraceutical

* Corresponding Author address, Email:[email protected].

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INTRODUCTION Cancer is a group of diseases characterized by the uncontrolled growth and spread of abnormal cells, it’s a complex chronic-degenerative disease characterized by a multistep process in which normal cells are transformed into malignant cells acquiring several properties such as abnormal proliferation and reduced apoptosis (Fabiani,2016). Worldwide, one in seven deaths is due to cancer; cancer causes more deaths than AIDS, tuberculosis, and malaria combined, when countries are grouped according to income, cancer is the second leading cause of death in high-income countries following cardiovascular diseases (American cancer society, 2012) and the third leading cause of death in low-and middle-income countries following cardiovascular diseases and infectious and parasitic diseases. According to estimates from the International Agency for Research on Cancer (IARC), there were 14.1 million new cancer cases in 2012 worldwide. Actually, the incidence and mortality of colon cancer is rising in developing countries (Jin et al.,2016), bladder cancer is becoming the first leading cause of death among urinary malignancies (Goodison et al., 2013), glioblastoma multiforme (GBM) is the most common and malignant type of astrocytoma of the central nervous system (CNS) (Chandana et al., 2008) and thyroid cancer represent the most common endocrine malignancy which incidence in several countries has been increasing during the last 30 years (Altekruse et al., 2013). Moreover a study of Hoffmann and Schwingshackl shows that during the mean follow-up of 4.8±1.7 years, 35 new cases of malignant breast cancer were accounted for via medical records and death certificates. However, in women maintaining a Mediterranean Diet (MD) specially supplemented with extra-virgin olive oil, risk of invasive breast cancer was reduced by approximately 68% (Hoffmann and Schwingshackl, 2016). Cancer incidence has shown many variations in different geographical regions. These variations are certainly related to the human exposure to different modifiable factors which may either increase or decrease cancer risk. Cancer is caused by external factors and internal factor that may act together or in sequence such as tobacco, infectious organisms, and an unhealthy diet, inherited genetic mutations, hormones, and immune conditions moreover it highlighted that environmental factors and food are of more importance in cancer growth than genetic susceptibility (Elmore et al., 2007). To overcome some of the therapeutic challenges in the treatment of cancer, novel strategies and approaches are required to prolong survival (Ramachandran et al., 2012). Over the past few years, there has been a growing interest in nutraceutical interventions. These approaches use the anti-inflammatory and chemopreventive properties of naturally occurring agents (Surh, 2003).

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Actually, a wide range of epidemiological data support the hypothesis that MD could have an important role in preventing several types of cancers. A recent meta-analysis in a multiethnic cohort showed that the highest adherence to MD including the intake of vegetables and some related micronutrients such as vitamins A, C, E and carotenoids was significantly associated to the reduction of cancer risk and in particularly olive oil consumption is inversely related to cancer prevalence. (Psaltopoulou et al., 2011; Bavaresco et al., 2013). It has been also evaluated that over 30% of all cancers may be avoidable by changing food intake. (Willet, 1995). Typically, MD is characterized by a high intake of fruits, plant proteins, whole grains, fish, low-fat dairy, moderate alcohol (red wine) intake and low red meat consumption with a peculiar characteristic which is the EVOO that represent an integral ingredient of the traditional MD and several studies addressed many of the health advantages of this diet to EVOO’s unique characteristics (Buckland et al., 2015). Since ancient times, extra virgin olive oil (EVOO) has been considered to have characteristics between a food product and a medicine. EVOO has been widely known for its benefic properties, its antioxidant and anti inflammatory effect. The chemopreventive ability of EVOO is due to its different constituents: fatty acids, tocopherols, squalene, phenolic compounds (Rafehi et al., 2012). Among all dietary factors, polyphenols: flavonoids, phenolic acids, phenolic alcohol and lignans, gain the greatest attraction due to their antioxidant properties because of their chemical structures (Le Marchand L et al., 2000). Some studies have focused on the effect of fatty acids,oleic acid (OA) has been reported to act synergistically with cytotoxic drugs, thus enhancing their antitumor effect (Schwartz et al., 2004). It has been also shown that OA promoted the growth of nonmalignant cells but, it had the opposite effect, in malignant cells (Zeng et al., 2010).Last years have seen a renewed importance in the use of olive oil and phenolic compounds, such as oleocanthal, an anti-inflammatory compound which has a similar chemical structure to ibuprofen that activates adenosine monophosphate-activated protein kinase to downregulate COX-2 expression in HT-29 colon cancer cells (Dixon, 2004), hydroxytyrosol (HT),which represent a potent antioxidant and has several biological activities (Rietjens et al., 2007) that control oxidative stress, inhibits proliferation in several tumor cell lines and act as a chemotherapeutic agent, and apoptosis promoter (Anter et al., 2016). On the other side, during gastrointestinal digestion, phenolic compounds undergo intense phase I and phase II metabolism as a result of digestive or hepatic activity (López de las Hazas, Piñol, Macià, & Motilva, 2017). During gastric digestion, complex EVOO phenols for example oleuropein aglycone derivatives or secoiridoids are transformed into HT (López de las Hazas et al., 2016). All thesetransformations of dietary phenolics could lead to more potent microbial-inhibitory compounds, such as phenolic acids that have other potent properties.

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With all this in mind, this chapter sheds new light not only on the preventive effect of olive oil consumption and MD adhesion but also the alternative natural therapeutic approach by using the different bioactive compounds of EVOO against cancer disease through investigating in the potential compounds effects in different cancer and in the mechanisms behind the apoptotic event induced. fifteen in vivo and in vitro studies meet the objectives and were included to focus on effect of EVOO on cancer risk and mortality. Queries of literature were performed using the electronic databases: Pubmed, Science direct and Google scholar with no restriction to the language until 20 march 2017 using the following terms: olive oil and cancer, olive oil induces apoptotic pathway, Mediterranean diet and cancer, from 2012 to 2017.

IN VIVO AND IN VITRO STUDIES HIGHLIGHTING THE POTENTIAL EFFECT OF OLIVE OIL ON CANCER Olive oil have gained much attention in this recent years and studies consistently support the concept that the Olive Oil-rich MD is compatible with healthier aging and anticancer, for this purpose different studies investigated in its efficacy, previous in vivo studies have focused on colon cancer. Actually Hashim and other demonstrated that a daily dose of phenolic extract given to mice with cancer colon decreased significantly not only tumour volume but also the number of metastases by 79% compared to control,(Hashim et al., 2014) another work of barone and other showed a reduction in polyp number and volume through a reduction of proliferation and a marked proapoptotic effect in intestinal cancer after following an olive oil-enriched diet mice fed for 10 week(Barone al.,2014). Furthermore a recent study of Pei et al., 2016) suggested that a different concentrations of (-)-oleocanthal for five weeks given to mice developing tumor metastasis reduce the tumor and make fewer and smaller metastases (Table 1). In accordance with the in vivo practices in vitro studies came to confirm these issues and similarly investigated the individual olive oil compounds potential and studying the different pathways action. Recently, (Toteda et al., 2016) showed that High doses of hydroxytyrosol, promotes mitochondrial apoptotic cell death in human papillary and follicular thyroid cancer cells, moreover it decreased cell viability of thyroid cancer cells in a concentration-dependent manner. These doses induce 50% cell growth inhibition (IC50) against TPC-1, FB-2 and WRO cells after 24 h. As well, a study of (Moran et al., 2016) shows obvious cytotoxic effects on human osteosarcoma cells in a concentrationand time-dependent manner, when using oleuropein treatment on Osteoarcinomic cells.

Table 1. Olive oil and cancer: in vivo studies Author, year

Animals studies/ Human studies

Type of cancer

Compounds source and treatment Extra virgin olive oil 250 μl/300 g every day (10day treatment).

(Di Francesco et al., 2014)

18 females Sprague–Dawley rats weighting 225–250 g

Colon cancer

(Hashim et al., 2014)

Female SCID Balb-c mice (8–10 weeks old, 20–23 g body weight)

Colon cancer

Phenolic extract was given by daily gavage 25 mg per kg per day

Barone al, 2014

Five-week-old C57BL/6J male mice with a heterozygote mutation for the Apc gene (ApcMin/+) Developing intestinal polyps

Intestinal cancer

olive oil-enriched diet fed for 10 weeks with (12.5% protein, 12% olive oil, 3% cellulose fiber)

Outcomes -Increase in cannabinoid receptors (CB1) expression in the colon of rats receiving dietary EVOO supplementation for 10 days. - CpG methylation of rat Cnr1 promoter, miR23a and miR-301a, that was shown to be involved in the pathogenesis of colorectal cancer and predicted to target CB1 mRNA, was reduced after EVOO administration down to ~50% of controls -Significant decrease not only in tumour volume but also in the number of metastases by 79% in SCID Balb-c mice compared to the control -Reduction of cancer development in the ApcMin/+ mouse model. -Reduction in polyp number and volume through a reduction of proliferation and a marked proapoptotic effect.

Table 1. (Continued) Author, year

(Pei et al., 2016)

Animals studies/ Human studies

Injection of luciferase expressing HCCLM3 cells into the tail veins of nude mice and monitored tumor metastasis using bioluminescence imaging.

Type of cancer

Hepatocellular Carcinoma

Compounds source and treatment

Different concentrations of (-)-oleocanthal for 05 weeks.

Outcomes - These biological effects were mediated by an inhibition of fatty acid synthase and HMG CoA reductase gene expression and activity -increase of ERβ/ERαratio. -lower FAS and HMGCoA reductase mRNA levels than the group fed with a standard diet (P< 0.05) -The treated group had fewer and smaller metastases. -Illumination signals were stronger in control group than in the (-)-oleocanthaltreated group.

Table 2. Olive oil and cancer: in vitro studies Author, year

Cancer cells

Moran et al., 2016

Osteocarcinoma

Rosignoli et al., 2015

-Breast cancer: MDA,MCF7--Prostate cancer: LNCap, PC3-Colon cancer: SW480,HCT116,cell lines

Treatment Compounds used source Oleuropein, established by Oleuropein the weighted Probit method for MG-63 and Saos2 cells lines at 24, 48 and 72 h of exposure. Incubation of different doses of hydroxytyrosolwith the different cancer cells at different concentrations

Hydroxytyrosol

Outcomes -Exhibition of obvious cytotoxic effects on human osteosarcoma cells in a concentration- and timedependent manner. -Statistical analysis of IC50 by the Probit regression method suggested that oleuropein had similar toxic effects on both cell lines tested -Treatment of different human cancer cells with 25 μM of 3,4DHPEA did not significantly affect proliferation while the higher doses induced a dose dependent reduction of cell growth. -HCT116, MDA and MCF7 cells were more sensitive to 3,4DHPEA than LNCap, PC3 and SW4 80 cells. -At 50 μM the proliferation of cancer cells was inhibited by 38%, 44% and 34% in HCT116, MDA and MCF7 cells respectively,

Table 2. (Continued) Author, year

Cancer cells

Treatment used

Compounds source

(Terzuoli, colorectal Giachetti, Ziche, adenocarcinoma cells & Donnini, 2016)

Incubation with hydroxytyrosl dose from 1to 300 µM.

Yan et al., 2015

Cells were treated with (0, oleuropein 20, 40, 60, 80 or 100 µM) oleuropein for 24 h

HepG2 human hepatoma cell line

Hydroxytyrosol

Outcomes while for the other cells the proliferation remained above 80%. -The increment of 3,4DHPEA concentration up to 75 and 100 μM reduced the proliferation in all cell types. -Down regulation of EGFR expression in human colorectal adenocarcinoma cells HT-29, CaCo2, and WiDr, and in HT-29 xenografts. -Acceleration of EGFR degradation by reducing its half-life. -HT induces EGFR ubiquitination that is mediated by phosphorylation at pY1045, the docking site for Cbl, thereby enabling receptor ubiquitination and degradation. -Oleuropein effectively inhibited cell viability and induced apoptosis in HepG2 human hepatoma cells in a dose-dependent manner, through activation of the caspase pathway

Author, year

Cancer cells

Treatment used

Compounds source

Elamin et al., 2013

Breast cancer cells

Cells were incubated with oleuropein 150µM,200µM,250µM, 300µM Oleuropein for (24,48 and72h)

Toteda et al., 2016

papillary (TPC-1, FB2) and follicular (WRO) thyroid cancer cell lines

cells were incubated with Hydroxytyrosol different concentrations of (HY) HY (65–973 µM)

Outcomes -Oleuropein was demonstrated to suppress the expression of activated AKT (protein kinase B) -oleuropein exhibits specific cytotoxicity against breast cancer cells, with higher effect on the basallike MDA-MB-231 cells than on the luminal MCF-7 cells through the induction of apoptosis via the mitochondrial pathway. -oleuropein inhibits cell proliferation by delaying the cell cycle at S phase and up-regulated the cyclin-dependent inhibitor p21. -oleuropein inhibited the anti-apoptosis and proproliferation protein NF-j B and its main oncogenic target cyclin D -High doses of HY, promotes mitochondrial apoptotic cell death in human papillary and follicular thyroid cancer cells. -HY decreased cell viability of thyroid cancer cells in a concentration-dependent manner

Table 2. (Continued) Author, year

Cancer cells

Le Gendre et al., -PC3 (prostate), 2015 -MDA-MB-231 (breast) -BxPC3 (pancreatic) cancer cells.

Treatment used

Prostante, breast and pancreatic cancer

Compounds source

Oleocanthal (OC)

Outcomes The doses inducing 50% cell growth inhibition (IC50) against TPC-1, FB-2 and WRO cells for HY after 24 h. -Agarose gel electrophoresis of chromosomal DNA extracted from TPC-1, FB-2 and WRO cells revealed, after 24 h of HY exposure, a marked DNA fragmentation consisting of multimers of approximately 180–200 bp - OC induced both primary necrotic and apoptotic cell death via induction of lysosomal membrane permeabilization (LMP)in cancer cells that have fragile lysosomal membranes. -OC promotes LMP by inhibiting acid sphingomyelinase (ASM) activity, which destabilizes the interaction between proteins necessary for lysosomal membrane stability.

Author, year

Cancer cells

Treatment used

(Acquaviva et al., 2012)

LNCaP and DU145 prostate cancer cell lines and on BPH-1 non-malignant cells.

The treatment of BPH-1, LNCaP and DU145 cells with 100-500 µM oleuropein for 72 h

(Coccia et al., 2016)

Bladderneoplasm. Cancer cells: T24

Incubation with 2.5yg-100µg/ml for 24h

Compounds source

Outcomes

-OC increased the levels of phosphorylated p44/42 (P-p44/42), but did not significantly increase the levels of cleaved poly-ADP-ribose polymerase (PARP) an indicator of apoptotic death in the absence of serum. oleuropein -Oleuropein significantly reduces a cell viability, particularly in LNCaP and DU145 cells cell viability and induces thiol group modifications, γglutamylcysteine synthetase, reactive oxygen species, pAkt and heme oxygenase-1 -Exposing cell cultures to oleuropein induces an antioxidant effect on BPH-1 cells and a prooxidant effect on cancer cells. phenols extracted -EVOO extract can significantly inhibit the from EVOO proliferation and motility of T24 bladder cells in a dose dependent manner. -The enzymatic activity of MMP-2 (metalloproteinases) was inhibited at non toxic EVOO extract doses only in T24 cells.

Table 2. (Continued) Author, year

(Fogli et al., 2016)

Cancer cells

Skin cancer: human malignant melanoma cells

Treatment used

Concentration range of 0.01–50 mM for 72h.

Compounds source

Outcomes

-The qRT-PCR revealed a decrease of the MMP-2 expression and a simultaneous increase of the tissue inhibitors of metalloproteinases expression (MMP-2) Oleocanthal -Oleocanthal induces cell growth inhibition in extracted and A375 and 501Mel cells in a concentrationpurified from dependent manner with IC50 mean values of 13.6 extra virgin olive ±1.5 and 20 ±1.5 mM, oil. -Significant inhibition of ERK1/2 and AKT phosphorylation and downregulation of Bcl-2 expression. -Oleocanthal specifically downregulated gene expression of the antiapoptotic Bcl-2 protein both in A375 and 501Mel cells without affecting Bax expression.

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Statistical analysis of IC50 suggested that oleuropein had similar toxic effects on all cell lines tested. Interestingly (Le Gendre et al., 2015) shows that Oleocanthal, a very promessing phenolic compounds, induced both primary necrotic and apoptotic cell death via induction of lysosomal membrane permeabilization (LMP) in cancer cells that have fragile lysosomal membranes on Prostante, breast and pancreatic cancer, it also promotes lysosomal membrane permeabilisation (LMP) by inhibiting acid sphingomyelinase (ASM). Furthermore (Fogli et al., 2016) confirmed the apoptotoc effect of oleocanthal on human skin cancer using extracted and purificated OC from extra virgin olive oil and outcomes shows that oleocanthal induces cell growth inhibition in A375 and 501Mel cells in a concentration-dependent manner, it also specifically downregulated gene expression of the antiapoptotic Bcl-2 protein both in A375 and 501Mel cells without affecting Bax expression (Table 2). All these outcomes make olive oil and phenolic compounds great tools that may make them a promising novel chemotherapeutic agents.

RELATIVE POTENTIAL COMPOUNDS IN OLIVE OIL THAT REDUCE CANCER RISKS The health promoting attributes associated with the adhesion to a traditional MD have been recognized for decades, particularly for the main fat in this diet: EVOO which is recognized as a contributing factor towards the favorable health profile that the Mediterranean population possesses (Tripoli et al.,2015).Importantly EVOO contains approximately 36 phenolic compounds, and it is this minor phenolic fraction that is partially responsible for the health benefits that is linked to its intake (Cicerale et al.,2010;Cicerale et al.,2012). This fraction is composed by phenolic antioxidants that are potent inhibitors of reactive oxygen species and are associated with a reduced risk for several types of human cancer (Giacosa et al., 2013).

Oleuropein Oleuropein is a phenylethanoid, a type of phenolic compound found in olive oil and leaf together with other closely related compounds such as 10-hydroxyoleuropein, ligstroside, and 10-hydroxyligstroside. Oleuropein aglycone and deacetoxyoleuropein aglycone isomers were the major compounds determined in olive oils (Talhaoui et al.,2016).Oleuropein is released from the olive fruit to VOO during the extraction process. It’s more abundant in high amounts in unprocessed olive leaves and fruit, (Morello et al., 2004). Nutraceutical properties have been attributed to secoiridoid

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oleuropein (OL) and its derivatives (Bendini et al., 2007).Actually, previous studies highlighted its ability to trigger cell death specifically in cancer cells avoiding normal ones (Warleta et al., 2011; Bulotta et al.,2013). Thus, Elamin et al. investigated the cytotoxic effect of oleuropein on normal and (MCF-7) and different breast cancer cell lines and showed that dose-dependent effect of oleuropein on breast cancer (MDA-MB231) cell lines exhibited significant sensitivity while the ‘normal’ MCF-10A cells showed only marginal sensitivity even when challenged with high doses of oleuropein (Elamin et al., 2013).

Oleuropein, C25H32O13. It has previously shown that Oleuropein has an outstanding safety profile with no side effect even at high dose 100mg/kg in rodents (Del Boccio et al., 2003)However, it may act only against cancer cell lines throw the modulation of pro and anti-oncogenic signaling pathway leading to cell apoptosis and growth arrest of several tumor cell line. It has been proposed by some authors that the anti-proliferative and pro apoptotic pathway of oleuropein on tumor cells may be mediated by their capability to induce the accumulation of hydrogen peroxide in the culture medium (Luo et al., 2013). OL and HT are considered the major candidates for a pharmacological use, both as single drug or after enrichment of olive oil or other food components. Moreover recent fining obtained with OL aglycone or some semisynthetic derivatives (Bulotta et al., 2013; Campolo et al., 2013; Impellizzeri et al., 2011) suggest that it is possible to improve the pharmacological properties of these compounds and consequently offer a wide utilization in human pharmacology.

Hydroxytyrosol Hydroxytyrosol is one of the main phenols in olive oil (Servili et al., 2009) with the highest amount, it’s an oleuropein metabolite coming from the hydrolysis of OL, it is present in the fruit, oil and olive leaf of the (Olea europaea L) while higher concentration

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of HT may be found in the fruit and particularly in olive oil, owing to chemical and enzymatic reactions that occur during maturation of the fruit (Brenes et al., 2001). Hydroxytyrosol is now a compound well known for its anti-oxidant properties, nevertheless an increasing number of studies have found that HY decreased proliferation and survival of cancer cells as a result of modulation of the apoptotic pathway and of its pro-oxidant properties (Casaburi et al., 2013). It has been demonstrated that polyphenols, can affect the overall process of carcinogenesis by several mechanisms, although Toteda and others demonstrated that hydroxytyrosol promotes mitochondrial apoptotic cell death in human papillary and follicular thyroid cancer cells by increasing in the percentage of late apoptotic/necrotic cells and a a marked DNA fragmentation consisting of multimers of approximately 180–200 bp compared to control. (Toteda et al., 2016). HT might have relatively low levels in olive oil, besides they are in the high micromolar concentration in colon, after the gastric hydrolysis and colonic fermentation of secoiridoids present in olive oil (Corona et al., 2006) which make it an optimal candidate to reduces colon tumour progression leading to apoptosis and growth arrest of tumour cell lines (Corona et al., 2009).

Hydroxytyrosol, C8H10O3.

Oleocanthal Oleocanthal is a type of natural phenolic compound found in extra-virgin olive oil. It is a aphenylethanoid; decarboxy methyl ligstroside aglycone also known as oleocanthal. (Beauchamp et al., 2015).This phenolic compound, sole irritant phenolic responsible for the peppery stinging sensation in EVOO stands alone in terms of sensory and antiinflammatory attributes. The concentration of oleocanthal contained in EVOO could vary between studies but it approximatively ranges from 284 to 711 mg/kg as evaluated in a variety of Greek oils (Karkoula et al., 2014). Discovered in the early 90’s, wide previous studies have reported the activity of Oleocanthal as a potent antioxidant; a nonsteroidal anti-inflammatory agent that inhibits COX-1 and COX-2; a neuroprotectant that alters the structure and function of the neurotoxins b-amyloid and Tau, which are associated with the debilitating effects of Alzheimer disease; an inhibitor of proliferation, migration, and invasion of human breast and prostate cancer cells through c-Met inhibition; an inhibitor of Activated Protein Kinase (AMPK) in colon cancer cells; and an inhibitor of

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macrophage inflammatory protein-1a in multiple myeloma (Busnena et al., 2013; El Nagar et al., 2011; Montit al.,2012; Scotece et al., 2013).

OleocanthalC17H20O5. Numerous in vitro studies have reported that the phenolic compounds in EVOO can inhibit the initiation and metastasis of several types of cancer, LeGendre and others have demonstrated that oleocanthal induced cell death in all cancer cells examined as rapidly as 30 minutes after treatment in PC3 (prostate), MDA-MB-231 (breast) and BxPC3 (pancreatic) cancer cells under serum withdrawal, 20 µM OC and resulted in 100% nonviability in all cancer cell lines after 24 h of treatment throw increasing the levels of phosphorylated p44/42 (also known as ERK1/ERK2) that its activation is a critical mediator of mitochondrial dysfunction and necrotic cell death (Le Gendre, O Breslin, P.A.S and Foster, 2017). In the same context, an in vivo and in vitro study of Pei and other have shown that shown that (-)-oleocanthal inhibits HCC tumor growth and metastasis in vitro and in vivo. Tumor growth was inhibited and gross tumor specimen sizes was reduced in (-)-oleocanthal-treated group compared to the control group (Pei et al., 2016). (-)-Oleocanthal may be useful in cancer prevention and treatment and it would seem that oleocanthal alone has significant pharmacological properties in vitro and is becoming recognized as a potential therapeutic agent (Parkinson & Keast, 2014). However Oleocanthal’s bioavailability in vivo is not yet fully established, and this is important to access the pharmacological efficiency of oleocanthal also clinical trials are needed to confirm these findings.

Oleic Acid Initially, the richness in monounsaturated fatty acids (MUFA), and in particular oleic acid (OA), was considered as the major healthful characteristic of EVOO. OA has attracted much attention, especially in the last few years, as a protective agent against tumoral cells. Thus, both epidemiological and animal studies have reported a protective role of oleic acid in several cancers. It has been previously demonstrated that OA promoted the growth of non-malignant cells but, it had the opposite effect, in malignant cells (Zeng et al.,2010).Several researchers have also reported an inhibition cell

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proliferation and an effects on tumor growth induced by OA in different tumor cell lines(Liu et al., 2009; Martínez et al., 2005).

Oleic acid C18:1. A study of Moon and other reveled that OA down-regulates cell proliferation and suppresses malignant potential of esophageal cancer cells, suggesting that OA is capable of influencing crucial processes responsible for esophageal cancer development by the up-regulatin of AMPK that was previously demonstrated to causes cell cycle arrest in certain cancer cell lines such ashepatomaHepG2, prostate carcinoma PC-3 and breast cancerMCF-7, and/or decreasing phosphorylation of S6. They also found that OA increases expression of the tumor suppressor genes (p53, p21 and p27) in OE19 and OE33 esophageal cancer cell lines (Moon, Batirel, & Mantzoros, 2014).Similar to these reports, a study showed that oleic acid downregulated COX2 expressions which play an important role in colorectal cancer development also induces apoptosis in HT-29 cells (Waterman and Lockwood, 2007). A recent study of Lamy and other investigated the chemo preventive properties of OA in human glioblastoma cells, they have found that OAinhibit TNF-α-induced COX-2 gene and protein expression, inhibit TNF- α-induced PGE2 secretion by human glioblastoma cells and blocked endothelial cell migration through different cellular mechanisms (Lamy, Ben Saad, Zgheib, & Annabi, 2016). Finally, in accordance with (Carrillo, Cavia, & Alonso-Torre, 2012) a new approach to chemotherapy using nutraceutical might have the potential to yield novel dietary-drug combinations that can offer additive or even synergistic protection against the progression of cancer and it is especially relevant when the etiology of disease development has varied mechanistic routes. OA has been reported to act synergistically with cytotoxic drugs, thus enhancing their anti-tumor effect (Zeng et al., 2010).

APOPTOTIC PATHWAYS The different components of EVOO have been widely suggested to induce apoptosis in several cancer cell lines. The mechanisms behind cell death are numerous and involve a complex set of pathways.

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Extrinsic pathway: Involves transmembrane receptors at the plasma membrane at the cell surface and activated by ligand binding, death receptors TNFα, Trail, DR5/DR4/CD95... These ligands and their receptors are the first factors that leads to caspase activation during apoptosis in mammals. TRAIL or (TNF-related apoptosis inducing ligand) has sparked growing interest in oncology due to its reported ability to selectively trigger cancer cell death. These will then associate with procaspase-8 via dimerization of the death effector domain, forming a death-inducing signalling complex (DISC) (Falschlehne et al., 2007). Caspase-8 becomes activated and, in turn, directly activates caspase-3 (effector protein) to initiate degradation of the cell. Intrinsic pathway: Many anticancer drugs induce apoptosis by activating intrinsic pathways throw the mitochondrial permeability changes that increase cellular ROS production. Juan et al. (2008) showed that HT-29 cells exposed to maslinic acid present in olive oil showed markedly increased levels of superoxide anion radicals in mitochondria (Juan, Planas, Ruiz-Gutierrez, Daniel, & Wenzel, 2008). ROS production occurs in an early phase suggesting a rapid release of cytochrome c from mitochondria into the cytosol that in turn activates procaspase-9 and the downstream effectors, including the pro-caspases -3, -6 and -7, which are synthesized as an inactive pro-enzyme that are processed in cells undergoing apoptosis by self-proteolysis and/or cleavage by another protease and results in the induction of the proapoptotic and antiapoptotic family members Bax/Bcl-2,followedfinally by the cleavage of proteins and DNA that characterize the final phase of apoptosis. Actually a recent study of Yan el showed that oleuropein markedly increased BAX gene expression, while decreasing that of the Bcl-2 gene (Yan, Chai, Cai, Miao, & Ma, 2015). The alteration in the ratio of BAX/Bcl-2 is correlated with apoptosis through a mitochondrial pathway. In the other hand the p53 tumor suppressor protein plays a central role in the regulation of apoptosis, cell cycle and senescence as a response to a broad range of stresses; Thus, p53 activates the transcription of genes that encode apoptotic effectors, such as PUMA, NOXA, BID, Bax, p53AIP1 proteins, p53 gene or its product was found to be inactivated in more than 50% of all human cancers (Scatena, Bottoni, & Giardina, 2012). Cardeno et al. reported that oleuropein limited cell growth and induced apoptosis in HT29 colorectal cancer cells through a p53-dependent pathway. Cardeno et al. 2013. Tumor cell proliferation requires upregulation of multiple intracellular signaling pathways, including cascades involved in survival, proliferation, and cell cycle progression. The most significant effects on signaling pathways have been observed in: The Phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) pathway: It's a pathway which is known to be an important survival mechanism that is activated in cancer (Hennessy at al., 2005). Oleuropein was demonstrated to induce apoptosis in human hepatocellular carcinoma cells via suppression of PI3K/AKT (Yan et al., 2015).

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Nuclear Factor-Kappa B (NF-KB) Signaling Pathway TNF α activates NFκβ and the subsequent gene expressions which regulate many inflammatory signaling pathways and promote cancer development (Lin and Karin, 2007; Harvey et al., 2011). In turn, NF-kB modulates the levels of inflammatory cytokines, such as interleukin- 8 (IL-8), of apoptosis suppressor proteins (Bcl-2), as well as of intercellular adhesion molecules (ICAMs), such as ICAM-1, highly expressed in the stomach (Aggarwal and Shishodia, 2006). The idea of dietary chemoprevention is usually applied in the context of protecting normal cells from initiating events that introduce oncogenic mutations. However, substantial literature is available to show that natural antioxidant agents can also disrupt the progression of carcinogenesis at any point so they can be considered also chemopreventive. Induction of apoptosis in different cancer cells may provide protection against cancer development and therefore, may provide the basis for a novel nutritional strategy for cancer prevention.

CONCLUSION In conclusion, a diet reflecting the Mediterranean populace, incorporating daily EVOO intake, has received significant attention over the last years both for the association of this diet to positive health profile and for the fail of some pharmacological drug and their link to different side effects. Research has demonstrated the advantageous effects of usapanifiable and saponifiable compounds of olive oil on health at both the epidemiologic and cellular level. The most important bioactive compounds in olive oil and their anti-proliferative and proapoptotic properties have been also demonstrated by investigating the effects of olive oil on the expression of different molecular markers. Both in vitro and in vivo studies have highlighted the involvement of EVOO in cancer prevention, suggesting their possible use as natural treatment or in synergic with other anti-cancer substituents and as an adjuvant agent in the treatment of prostatitis, to prevent the transformation of hypertrophic to cancerous cells.

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Morello, J.R., Motilva, M.J., Tovar, M.J., Romero MP., (2004). Changes in commercial virgin olive oil (cv. Arbequina) during storage, with special emphasis on the phenolic fraction. Food. Chem. 85,357–364. Monti, M.C., Margarucci, L, Riccio, R., Casapullo, A., (2012). Modulation of tau protein fibrillization by oleocanthal. J. Nat. Prod.75, 1584–8 Moon, H. S., Batirel, S., Mantzoros, C. S., (2014). Alpha linolenic acid and oleic acid additively down-regulate malignant potential and positively cross-regulate AMPK/S6 axis in OE19 and OE33 esophageal cancer cells. Metabolism: Clinl. Exper. 63, 1447– 1454. Parkinson, L., Keast, R., (2014). Oleocanthal, a Phenolic Derived from Virgin Olive Oil: A Review of the Beneficial Effects on Inflammatory Disease. Inter.J. Mol.Sci. 15,12323-12334. Pei, T., Meng, Q., Han, J., Sun, H., Li, L., Song, R.,Liu, L., (2016). Oleocanthal inhibits growth and metastasis by blocking activation of STAT3 in human hepatocellular carcinoma. Oncotarget. 7, 43475–43491. Psaltopoulou, T., Kosti, R.I., Haidopoulos, D., dimopoulos, M., Panagiotakos, D.B., (2011). Olive oil intake is inversely related to cancer prevalence: A systematic review and a meta-analysis of 13,800 patients and 23,340 controls in 19 observational studies. Lipids. Health. dis. 10: 127- 131. Ramachandran, C., Nair, S.M., Escalon. E., Melnick, S.J., (2012). Potentiation of etoposide and temozolomide cytotoxicity by curcumin and turmeric force in brain tumor cell lines. J. Complement. Integr.med.9:255-262. Rafehi, H., Ververis, K., Karagiannis, T.C., (2012). Mechanisms of action of phenolic compounds in olive. J. Diet. Suppl. 9, 96-109. Rietjens, S. J., Bast, A., Haenen, G. R. M., (2007). New insights into controversies on the antioxidant potential of the olive oil antioxidant hydroxytyrosol. J. Agric. Food Chem. 55, 7609−7614. Scatena, R., Bottoni, P., Giardina, B., (2012). Advances in Mitochondrial Medicine. Adv. Exp. Med.Biol. 942, 311–327. Servili, M., Esposto, S., Fabiani, R., Urbani, S., Taticchi, A., Mariucci, F., (2009). Phenolic compounds in olive oil: antioxidant, health and organoleptic activities according to their chemical structure. Inflam. Pharmacol.17, 76–84. Surh, Y.J., (2003). Cancer chemoprevention with dietary phytochemicals. Nat. Rev. Cancer. 3,768-80. Schwartz, B., Birk, Y., Raz, A., Madar, Z., (2004). Nutritional-pharmacological combinations - a novel approach to reducing colon cancer incidence. Eur. J. Nutr. 43, 221-9. Scotece, M., Gomez, R., Conde, J., Lopez, V., Gomez-Reino, J.J., Lago, F., Smith, A.B., Gualillo, O., (2013). Oleocanthal inhibits proliferation and MIP-1a expression in human multiple myeloma cells. Curr Med Chem. 20, 2467–75.

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Talhaoui, N, Gómez-Caravaca, A.M, León L, De la Rosa, R, Fernández-Gutiérrez, A andSegura-Carretero A. (2016). From Olive Fruits to Olive Oil: Phenolic Compound Transfer in Six Different Olive Cultivars Grown under the Same Agronomical Conditions.Int. J. Mol. Sci.17, 337-347. Terzuoli, E., Giachetti, A., Ziche, M.,Donnini, S., (2016). Hydroxytyrosol, a product from olive oil, reduces colon cancer growth by enhancing epidermal growth factor receptor degradation. Mol. Nutr.Food.Res. 60, 519–529. Toteda, G., Lupinacci, S., Vizza, D., Bonofiglio, R., Perri, E., Bonofiglio, M., Perri, A., (2016). High doses of hydroxytyrosol induce apoptosis in papillary and follicular thyroid cancer cells. J. Endocrinol. Invest. 40,153-162. Tripoli, E., Giammanco, M., Tabacchi, G., Di Majo, D., Giammanco, S., la Guardia, M., (2005). The phenolic compounds of olive oil: Structure, biological activity and beneficial effects on human health. Nutr. Res. Rev. 18, 98–112. Willet, W.C., Diet, nutrition, and avoidable cancer, (1995). Environ. Health Perspect, 103, 165– 170. Warleta, F., Quesada, C.S., Campos, M., Allouche, Y., Beltrán, G., Gaforio, J.J., (2011). Hydroxytyrosol protects against oxidative DNA damage in human breast cells. Nutrients. 3,839–857. Waterman, E., Lockwood, B., (2007) Active components and clinical applications of olive oil. Alt. Med. Rev. 12, 331-342. Yan, C. M., Chai, E. Q., Cai, H. Y., Miao, G. Y., Ma, W., (2015). Oleuropein induces apoptosis via activation of caspases and suppression of phosphatidylinositol 3kinase/protein kinase B pathway in HepG2 human hepatoma cell line. Mol. Med. Rep, 11, 4617–4624. Zeng, L., Biernacka, K.M., Holly, J.M., Jarrett, C., Morrison, A.A., Morgan, A., (2010) Hyperglycaemia confers resistance to chemotherapy on breast cancer cells: the role of fatty acid synthase. Endocr. Relat. Cancer. 17,539-514.

In: Handbook of Olive Oil Editor: Jozef Miloš

ISBN: 978-1-53612-356-2 © 2017 Nova Science Publishers, Inc.

Chapter 13

HYDROXYTYROSOL AND TYROSOL, MAIN PHENOLS OF VIRGIN OLIVE OIL, AS HEALTHY NATURAL PRODUCTS IN HUMAN BREAST CANCER PREVENTION: A REVIEW Cristina Sánchez-Quesada, PhD1,2,3 and José J. Gaforio, MD, PhD1,2,3,4, 1

Center for Advanced Studies in Olive Grove and Olive Oils, University of Jaén, Spain 2 Immunology Division, Department of Health Sciences, Faculty of Experimental Sciences, University of Jaén, Jaén, Spain 3 Agrifood Campus of International Excellence, ceiA3, Spain 4 CIBER-ESP, Instituto de Salud Carlos III, Madrid, Spain

ABSTRACT Hydroxytyrosol (HT) and tyrosol (TY) are two of the main phenolic compounds present in several plants of the vegetable kingdom, but with major presence in the product of olive trees. These two compounds appear from the secoiridoid hydrolysis of virgin olive oils during storage. While the concentration of TY is always higher than HT, the quantity of both depend on the olive tree variety, climatic and agronomic conditions. Multiple health claims are attributed to these two compounds (as cardioprotectives, antioxidants, protection against DNA…). Therefore, recent studies show us that they may 

Correspondence: [email protected]; Tel.: +34-953-212-002.

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Cristina Sánchez-Quesada and José J. Gaforio play a key role in the development and prevention of different kind of cancers, among them breast cancer. This chapter explain and describe the more notably beneficial effects of these two compounds in the prevention, appearance and development of breast cancer. In vivo and in vitro studies demonstrate that these two phenols could aid in chemotherapies against breast cancer and in the prevention of it. Nevertheless, although they are natural compounds, special attention should be paid to the concentration administrated, this is the main reason why more deeply studies are needed for asseverate their preventive and antitumoral activities.

INTRODUCTION Nowadays there is a general concern about healthy and natural products present in food, which could improve or be protective against several diseases. Certainly, several natural compounds exert beneficial action against different illness. Phenols are one of the plant kingdom groups more studied until now. These compounds are present in different plants, but the main phenols of olive tree (Olea europaea spp.) appear to attract the main attention of the recent biomedical studies because of the effects that promote on health and in the development of an amount of diseases [1-5]. Cancer is a chronic-degenerative disease characterized by a multistep process in which normal cells are transformed into malignant cells, who acquires different mutations and chromosomal aberrations, with uncontrolled proliferation. Breast cancer disease is one of the top five cancer deaths in 2015, below lung, liver, colon and stomach cancers according to World Health Organization [6]. Breast cancer is described, because of that, as the first cause of death by cancer in women in 2015 [6]. Recent studies showed that breast cancer incidence has been affected by geographical regions. These variations are certainly related to the human exposure to different modifiable factors which may either increase or decrease cancer risk. One of the modifiable factor is diet. It has been described the beneficial effects of consume a Mediterranean Diet against others for prevention and treatment of breast cancer [2, 7]. In general, it has been estimated that over 30% of all cancers may be avoidable by changing food intake [3]. Because of that, diet is a key point that has to be studied and adapted to each individual. As we mentioned before, Mediterranean diet is related with prevention of breast cancer disease [8], an virgin olive oil is the main fat that conforms this diet. Virgin olive oil (VOO), obtained directly from olive tree pomace, contain about 230 different compounds denominated “minority compounds”. A fat acid named oleic, composes the 98% of VOO and the remaining (2%) correspond to these minority compounds. The phenols are included in the minor compounds of VOO, mainly represented by hydroxytyrosol (3,4-dihydroxyphenylethanol: 3,4-DHPEA) (HT) and tyrosol (p-hydroxyphenylethanol: p-HPEA) (TY) [9]. In the present chapter, we shows the active role that both phenols exert in breast cancer prevention and treatment.

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HYDROXYTYROSOL AND TYROSOL SYNTHESYS IN OLIVES AND BIOAVAILABILITY Virgin olive oil (VOO) is produced by centrifugation or hydraulic pressing of malaxed olive drupes (pomace) harvested from the olive groves of Europe and northern African countries mainly [10]. VOO contains relatively high amounts of minor compounds compared to other oils (refined olive oil or seed oils). Minor components can be grouped in classes as hydrocarbons (satured, unsatured, linear, branched); esters (fatty acid derivates of shortchain alohols, long-chain alcohols, sterols, triterpenic alcohols, monoterpenic alcohols, phenols); aldehydes (medium- and long-chain, monoterpenic); phenols (tocopherols, epoxyphenols); acids (in addition to free fatty acids, triterpenic acids, phenoxy acids) and chlorophyll [9]. Among these, phenolic compounds are present at levels between 200 and 1500 mg/kg [11] depending on the olive tree variety, climatic and agronomic conditions, degree of maturation at harvest, and the manufacturing process [11]. The major phenolic compounds in olive oil comprise simple phenols, polyphenols, secoiridoids (SID) and lignans [10]. In particular, the phenolic alcohols, hydroxytyrosol (HT) and tyrosol (TY) are abundantly and exclusively present in olives, olive leaves and olive oil as both free compounds and linked to either elenolic acid (EA) or its dialdehydic form (EDA) giving rise to the following secoiridoid derivates: 3,4-DHPEA-EDA (oleuropein aglycon), pHPEA-EDA (ligstroside aglycon), 3,4-DHPEA-EDA, p-HPEA-EDA (oleocanthal), and oleuropein (Figure 1) [3]. The three phenolic compounds in highest concentration in olive oil are the glycoside oleuropein, HT and TY. Oleuropein is the major phenolic compound in olive fruit, which can be as much as 14% in dried fruit, HT is the major phenolic component in olive oil. As the olive fruit matures, the concentration of oleuropein decreases and HT (the hydrolysis product of oleuropein) increases [12]. A correlation between the concentration of HT and the stability of olive oil exists, however the same does not apply for TY [12]. Because of that as much as HT is present in VOO, this oil will be more stable that the oil that does not content high amount of HT. The availability in which cells can use these compounds after olive oil consumption is a matter of discuss nowadays. Nevertheless, the studies that describe the bioavailability of both compounds affirm that the doses absorbed by olive oil are realistic and could be measured [13]. After a single dose of virgin olive oil ingestion, the half-life of TY and HT was estimated to be around 8h.

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Figure 1. Chemical structures of phenolic alcohols (hydroxytyrosol and tyrosol) and their secoiridoid derivates present in olive oil.

Although continuous exposure to olive oil could result in a long-term HT accumulation, the urinary HT concentrations and the urinary recovery observed after sustained doses of 25mL of olive oil could be explained with difficulty only by the steady-state reached. It seems that differences in the phenolic compounds metabolism exist. Levels of urinary TY obtained after one week of sustained doses (25mL/day) of virgin olive oil were lower than those obtained after a single 50mL dose. Urinary HT

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levels, however, were similar at both intervention periods, being the estimated recovery of HT in urine after sustained doses of 25mL of virgin olive oil greater than 100% [13]. Covas et al. have studied bioavailability of TY in men and women who showed a different pattern of urinary excretion, but TY was absorbed in a dose-dependent manner after sustained and moderate doses of virgin olive oil in both sex [14].

HEALTH BENEFITS OF HYDROXYTYROSOL AND TYROSOL IN BREAST CANCER Breast cancer is a complex chronic-degenerative disease characterized by a multistep process in which normal cells are transformed into malignant cells acquiring several properties such as abnormal proliferation and reduced apoptosis. It is actually responsible of the major index of death in women, and its incidence is directly correlated with developed countries [15] (Figure 2). Numerous studies have shown that diet intake is one of the main risk factor in breast cancer [8, 16-18]. In this context, of particular relevance is the observation that populations living in the Mediterranean area have a lower cancer mortality and/or incidence compared to other regions. This fact has been attributed to a Mediterranean Diet adherence [8]. Specially, the role of virgin olive oil remains clear for the scientific community [2, 19, 20], thus the minor compounds found in this Mediterranean fat appear to be the key in several cancers [21-23].

GLOBOCAN, 2012. Figure 2. Estimated breast cancer incidence in 2012.

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Hydroxytyrosol The antitumoral activity of Hydroxytyrosol (HT) has been studied in vivo and in vitro in different cancer cellular models. In vitro studies has described an antiproliferative and apoptotic role on MCF-7 ER+ breast cancer cells [24, 25], but the authors did not identify the pathways used by HT to promote cell death. Sirianni et al. [26] described that HT could manage tumor cell growth by the inhibition of estrogen-dependent rapid signals, mainly inhibiting the ERK1/2 activation. In SKBR3 ER- breast cancer cells, HT acted as a GPER (G-protein-coupled receptor) inverse agonists in ER-negative and GPERpositive SKBR3 cells, which in fact showed a sustained ERK1/2 activation triggering an intrinsic apoptotic pathway [27]. Indeed, HT could act in the environment of breast tumor, inhibiting CCL5 (Chemokine C-C motif ligand 5) expression in aging quiescent normal human fibroblasts. CCL5 is associated with the impossibility of fibroblasts to activate the ERK1/2-cyclin D1 pathway and consequently the enhancement of breast cancer cells proliferation. Fibroblast nearing the end of their chronological life span promote proliferation of human breast epithelial cancer cells and HT could inhibit this process [28]. Studies realized in rats showed that HT alters several genes associated with cell proliferation, inhibiting mammary tumor growth and proliferation in Sprague-Dawley rats, even with better results that doxorubicin, a chemotherapeutic used in breast cancer patients [29]. Among these gens we can observed a downregulation of oncogenes as cJun and JunB and the Cryab protein, related with the cell cycle progression in breast carcinogenesis. These authors also described later the improvement of cardiac disturbances enhanced by doxorubicin in these rats thanks to HT. In this sense, HT act as protector of heart damage in rats treated with this antitumoral drug, decreasing oxidative damage and mitochondrial alterations [30]. Interestingly, HT also prevent breast cancer appearance, which could explain the low incidence of breast cancer in Mediterranean countries. Warleta et al. [21] described the preventive role that HT exerted in human breast epithelial cells. HT could prevent oxidative damages induced by H2O2 in DNA. In this sense, HT also acted as antioxidant in normal epithelial cells, while did not show any of this antioxidant action in breast cancer cells. This way, this natural compound act as chemopreventive in breast cancer cells, focusing its action in prevent the appearance of oncogenes activated by oxidative mutations in the DNA. Furthermore, HT has been described as antioxidant in many studies [9, 31]. This antioxidant role could aid in the prevention not only of cancer, cardiovascular or neurologic diseases could be prevented also.

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Tyrosol Tyrosol (TY) is one of the three main phenols in olives, and olive juice. Many studies remark the chemical antioxidant activity that it can achieve [21], but the effect appears always be more potent with HT. In this article, TY also acted as antioxidant in normal epithelial cells while in breast cancer cells did not. Unless it was as antioxidant as HT, TY did not achieve the same activity at the same concentration, on the contrary an increment in the concentration of TY was necessary for it. This found appear to repeat along the same studies that studied HT. TY acts as chemopreventive as well, but not with the same strength that HT. Nevertheless further studies are needed for contrast and explain the chemopreventive effects that both compound seem to have in breast cancer appearance. In conclusion, natural components of many vegetables consumed in different diets could contribute to prevent and aid to treat many diseases, among them breast cancer. Hydroxytyrosol and tyrosol may play an essential role in the prevention of breast cancer as long as they act as antioxidant compounds. Moreover, they could contribute in breast cancer treatment as many studies described their antitumoral activities in different breast cancer cells in vitro.

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Cardeno A., Magnusson M. K., Strid H., Alarcon de La Lastra C., Sanchez-Hidalgo M., Ohman L. The unsaponifiable fraction of extra virgin olive oil promotes apoptosis and attenuates activation and homing properties of T cells from patients with inflammatory bowel disease. Food Chem., 2014 Oct. 15;161:353-360. Escrich E., Moral R., Solanas M. Olive oil, an essential component of the Mediterranean diet, and breast cancer. Public Health Nutr., 2011 Dec.; 14(12A):2323-2332. Fabiani R. Anti-cancer properties of olive oil secoiridoid phenols: a systematic review of in vivo studies. Food Funct., 2016 Oct. 12; 7(10):4145-4159. Martinez-Gonzalez M. A., Toledo E., Aros F., Fiol M., Corella D., Salas-Salvado J., et al. Extra-Virgin Olive Oil Consumption Reduces Risk of Atrial Fibrillation: The PREDIMED Trial. Circulation, 2014 Apr. 30; 130(1):18-26. Muscoli C., Lauro F., D'Agostino C., Ilari S., Giancotti L. A., Gliozzi M., et al. Olea Europea-derived phenolic products attenuate antinociceptive morphine tolerance: an innovative strategic approach to treat cancer pain. J. Biol. Regul. Homeost. Agents, 2014 Jan.-Mar.; 28(1):105-116. World Health Organization. 2015; Available at: http://www.who.int/.

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Cristina Sánchez-Quesada and José J. Gaforio Trichopoulou A., Lagiou P., Kuper H., Trichopoulos D. Cancer and Mediterranean dietary traditions. Cancer Epidemiol. Biomarkers Prev., 2000 Sep.; 9(9):869-873. Buckland G., Travier N., Cottet V., Gonzalez C. A., Lujan-Barroso L., Agudo A., et al. Adherence to the mediterranean diet and risk of breast cancer in the European prospective investigation into cancer and nutrition cohort study. Int. J. Cancer, 2013 Jun. 15; 132(12): 2918-2927. Owen R. W., Giacosa A., Hull W. E., Haubner R., Spiegelhalder B., Bartsch H. The antioxidant/anticancer potential of phenolic compounds isolated from olive oil. Eur. J. Cancer, 2000 Jun.; 36(10):1235-1247. Owen R. W., Haubner R., Wurtele G., Hull E., Spiegelhalder B., Bartsch H. Olives and olive oil in cancer prevention. Eur. J. Cancer Prev., 2004 Aug.; 13(4):319-326. Allouche Y., Jimenez A., Gaforio J. J., Uceda M., Beltran G. How heating affects extra virgin olive oil quality indexes and chemical composition. J. Agric. Food Chem., 2007 Nov. 14; 55(23):9646-9654. Tuck K. L., Hayball P. J. Major phenolic compounds in olive oil: metabolism and health effects. J. Nutr. Biochem., 2002 11; 13(11): 636-644. Miro-Casas E., Covas M., Fito M., Farre-Albadalejo M., Marrugat J., de lT. Tyrosol and hydroxytyrosol are absorbed from moderate and sustained doses of virgin olive oil in humans. Eur. J. Clin. Nutr., 2003 print; 57(1):186-190. Covas M., Miro-Casas E., Fito M., Farre-Albadalejo M., Gimeno E., Marrugat J., et al. Bioavailability of tyrosol, an antioxidant phenolic compound present in wine and olive oil, in humans. Drugs Exp. Clin. Res., 2003; 29(5-6):203-206. GLOBOCAN I. 2012; Available at: http://globocan.iarc.fr/Pages/ fact_sheets_cancer.aspx. Fung T. T., Hu F. B., Holmes M. D., Rosner B. A., Hunter D. J., Colditz G. A., et al. Dietary patterns and the risk of postmenopausal breast cancer. Int. J. Cancer, 2005 Aug. 10; 116(1):116-121. Sieri S., Krogh V., Pala V., Muti P., Micheli A., Evangelista A., et al. Dietary patterns and risk of breast cancer in the ORDET cohort. Cancer Epidemiol. Biomarkers Prev., 2004 Apr.; 13(4):567-572. Toledo E., Salas-Salvado J., Donat-Vargas C., Buil-Cosiales P., Estruch R., Ros E., et al. Mediterranean Diet and Invasive Breast Cancer Risk Among Women at High Cardiovascular Risk in the PREDIMED Trial: A Randomized Clinical Trial. JAMA Intern. Med., 2015 Nov. 1; 175(11):1752-1760. Colomer R., Menendez J. A. Mediterranean diet, olive oil and cancer. Clin. Transl. Oncol., 2006 Jan.; 8(1):15-21. Escrich E., Ramírez-Tortosa M. C., Sánchez-Rovira P., Colomer R., Solanas M., Gaforio J. J. Olive oil in cancer prevention and progression. Nutr. Rev., 2006; 64(10 SUPPL. 1):S40-S52.

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[21] Warleta F., Quesada C. S., Campos M., Allouche Y., Beltran G., Gaforio J. J. Hydroxytyrosol protects against oxidative DNA damage in human breast cells. Nutrients, 2011 Oct.; 3(10):839-857. [22] Smith T. J. Squalene: potential chemopreventive agent. Expert Opin. Investig. Drugs, 2000 Aug.; 9(8):1841-1848. [23] Sanchez-Quesada C., Lopez-Biedma A., Warleta F., Campos M., Beltran G., Gaforio J. J. Bioactive properties of the main triterpenes found in olives, virgin olive oil, and leaves of Olea europaea. J. Agric. Food Chem., 2013 Dec. 18; 61(50):12173-12182. [24] Han J., Talorete T. P., Yamada P., Isoda H. Anti-proliferative and apoptotic effects of oleuropein and hydroxytyrosol on human breast cancer MCF-7 cells. Cytotechnology, 2009 Jan.; 59(1):45-53. [25] Bouallagui Z., Han J., Isoda H., Sayadi S. Hydroxytyrosol rich extract from olive leaves modulates cell cycle progression in MCF-7 human breast cancer cells. Food Chem. Toxicol., 2011 Jan.; 49(1): 179-184. [26] Sirianni R., Chimento A., De Luca A., Casaburi I., Rizza P., Onofrio A., et al. Oleuropein and hydroxytyrosol inhibit MCF-7 breast cancer cell proliferation interfering with ERK1/2 activation. Mol. Nutr. Food Res., 2010 Jun.; 54(6):833840. [27] Chimento A., Casaburi I., Rosano C., Avena P., De Luca A., Campana C., et al. Oleuropein and hydroxytyrosol activate GPER/ GPR30-dependent pathways leading to apoptosis of ER-negative SKBR3 breast cancer cells. Mol. Nutr. Food Res., 2014 Mar.; 58(3): 478-489. [28] Sarsour E. H., Goswami M., Kalen A. L., Lafin J. T., Goswami P. C. Hydroxytyrosol inhibits chemokine C-C motif ligand 5 mediated aged quiescent fibroblast-induced stimulation of breast cancer cell proliferation. Age (Dordr), 2014 Jun.; 36(3):9645. [29] Granados-Principal S., Quiles J. L., Ramirez-Tortosa C., Camacho-Corencia P., Sanchez-Rovira P., Vera-Ramirez L., et al. Hydroxytyrosol inhibits growth and cell proliferation and promotes high expression of sfrp4 in rat mammary tumours. Mol. Nutr. Food Res., 2011 May; 55 Suppl. 1:S117-26. [30] Granados-Principal S., El-Azem N., Pamplona R., Ramirez-Tortosa C., PulidoMoran M., Vera-Ramirez L., et al. Hydroxytyrosol ameliorates oxidative stress and mitochondrial dysfunction in doxorubicin-induced cardiotoxicity in rats with breast cancer. Biochem. Pharmacol., 2014 Jul. 1; 90(1):25-33. [31] Visioli F., Caruso D., Plasmati E., Patelli R., Mulinacci N., Romani A., et al. Hydroxytyrosol, as a component of olive mill waste water, is dose- dependently absorbed and increases the antioxidant capacity of rat plasma. Free Radic. Res., 2001 01/01; 34(3):301-305.

In: Handbook of Olive Oil Editor: Jozef Miloš

ISBN: 978-1-53612-356-2 © 2017 Nova Science Publishers, Inc.

Chapter 14

CONSUMPTION OF EXTRA VIRGIN OLIVE OIL AND THE COMPONENTS OF THE METABOLIC SYNDROME Hady Keita1,* and María del Rosario Ayala Moreno2 1

Dirección de Carrera de Paramédico y Protección civil, Universidad Tecnológica del valle de Toluca, Toluca, México 2 Grupo de Investigación en Alimentos y Salud, Facultad de Ciencias Químicas, Universidad La Salle Ciudad de México, México

ABSTRACT Chronic-degenerative diseases are currently a public health problem worldwide, including dyslipidemias, alterations in glucose metabolism, arterial hypertension and abdominal obesity, which together characterize the so-called metabolic syndrome. The risk of developing cardiovascular disease is estimated to be approximately double in subjects with metabolic syndrome and three times the risk of developing type 2 diabetes, which represent the main causes of death in the population. Although metabolic syndrome is a complex and multifactorial entity, the alarming increase in the components of metabolic syndrome is mainly associated with two etiological factors, the decrease in physical activity and the dietary pattern in the population, particularly the high consumption of energy from simple carbohydrates and fats. We know that maintaining a healthy diet is essential to prevent metabolic alterations, not only in terms of total caloric intake, but also the “quality” of fats consumed, i.e., the type of fatty acids that characterize the individual’s diet is determinant for the development of the disease. The Mediterranean diet has become in recent decades a reference icon for healthy eating, and its beneficial effects have been recognized by scientists, doctors, nutritionists and *

[email protected].

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Hady Keita and María del Rosario Ayala Moreno international organizations such as the World Health Organization (WHO), the organization of the United Nations for Food and Agriculture (FAO), among others. It is now known that extra virgin olive oil is one of the fundamental components of the Mediterranean diet, and that it is characterized by a high content of essential fatty acids and phenolic components, whose biological effect on the body gives it preventive properties and/or the ability to reduce the incidence of chronic-degenerative diseases associated with metabolic syndrome. The results summarized in this chapter have been obtained from population studies, as well as experimental animal models, which demonstrate some of the mechanisms of action of the main components of extra virgin olive oil, on metabolic processes related to the pathophysiology of metabolic syndrome, that confer the beneficial effects to this oil. The above highlights the importance of the Mediterranean diet on the prevention and/or control of metabolic syndrome.

Keywords: metabolic syndrome, olive oil, CVD

1. INTRODUCTION Metabolic syndrome (MetS) is defined as a cluster of closely related clinical alterations that include abdominal obesity, hyperglycemia, dyslipidemia, and elevated systolic or diastolic blood pressure. It is also associated with other comorbidities including the prothrombotic state, proinflammatory state, nonalcoholic fatty liver disease, and reproductive disorders (Cornier et al., 2008). In general, the reported prevalence of MetS in adult population is between 20 and 30% (Grundy, 2008). Moreover, subjects with MetS have double the risk of developing cardiovascular disease (Grundy et al., 2005) and triple the risk of developing type 2 diabetes (Ford, 2005), one of the leading causes of death worldwide. The physiopathology of MetS is complex, and despite the vast amount of scientific information obtained from research, several biochemical and molecular mechanisms that are key for MetS development are still not fully elucidated. However, abdominal obesity and insulin resistance (IR) are two fundamental factors recognized in MetS etiology (Ayala-Moreno, 2017). The causes of MetS are multifactorial, with an important genetic component that is considerably determined by environmental factors. In this sense, the diet as a promoter of obesity and IR becomes an assencial factor to prevent MetS development. Therefore, promoting a new “dietary transition” is crucial to reduce the prevalence of this syndrome. Many clinical studies and investigations have noted that consuming a Mediterranean diet, the main components of which is olive oil, may have cardioprotective effects that counteract the distinctive clinical characteristics of MetS (Kastorini et al., 2011). This chapter emphasizes the importance of the Mediterranean diet in the prevention and/or control of metabolic syndrome.

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2. MEDITERRANEAN DIET Several proposed therapeutic diets that counteract metabolic alterations are based on adopting a dietary pattern similar to that of the Mediterranean diet, as the latter reduces morbidity and mortality from cardiovascular disease (CVD) in Mediterranean population (Sofi et al., 2014; Babio et al., 2009). In contrast, populations that consume a Western diet have higher incidences of MetS and IR (Esmaillzadeh et al., 2007; Cho et al., 2011; Rodríguez-Monforte et al., 2015 and 2016). In this context, important population intervention studies, such as the PREDIMED (Prevention with Mediterranean Diet) study in Spain, have demonstrated that the Mediterranean diet can reduce CVD by up to 30% and prevent MetS (Dussaillant et al., 2016). Interest in the Mediterranean diet began 30 years ago, when Ancel Keys published in 1945 the results of the famous Seven Countries Study. There are some countries with coasts on the Mediterranean sea that are known for following this dietary pattern: Spain, southern France, Italy, Malta, Croatia, Bosnia, Albania, Greece, Turkey, Syria, Lebanon, Cyprus, Egypt, Libya, Tunisia, Algeria, and Morocco (Noah and Truswell, 2001). The Mediterranean diet is a dietary pattern characterized by the following: consumption of large amounts of fruits and vegetables, use of olive oil as a principal source of fat, consumption of wholegrain breads and cereals, legumes and nuts, preference for fish, dairy such as yogurt and cheese, sporadic consumption of red meat, and minimum consumption of sugar or sweetened products (Serra-Majem et al., 2004). Clearly, this dietary pattern provides a significant amount of olive oil, which has been recognized for its beneficial effects on health (Buckland and González, 2015), especially those that protect against CVD, dyslipidemia, IR, and cancer. Although these effects have been attributed to high levels of monounsaturated fatty acids, there is currently more evidence that minor compounds with antioxidant activity are responsible for most of the health benefits of olive oil (Ghanbari et al., 2012).

3. OLIVE OIL AND ITS PROPERTIES Extra-virgin olive oil contains 98 to 99% triacylglycerol (Viola and Viola, 2009) and is particularly characterized by its high content of monounsaturated fatty acids (MUFAs). It also contains 1 to 2% of minor components (Viola and Viola, 2009) that have a high nutritional value, including phenolic compounds, phytosterols, tocopherols, carotenoids, chlorophyll and squalene. Although these components have various biological activities, they are mostly antioxidant. The composition of olive oil may be influenced by the variety of olive, the region of cultivation, climatic conditions, and processing techniques to obtain the oil (Viola and Viola, 2009; Ghanbari et al., 2012). Both MUFAs and antioxidant compounds in olive oil are highly stable, not only during oil extraction

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process but also throughout its shelf life, which maintains the oil’s health benefits (Talhaoui et al., 2016).

4. FATTY ACID COMPOSITION According to the report by Ghanbari et al., 2012, which analyzes different studies on the fatty acid profile of olive oil, oleic acid (18:1) is the main component and makes up 55.0 to 78.3% of olive oil. In relation to saturated fatty acids (SFAs), palmitic acid (C16:0) makes up 7.5 to 20% and stearic acid (C18:0) 0.5 to 5.0% of olive oil. Polyunsaturated fatty acids (PUFAs) content varies: linoleic acid (C18:2n6) content ranges between 3.5 and 21.0%, linolenic acid (C18:3n3) has been reported only in small fractions (