Fruit and Pomace Extracts Biological Activity,

FOOD AND BEVERAGE CONSUMPTION AND HEALTH

FRUIT AND POMACE EXTRACTS BIOLOGICAL ACTIVITY, POTENTIAL APPLICATIONS AND BENEFICIAL HEALTH EFFECTS

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FOOD AND BEVERAGE CONSUMPTION AND HEALTH

FRUIT AND POMACE EXTRACTS BIOLOGICAL ACTIVITY, POTENTIAL APPLICATIONS AND BENEFICIAL HEALTH EFFECTS

JASON P. OWEN EDITOR

New York

Copyright © 2015 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.

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Published by Nova Science Publishers, Inc. † New York

CONTENTS Preface Chapter 1

Chapter 2

Chapter 3

Chapter 4

Chapter 5

Chapter 6

Chapter 7

Chapter 8

vii Fruit and Pomace Extracts: Applications to Improve the Safety and Quality of Meat Products P. G. Peiretti and F. Gai Valorization of Liquid Effluents from Olive Oil Extraction Activity in the Production of Ceramic Bricks: Influence of Conformation Process D. Eliche-Quesada and F. A. Corpas-Iglesias

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Fruit and Pomace Extracts: Applications to Improve the Safety and Quality of Fish Products F. Gai and P. G. Peiretti

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Supercritical Fluid Extraction of Pharmaceutic Compounds from Waste Materials Derived from Vinification Processes Cleofe Palocci and Laura Chronopoulou

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Passion Fruit Pomace Powder: Potential Applications of Emerging Technologies for Extraction of Pectin Cibele Freitas de Oliveira and Poliana Deyse Gurak

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Hesperetin: Simple Natural Compound with Multiple Biological Activity José Valdo Madeira Junior, Vânia Mayumi Nakajima, Fabiano Jares Contesini, Camilo Barroso Teixeira, Juliana Alves Macedo and Gabriela Alves Macedo A Review of the Antimicrobial Activity of Various Solvent Type Extracts from SOME Fruits and Edible Plants R. C. Jagessar, N. Ramchartar and O. Spencer Coconut Water: An Essential Health Drink in Both Natural and Fermented Forms Mansi Jayantikumar Limbad, Noemi Gutierrez-Maddox and Nazimah Hamid

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vi Chapter 9

Chapter 10

Contents Extraction, Characterization and Potential Health Benefits of Bioactive Compounds from Selected Cornus Fruits Luminiţa David and Bianca Moldovan Kumquat (Fortunella Spp.): Biochemical Composition and Prophylactic Actions Theeshan Bahorun, Darshini Narrain, Piteesha Ramlagan and Chandra Tatsha Bholah

Chapter 11

Aloe Vera Extracts: From Traditional Uses to Modern Medicine Taukoorah Urmeela and Mahomoodally Mohamad Fawzi

Chapter 12

Elderberries Extracts: Biologic Effects, Applications for Therapy: A Review Mihaela Mirela Bratu and Ticuta Negreanu-Pirjol

Chapter 13

Tumor Cell Growth Activity of Fruit and Pomace Extracts Dragana Četojević-Simin

Chapter 14

Influence of Two Maturation Stages and Three Irrigation Regimes on Fatty Acid Composition of cv. Arbequina Produced under Tunisian Growing Conditions Faten Brahmi, Chehab Hechmi, Imed Chraief and Mohamed Hammami

Index

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211

227 241

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PREFACE The use of natural or naturally-derived antioxidants, instead of synthetic antioxidants, to produce foods with a longer shelf life and a higher degree of safety is a growing trend. Fruit and fruit-processing by-products are considered to be an important source of bioactive molecules (vitamins C, E, carotenoids, phenolic compounds and dietary fiber) of great interest for the food industry, although their content varies greatly depending on origin, source, type of extract and concentration levels. This book discusses biological activity, potential applications and beneficial health effects of fruit and pomace extracts. Chapter 1 – Meat and meat products are prone to both microbial and oxidative spoilage; therefore, it is desirable to use a natural preservative with both antimicrobial and antioxidant properties. This chapter aims to critically review the use of fruit and pomace extracts in order to improve the safety and quality of meat and meat products, as described in studies recently carried out worldwide. In particular, the antimicrobial and antioxidant effects of these natural food additives in fresh or frozen beef, pork and chicken meat products are evaluated. Chapter 2 – Olive oil production industry is characterized by relevant amounts of byproducts that represent an important environmental problem in Mediterranean areas where they are generated in huge quantities in short periods of time. In this work the feasibility of using olive wastewater (OW) or olive oil wastewater (OOW), in bricks, were reported. In order to evaluate how it affects the method of forming the bricks on the microstructure and properties of ceramic materials, bricks have been molded by compression or extrusion. The influence of the replacement of fresh water (FW) by wasted was analyzed. The samples containing FW, OW or OOW (22 wt %) was added to the clay in order that it acquires enough plasticity to the stage of molding by extrusion. The specimens molded by extrusion and compression were dried at 110 ºC (24 hours) and fired at 950 ºC (3 ºC/min) for 4 h. Loss on ignition, linear shrinkage, bulk density, water absorption, water suction, compressive strength, thermal conductivity and microstructural properties values of the fired samples were investigated depending on the type of waste and method of forming. Results show that the bricks obtained with olive and olive oil wastewaters are comparable and slightly better to traditional bricks used fresh water as mixing water in terms of forming and technological properties of end products. The use of waste decreases bulk density, water absorption and thermal conductivity, while slightly increases the mechanical strength of bricks, because of the closed porosity that originates during the combustion process the small content of organic matter from waste. In addition, the forming by extrusion process turns out to be more appropriate than the process of compression.

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The incorporation of OW and OOW wastewaters in bricks can represent a promising way to valorize these effluents, can alleviate the environmental impact generated by the industry of extraction of olive oil and, at the same time, represent an economic and water saving for the ceramic industry. Chapter 3 – The use of natural or naturally-derived antioxidants, instead of synthetic antioxidants, to produce foods with a longer shelf life and a higher degree of safety is a growing trend. Fruit and fruit-processing by-products are considered to be an important source of bioactive molecules (vitamins C, E, carotenoids, phenolic compounds and dietary fiber) of great interest for the food industry, although their content varies greatly depending on origin, source, type of extract and concentration levels. After a brief introduction, this chapter aims to critically review the applications of fruit and pomace extracts from processing by-products of grape, pomegranate and berry fruits, in improving the safety and quality of fish products, as described in studies recently carried out worldwide. In particular, the antioxidant and antimicrobial effects of these natural food additives in the minced muscle of various marine and freshwater fish species are evaluated. Chapter 4 – Grape cultivation dates back to approximately 6000-8000 years ago. Nowadays it is still one of the major crops produced worldwide, mostly for wine production. Accordingly, grape pomace, the solid remain of the wine making process, is produced in large quantities. The disposal of such waste material is an issue of great ecologic and economic importance. Some wineries use the material as a fertilizer, while others are selling it to biogas companies for energy production. However, grape pomace possesses a much higher potential. Pomace is composed of grape seeds, stems, pulps and skins and contains pharmaceutically interesting polyphenolic compounds such as catechin, epicatechin, transresveratrol and procyanidin B1. Such compounds have beneficial effects on human health including antioxidant, anti-inflammatory, antidiabetic and anticarcinogenic activities. Such interesting compounds may be extracted from grape pomace by the use of organic solvents, however this procedure has several limitations, including solvent toxicity and the non-selectivity of the extraction towards lipophilic compounds. Alternative extraction technologies focus on the use of supercritical fluids. Supercritical CO2 is the most commonly used solvent, since it is non-toxic, inert and has modest critical values in terms of temperature and pressure, making its use industrially appealing. By the use of supercritical fluids extraction, high-quality extracts can be obtained from a variety of raw materials, including grapes, grape seeds and grape pomace. Chapter 5 – The term ‗passion fruit‘ comprises several species from the genus Passiflora L., family Passifloraceae; the genus Passiflora consists of approximately 400 species, with over 150 being native from Brazil. The most important variety cultivated in Brazil for commercial purposes is the yellow passion fruit, Passiflora edulis Sims f. flavicarpa Degener, which is used for pulp and juice processing. Passion fruit are climacteric fruits classified botanically as fleshy fruit with round shape; the edible part of passion fruit (40 %) consists of pulp with seeds, and approximately 60 % of the peel consists of mesocarp and epicarp. The valorization of agricultural residues is receiving more attention nowadays, and many researchers have been evaluating the conversion of by-products into food ingredients and other value-added materials. Residues obtained from fruits represent an imminent environmental risk due to the high quantity generated in a short period and their polluting characteristics. Meanwhile, passion fruit pomace has been highlighted for reuse for its

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interesting composition, overcoming environmental issues and adding value to this raw material. The yellow passion fruit pomace contains bioactive compounds and high levels of dietary fiber, such as pectin. Pectin is a complex polysaccharide material that can be extracted from the cell walls of non-graminaceous plants. The structure of pectin is based on 1,4-linked α-D-galacturonic acid and has L-rhamnose residues with side-chains of neutral sugars (mainly D-galactose and L-arabinose). Pectin is a soluble fiber, and it can be used as a gelling agent and stabilizer in a variety of food, pharmaceutical, and cosmetic products. The utilization of a suitable method for pectin extraction is significant to maximize its extraction yield and improve the product quality. Numerous scientific publications have studied the influence of extraction conditions on the physicochemical characteristics and functional properties of pectin extracted from various plant tissues. Pectin can be extracted from apple pomace (1520% dry matter), citrus albedo (30-35% dry matter), beet pulp (15-20% dry matter) and passion fruit pomace (10-20% dry matter). The most commonly used method for the extraction of pectin is direct boiling, named conventional method, which takes up to approximately two hours to obtain a good yield of pectin. Due to a relatively long period of direct heating, the extracted pectin undergoes thermal degradation and a lot of time and energy is spent. Several kinds of new technologies have been studied for enhance extraction of pectin. Moderate electric field and high pressure are emerging technologies that can be use to extract the pectin using less time and low temperature. For these reason, this review will explorer extraction mechanism of these technologies. Chapter 6 – Bioactive compounds are extra nutritional constituents that naturally occur in small quantities in plant and food products. Most common bioactive compounds include secondary metabolites, such as antibiotics, mycotoxins, alkaloids, food grade pigments, plant growth factors, and phenolic compounds. Flavonoids constitute the largest group of plant phenolics, accounting for over half of the eight thousand naturally occurring phenolic compounds. Currently, flavanones are obtained by chemical synthesis or extraction from plants, and these processes are only produced in the glycosylated form. However, there are environmentally friendly bioprocesses that deserve attention regarding phenolic compound production, especially in aglycon forms. One of these flavonoids is the hesperetin, that has recently been recognized for their influence on human metabolism, acting in the prevention of some chronic diseases, as well as proving to be an important antioxidant in food. In the last few years, great attention has been paid to bioactive phenolic compounds due to their ability to promote benefits for human health. Hesperetin is reported to be a powerful radical scavenger and a promoter of cellular antioxidant defense-related enzyme activities. This compound exhibited anti-inflammatory activity by inhibiting of LPS-induced expression of the COX-2 gene in RAW 264.7 macrophages. Hesperetin is a potent chemopreventive agent; its supplementation during the initiation, post-initiation, and entire period stages of colon carcinogenesis in the male rat model in vivo significantly reversed these activities. In addition, the aglycon flavanone presents activity against parasites from tropical diseases. Considering the folk claims, several medicinal compounds (including hesperetin) have been evaluated for this antifilarial activity. Recent studies showed that hesperetin inhibited (>60%) the adult worms growth (Wuchereria bancrofti) at 7.8 and 31.2 μg/ml concentration. The bioactive aglycon phenolic compound demonstrates antiviral activity. Experimental tests showed hesperetin presents inhibition activities of genotype 2 (DENV-2) virus replication. This flavonoid seems to be usefull also in the treatment of some non-communicable diseases, such as cardiac diseases, diabetes, hypertension. A hesperetin suspension administered in

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adult male C57BL/6 mice inhibited cardiac hypertrophy, fibrosis, oxidative stress and myocytes apoptosis induced by pressure overload and protected against cardiac dysfunction. In another study, hesperetin enhanced ApoA-I-mediated cholesterol efflux in THP-1 macrophages, which was accompanied by an induction of the ABCA1 gene, which is critical for cholesterol metabolism. The effect of hesperetin on ABCA1-dependent cholesterol efflux may be explained by its potency of activation of LXRα and PPARγ enhancers. In a study conducted with Streptozotocin induced diabetic rats, hesperitin reduced vascular leakage, dilatation of retinal vessels and basement membrane thickening. In another study also with Streptozotocin induced diabetic rats, hesperitin treatment rescued retinal neuroinflammation, oxidative stress, apoptosis and oedema as a result of chronic uncontrolled hyperglycaemic state. These studies indicate that hesperitin can be used for the prevention of induced neurovascular complications caused by descompansated diabetes. Intravenous administration of hesperetin-7-O-b-D-glucuronide decreased blood pressure in anesthetized spontaneously hypertensive rat. Furthermore, it enhanced endothelium-dependent vasodilation in response to acetylcholine, decreased hydrogen peroxide-induced intracellular adhesion molecule-1 and monocyte chemoattractant protein-1 mRNA expression in rat aortic endothelial cells. Hesperitin can also be used in management of obesity due to its influence in the control of hunger and satiety. In this context, the flavanone aglycone caused an increase in the secretion of cholecystokinin (CCK) in STC-1 cells through increase in intracellular calcium concentration by the TRP (transient receptor potential) and TRP 1 ankirin channels. The addition of hesperidin analytical standard in the same model caused no effect. The increase in CCK would be interesting because this hormone assists in the control of food intake. The purpose of this chapter is to provide an overview of the study of obtainment and biological properties of hesperetin. Chapter 7 – As part of a research initiative to evaluate plants used for their nutritional and herbal values, the antimicrobial activity of the n-C6H14, CH2Cl2 and CH3CH2OH extract of Brassica rapa chinensis vegetable, Artocarpus altilis and Solanum melongena fruit and leaves of Moringa oleifera were investigated. Each plant part was subjected to selective extraction using solvents of varying polarity: n-C6H14, CH2Cl2, EtOAc and CH3CH2OH. using the Disc Diffusion Assay under asceptic conditions at a concentrations of 0.025g/ml, 0.05g/ml and 0.1g/ml against pathogens: E.coli, S.aureus, Bacillus species and C. albicans. Also, the combined CH3CH2OH and n-C6H14 extracts of A. altilis plus Brassica rapa chinensis at high concentrations were investigated. For each concentration, experimental discs on a single plate were prepared in triplicates versus a single reference disc. The diameter of the zone of inhibition, DZOI was measured from which the Area of Zone of Inhibition (AZOI) was calculated. The highest AZOI of 209.34 mm2 was induced by the CH3CH2OH extract of Brassica rapa chinensis against E. coli at a concentration of 0.025g/ml and the CH3CH2OH extract of A. altilis at a low concentration of 0.025g/ml which induces AZOI of 94.89 mm2. The lowest AZOI of 12.56 mm2 was induced by Brassica rapa chinensis against Bacillus at a concentration of 0.025g/ml. Zero AZOI was induced by n-C6H14 extract of A. altilis against all four pathogens at a low concentration of 0.025g/ml. Zero AZOI was also induced by the n-C6H14 extract of A. altilis at a low concentration of 0.025g/ml against all four pathogens and the CH3CH2OH extract of A. altilis at a high concentration against all pathogens. Selective antimicrobial activity were observed in several instances. Interestingly, the CH2Cl2 and CH3CH2OH extract at low concentration were more antimicrobial than that at high concentration of A. altilis. A similar trend was noted for the n-C6H14 and CH3CH2OH

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extract of Brassica rapa chinensis. Thus these two plants can be used as both antimicrobial and nutritional agents. The n-C6H14 and CH3CH2OH extract of Solanum melongena fruit and leaves of Moringa oleifera were tested for their antimicrobial activity at three different concentrations of 5%, 10% and 20% of crude extracts against Eschericia coli, Staphyloccocus aureus and Klebsiella pneumoniae. Both the n-C6H14 and CH3CH2OH extracts of Solanum melongena fruit and Moringa oleifera leaves showed antibacterial activity at a higher concentration of 20% of crude extract. The order of bacteria susceptibility to Moringa oleifera extract been S. aureus > K. pneumoniae > E.coli whereas that for Solanum Melongena extract been S. aureus > E.coli > K. pneumonia. The area of zone of inhibition ranging from 44.15 mm2 to 53.55 mm2. These investigations suggest that the extracts of Brassica rapa chinensis, Artocarpus altilis, Moringa oleifera and Solanum Melongena can be used as antibacterial agents in addition to their nutritional value. Chapter 8 – Coconut water is the liquid endosperm fluid of the coconut fruit which contains high amounts of essential nutrients and minerals. This endosperm fluid is a widely consumed as a beverage in many parts of the world as it provides hydration along with increased nutritional, health and medicinal benefits. In addition to being used as a medium constituent, it also acts as a natural biocatalyst. One of the fermented products of coconut water, coconut water kefir, is made by fermenting coconut water with the kefir granules which contain essential lactic acid bacteria and yeast spp. known to have health benefits for a disease-free life. It has many applications in the food industry and functional food market. It is used as one of the important constituents in a variety of products or can be consumed ‗as-itis‘. It is known to have no undesirable side effects and is said to improve digestion. This paper reviews the functional properties of coconut water, its applications in the food industry and recent advancements in this area. Chapter 9 – Cornus is a genus of the Cornaceae plant family, represented by about 30-60 species of woody plants commonly named Dogwoods, widely spread in Europe, Asia and North America. From about 2000 years ago, traditional Chinese medicine used different parts of plants belonging to Cornus genus for treatment of various diseases such as kidney and gastrointestinal disorders, diabetes, uterine bleeding and bladder incontinence. The fruits and the bark of Cornus species have been widely used for their analgesic, anti-inflammatory, antimalarial, anti-bacterial, anti-histamine, anti-allergic, anti-microbial, anti-parasitic, tonic, febrifuge and vulnerary properties as well as for their inhibitory effect on tumor cell proliferation. A high number of bioactive compounds have been identified in Cornus fruits, including ascorbic acid, phenolic compounds, anthocyanins, flavonoids, iridois, terpenoids, compounds that exert health effects especially by acting as potent antioxidants. This review will focus on the recent data reported on the extraction, characterization and biological activities of bioactive compounds isolated from fruits of selected Cornus plants in order to understand the high nutritional value of these fruits and their possible use as source of bioactive compounds for developing new pharmacological products. Chapter 10– Natural plant products continue to be of increasing interest due to the wide range of health benefits they confer to humans. Citrus fruits have been extensively studied for their health-promoting potential and have been widely applied in the medical and food industry. Kumquat, a tropical fruit originally included in the genus Citrus has been classified

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a century ago in the genus Fortunella. The latter has so far been poorly studied compared to the genus Citrus, most probably due to its limited distribution and consumption. This chapter reviews selected interesting findings on phytochemical content of kumquats with emphasis on their prophylactic effects at biochemical and molecular levels. Chapter 11 – Aloe vera, one of nature‘s most curative medicinal plants, has been traditionally used as alternative treatment against a plethora of human ailments in various countries like China, India, and Egypt, amongst others. Its therapeutic attributes have been well investigated and proven by numerous in vitro, in vivo and clinical studies. Native to North Africa, this succulent plant has been shown to be beneficial in the treatment and management of a wide range of conditions including skin disorders, constipation, non-insulin dependent diabetes mellitus, cardiovascular disorders, cancer and even AIDS. During the past recent years, the commercialisation of crude Aloe vera extracts and/or formulated products has experienced a boom in the pharmaceutical, food, cosmetic and the wellness industries. The beneficial effects of Aloe vera can be attributed to the panoply of phytonutrients and phytochemicals including non-nutritive constituents like phenolic compounds present in the plant. This chapter attempts to give an updated overview of the therapeutic uses of Aloe vera extracts and related formulation in the treatment and manage of human diseases. Chapter 12 – As many berries, the fruits of Sambucus nigra (L.) contain large amounts of flavonoids with different structures, mostly anthocyanins (mainly cyanidin-3-glucoside and cyanidin-3-sambubioside) and small quanities of flavonols and flavonol ester. Flavonoids are a broad class of low-molecular-weight secondary metabolites encompassing more than 10,000 scaffolds, and are commonly found in leaves, seeds, bark and flowers. Their role in plants is to afford protection against ultraviolet radiation, pathogens and herbivore animals. Due to their activity as safe and potent antioxidants, they are considered as important nutraceuticals. Due to the content in anthocyanins, elderberries have an attractive bright purple color, which make elderberry anthocyanins extracts valuable foodstuff colorants but also therapeutic agents. There are many studies showing the biologic effects of certain elderberries extracts, such as: in vitro and in vivo antioxidant activities, anti-inflammatory properties, stimulant of cell division. Some of them offers contradictory information. There are also reports concerning attempts to formulate and develop new pharmaceutical/nutraceutical products. This chapter tries to join together the information concerning the main therapeutic effects of elderberries extracts as they are presented in the recent publications. Also, it presents some attempts to apply the elderberries extracts in pharmacy as active principles. Chapter 13 – Fruit and fruit waste by-products that are usually obtained after industrial processing should be regarded as a potential nutraceutical resource capable of offering lowcost, nutritional and health promoting dietary supplements. They can contain significant amounts of carotenoids, phenolics, flavonoids, anthocyanins and other bioactive phytochemicals that can modulate cell proliferation, oxidative reactions in cellular systems and exert excellent anti-oxidative, anti-microbial, anti-proliferative and pro-apoptotic activities. Fruit and fruit pomace extracts of different genotypes of tomato, pepper, raspberry, bilberry and rosehip exerted pronounced and selective tumor cell growth inhibition effects in cervix, breast and colon tumor cells. They also demonstrated favorable non-tumor/tumor cell

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growth ratios and increased apoptosis/necrosis ratios. These are the qualities that favor their use as healthy food and promote the development of dietary supplements on their basis. Antitumor activity of different fruit species, genotypes and their waste by-products was compared and discussed with regard to different extraction procedures and bioactive phytochemicals content. Chapter 14 – The effects of three-irrigation managements (50% evapotranspiration [ETc], 75% ETc and 100% ETc) and two-maturation degrees (maturation I and maturation II) on the fatty acid composition of fruits from olive grown in Tunisian conditions were evaluated. At maturation grade I, at the highest level of water supplied to the variety arbequina of olive produced under Tunisian growing conditions, a statistically significant decrease of oleic acid percentage (from 66.71 to 64.73%) and an increase of gadoleic and linolenic acids levels (from 0.6 to 1.53% and from 0.84 to 1.1% respectively) were observed. At the second maturation stage, an inverse trend of the fatty acids composition at the different water managements was noted for the linolenic acid. Hence, when the percentage of palmitoleic acid increased (from 2.42 to 3.16%) the percentage of oleic acid decreased (from 64.94 to 63.35%) as the amount of water supplied to the olive tree increased. These results could be due the fact that the levels of saturated, polyunsaturated, monounsaturated fatty acids and oleic to linoleic acid ratio may have undergone some changes during ripening and also to the three different amounts of water supplied to the olive tree. Therefore, the authors noticed that, the oleic linoleic acid ratio in the second stage of maturation increased proportionally with water managements and proportionally with maturation.

In: Fruit and Pomace Extracts Editor: Jason P. Owen

ISBN: 978-1-63482-497-2 © 2015 Nova Science Publishers, Inc.

Chapter 1

FRUIT AND POMACE EXTRACTS: APPLICATIONS TO IMPROVE THE SAFETY AND QUALITY OF MEAT PRODUCTS P. G. Peiretti and F. Gai Institute of Sciences of Food Production, Italian National Research Council, Grugliasco, Italy

ABSTRACT Meat and meat products are prone to both microbial and oxidative spoilage; therefore, it is desirable to use a natural preservative with both antimicrobial and antioxidant properties. This chapter aims to critically review the use of fruit and pomace extracts in order to improve the safety and quality of meat and meat products, as described in studies recently carried out worldwide. In particular, the antimicrobial and antioxidant effects of these natural food additives in fresh or frozen beef, pork and chicken meat products are evaluated.

Keywords: Meat, citrus, apple, grape, pomegranate, plum, berry, antioxidant, antimicrobial

INTRODUCTION Fruits and pomace extracts are rich sources of antioxidants and can serve as a source of natural antioxidants for meat products. These antioxidants include fat-soluble vitamins and precursors, such as carotenoids and tocopherols, as well as flavonoids and the water-soluble vitamin C (Banerjee et al., 2012). The high content of bioactive compounds (vitamin C, carotenoids, tocopherols, phenolic compounds and dietary fiber) present in fruit by-products



Tel.: +39 011 6709232; fax: +39 011 6709297.E-mail address: [email protected]

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can be used as natural food additives (antioxidants, antimicrobials, colorants, flavorings, and thickening agents) (Schieber et al., 2001; Abd El-Khalek & Zahran, 2013). Application of various fruits and their by-products to meat products as natural antioxidants has been attempted by many researchers. Introducing natural antioxidants into meat products increases their nutritional value by bringing a health benefit to consumers, and reducing the doses of synthetic antioxidants currently being used (Bisboaca & Bara, 2011). By-products of plant food processing are a major disposal problem for the industry concerned, but they are also promising sources of compounds which may be used because of their favorable technological or nutritional properties (Schieber et al., 2001). Significant interest has recently focused on the addition of natural antioxidants to foods to replace synthetic antioxidants, due to their potential to prolong the shelf life of food products by inhibiting and delaying lipid oxidation (Rey et al., 2005). Synthetic additives can reduce food spoilage, but consumers are concerned about chemical residues in food; therefore, one of the major emerging technologies is the application of natural additives (Abd El-Khalek and Zahran, 2013). Full utilization of fruits could transform the industry into a lower-waste agribusiness, increasing industrial profitability (Ayala-Zavala and GonzálezAguilar, 2011), but application of these natural plant extracts at higher levels might be limited if the sensory quality of the meat were to be affected. The discovery of new compounds with specific roles in human metabolism has encouraged food technologists to develop new processes and soft technologies to preserve the beneficial characteristics of these compounds (González-Aguilar et al., 2008). However, different practical aspects should be borne in mind concerning their possible application in meat products: extraction efficiency, availability of sufficient material for subsequent application, and health and safety considerations (ViudaMartos et al., 2009). The use of plant-derived nutraceuticals may afford meat processors the opportunity to develop novel meat products with enhanced nutritional and health benefits (Carpenter et al., 2007). But sometimes, when these ingredients are added at high concentrations, their use results in products of lower sensory and physicochemical quality (Fernández-Ginés et al., 2005). The antioxidant potential of fruit and berry extracts against muscle lipid oxidation has been profusely documented. Therefore, the purpose of this paper is to review the information and studies on fruit and pomace extracts and their applications in meat.

APPLE Apple (Malus domestica Borkh) is a good source of total phenolics, carbohydrates, pectin, minerals and fiber with a well-balanced proportion between soluble and insoluble fractions (Gorinstein et al., 2001). Apple pomace is a co-product of the apple juice industry, abundantly available, safe and can be implemented without further fractionation or purification (Lantto et al., 2006) making it a potential fiber source for food enrichment (Figuerola et al., 2005) and giving it potential in restructured meat products (Huda et al., 2014). Furthermore, apple pomace powder, a recovered co-product of an industrial process, may contain suitable enzyme activities for food protein stabilization (Lantto et al., 2006). The

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incorporation of apple pomace into meat products (Table 1) could help to overcome the fiber deficit in the current human diet (Huda et al., 2014). Fernández-Martín et al., (2000) studied the addition of three non-meat ingredients: apple fiber, potato starch and plasma proteins to pork meat (low-fat) batters. They processed batters by cooking alone (70 °C) and by a high-pressure/temperature combination (400 MPa/70 °C) and determined some batter characteristics such as water holding and various texture parameters. No particular interactions were detected between meat batter proteins and nonmeat ingredients. Apple fiber behaved as an inert filler in both kinds of processed batter, increasing hardness but proved ineffective at improving cohesiveness and water holding in cooked-only batters. Lantto et al., (2006) studied the effects of a co-product of an industrial process (freezedried apple pomace powder) containing both tyrosinase and transglutaminase enzyme activities on heat-induced rheological changes, and on gel hardness in unheated pork meat. The efficiency of the apple pomace powder was compared with commercial microbial transglutaminase, mushroom tyrosinase and polyphenol oxidase. All the enzymes studied were able to improve the gel hardness of unheated meat homogenate at 4°C to a certain extent. These authors concluded that apple pomace powder containing protein-modifying enzymes other than proteinase has the potential to improve gel formation during heating in pork meat homogenate. Table 1. Recent articles about meat and meat products with apple by-product Meat product Pork meat batters Mutton nuggets Pork meat homogenate

Raw pork sausages Low fat chicken nuggets

Type of ingredient Impact on product Apple fiber Behave as an inert filler & Increase hardness Apple pomace Reduce hardness, texture, flavor & overall acceptability scores Apple powder Improve gel hardness & may contain suitable enzyme activities for food protein stabilization Apple puree Together with plum, decrease fat & increased moisture Apple pulp Increase dietary fiber content, redness, yellowness & chroma index

Reference Fernández-Martín et al., 2000 Huda et al., 2014 Lantto et al., 2006

Nuñez de Gonzalez et al., 2008a Verma et al., 2010

Huda et al., (2014) determined pH, cooking yield, emulsion stability, proximate composition, texture analysis and sensory properties of mutton nuggets produced with the addition of apple pomace at levels of 0%, 5%, 10%, and 15%. The results of this study indicate that mutton nuggets containing apple pomace had improved cooking yield and emulsion stability compared to the control, while pH values were significantly higher for the control than in the treated samples. Obviously, crude fiber content increased significantly with increasing levels of apple pomace, while protein, ash and fat contents were significantly higher in the control and decreased in the treated samples. Among these samples, the mutton nuggets with 15% apple pomace had significantly higher moisture content. Among textural properties, springiness, cohesiveness, chewiness and gumminess did not change for the apple pomace-incorporated treatments, whereas the addition of this by-product significantly

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decreased hardness in the meat products. Sensory evaluation showed significant reductions in flavor, texture, and overall acceptability scores in the treated samples; however, the scores were in the range of acceptability and 5% apple pomace showed the best acceptability among the treated samples. Nuñez de Gonzalez et al., (2008a) evaluated the antioxidant properties of 3% or 6% dried plum and apple purees in both raw and precooked pork sausages, stored either refrigerated or frozen. The results of objective color evaluations showed that the addition of dried plum and apple purees together and dried plum puree alone changed the internal color attributes of raw pork sausage to a small degree by darkening the samples, slightly diluting internal redness, and increasing yellowness. Consumer sensory evaluations indicated that pork sausage patties with 3% dried plum and apple purees together or 3% dried plum puree alone were liked as much as the butylated hydroxyanisole (BHA)/ butylated hydroxytoluene (BHT) treatment or control. Verma et al., (2010) studied the effect of adding apple pulp, at levels of 8%, 10% and 12%, and of a formulation replacing 40% of the common salt with a salt-substitute blend consisting of potassium chloride, citric acid, tartaric acid and sucrose, on the physicochemical, textural and sensory properties of low-fat chicken nuggets. Addition of apple pulp and replacement of common salt resulted in lower pH, cooking yield, emulsion stability, ash and protein contents and in higher moisture, dietary fiber and color parameters (redness, yellowness and chroma index) when compared to control. Textural properties (hardness, springiness, cohesiveness, gumminess and chewiness values) of chicken nuggets were affected by addition of apple pulp and common salt replacement. Sensory evaluation showed significant reductions in the texture, flavor and overall acceptability scores in treated samples, while their appearance, saltiness and juiciness scores were almost similar to the control.

CITRUS FRUIT Citrus fruits are mainly used for juice, oil and pectin production. The by-products obtained during the processing of citrus fruit to obtain juice are promising new sources of phenolic antimicrobial and antioxidant compounds (myricetin, mangiferin, gallic acid and hydrolysable tannins, which are most likely gallotannins, constitute the major antioxidant polyphenolics) and offering new commercial opportunities to the food industry (GonzálezAguilar et al., 2008). The antimicrobial activity of citrus by-products obtained from industrial manipulation of citrus fruit depended on the volatile oils present in their rinds; indeed, mandarin rind powder was the most effective, followed by orange rind powder and then grapefruit rind powder. Since limonene was present at very high and similar concentrations in the three citrus peels, the greater antimicrobial activity of mandarin essential oil might not be attributable to limonene, but probably to the presence of other essential oil constituents, given the higher proportion of oxygenated monoterpenes in mandarin. Citrus fruits are an important source of flavonoids (hesperidin, narirutin, naringin and eriocitrin) and vitamin C (Schieber et al., 2001). Kinnow or Tangerine (Citrus reticulata) is a citrus fruit variety grown in northern Indian states. In the process of juice extraction, 30–34% of kinnow peel is obtained as a major by-product. Kinnow peel is a rich source of vitamin C, carotenoids, limonene, and polyphenolic antioxidants (Anwar et al., 2008). Hesperidin was selected by Fernández-López

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et al., (2007) as the most suitable compound for monitoring polyphenol changes in sausages added with citrus fiber during processing. Citrus bioflavonoids reportedly have wide-ranging antimicrobial properties effective against a broad range of human pathogens, fungi and food spoilage organisms (Fernández-López et al., 2005). Citrus by-products can be considered as potential ingredients of meat products (Table 2), because of their ability to reduce residual nitrite levels, thus avoiding the possible formation of nitrosamides and nitrosamines (ViudaMartos et al., 2009). Health concerns relating to the use of nitrates and nitrites in cooked and dry cured meats tend toward decreased usage to alleviate the potential risk to consumers from formation of carcinogenic compounds. Alesón-Carbonell et al., (2005) assessed that albedo of citrus fruits could be an interesting functional ingredient to improve the cooking properties of beef patties, because better fat and water retention reduces cooking losses in meats. Furthermore, if an increase in dietary fiber is normally recommended in some specific diets, the increased fiber content constitutes an additional nutritional benefit for the consumer. The use of citrus fiber could be attractive to some consumers as a positive alternative to conventional fillers in meat-based products. The effects of citrus fruit (lemon, orange, mandarin, etc.) extracts and their byproducts (albedo, rind and fiber powder, etc.) have been reported on lipid oxidation of meat products, whether fresh (Alesón-Carbonell et al., 2005), cooked (Viuda-Martos et al., 2009) or dry cured (Fernández-López et al., 2008). Abd El-Khalek and Zahran (2013) evaluated the use of fruit by-products such as mandarin rind powder, orange rind powder, and grapefruit rind powder, with or without γ irradiation on color change, microbial growth and lipid oxidation of raw ground beef meat stored at 4±1°C. Table 2. Recent articles about meat and meat products with citrus by-products Meat product Ground beef meat

Type of ingredient Citrus by-products

Dry-cured sausages Beef burger

Raw & cooked lemon albedo Lemon albedo

Raw ground goat meat Goat meat patties Swedish-style meatballs Dry-cured sausages Dry-fermented sausages Fresh ground chicken Dry-cured sausage & Bologna sausage Bologna sausage

Kinnow rind powder Kinnow rind powder Orange & lemon extracts Orange dietary fiber Orange dietary fiber Citrus extract Lemon albedo & orange dietary fiber Orange dietary fiber

Impact on product Increase nutritive value, preserve & extend shelf life Decrease nitrite levels & delay oxidation development Improve cooking properties & increase fiber content Antioxidant effect

Reference Abd El-Khalek & Zahran, 2013 Alesón-Carbonell et al., 2003, 2004 Alesón-Carbonell et al., 2005 Devatkal & Naveena, 2010 Antioxidant effect Devatkal et al., 2010 Control rancidity & off-flavor Fernández-López et al., development 2005 Decrease residual nitrite levels Fernández-López et al., 2007 Decrease residual nitrite levels & Fernández-López et al., favor micrococcus growth 2008 Slight preservative effect Mexis et al., 2012 Reduce nitrite levels, thus avoiding the formation of nitrosamines & nitrosamides Improve shelf life of meat products

Viuda-Martos et al., 2009 Viuda-Martos et al., 2010a,b

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They found that color parameters were significantly affected by the additives used. All treatments increased lightness values significantly compared to the control over the 21 days of storage, while treatment with 2% grapefruit rind powder and control had the highest redness values and gave greater stability to the samples with regards to red discoloration of ground meat compared to other treatments. The results show that at day 0 different treatments caused a significant increase in yellowness values over the control value. All by-product additives significantly extended the shelf life of ground meat compared with the control, reducing total bacterial, lactic acid bacteria and total mold and yeast counts. Concerning lipid oxidation, control meat showed significantly higher malonaldehyde content throughout the storage period than treated meat. Abd El-Khalek and Zahran (2013) concluded that citrus byproducts combined with NaCl or γ irradiation preserved ground meat and extended its shelf life for more than 21 days and can therefore be used in biotechnological fields as natural preservatives for the food industry. In contrast, Mexis et al., (2012) found that the addition of citrus extract had only a slight preservative effect on fresh ground chicken meat. Alesón-Carbonell et al., (2003, 2004) studied the effect on compositional, textural, and sensory characteristics of different types of lemon albedo (raw and cooked) when these byproducts were added at different concentrations (0%, 2.5%, 5%, 7.5% and 10%) to dry-cured sausages. Products that contained 2.5%, 5%, and 7.5% of cooked albedo and 2.5% of raw albedo demonstrated sensory properties similar to conventional sausages (Alesón-Carbonell et al., 2003). Addition of 7.5 % of dehydrated cooked albedo or 5% of dehydrated raw albedo yielded products with sensory properties similar to those of control sausages (AlesónCarbonell et al., 2004). These authors concluded that the addition of lemon albedo to drycured sausages improves their nutritional properties and may have beneficial effects due to the presence of active biocompounds that decrease residual nitrite levels and delay oxidation development. Furthermore, they suggested that a good source of dietary fiber, such as lemon albedo, could be successfully used in other processed meats or other food products, including dairy and bakery products. Alesón-Carbonell et al., (2005) studied the effect of adding four concentrations (0%, 2.5%, 5% and 7.5%) of lemon albedo prepared using four different methods (either cooking and/or drying and mincing) on the quality attributes of beef burgers including: pH, fat oxidation, compositional analysis, cooking characteristics, color, texture profile analysis and a range of sensory attributes. These authors found that pH and lipid oxidation of samples were slightly affected by the type of albedo, while some treatment types significantly improved the cooking properties of meat patties when compared with the controls. Color parameters showed differences in lightness, yellowness and redness, while gumminess, springiness, hardness and chewiness grew as albedo concentration increased. Devatkal & Naveena (2010) studied the effect of 2% kinnow fruit by-product powder + 2% salt on color and oxidative stability of raw ground goat meat stored at 4°C. Addition of salt resulted in a reduction in redness scores. Lightness increased in controls and was unchanged in treated samples during storage, while redness scores declined and yellowness showed inconsistent changes. Thiobarbituric acid reactive substances (TBARS) values in meat treated with kinnow fruit was lower than control meat throughout storage and the percentage reduction in TBARS values was 123%. Salt accelerated TBARS formation, and by-products of kinnow fruit counteracted this effect. Therefore, they concluded that this powder had the potential to be used as a natural antioxidant to minimize autooxidation and salt-induced lipid oxidation in raw ground goat meat. Devatkal et al., (2010) evaluated the

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anti-oxidant effect of extracts of kinnow rind powder in goat meat patties. The results of this study showed that this extract was a rich source of phenolic compounds with free radicalscavenging activity; the authors concluded that extracts of this powder had potential for use as a natural anti-oxidant in meat products. Fernández-Ginés et al., (2003) showed that the addition of orange fiber powder (0.5, 1, 1.5, and 2%) to cooked Bologna sausage improved its nutritional value, decreased residual nitrite levels, and delayed the oxidation process. These authors reported that microbial growth was not modified by citrus fiber during storage and the products were harder and less springy and chewy at all concentrations of citrus fiber in comparison with untreated samples. All samples had similarly satisfactory quality scores except sausage with 2% orange fiber powder, which scored the lowest. Fernández-López et al., (2005) evaluated the antioxidant and antibacterial effect of orange and lemon extracts in cooked Swedish-style meatballs in comparison with rosemary and garlic extracts. They found that the application of citrus extracts and rosemary improved the acceptability of the product. Activity in a lard system was established for all the extracts and further determination of the development of rancidity measured as TBARS consistently showed that about 50% of rancidity can be controlled by the citrus preparations, while watersoluble and oil-soluble rosemary extracts were more effective, almost completely eliminating rancidity. They concluded that the application of orange and lemon extracts could serve to control the development of rancidity and off-flavors, and could have additional effects such as water binding. The use of orange fiber at five concentrations (0%, 0.5%, 1%, 1.5% and 2%) as an ingredient in dry-cured sausages was studied by Fernández-López et al., (2007). They found that TBARS values increased in all samples during drying, with higher increases in control than in treatment samples and concluded that this juice industry by-product has a protective effect from oxidation and due to the decrease in residual nitrite level could prevent nitrosamide and nitrosamine formation in meat products. The authors supposed that the high reactivity of nitrites could lead to a reaction with the polyphenols present in orange fiber. They also determined the polyphenol composition of each formulation and its evolution during dry-curing, and found that hesperidin was the most important phenolic compound in orange fiber and in sausages to which this fiber has been added. Fernández-López et al., (2008) studied the effect of adding three concentrations (0%, 1% and 2%) of orange fiber to Spanish dry-fermented sausages on their stability. Microbiological (aerobic mesophilic bacteria, lactic acid bacteria, Enterobacteriaceae, Micrococcaceae and mold and yeast counts), chemical (moisture, lactic acid and residual nitrite level), physicochemical (pH and water activity) and sensory analyses were performed by these authors. They concluded that the use of orange fiber as an ingredient has no negative effects upon the fermentation or dry-curing processes of dry-fermented sausages. They found that orange fiber addition during fermentation affected residual nitrite levels and counts of micrococcus, while fiber addition during dry-curing affected pH and water activity, while decreasing residual nitrite level and favoring micrococcus growth. Both effects have a positive impact on sausage quality and safety. Finally, similar scores for all sensory attributes were found for control sausages and sausages with 1% orange fiber, while the excessively low pH reached in sausages with 2% orange fiber could cause changes in texture and color that could affect the perception of taste, appearance and color.

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Mexis et al., (2012) investigated the combined effect of a citrus extract (0.1% and 0.2%) and an oxygen absorber (Ageless® FX type) on shelf-life extension in fresh ground chicken stored at 4 °C. The authors monitored microbiological changes (total viable count, lactic acid bacteria, Enterobacteriaceae, Pseudomonas, and Clostridium spp.), physicochemical changes (pH, total volatile nitrogen, and color) and sensory changes (odor, color, and taste) as a function of treatment and storage time. Results showed that addition of the citrus extract led to a shelf-life extension of about 2 days, while the use of the oxygen absorber substantially increased product shelf life by approx. 3 days as compared to control samples. A 4-5 day product shelf-life extension was achieved using the combination of 0.1% citrus extract and oxygen absorber. Viuda-Martos et al., (2009) described the latest advances in the use of citrus by-products (albedo, dietetic fiber obtained from the whole co-product, and washing water used in the process to obtain the dietetic fiber) in meat products as a potential ingredient to reduce sodium or potassium nitrite content. These salts are widely used as a curing agent in cured meat products, because they develop the characteristic flavor, inhibit outgrowth and neurotoxin formation by Clostridium botulinum, delay the development of oxidative rancidity, react with myoglobin and stabilize the red meat color. Citrus fiber shows the highest potential to reduce any nitrites that have not reacted with myoglobin, followed by albedo and finally washing water. Viuda-Martos et al., (2010a) found that 1% orange dietary fiber and spice essential oils (0.02% rosemary essential oil or 0.02% thyme essential oil) could find a use in the food industry to improve the shelf life of a Bologna-type sausage called mortadella. Fibre content affected the moisture, fat, ash content and color coordinates of lightness and yellowness. The treatments analysed lowered the extent of lipid oxidation and the levels of residual nitrite, while analysis of the samples revealed the presence of hesperidin and narirutin. The treated samples stored in vacuum packaging showed the lowest aerobic and lactic acid bacteria counts and no psychotropic bacteria or enterobacteria were found in any of the treatments. Sensorially, the most appreciated sample was the one containing orange dietary fiber and rosemary essential oil, stored in vacuum packaging. Viuda-Martos et al., (2010b) studied the effect of adding 1% orange dietary fiber and 0.02% oregano essential oil and of various storage conditions (air, modified atmosphere and vacuum) on the shelf-life of Bologna sausage. These authors found that samples with orange fiber and oregano essential oil showed the lowest aerobic and lactic acid bacteria counts and lowest TBARS values when they were stored in vacuum packaging, while samples with orange fiber and oregano essential oil showed similar sensory evaluation scores when stored either in air or in vacuum packaging. Viuda-Martos et al., (2010a,b) concluded that orange dietary fiber and essential oils could find a use in the food industry to improve the shelf life of various meat products.

GRAPE Grape (Vitis vinifera L.) seed extract has been investigated for use as an antioxidant in a few meat types and has been reported to improve the oxidative stability of goat meat (Rababah et al., 2011), turkey patties, and cooled stored turkey meat (Lau & King, 2003; Mielnik et al., 2006). Many studies have shown that grapes are used for increasing shelf life

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in meat and meat products (Ahn et al., 2002; Ahn et al., 2007; Kulkarni et al., 2011). Grape extract would probably be a more effective preservative in precooked or cooked meat products (Bañón et al., 2007), especially when lipid oxidation of high-fat ground meat products compromises quality (Tables 3a and 3b). Table 3a. Recent articles about meat and meat products with grape by-product Meat product Cooked ground beef

Type of ingredient Grape seed extract

Cooked ground beef

Grape seed extract

Raw beef patties Grape seed extract Ground chicken thigh Grape seed extract meat Ground chicken thigh Grape seed extract & breast Cooked pork patties Grape seed extract Pork burger Red grape pomace extract Fried beef patties

Grape seed extract

Pre-cooked, frozen, re-heated beef sausage Ground dark turkey meat

Grape seed extract

Grape seed extract

Impact on product Improve oxidative stability & reduce warmed-over flavor development Positive effect on microbial growth, color change & lipid oxidation Increase shelf life Inhibit TBARS formation & mitigate the prooxidative effects of NaCl Inhibit intensity of musty & rancid odor, & rancid flavor Decrease lipid oxidation Increase color stability & acceptability & decrease lipid oxidation Inhibit formation of heterocyclic amines Protect against oxidation & retain fresh odor & flavor longer

Reference Ahn et al., 2002

Inhibit development of TBARS

Lau & King, 2003

Ahn et al., 2007

Bañón et al., 2007 Brannan, 2008

Brannan, 2009 Carpenter et al., 2007 Garrido et al., 2011

Gibis & Weiss, 2012 Kulkarni et al., 2011

Ahn et al., (2002) evaluated the effectiveness of selected natural antioxidants added to meat samples at levels of 0.02%, 0.05% and 0.1% to reduce warmed-over flavor development in cooked ground beef. They found that 0.1% grape seed extract reduced hexanal content by 97% after 3 d of refrigerated storage, while treated meat showed significantly lower TBARS values than control meat. These authors reported no adverse effects of this natural plant extract on flavor and aroma at the 0.02% level. Ahn et al., (2007) studied the effects of 1% grape seed extract on the growth of foodborne pathogens, color changes, and lipid oxidation of cooked ground beef compared to untreated and butylated hydroxyanisole/butylated hydroxytoluene-treated meat. When compared to the control, grape seed extract effectively reduced numbers of Escherichia coli and Salmonella Typhimurium, and retarded the growth of Listeria monocytogenes and Aeromonas hydrophila. The color of cooked beef treated with grape seed extract was less light, more red, and less yellow than those treated with butylated hydroxyanisole/butylated hydroxytoluene and other plant extracts (pine bark and oleoresin rosemary). The control showed significantly higher hexanal content and TBARS during storage than cooked ground beef treated with plant extracts. Indeed, grape seed extract

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retarded the formation of TBARS by 92% after 9 days, and significantly lowered hexanal content throughout the storage period. Table 3b. Recent articles about meat and meat products with grape by-product Meat product Pork patties

Type of ingredient Grape extract

Cooked turkey breast Grape seed extract meat Cooked pork patties Grape skin Pig liver pâté

Grape seed extract

Irradiated & nonirradiated chicken breast meat Irradiated & nonirradiated chicken breast meat Baladi Goat Meats Cooked beef & pork patties Raw beef & pork patties Beef patties

Grape seed extract

Raw & cooked chicken meat

Grape seed extract

Grape seed extract Grape seed extract Grape seed extract Grape pomace extract Grape seed & peel extracts

Impact on product Increase the quality & extend the shelf-life Improve oxidative stability during heat treatment & storage Provide partial protection against lipid oxidation Reduce the oxidative deterioration of lipid Prevent & minimize major sensory changes during irradiation Decrease the amount of TBARS, hexanal & pentanal values Minimize lipid oxidation Reduce oxidative rancidity & improve shelf life Provide minimal protection against oxidation Inhibit some foodborne pathogens Prevent lipid oxidation & alter color of cooked meat

Reference Lorenzo et al., 2014 Mielnik et al., 2006 Nissen et al., 2004 Pateiro et al., 2014 Rababah et al., 2005

Rababah et al., 2006

Rababah et al., 2011 Rojas & Brewer, 2007 Rojas & Brewer, 2008 Sağdıç et al., 2011 Selani et al., 2011

Bañón et al., (2007) proposed grape seed and green tea extracts as preservatives for increasing the shelf life of low-sulphite raw beef patties, comparing the antimicrobial and antioxidant activities of both extracts with ascorbate. These authors evaluated meat spoilage (total viable and coliform counts, pH, color parameters, metmyoglobin and TBARS) and pointed to the possibility of using low-sodium metabisulphite/vegetable extract combinations to preserve raw-meat products. In particular, they found that ascorbate, grape seed and green tea extracts delayed microbial spoilage, redness loss and lipid oxidation, and improved the preservative effects of SO2 on beef patties, especially against meat oxidation. No anomalous sensory traits were caused by either extract. Brannan (2008) examined the effect of 0.1% grape seed extract and 1% NaCl on ground chicken thigh meat during refrigerated storage at different relative humidity levels. Grape seed extract delayed the reduction of water activity that occurred during refrigerated storage, but had no effect on pH or moisture content compared to the untreated control. This extract is an effective antioxidant in ground chicken thigh meat that inhibits the formation of TBARS compared to the untreated control, helps to mitigate the prooxidative effects of NaCl, and may alter the effects of NaCl on protein solubility in salted chicken patties. Brannan (2009) performed sensory, instrumental color, yield, pH, water activity, and binding strength analyses on ground chicken thigh and breast with or without grape seed extract during refrigerated storage. This author concluded that grape seed extract may be an

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effective antioxidant in precooked chicken breast systems. Indeed, this extract inhibited the intensity of musty and rancid odor, and rancid flavor compared to control patties, but in chicken thigh and breast, grape seed extract caused significantly darker, redder, and less yellow patties, while the differences in sensory scores were only due to storage time or precooking. Carpenter et al., (2007) assessed the effect of grape seed extract (0–1000 μg/g muscle) on lipid oxidation, color, pH, microbial status and organoleptic properties of raw and cooked pork patties during chilled storage. The addition of grape seed extract resulted in minor increases in the surface color of raw and cooked pork and decreases in TBARS in raw pork patties on days 9 and 12 of storage, relative to controls. The redness value of raw and cooked pork patties increased marginally with increasing grape seed extract concentration. The eating quality of cooked pork, mesophilic plate counts and pork pH was unaffected by grape seed extract addition. Garrido et al., (2011) studied the effect on meat quality (pH, microbial spoilage, lipid oxidation and color parameters) of two different types of red grape pomace extracts (0.06 g/100 g final product) obtained by different extraction systems in pork burgers packed under aerobic conditions. The addition of these two extracts did not affect their microbial spoilage and pH value. The lightness value of pork burgers decreased (darker meat) on day 6 when grape pomace extract was added. These authors concluded that the new extraction system (methanolic extraction + High-Low Instantaneous Pressure) could be a valid alternative to optimize the purity of the grape pomace extracts in order to use them as a preservative in meat foodstuffs. Gibis & Weiss (2012) assessed the ability of water-in-oil marinades containing grape seed extract (0.2, 0.4, 0.6 and 0.8 g/100 g) to reduce formation of heterocyclic amines in fried beef patties. These authors found four heterocyclic amines (MeIQx, PhIP, Harman, and Norharman) in low concentrations in fried patties and the content of MeIQx (2-amino-3,8dimethylimidazo[4,5-f]quinoxaline) and PhIP (2-amino-1-methyl-6-phenylimidazo [4,5b]pyridine) reduced significantly, by 57% and 90%, respectively, after use of marinades containing the highest extract concentration. The antioxidant capacity of grape seed was also compared with rosemary extract and resulted about two times greater. They concluded that both lipophilic and hydrophilic fractions of these extracts contain polyphenols that are apparently able to partition to the reaction site, thereby inhibiting heterocyclic amine formation. Marinating is thus a useful pre-treatment for meats prior to heating, and it should be considered as a recommended method for decreasing daily exposure of consumers to heterocyclic amines. Kulkarni et al., (2011) compared grape seed extract (100, 300, and 500 ppm) to common antioxidants (ascorbic acid at 100 ppm of fat and propyl gallate at 100 ppm of fat) in a precooked, frozen, stored meat model system sausage (70% lean beef, 28% pork fat and 2% salt). After addition of grape seed extract or common antioxidants, the meat product was formed into rolls, frozen, sliced into patties, cooked on a flat griddle to 70 °C, overwrapped in PVC, then frozen at −18 °C for 4 months. Based on sensory characteristics, instrumental color and TBARS values, grape seed extract at concentrations of 100 and 300 ppm generally performed as well as propyl gallate in maintaining product quality throughout the storage period and these samples retained their fresh cooked beef flavor and odor longer than controls during the 4-month storage period.

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Lau & King (2003) reported that the addition of 1% and 2% grape seed extract with 85.4 g of gallic acid equiv/100 g to dark poultry meat patties effectively inhibited the development of TBARS, with treated samples having 10-fold lower TBARS values compared to untreated controls. Lorenzo et al., (2014) evaluated four natural extracts from grape, tea, chestnut and seaweed with potential antioxidant activity. The addition of these natural antioxidants had a preservative effect in porcine patties during 20 days of storage in modified atmosphere packs at 2 °C. Among the four natural compounds tested, grape and tea extracts showed the most potential as alternatives to commercial antioxidants and both led to a decrease in Pseudomonas, total viable counts, lactic acid and psychotropic aerobic bacteria compared to the control. In particular, grape extract inhibited discoloration in refrigerated patties by reducing the increase in yellowness and loss of redness. These authors stated that the protective effect on the desirable red color of raw patties may influence consumer purchase decisions. Mielnik et al., (2006) tested the efficiency of four concentrations of grape seed extract (0, 0.4, 0.8, and 1.6 g/kg) in retarding the oxidative rancidity of cooked turkey breast meat. Development of lipid oxidation over the 13 days of refrigerated storage was evaluated by means of TBARS and volatile compound (hexanal, pentanal, octanal, 2-octenal, 1-octen-3-ol, 2-octen-1-ol, and 1-penten-3-ol) formation. The authors found that the ability of this extract to prevent lipid oxidation was concentration-dependent and concluded that the addition of grape seed extract combined with vacuum-packaging should be considered as a good method for improving lipid stability in cooked poultry meat. Nissen et al., (2004) compared the antioxidative efficiency of extract of grape skin with rosemary, green tea, and coffee extracts in precooked pork patties over 10 days of storage at 4°C in atmospheric air. They used descriptive sensory profiling following reheating and quantitative measurements of hexanal, TBARS and vitamin E as indicators of lipid oxidation. All extracts retarded lipid oxidation during processing of the pork patties, because their initial oxidative status showed a significantly lower level of secondary oxidation products and higher levels of vitamin E when extracts were incorporated. The effect of the extracts incorporated in the meat was clearly related to the degree of lipid oxidation and an overall ranking of the antioxidative efficiency of extracts in increasing order became apparent: Coffee60%) the adult worms growth (Wuchereria bancrofti) at 7.8 and 31.2 μg/ml concentration. The bioactive aglycon phenolic compound demonstrates antiviral activity. Experimental tests showed hesperetin presents inhibition activities of genotype 2 (DENV-2) virus replication. This flavonoid seems to be usefull also in the treatment of some non-communicable diseases, such as cardiac diseases, diabetes, hypertension. A hesperetin suspension administered in adult male C57BL/6 mice inhibited cardiac hypertrophy, fibrosis, oxidative stress and myocytes apoptosis induced by pressure overload and protected against cardiac dysfunction. In another study, hesperetin enhanced ApoA-I-mediated cholesterol efflux in THP-1 macrophages, which was accompanied by an induction of the ABCA1 gene, which is critical for cholesterol metabolism. The effect of hesperetin on ABCA1-dependent cholesterol efflux may be explained by its potency of activation of LXRα and PPARγ enhancers. In a study conducted with Streptozotocin induced diabetic rats, hesperitin reduced vascular leakage, dilatation of retinal vessels and basement membrane thickening. In another study also with Streptozotocin induced diabetic rats, hesperitin treatment rescued retinal neuroinflammation, oxidative stress, apoptosis and oedema as a result of chronic uncontrolled hyperglycaemic state. These studies indicate that hesperitin can be used for the prevention of induced neurovascular complications caused by descompansated diabetes. Intravenous administration of hesperetin-7-O-b-D-glucuronide decreased blood pressure in anesthetized spontaneously hypertensive rat. Furthermore, it enhanced endothelium-dependent vasodilation in response to acetylcholine, decreased hydrogen peroxide-induced intracellular adhesion molecule-1 and monocyte chemoattractant protein-1 mRNA expression in rat aortic endothelial cells. Hesperitin can also be used in management of obesity due to its influence in the control of hunger and satiety. In this context, the flavanone aglycone caused an increase in the secretion of cholecystokinin (CCK) in STC-1 cells through increase in intracellular calcium concentration by the TRP (transient receptor potential) and TRP 1 ankirin channels. The addition of hesperidin analytical standard in the same model caused no effect. The increase in CCK would be interesting because this hormone assists in the control of food intake. The purpose of this chapter is to provide an overview of the study of obtainment and biological properties of hesperetin.

1. INTRODUCTION Flavonoids correspond to an important group of plant-derived heterocyclic organic compounds. They are divide into 14 different subgroups [1], based on their chemical nature and position of substituents on the A, B and C rings. Their relavance are due to many biological properties that have been reported, including antimicrobial, antioxidant and vascular activities [2]. Flavonoids are usually found in the form of glycosides in foods of plant origin, in particular in vegetables, beverages and citrus fruits [3]. The therapeutical effects of flavonoids are due to their hydrogen-donating antioxidant activity and their capability to complex the divalent transition metal cations involved in processes forming radicals. These compounds have two aromatic rings enclosing a heterocyclic six-membered ring with oxygen. Different classes of flavonoids are based on modifications of this central C-ring: flavones, flavonols, flavanones, isoflavonoids, anthocyanins, flavanols, chalconoids, dihydrochalcones and aurones [4]. This chapter is divided into three parts: sources of hesperetin; methods of extraction/obtantion; and biological potential.

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2. SOURCES OF HESPIRITIN Hesperetin (4′-methoxy-3′,5,7-trihydroxyflavanone), which is a bioactive plant flavonoid belonging to the chemical class ‗flavanone‘ (abundantly present in citrus fruits), is rapidly emerging as an especially attractive therapeutic agent with an enormous spectrum of activities. This flavonoid corresponds to the aglycone form of hesperidin. Although hesperetin can be considered much more biologically active, firstly hesperidin is obtained, which is the natural form of these compounds. Hesperidin (6''-O-(α-L-rahmnopyranosyl)-D-glucose flavonoid) consists of the hesperetin bound at the C-7 position (on ring A) to rutinose (C12H22O10), a disaccharide composed of one molecule of rhamnose and one of glucose. However, one important drawback is the limited bioavailability of many flavonoids, and in fact the sugar moiety has been proposed as the major determinant of the absorption of dietary flavonoids in humans, whereas the rutinoside moiety is poorly absorbed in comparison with the aglycone and glucoside forms [5]. Within this context, the enzymatic de-glycosylation of flavonoids has been reported as a good alternative for increasing antioxidant activity of these compounds [6]. Hesperidin is the predominant flavanone glycoside of sweet oranges and is extracted from citrus peel [7] and applied in pharmaceutical industries for its therapeutic importance to many diseased capillary conditions [8]. Orange peel flavedo and albedo are interesting sources of phenolic compounds, more especifically flavonoids including hesperidin and hesperetin. Furthermore, orange peel is the primary waste fraction in the production of orange juice, and therefore it has been used as a source of hesperidin because of its high concentration in this material [9]. Taking into account flavonoids are mainly abundant in plant species from the genus Citrus, they present significant impact on nearly every aspect of citrus fruit production and processing. They are responsible for some unpleasant characteristics of fruit juices, such as turbidity and bitterness [9] and particularly hesperidin clogs the steel pipes of the citrus juice plants. In addition, they are abundant in the by-products, mostly in peels (albedo + flavedo), accounting for 4–12% of the dry weight [10]. Its recovery from citrus industry by-products is attractive because of two main reasons: its bioactive properties and the reduction of the amount of residues. Moreover, worldwide industrial wastes may be estimated at more than 1.5 × 106 tons, as the amount of residue obtained from the fruits accounts for 50% of the original whole fruit mass [11]. From citrus flavonoids, hesperidin is the most abundant in lemons, limes, sweet oranges, tangors and tangelos (∼15 mg/100 g edible fruit) [12]. Owing to the importance of hesperidin for food and pharmaceutical industries, several efforts have been made for its extraction and purification. In the paper of Di Mauro et al. [13] a procedure for recovering hesperidin from the waste water of orange juice processing by concentration of diluted extracts on styrene−divinylbenzene resin was reported, resulting in high concentration of hesperidin in selected fractions (10−78 g/L). On the other side, in the work of Ma et al. [14] hesperidin was extratced from penggan (Citrus reticulata) peels by ultrasound-assisted extraction, with interestng results. Kanaze et al. [15] investigated orange peel (Citrus sinensis) cultivated in Greece–Crete as an a new commercial source of hesperidin. The flavonoid content of several methanolic

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extract fractions of Navel orange peel (flavedo and albedo of Citrus sinensis) cultivated in Greece was first analysed phytochemically and then assessed for its antioxidant activity in vitro. The main flavonoid groups found within the fractions examined were polymethoxylated flavones, O-glycosylated flavones, C-glycosylated flavones, O-glycosylated flavonols, Oglycosylated flavanones and phenolic acids along with their ester derivatives. Furthermore, the quantitative HPLC analysis confirmed that hesperidin is the major flavonoid glycoside found in the orange peel. The authors concluded that quantity of hesperidin at 48 mg/g of dry peel permits the commercial use of orange peel as a source for the production of this compound. Although the main source of hesperidin is citrus peel, literature reports other different sources of hesperidin, such as Cyclopia species (Fabaceae) [16] and Rosemary (Rosmarinus officinalis, Lamiaceae) [17].

3. METHODS OF EXTRACTION/OBTAINTION The solvent extraction is the most used method for phenolic compounds obtainment from plant tissue. The main factor is the phenolics solubility, which depends on its chemical structure. Plant materials may contain different concentrations of phenolic acids, phenylpropanoids, anthocyanins and tannins. It is possible to occur interactions between phenolics and other plant components, such as carbohydrates and proteins which form complexes responsible for insolubility. Besides that, the polarity of solvent affects the solubility and therefore, it is considered difficult to develop a extraction method suitable for all plant phenolics [18]. Ethanol and methanol are the most used solvents for the citrus flavonoids extraction, as hesperidin, narigenin, narirutin and neohesperidin. It is usually extracted from byproducts residues [19, 20, 21]. Nevertheless, the two solvents present some limitations, such as low efficiency recovery, long extraction time and degradation of unsaturated compounds [21]. To solve this, many technologies have been studied to improve solvent extraction. Some new ―green‖ extraction techniques, aimed at sparing energy and reducing costs, such as solid state and submerse fermentation, enzymatic, microwave- or ultrasound-assisted extraction, ultrafiltration, flash distillation and controlled pressure drop processing [22, 23] have been studied to improve solvent extraction. For phenolics extraction it is first necessary to release it from the vegetable matrix. Most of the technologies cited above are used aiming to improve release of phenolics from other compounds complexes as pectin and cellulose; and diffusion of the specific composites into extraction solvent.

3.1. Subcritical Water Subcritical water has been used to flavonoids extraction, such as citrus flavanones with selectivity capacity modelled by temperature solubility dependence of the phenolics [24, 21]. The subcritical water extraction (SWE) is based on solubility enhacement of phenolics compounds in high temperature water (100-374ºC). To keep the liquid state of water, a high

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pressure is applied (>40 atm) [25, 21]. With high temperatures it is possible to change the water polarity, which permits the solubility of not so polar molecules [26, 21]. The performance of SWE in hesperidin extraction from Citrus unshiu peel was evaluated by Cheigh et al. [21]. Varying the extraction temperature (110–200 °C) and time (5–20 min) under high pressure (100 ± 10 atm), they obtained almost 99% of extraction yield in 10 minutes at 170ºC. When compared with ethanol, methanol and hot water, the extraction yield of SWE was 1.9-, 3.2-, and 34.2-fold higher, respectively.

3.2. Ultrasound Plant material extraction using ultrasound technique in a laboratory scale has been widely used. Review papers were published dealing with the extraction of plant origin metabolites [27, 28], food flavonoids with different solvents [29, 28] and bioactives from herbs [30, 28]. The ultrasonic technology in food processing has attracted widely attentions nowadays [31] and also many papers had described the ultrasonic extraction of flavonoids from citrus peel [31, 32, 33]. Ultrasound generates energy throught a sound wave which is transferred to the medium resulting in a continuous wave type motion with longitudinal waves creating alternative compression and rarefaction of the medium [34, 35]. This wave type motion forms cavitation bubbles and are classified in two types of cavitation: a stable one with increasing and decreasing size behavior giving rise to the so-called ―stable cavitation‖ generating a microagitation of the medium. The second one called ―transient cavitation‖ can also grow and collapse generating very high local temperatures (5000 K) and pressures (1000 atm) with high energy shear waves and turbulence in the cavitation zone [36, 37]. The effects of ultrasound depends on the frequency used and the sound wave amplitude applied contributing to a diverse number of physical, chemical and biochemical effects observed, which permits a variety of applications. Shock waves are generated due to cavitation, which are contributed to the ultrasound effect. Formation and behaviour of the bubble of cavitation upon the propagation of the acoustic waves constitute the essential events which induce the majority of the acoustic effects [38, 39, 36, 40, 35], including the catalysis of solvent extraction, considering that the diffusion through the cell walls and washing out (rinsing) the cell contents are the two types of physical phenomena involved on extraction mechanism. Both phenomena are significantly affected by ultrasonic irradiation [30].

3.3. Microwaves The Microwave-assisted extraction (MAE) is another technology used to improve solvent extraction of bioactive compounds with high efficiency in extraction time and environmentalfriendliness [41, 42]. The mechanism is based on heating dipolar compounds by microwaveirradiation generating a cell wall destruction, release of compounds and diffusion into extraction solvent. Microwaves are transmitted as waves, defunding into biomatrices and interact with polar molecules, such as water in the biomaterials to create heat. Consequently, microwaves can heat a whole material to penetration depth simultaneously. This behavior makes the effect of microwave energy strongly dependent on the dielectric susceptibility of

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both solvent and solid plant matrix[41]. The vantages of MAE are the prevention of extracted materials decomposition, reduction in heating period and in volume of solvent demand [42]. MAE is considered a potential technology to improve traditional solid–liquid extraction for the metabolites extraction from plants. Many advantages for MAE application for nutraceuticals includes reduced extraction time, reduced solvent utilization generating improved extraction yield. MAE is also comparable to other modern extraction techniques such as supercritical fluid extraction due to its process simplicity and low cost. By considering economical and practical aspects, MAE is a strong novel extraction technique for the extraction of nutraceuticals. Nevertheless, MAE when compared to SFE, requires an additional filtration or centrifugation to remove the solid residue during MAE. Moreover, the efficiency of microwaves depends on target compounds solvent polarity decreasing its efficiency on bioactive compounds extraction [41]. Previously, [43] employed MAE for extraction of hesperidin from pericarpium citri reticulate (dried pericarp of the ripe fruit of C. reticulata), by using 70% aqueous methanol as a solvent, and showed that MAE is a fast, efficient and energy-saving extraction method [42]. In another published study [44] compared MAE, ultrasound and rotary methods to extract phenolic acids from citrus mandarin peels. They concluded that MAE is a better approach showing many advantages, such as shorter time, less solvent, higher extraction rate, savings of energy and better products with lower cost.

3.4. Microbial Transformation Microbial fermentation has appeared as a biotechnology alternative for biomaterial pretreat and for obtaining bioproducts metabolized by microorganism. Enzymes present in microbial fermentation are responsible for hydrolysis of glucosidic phenolics, increasing the release with increased solubility. Comparing with other processes that use high temperatures and therefore generate high energy costs, this could be a great advantage. This difference in clearance of phenolic compounds is due to the metabolic activity of each microorganism. This case is related to various types of microbial enzymes and their activities [45, 46]. Georgetti et al. [47] evaluated the biotransformation of polyphenol glycosides from soybeans to form aglycones through Aspergillus awamori solid-state fermentation. This result was direct correlated with β-glucosidase enzyme production. The greater number of free hydroxyl groups present in the non-glycoside form is responsible for the increae on biological activity. The microbial biotransformation of phenolic compounds seems to be a promising way to increase the concentration of phenolics with high biological potential [45]. Madeira et al. [45] developed a bioprocess for phenolics obtainment from Brazilian Citrus residues by Paecilomyces variotii solid-state fermentation. Using 10g of Citrus residues (2.0 mm of substrate particle size), 20mL distilled water, at 32 ºC after 48h of incubation were the optimum conditions which generated, simultaneously, an increase of 900, 1400 and 1330% of hesperetin, naringenin and ellagic acid concentration, respectively, and an increase of 73% of the antioxidant capacity.

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3.5. Enzymatic Extraction Enzyme-assisted extraction has been reported for extraction of carotenoids from marigold flower [48], vanillin from vanilla green pods [49], oil from coconut or seeds [48, 50] and phenols from black currant and herb [51, 52]. The enzyme-assisted extraction mechanism is based on cell wall degrading capacity of enzymes glucanases and pectinases which can weaken or break down the cell wall permitting the intracellular materials release and more accessible for extraction. The β -glucosidase (β-D-glucoside glucohydrolase, EC 3.2.1.21) catalyzes the hydrolysis of disaccharide glycosides and conjugates from the non-reducing end. It has several applications in the pharmaceutical industries with hydrolysis of cellobiose to glucose. The βglucosidase enzyme has numerous applications in the food and pharmaceutical industries, working in the hydrolysis of cellobiose to glucose, cellulose to glucose in combination with other cellulolytic enzymes, and the release of aroma compounds in fruit juices and wine. This enzyme is also used in the hydrolysis of cyanogenic compounds present in plants for hormone replacement therapy [53, 54, 46]. Li et al., [20] studied the enzymatic treatment for aqueous extraction of the total phenolic contents of five citrus peels (Yen Ben lemon, Meyer lemon, grapefruit, mandarin and orange). The highest recovery using Celluzyme MX (cellulase) in the enzyme-assisted extraction process was up to 65.5% (about 87.9% of the solvent extraction). The phenolics in grapefruit peels had the highest total antioxidant activity, followed by Yen Ben lemon, mandarin, orange and Meyer lemon according to the total antioxidant activity (FRAP). Moreover, Mandalari et al. [55] evaluated the effect of pectinases and cellulases on hydrolysis of hesperidin in Bergamot (Citrus bergamia Risso) peel and obtained more than 90% of glycosidic cleavage generating the aglycone form (hesperetin).

4. BIOLOGICAL POTENTIAL Hesperetin has a variety of biological effects in numerous mammalian cell systems, in vitro as well as in vivo. They have been shown to exert antimicrobial, antiviral, antiulcerogenic, cytotoxic, antineoplastic, mutagenic, anti-inflammatory, antioxidant, antihepatotoxic, antihipertensive, hypolipidemic and antiplatelet activities. The next topic will discuss the biological potential of hesperetin: combating tropical diseases, anti-tumor, obesity, diabetes and cardiovascular diseases. Filariasis is an endemic disease in tropical and sub-tropical regions of Asia, Africa, Central, South America and Pacific Island nations. Lymphatic Filariasis is caused by the worms Wuchereria bancrofti, Brugia malayi, and Brugia timori, which occupy the lymphatic system and in chronic cases lead to the disease Elephantiasis. Flavonoids like naringenin, hesperetin, and naringin were evaluated against the human lymphatic filarial parasite, using an in vitro motility assay with adult worms and microfilariae. Naringenin and hesperetin killed the adult worms and inhibited (>60%) at 7.8 and 31.2 μg/ml concentration, Microfilariae (mf) were killed at 250–500 μg/ml. Thus hesperetin may provide a lead for the design and development of new antifilarial agent [56].

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Hesperetin is reported to be a powerful radical scavenger and a promoter of cellular antioxidant defense-related enzyme activities. This compound exhibited anti-inflammatory activity by inhibiting of LPS-induced expression of the COX-2 gene in RAW 264.7 macrophages. Hesperetin is a potent chemopreventive agent; its supplementation during the initiation, post-initiation, and entire period stages of colon carcinogenesis in the male rat model in vivo significantly reversed these activities. Administration of hesperetin to 1,2dimethylhydrazine (DMH)-treated rats decreased the tumor incidence and the number of aberrant crypt foci with simultaneous enhancement of tissue lipid peroxidation, glutathione Stransferase (GST), GPx, SOD, and CAT activities. Hesperetin induced Notch homolog 1 (NOTCH1) expression in human gastrointestinal carcinoid (BON) cells, subsequently suppressing tumor cell proliferation and bioactive hormone production. Furthermore, results of anti-carcinogenesis experiments indicated that hesperetin inhibited aflatoxin B1-induced carcinogenesis and that hesperetin caused cytotoxicity and apoptosis via a transient induction of caspase-3 activity in HL60 cells. Additionally, it exhibited strong antiproliferative activity in various cancer cells, and its treatment dose showed no toxic effect on normal cells [56, 57]. There are also some evidence that this flavonoid might be usefull in the treatment of some other non-communicable diseases, such as cardiac diseases, diabetes, hypertension. Considering hypertension, hesperetin and hesperetin-7-O-β-D-glucuronide (HPT7G) enhanced nitric oxide (NO) release by inhibiting NADPH oxidase (nicotinamide adenine dinucleotide phosphate-oxidase) activity in human umbilical vein endothelial cell culture, indicating that hesperetin metabolites in plasma can improve vasodilatation in the vascular system. In the same work, the authors treated women with cold sensitivity, and a single dose of water-dispersible hesperetin was effective on peripheral vasodilatation. These results strongly suggest that hesperetin exert a potential vasodilatation effect by the endothelial action of its plasma metabolites [58]. Another group of researchers investigated the effects of HPT7G and hesperetin-30-O- βD-glucuronide (HPT30G), which are the predominant hesperetin metabolites in rat plasma, on blood pressure and endothelial function. Intravenous administration of hesperetin and HPT7G (5 mg/kg) decreased blood pressure in spontaneously hypertensive rats (SHRs) compared to the control group. HPT7G enhanced endothelium-dependent vasodilation in response to acetylcholine, but had no effect on endothelium independent vasodilation in response to sodium nitroprusside (SNP) in aortas isolated from SHRs. HPT7G and hesperetin decreased ICAM-1 (intracellular adhesion molecule-1) and MCP-1 (monocyte chemoattractant protein1) mRNA expression induced by hydrogen peroxide in rat aortic endothelial cells. In contrast, HPT30G had little effect on these parameters. In conclusion, HPT7G exerted hypotensive, vasodilatory and anti-inflammatory activities, similar to hesperetin, indicating that this flavanone could improve hypertension and endothelial dysfunction [59]. A hesperetin [(95 %) from Sigma-Aldrich] suspension was administered for adult male C57BL/6 mice (8–10 weeks old) at a constant volume of 1 ml/100 g body weight by oral gavage once a day. The animals were submitted to aortic banding leading to cardiac remodeling induced by pressure overload. The results indicate that hesperetin inhibited cardiac hypertrophy and myocite cross-sectional area. In response to pressure overload, it was observed the activation of PKCα/βπ, Akt, GSK3β, mTOR, FOXO3a, CaN, GATA4 and JNK. However, hesperetin supplementation almost completely blocked the activation of these factors. Also, aortic banding caused perivascular and intersticial fibrosis that was remarkably reduced in hesperetin-fed mice. mRNA levels of the fibrotic mediators TGFβ1 (transforming

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growth factor-β1), CTGF (connective tissue growth factor) and collagen I were high in animals submitted to aortic banding, but hesperetin consumption significantly reduced their expression. The flavanone also attenuated oxidative stress acting in the reduction of NADPH oxidase activity and recovery of SOD1 and SOD2 mRNA expression [60]. In another study, hesperetin enhanced ApoA-I-mediated cholesterol efflux in THP-1 macrophages, probably due to a greater transcription of ABCA1 gene, which is critical for cholesterol metabolism. The effect of hesperetin on ABCA1-dependent cholesterol efflux may be explained in part by its LXRα and PPARγ agonist action. These results indicates the potential of this flavonoid in the prevention and treatment of atherosclerosis [61]. In a study conducted with Streptozotocin induced diabetic rats, hesperetin (200mg/kg body weight by oral gavage) reduced vascular leakage, dilatation of retinal vessels and retinae basement membrane thickening. Diabetic rats treated with hesperetin had lower values of VEGF and PKC-β (angiogenic factors), when compared to untreated diabetic rats. These results indicate that retinal vasoprotective effects of hesperetin are due to its anti-angiogenic properties, preventing early or late stage micro-vasculopathy [62]. In another study developed by the same group also in Streptozotocin induced diabetic rats, hesperetin treatment reduced retinal neuroinflammation with lower levels of TNF-α and IL-1β; reduced oxidative stress with higher levels of glutathione, superoxide dismutase and catalase; inhibited apoptosis via caspase-3 and reduced edema [63]. In both studies, hesperetin treatment in diabetic rats caused a glycaemia reduction, however the glucose levels remained high. These results indicate that hesperetin can be used for the prevention of induced neurovascular complications caused by decompensated diabetes. Another complication observed in diabetes is the synthesis of advanced glycation endproducts, such as pentosidine. These compounds contribute to the lesions characteristic of microvascular complications and alter glomerular permeselectivity to proteins in diabetes. In a collagen advanced glycation in vitro study, hesperetin treatment (250µmol/L in 2% ethanol) inhibited 60% pentosidine formation in collagen incubated with glucose. Aminoguanidine and pyridoxamine, known glycoxidation inhibitors, prevented pentosidine formation by 86% and 89% respectively. These results indicate the promising potential of hesperetin in glycoxidation treatment [64]. Hesperetin can also be used in management of obesity due to its influence in the control of hunger and satiety. In this context, hesperetin analytical standard (0.1 – 1.0 mM) has shown the increase of secretion of cholecystokinin (CCK) in STC-1 cells. This phenomenon was caused by higher intracellular calcium concentration due to the TRP (transient receptor potential) and TRP 1 ankirin channels work. The addition of hesperidin analytical standard in the same model caused no effect, indicating that only the aglycone form influences hormone secretion [65]. The increase in CCK would be interesting because this hormone, secreted from endocrine cells in the small intestine, assists in the control of food intake [66]. Also, Yoshida et al. [67] observed that 3T3-L1 adipocytes cell culture treatment with hesperetin and naringenin analytical standards showed anti-inflammatory effect. NFκB activation through TNF-α was inhibited with a consequent reduction in the secretion of interleukin-6 (IL-6). There was also observed an inhibition of ERK (extracellular signal regulated kinase) pathway causing a decreased activation of hormone sensitive lipase (HSL); contributing to reduce the insulin resistance. Subash-Babu et al., [68] studied the effects of hesperetin in immortalized human bone marrow mesenchymal stem-cell (TERT20) differentiated with dexamethasone, IBMX,

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indomethacin and insulin. Hesperetin was added in two different situations: in group 1 the flavanone was administered in the differentiation medium; in group 2 the compound was added after the differentiation in the maintenance medium. In both cases there were a reduction on lipid accumulation by staining with Oil Red O, even though the effect was more pronounced in group 2, with almost 50% reduction. The glycerol release results shows that the less amount of lipid could be caused by stimulation of lipolysis. Hesperetin treatment also reduced triglyceride levels and GPDH activity, enzyme essential for glycerol-3phosphate synthesis, precursor of triacylglycerol. Only in group 1 there was a reduction in protein expression of PPAR-γ and C / EBPα, transcription factors necessary for the differentiation of pre-adipocytes into mature adipocytes. Adiponectin levels reduced after cell differentiation; however, treatment with hesperetin increased the mRNA expression of this adipokine. Resisitn, TNF-α and LPL mRNA expression reduced with hesperetin treatment. In addition, there was an increase in Bax, Bcl and p21 mRNA expression with hesperetin, especially in group 2, indicating the possible action of the flavanone in programmed cell death of differentiated adipocytes. For the authors, hesperetin could inhibit pre-adipocyte differentiation.

CONCLUSION Hesperetin belongs to one of the largest group of plant phenolics, accounting for over half of the eight thousand naturally occurring phenolic compounds. Currently, most of phenolic compounds are obtained by chemical synthesis or extraction from plants, and these processes are only produced in the glycosylated form. However, there are environmentally friendly bioprocesses that deserve attention regarding phenolic compound production, especially in aglycon forms. These bioprocesses are clean technologies with great potential for obtaining biologically active compounds from natural sources, such as hesperetin. The studies performed both in vitro and in vivo have shown that hesperetin play an important role in the prevention of degenerative and infective diseases, which is related to particular chemical structures. Hesperetin belongs to flavanones, which is a widely distributed group of polyphenolic compounds, called ―nutraceutical substances‖, with anticancer, antiatherogenic, antimicrobial and anti-inflammatory properties.

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[53] Van Den Brink, J.; De Vries, R.P. Fungal enzyme sets for plant polysaccharide degradation. Appl. Microbiol. Biotechnol. v. 91, p. 1477-1492, 2011. [54] Puri, M.; Sharma, D.; Barrow, C.J. Enzyme-assisted extraction of bioactives from plants. Trends in Biotechnol. v. 30, p. 37-44, 2012. [55] Mandalari, G.; Bennett, R.N.; Kirby, A.R.; Lo Curto, R.B.; Bisignano, G.; Waldron, K.W.; Faulds, C.B. Enzymatic hydrolysis of flavonoids and pectic oligosaccharides from bergamot (Citrus bergamia Risso) peel. J. Agric. Food Chem. v. 18, p. 8307-13, 2006. [56] Lakshmi, V.; Joseph, SK.; Srivastava, S.; Verma, S.K.; Sahoo, M.K.; Dube, V.; Mishra, S.K.; Murthy, P.K. Antifilarial activity in vitro and in vivo of some flavonoids tested against Brugia malayi. Acta Tropica. v. 116, p.127-133, 2010. [57] Hirata, A.; Murakami, Y.; Shoji, M.; Kadoma, Y.; Fujisawa, S. Kinetics of radicalscavenging activity of hesperetin and hesperidin and their inhibitory activity on COX-2 expression. Anticancer Res. v. 25, p. 3367-3374. [58] Takumi, H.; Nakamura, H.; Simizu, T.; Harada, R.; Kometani, T.; Nadamoto, T.; Mukai, R.; Murota, R.K.; Kawaix, Y.; Terao, J. Food Funct. 3, 389 (2012). [59] M. Yamamoto, H. Jokura, K. Hashizume, H. Ominami, Y. Shibuya, A. Suzuki, T. Hase, A. Shimotoyodome, Food Funct. 4, 1346 (2013). [60] W. Deng, D. Jiang, Y. Fang, H. Zhou, Z. Cheng, Y. Lin, R. Zhang, J. Zhang, P. Pu, Y. Liu, Z. Bian, Q. Tang, J. Mol. Hist. 44, 575 (2013). [61] A. Iio, K. Ohguchi, M. Iinuma, Y. Nozawa, M. Ito, J. Nat. Prod. 75, 563 (2012). [62] B. Kumar, S. K. Gupta, B.P. Srinivasan, T. C. Nag, S. Srivastava, R. Saxena, Vascular Pharmacology 57, 201 (2012). [63] 63.B. Kumar, S. K. Gupta, B.P. Srinivasan, T. C. Nag, S. Srivastava, R. Saxena, K. A. Jha, Microvascular Research 87, 65 (2013). [64] P. Urios, I. Kassab, A.M. Grigorova-Borsos, R. Guillot, P. Jacolot, F. Tessier, J. Peyroux, M. Sternberg, Diabetes Research and Clinical Practice 105, 373 (2014). [65] H. Y. Kim, M. Park , K. Kim, Y. M. Lee, M. R. Rhyu, Biomolecules & Therapeutics 21(2), 121–125 (2013). [66] H. E. Raybould, Current Opinion in Pharmacology 7(6), 570 (2009). [67] H. Yoshida, N. Takamura, T. Shuto, K. Ogata, J. Tokunaga, K. Kawai, H.Kai, Biochemical and Biophysical Research Communications 394, 728 (2010). [68] P. Subash-Babu, .A. A. Alshatwi, J. Biochem Molecular Toxicology 00 (2014).

In: Fruit and Pomace Extracts Editor: Jason P. Owen

ISBN: 978-1-63482-497-2 © 2015 Nova Science Publishers, Inc.

Chapter 7

A REVIEW OF THE ANTIMICROBIAL ACTIVITY OF VARIOUS SOLVENT TYPE EXTRACTS FROM SOME FRUITS AND EDIBLE PLANTS R. C. Jagessar1*, N. Ramchartar2 and O. Spencer2 1

Department of Chemistry, Department of Biology, University of Guyana, South America

2

ABSTRACT As part of a research initiative to evaluate plants used for their nutritional and herbal values, the antimicrobial activity of the n-C6H14, CH2Cl2 and CH3CH2OH extract of Brassica rapa chinensis vegetable, Artocarpus altilis and Solanum melongena fruit and leaves of Moringa oleifera were investigated. Each plant part was subjected to selective extraction using solvents of varying polarity: n-C6H14, CH2Cl2, EtOAc and CH3CH2OH. using the Disc Diffusion Assay under asceptic conditions at a concentrations of 0.025g/ml, 0.05g/ml and 0.1g/ml against pathogens: E.coli, S.aureus, Bacillus species and C. albicans. Also, the combined CH3CH2OH and n-C6H14 extracts of A. altilis plus Brassica rapa chinensis at high concentrations were investigated. For each concentration, experimental discs on a single plate were prepared in triplicates versus a single reference disc. The diameter of the zone of inhibition, DZOI was measured from which the Area of Zone of Inhibition (AZOI) was calculated. The highest AZOI of 209.34 mm 2 was induced by the CH3CH2OH extract of Brassica rapa chinensis against E. coli at a concentration of 0.025g/ml and the CH3CH2OH extract of A. altilis at a low concentration of 0.025g/ml which induces AZOI of 94.89 mm2. The lowest AZOI of 12.56 mm2 was induced by Brassica rapa chinensis against Bacillus at a concentration of 0.025g/ml. Zero AZOI was induced by n-C6H14 extract of A. altilis against all four pathogens at a low concentration of 0.025g/ml. Zero AZOI was also induced by the n-C6H14 extract of A. altilis at a low concentration of 0.025g/ml against all four pathogens and the CH3CH2OH extract of A. altilis at a high concentration against all pathogens. Selective antimicrobial activity were observed in several instances. Interestingly, the CH 2Cl2 and CH3CH2OH extract at low concentration were more antimicrobial than that at high concentration of A. altilis. A similar trend was noted for the n-C6H14 and CH3CH2OH extract of Brassica *

[email protected].

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R. C. Jagessar, N. Ramchartar and O. Spencer rapa chinensis. Thus these two plants can be used as both antimicrobial and nutritional agents. The n-C6H14 and CH3CH2OH extract of Solanum melongena fruit and leaves of Moringa oleifera were tested for their antimicrobial activity at three different concentrations of 5%, 10% and 20% of crude extracts against Eschericia coli, Staphyloccocus aureus and Klebsiella pneumoniae. Both the n-C6H14 and CH3CH2OH extracts of Solanum melongena fruit and Moringa oleifera leaves showed antibacterial activity at a higher concentration of 20% of crude extract. The order of bacteria susceptibility to Moringa oleifera extract been S. aureus > K. pneumoniae > E.coli whereas that for Solanum Melongena extract been S. aureus > E.coli > K. pneumonia. The area of zone of inhibition ranging from 44.15 mm2 to 53.55 mm2. These investigations suggest that the extracts of Brassica rapa chinensis, Artocarpus altilis, Moringa oleifera and Solanum Melongena can be used as antibacterial agents in addition to their nutritional value.

Keywords: Antimicrobial, Brassica rapa chinensis, Artocarpus altilis, Solanum melongena fruit, Moringa oleifera leaves, E.coli, S.aureus, Bacillus species, K. pneumonia, C. albicans, antimicrobial selectivity, bacteria susceptibility

INTRODUCTION Research in the design and syntheses of antimicrobials will continue to be problematic on our planet, considering the fact that bacteria and fungus developed resistance to antimicrobials over a period of time [1-7]. Antibiotic resistance has become a global concern [5-7]. This is primarily due to indiscriminate use of commercial antimicrobial drugs used for the treatment of infectious diseases. This has led to the search for new antimicrobials, both herbal and synthetic. However, synthetic drugs/medicine have several adverse side effects which are usually irreversible when administered and the cost of synthesizing drugs in most cases is an expensive endeavour [1-5]. In addition, phytochemical screening and natural products isolation can lead to novel and know natural products whose in vitro antimicrobial activity can be correlated with that of the crude plant extract [8-9]. Guyana has a rich bio diversified flora whose organic and aqueous extract have been shown to possess potent and selective antimicrobial activity compared with standard antibiotics: penicillin, nystatin and ampicillin [10-16] etc. In addition, there is also a need to assess the medicinal values of plant used as food source. Thus, efforts should be made to intensify the production of food crops in the agro-industry that have antimicrobial properties in addition to their nutritional properties. Once, the antimicrobial efficacy of nutritious vegetables and fruits have been established, their commercialisation will be realized, fostering the Agro Economic Growth (AEG) of a country and also a boost to the Health Sector. Extracts from fruits and vegetables can also be incorporated in soaps, detergents and cough syrups to boost their antimicrobial potency, a significant impetus to Pharmaceutical companies locally and internationally. In search of antimicrobials that have nutritional values (neutraceuticals), the use of the solventless C6H14 and CH3CH2OH extracts of Solanum melongena (Solanaceae), Moringa oleifera (Moringaceae), Brassica rapa chinensis (Brassicaceae), Artocarpus altilis (Moraceae), against human pathogenic microorganisms: E. coli, S.aureus, K. pneumoniae and C. albicans are reported in this Chapter.

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FOLKLORE AND NATURAL PRODUCTS CONSTITUENTS Moringa oleifera is widely cultivated species of the genus Moringa, the only genus in the Moringaceae. This plant is rich in unique compounds such as glucosinolates and isothiocyanates. Natural products such as 4-(4'-O-acetyl--L-rhamnopyranosyloxy)benzyl isothiocyanate, 4-(-L-rhamnopyranosyloxy)benzyl isothiocyanate, niazimicin, benzyl isothiocyanate (1) pterygospermin (2), and 4-(-L-rhamnopyranosyloxy)benzyl glucosinolate isolated from Moringa species have been reported to have hypotensive and anticancer activity. Phytochemicals such as the carotenoids (-carotene or pro-vitamin A have also been N C Sare shown in Figure 1. isolated [17, 18]. The structure of two of these compounds (1) N

C

S

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O

O

S

S

O

N

N

O

O

(2)

O

N

O

N

O

S

S

Figure 1. Benzyl isothiocyanate (1) and Pterygospermin (2) from Moringa oleifera. (2)

The leaves are the most nutritious and contain significant amount of vitamin B6, vitamin C, provitamin A, -carotene, magnesium and protein. Calcium in Moringa oleifera leaves are usually complexed as crystals of calcium oxalate. Moringa oleifera provides a rich and rare combination of zeatin, quercetin, kaempferom and many other phytochemicals such as hexadecanoic acid, ethyl palmitate, palmitic acid, ethyl ester, 2,6-Dimethyl-1, 7-octadiene-3-ol, 4-Hexadecen-6-yne, 2-hexanone, and 3cyclohexyliden-4-ethyl - E2- Dodecenylacetate [17]. It is very important for its medicinal value. Various parts of the plant such as the leaves, roots, seed, bark, fruit, flowers and immature pods act as cardiac and circulatory stimulants, possess antitumour, antipyretic, antiepileptic, antinflammatory, antiulcer [17-18] Moringa oleifera preparations have been used for its antitrypanosomal, hypotensive, antispasmodic, antiulcer, anti-inflammatory, hypocholesterolemic, and hypoglycemic activities, as well as having considerable efficacy in water purification by flocculation, sedimentation, antibiosis and even reduction of Schistosome cercariae titer [19]. A new biflavonol glycoside, Solanoflavone was isolated from aerial part of Solanum melongena. The chemical structure was elucidated as isorhamnetin-3-O-beta-Dglucopyranoside-(4'->O->4''')-galangin-3''-O-beta-D-glucopyranoside on the basis of physicochemical and spectroscopic techniques, including 2D NMR spectral techniques [20].

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Flavanoids, isolated from Solanum melongena have been shown to possess potent antioxidant activity. Concentrations of malondialdehyde, hydroperoxides and conjugated dienes were lowered significantly [21]. Phenylethyl cinnamides, potential alpha-glucosidase inhibitors were isolated from the roots of Solanum melongena (Solanaceae). Bioassay-guided fractionation against alphaglucosidase resulted in isolation and identification of six phenolic compounds from the 70% EtOH extract of the roots. Three of the phenylethyl cinnamides, N-trans-feruloyl tyramine, N-trans-p-coumaroyl tyramine and N-cis-p-coumaroyl tyramine possessed inhibitory activity against alpha-glucosidase with IC50 values of 500.6, 5.3 and 46.3 microM, respectively. Mechanisistic studies revealed these phenylethyl cinnamides as non-competitive inhibitors. The above is the first study of the alpha-glucosidase inhibitory activities of the roots of Solanum. melongena, suggesting potential medicinal use of this herb [22] Phytochemical screening of the methanolic and aqueous extracts of the fruit and crown of Solanum Melongena revealed the presence of alkaloids, saponins, steroids, tannins/ phenolics, flavonoids, proteins and carbohydrates. Ascorbic acid and phenolics both which are powerful antioxidants were also present in fruit. The presence of saponins and glycoalkaloids protect the plant from microbial pathogens [23]. Various parts of Solanum melongena (Solanaceae) are useful in the treatment of inflammatory conditions, cardiac debility, neuralgia, ulcers of nose, cholera, bronchitis and asthma. Roots are used as antiasthmatic and general stimulant, juice is employed for otitis, applied to ulcers of the nose. Leaves are used in the treatment of bronchitis, asthma and dysuria, also given in liver complaints and they stimulate the inter hepatic metabolism of cholesterol. The fruit of Solanum melongena has a high percentage of Vitamin B2. The fruit is also used in the treatment of diabetes [23]. Brassica rapa chinensis, Artocarpus altilis and their related species have medicinal uses and Natural Products/Phytochemicals with medicinal properties have been isolated from both plants or related species. Brassica rapa chinensis and related species have antirheumatic, antiarthritic, antiscorbutic and resolvent properties [25]. Brassica rapa vegetables have been shown to possess glucosinolates with antioxidant properties [26]. The juice from the leaves of Brassica rapa species such as Turnip (Brassica rapa L.) have been shown to have hepatoprotective action through its antioxidative potentials [26-27]. Compounds isolated from related species of turnip (Brassica rapa ssp. campestris (Brassicaceae) have been shown to exhibit high inhibitory activity against the growth of human cancer lines, HCT-116, MCF-7, and HeLa, with IC50 values ranging from 15.0 to 35.0 μM and against LDL-oxidation with IC50 values ranging from 2.9 to 7.1 μM [28] Phenolic natural products were isolated from Pak choi (Brassica rapa chinensis) and seven other vegetables. These compounds were found to be hydroxybenzoic acids, hydroxycinnamic acids and flavonoids. Salicylic acid was found to be the most common hydroxybenzoic acid, ranging from 4.40 to 117.36 μg/g fresh frozen weight (ffw). Vanilic, gallic, caffeic, chlorogenic, p-coumaric, ferulic and m-coumaric acids were also found in all of these vegetables. Isoquercetin and Rutin, the most common flavonoids, ranged from 3.70 to 19.26 and 1.60 to 7.89 μg/g ffw, respectively [28]. Phytochemical, and spectroscopic investigations of related species of turnip (Brassica rapa ssp. campestris (Brassicaceae) revealed the presence of a novel phenanthrene derivative, 6-methoxy-1-(10-methoxy-7-(3-methylbut-2-enyl)phenanthren-3-yl)undecane-2,4-

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dione, brassicaphenanthrene, along with two known diarylheptanoid compounds, 6-paradol and trans-6-shogaol. These compounds have been reported to have anticancer activity [28]. The breadfruit (Artocarpus altilis) is edible. The leaves and the sap have been used for various medicinal purposes. The tea of breadfruit leaves are used to lower blood pressure and treat diabetes. The sap is applied to contagious skin ailments to prevent their spreading and promote healing [29]. Artocarpus altilis leaf extracts have been shown to have cytoprotective, antiinflammatory, cytotoxic, negative inotropic effect, anti-cancer, antitubercular and antiplasmodial activities. An ethanol extract of the leaves showed potent ACE (Angiotensinconverting enzyme) inhibitory activity, supporting its use in folk medicine for the treatment of hypertension. The isolated compounds exhibited antitubercular and antiplasmodial activities [30]. Artocarpus altilis leaf extracts were investigated against angiotensin-converting enzyme (ACE) activity. Amongst the extracts tested, hot ethanol extract exhibited a potent ACEinhibitory activity with an IC₅₀ value of 54.080.29µgmL⁻¹, followed by cold EtOAc extract (IC₅₀ of 85.44±0.85µgmL⁻¹). In contrast, the hot aqueous extracts showed minimum inhibition with the IC₅₀ value of 765.5211.97µgmL⁻¹ at the maximum concentration tested. The high content of glycosidic and phenolic compounds could be involved in exerting ACEinhibitory activity, supporting the utilisation of A. altilis leaf in the folk medicine for the better treatment of hypertension [30-31]. Phytochemical and spectroscopic studies of the methanol extract of Artocarpus altilis resulted in the isolation and spectroscopic characterisation of a new prenylated aurone, artocarpaurone, together with eight known compounds, including two prenylated chalcones, three prenylated flavanones, and three triterpenes. The structure of the new compound was elucidated as 6-hydroxy-2-[8-hydroxy-2-methyl-2-(4-methyl-3-pentenyl)-2H-1-benzopyran5-ylmethylene)-3(2H)-benzofuranone. It showed moderate nitric oxide radical scavenging activity, whereas two compounds had moderate 2,2-diphenyl-1-picrylhydrazyl radical scavenging effect, compared with the positive control (+)-catechin [32]. Antitubercular and antimalarial activity-guided study of the roots of Artocarpus altilis led to the isolation of nine prenylated flavones. Cycloartocarpin (1), Artocarpin (2), and Chaplashin (3) were isolated from the CH2Cl2 extract of the root stems, whereas Morusin (4), Cudraflavone (5), Cycloartobiloxanthone (6), Artonin (7), Cudraflavone (8) and Artobiloxanthone (9) were found in the root barks. The isolated compounds exhibited antitubercular and antiplasmodial activities, and also showed moderate cytotoxicity against KB (human oral epidermoid carcinoma) and BC (human breast cancer) cell lines [33-34]. The cytoprotective effects of various solvent extracts of Artocarpus altilis (Parkinson) Fosberg were evaluated. These effects were determined in human U937 cells incubated with oxidized LDL (OxLDL) using the 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1, 3-benzene disulfonate (WST-1) assay. Results demonstrated that the EtOAc extract showed cytoprotective activities. To identify the main cytoprotective components, a bioassay guided isolation of the ethyl acetate extract afforded -sitosterol and six flavonoids. Their chemical structures were established on the basis of spectroscopic evidence and comparison with literature data. One of these compounds was obtained from A. altilis for the first time. The cytoprotective effect offers good prospects for the medicinal applications of A. Altilis [34].

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OH H3CO

H3CO

O

O

OH

O OH OH

O

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(1) Artocarpin Cycloartocarpin

OH

OH O H3CO

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OH

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Morusin Chaplashin HO

OH HO

OH

(6) O

O

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O

O

OH OH

O

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(5) Cycloartobiloxanthone HO

Cudraflavone

OH

(7) O

O O

OH

O

O

Artonin

Figure 2. Isolates from Artocarpus altilis.

Flavonoids, 10-oxoartogomezianone, 8-geranyl-3-(hydroxyprenyl)isoetin, hydroxylartoflavone, isocycloartobiloxanthone, and furanocyclocommunin, together with 12 known compounds, were isolated from heartwood and cortex of Artocarpus altilis, and were

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spectroscopically characterised. The flavonoids isolated from A. altilis may be suspected candidate antioxidants and/or skin-whitening agents [35].

MATERIALS AND METHODS Reagents and materials: Antibiotics, Ampicillin, Mueller Hinton Agar, agar plates were purchased from Meditron Scientific Limited. Bacterial and fungal cultures were obtained from the Georgetown Public hospital, GPHC.

Collection of Plant Material Fresh leaves of Moringa oleifera and fruits of Solanum meologena were handpicked and placed in bags. These were washed with distilled water and dried for four (4) hours. They were further air dried for one week and sent for authentication at the Centre for the Study of Biological Diversity, University of Guyana. Breadfruit and Pak choi were collected from a local farm and were subjected to aerial drying. Dried leaves of Moringa oleifera, Solanum melogena, breadfruit and Pak Choi were severed into small pieces and weighed prior to solvent extraction.

Procedure (a) Preparation of Herbal Extracts: Solvent Extraction: n-C6H14, CH2Cl2 and CH3CH2OH solvents were freshly distilled prior to use. Approximately six hundred grams (387 g) of Solanum melongena fruit and Moringa oleifera leaves (350g) were extracted thrice in six hundred milliliters (600 ml) of nC6H14. The procedure was repeated using freshly distilled CH3CH2OH. The contents for each extraction was filtered, solvents dried over anhydrous Na2SO4 and removed in vacuo resulting in viscous extracts whose state are shown in Table 1.0.. The weighed plant parts of Brassica rapa chinensis (375g) and Artocarpus altilis (361g) were also placed in extraction jars and extracted sequentially with solvents of varying polarity: n-C6H14, CH2Cl2 and CH3CH2OH. After extraction, solvents were filtered and dried over anhydrous Na2SO4. Solvents were removed in vacuo, resulting in viscous extracts and solids, Table 1.0. (b) Preparation of Extract solution for Antimicrobial activity: Antimicrobial properties of Solanum melogena and Moringa oleifa C2H5OH and n-C6H14 extracts were investigated in vitro at concentrations of 5%, 10% and 20% of extract per solvent. For Brassica rapa chinensis and Artocarpus altilis, each extract was prepared in concentrations of 0.025g/ml, 0.05g/ml and 0.1 g/ml respectively. For the CH2Cl2 extract, only concentrations of 0.025g/ml and 0.05 g/ml were used. Solutions containing varying concentration of Solanum melongena, Moringa oleifera, Brassica rapa chinensis and Artocarpus altilis extracts were subjected to antimicrobial

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R. C. Jagessar, N. Ramchartar and O. Spencer susceptibility tests against human pathogens: E.coli, S. aureus, K. pneumoniae and C. albicans. (c) Antimicrobial Susceptibility Tests: 40g of Mueller Hinton Agar was placed into 1000ml of distilled water. This was mixed thoroughly. The mixture was then heated with frequent agitation while in a conical flask. The mixture was boiled for one minute to completely dissolve the agar powder. It was then autoclaved at 121 0ºC for 15 minutes. The Molten agar was then poured into 90 mm sterile Petri dishes, to a depth of 4mm. These plates were allowed to cool and refrigerated for use the following day. The plates were labelled and inoculated with the respective bacterial colonies. Three disc impregnated with the antimicrobial plant extracts at appropriate concentrations were placed on the MHA plates. Four separate plates were prepared in a similar manner for the positive controls for the bacterial strains respectively. The plates containing the bacterial colonies were incubated for 24hrs at 37 ºC. The plates containing the fungi was incubated for 48hrs at 37 ºC. The Disc diffusion method was used to screen plant extracts for its in vitro antimicrobial activity. Plates were labelled according to extract, concentration and bacteria. Using the Disc diffusion assay [36, 37], an inoculum containing bacteria cells were applied onto Mueller Hinton agar plates. A sterile swabbed was dipped into the bacteria culture and was uniformly spread on the surface of the Mueller Hinton agar. This was allowed to dry for 10 minutes. On each plate, four discs were placed equidistant using a sterilized tweesor. One of these is the reference disc onto which antibiotic was also applied and was used as the positive control: ampicillin for the bacteria. The reference antibiotic disc contained 200mg antibiotic/ml. The discs were made by cutting discs (5-6mm) from a filter paper with a sterilised perforator. Each disc was impregnated with the anticipated antimicrobial plant extract of Solanum Melongena and Moringa oleifera at appropriate concentrations of 5%, 10% and 20 % of n-C6H14 or CH3CH2OH extract using a microlitre syringe. For Brassica rapa chinensis and Artocarpus altilis, these were at concentration of 0.025mg/L, 0.5g/L and 0.1 g/L respectively. The plates were then incubated with the test organism: Bacteria at 37ºC for 24 hours. The antimicrobial compound diffuses from the disc into the medium. Following overnight incubation, the culture was examined for areas of no growth around the disc (zone of inhibition, ZOI). The diameter of the zone of inhibition, DZOI, was measured using a transparent plastic ruler. Each experiment was done in triplicates.

Reference and Control Ampicillin was choosen as the reference for all bacteria species used: E.coli, S. aureus and Klebsiella pneumonia, whereas Nystatin was used for fungal species. The Control experiment consists of a plate of solidifying agar onto which was inoculated pure solvent with microorganism mixed in a 1:1 portion, [36, 37].

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Source of Microorganisms Gram negative (-) E. coli, Gram positive (+) strains, Staphylococcus aureus (ATCC 25923), Klepsiella pneumoniae and Gram positive (+) strains were obtained from the Georgetown Public Hospital, GPH and stored in a refrigerator until required.

Positive Control Tetracycline was used as a positive control to screen and analyze for the antimicrobial properties of the different medicinal plants. This antimicrobial drug is clinically effective against both gram- negative as well as gram positive microbes.

Results Table 1. State and % yield of solvent type extract for S. melongena, M. oleifera, Brassica rapa chinensis and Artocarpus altilis Name of Plant

Type of extract

State of Extract

Solanum melongena

Weight of ground plant material (g) 387 g

Weight of Extract (g)

% yield of Extract

n-C6H14

3.6

0.9

4.1

1.1

3.7

1.1

4.5

1.2

2.5

0.7

CH2Cl2

Black viscous extract Green Viscous Extract Green semi viscous Extract Green Viscous Extract Green viscous solid Light green

Solanum melongena

387g

CH3CH2OH

Moringa oleifera

350g

n-C6H14

Moringa oleifera

361g

CH3CH2OH

Brassica rapa Chinensis Brassica rapa Chinensis Brassica rapa Chinensis Altocarpus altilis

375g

n-C6H14

375g

0.7

0.2

375g

CH3CH2OH

Dark green

21.9

5.9

361g

n-C6H14

2.5

0.69

361g 361g

CH2Cl2 CH3CH2OH

Off-White yellow Green Viscous light brown

1.4 22.5

0.39 6.23

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Table 2. TLC profile for Solanum Melongena, Moringa oleifera, Brassica rapa chinensis and Artocarpus altilis Solvent Extract

Solanum melongena Rf CH3CH2OH 0.21, 0.35, 0.56, 0.61 C6H12 0.35, 0.5, 0.75, 0.81 Rf: Retention factor.

Moringa oleifera Rf 0.35, 0.41, 0.61, 0.75 0.25, 0.41, 0.49, 0.61

Brassica rapa Chinensis Rf 0.8, 1.88

Altocarpus altilis Rf

0.32, 0.44,0.88,0.96

0.30, 0.51, 0.63

0.8, 0.6

Table 3. Mean, Standard Deviation and Area of Zone of Inhibition for the n-C6H14 and CH3CH2OH extract of Solanum Melongena and Moringa oleifera Sample

Pathogenic Concentration Mean Mean Diameter with Area of Zone of Microorganism (%) Diameter Standard deviation Inhibition (mm2) 5 4.43 4.43 ±3.85 15.04 Solanum melogena E.coli Hexane 10 4.46 4.46 ± 2.97 15.65 20 7.03 7.03 ± 0.25 38.79 S. aureus 5 6.77 6.77 ± 1.04 35.87 10 7.1 7.1 ± 0.22 39.57 20 5.03 5.03 ± 2.53 19.86 Klebsiella 5 2.33 2.33 ± 1.04 4.26 pneumoniae 10 7.97 7.97 ± 3.87 48.99 20 7.17 7.17 ± 0.25 40.24 E.coli 5 7.2 7.2 ± 0.71 40.69 Solanum melongena Ethanol 10 7.43 7.43 ± 0.30 43.33 20 7.63 7.63 ±0.42 45.7 S.aureus 5 7.87 7.87 ± 0.32 48.49 10 7.73 7.73 ± 0.64 46.9 20 8.27 8.27 ± 0.21 53.55 Klebsiella spp 5 7.03 7.03 ± 0.11 38.79 10 7.53 7.53 ± 0.32 44.51 20 7.5 7.5 ±0.17 44.15 E.coli 5 4.4 4.4 ±3.81 15.19 Moringa oleifera Hexane 10 7 7 ±0.2 38.46 20 7.06 7.06 ±0.11 39.12 S.aureus 5 4.66 4.66 ±4.07 17.04 10 7.4 7.4 ±0.52 42.98 20 7.53 7.53 ±0.49 44.51 klebsiella 5 7.33 7.33 ± 0.28 42.17 pneumoniae 10 7.26 7.26 ± 0.20 41.37

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Moringa oleifera Ethanol

Pathogenic Concentration Mean Mean Diameter with Area of Zone of Microorganism (%) Diameter Standard deviation Inhibition (mm2) 20 4.86 4.86 ± 4.23 18.54 E.coli 5 6.73 6.73 ±0.25 33.55

S.aureus

Klebsiella pneumoniae

10 20 5 10 20

4.76 7.73 5 8.1 8.1

4.76 ± 4.12 7.73 ± 0.11 5 ± 4.35 8.1 ±0.79 8.1 ±0

17.78 46.9 38.46 51.5 51.5

5

6.93

6.93 ±0.05

37.69

10 20

7.33 7.93

7.33 ±0.05 7.93 ±0.11

42.17 49.36

Positive Control Table 4. Area of Zone of Inhibition, AZOI for the positive control, tetracycline against human pathogens Microorganism Escherichia.coli Staphylococus. aureus Klebsiella. pneumoniae

Area of zone of inhibition (mm2) 36cm2 37cm2 35cm2

Table 5. Plant Extracts Hexane extract of Brassica rapa chinensis at low concentration (0.01 g/ml)

Hexane extract of Brassica rapa chinensis at high concentration 0.1g/ml

Tested Microorganism E. coli

Diameter of DZOI 10mm, 9mm,14mm

Mean Diameter of ZOI 11± 2.65

S. aureus

13mm, 14mm, 8mm

11.67± 3.22

Area of ZOI 94.9

106.9 Bacillus subtilis

8mm, 6mm, 6mm

6.67 ± 1.16

34.9

C. albicans E. coli

18mm, 8mm, 11mm No Inhibition

12.33 ± 5.13

119.3 0

S. aureus

8mm, 6mm, 0

7 ± 4.16

38.5

Bacillus subtilis

No Inhibition

C. albicans

11mm, 10mm, 8mm

0 9.67 ± 1.53

73.4

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R. C. Jagessar, N. Ramchartar and O. Spencer Table 6. Antimicrobial activity of n-C6H14 extract of A. altilis at low and high concentration

Plant Extracts Hexane extract of A. altilis at low concentration (0.01g/ml) Hexane extract of A. altilis at high concentration (0.1 g/ml)

Tested Microorganism E. coli S. aureus Bacillus subtilis C. albicans E. coli S. aureus Bacillus subtilis C. albicans

Diameter of ZOI No Inhibition No Inhibition No Inhibition No Inhibition No Inhibition No Inhibition 12mm, 10mm, 10mm No Inhibition

Mean Diameter of ZOI 0 0 0 0 0 0 10.67 ± 1.16 0

AZOI 0 0 0 0 0 0 89.2 0

Table 7. Antimicrobial activity of CH2Cl2 extract of A. altilis at low, high and very high concentration Plant Extracts Dichloromethane Extract of A. altilis At low concentration (0.01g/ml) Dichloromethane Extract of A. altilis At high concentration (0.05 g/ml) Dichloromethane Extract of A. altilis At a very high concentration 0.1g/ml

Tested Microorganism E. coli S. aureus Bacillus subtilis C. albicans E. coli S. aureus Bacillus subtilis C. albicans E. coli S. aureus Bacillus subtilis C. albicans

Diameter of ZOI

Mean Diameter

No Inhibition No Inhibition 9mm, 8mm, 9mmm No Inhibition No Inhibition 7mm, 9mm, 0 8mm, 7mm, 8mm No Inhibition No Inhibition 8mm, 10mm, 7mm No Inhibition No Inhibition

0 0 8.67 ± 0.58 0 0 5.33 ± 4.73 7.67 ± 0.58 0 0 8.3 ± 1.5 0 0

Area of ZOI 0 0 59.0 0 0 22.3 46.2 0 0 54.1 0 0

Table 8. Antimicrobial activity of CH3CH2OH extract of Brassica rapa chinensis at low and high concentratio Plant Extracts

Tested Microorganism Ethanol extract of E. coli Brassica rapa chinensis at low S. aureus concentration 0.01g/ml Bacillus subtilis C. albicans Ethanol extract of E. coli Brassica rapa

Diameter of ZOI Mean Diameter of ZOI 17mm, 17mm, 16.33 ± 2.7 15mm 9mm, 17mm, 13.67 ± 3.73 15mm 7mm, 5mm, 0 4 ± 3.61 16mm, 15mm, 14 ± 2.6 11mm 9mm, 8mm, 7mm 8 ± 1

Area of ZOI (mm2) 209.3 146.7 12.6 153.9 50.2

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Tested Microorganism S. aureus Bacillus subtilis C. albicans

Diameter of ZOI Mean Diameter of ZOI 15mm, 13mm, 12.33 ± 3.05 9mm 5mm, 9mm, 9.67± 5.03 15mm 15mm, 14mm 12.67± 3.27 9mm

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Area of ZOI (mm2) 119.3 73.4 126.0

Table 9. Antimicrobial activity of CH3CH2OH extract of A. altilis at low and high concentration Plant Extracts

Tested Microorganism Ethanol extract of E. coli A. altilis at low S. aureus concentration Bacillus subtilis 0.01g/ml C. albicans Ethanol extract of E. coli A. altilis at high S. aureus concentration Bacillus subtilis 0.1g/ml C. albicans

Diameter of ZOI 9mm, 8mm, 9mm 13mm, 11mm, 9mm 9mm, 8mm, 6mm No Inhibition No Inhibition No Inhibition No Inhibition No Inhibition

Mean Diamter ZOI 8.67 ± 0.6 11 ± 2 7.66 ± 1.5 0 0 0 0

Area of ZOI 59.00 94.9 46.7 0 0 0 0

Table 10. Combined fruit extracts of Brassica rapa chinensis and Artocarpus altilis Plant Extracts

Tested Microorganism Ethanol extracts of E. coli A. altilis + Brassica rapa chinensis at high concentration S. aureus 0.1g/ml

Hexane extracts of A. altilis + Brassica rapa chinensis at high concentration, 0.1g/ml

Diameter of ZOI 11mm, 11mm, 10mm

Mean Diameter of Area of ZOI ZOI 10.67 ± 0.58 89.4

19mm, 12mm, 11mm

14

153.9

Bacillus subtilis

10mm, 10mm, 8mm

9.3 ± 1.16

67.8

C. albicans

18mm, 13mm, 13mm

14.67 ± 5.78

168.9

E. coli S. aureus Bacillus subtilis

No Inhibition No Inhibition 13mm, 8mm, 8mm No Inhibition

0 0 9.67 ± 2

0 0 73.4

0

0

C. albicans

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DISCUSSION The % yield of the solvent type extract follows the sequence: CH3CH2OH > n-C6H14 > CH2Cl2, in accordance with solvent increasing polarity. These range from 0.2 to 6.23 % and are generally low yielding. TLC analysis of the CH3CH2OH extract of Brassica rapa chinensis and A. altilis recorded the presence of two and three spots with Rf value of 0.8, 1.88 and 0.8, 0.7 and 0.6 respectively. The n-C6H14 extract of Brassica rapa chinensis and A. altilis revealed the presence of 4 and 3 spots respectively. Rf value being 0.32, 0.44, 0.88, 0.96 and 0.30, 0.51 and 0.63 respectively. TLC analyses of S. Melongena and M. oleifera revealed the presence of four and five spots respectively for the ethanol extracts. The Rf values of these being 0.21, 0.35, 0.50, 0.61 and 0.35, 0.81, 0.61, 0.75 respectively. For the n-C6H14 extract, TLC analyses revealed the presence of four and six spots respectively. These being: 0.35, 0.5, 0.75, 0.81 and 0.25, 0.41, 0.49 and 0.61 respectively. Each spot is probably due to a pure phytochemical constituent. Antimicrobial properties of Solanum melogena and Moringa oleifa C2H5OH and n-C6H14 extracts were investigated in vitro at concentrations of 5%, 10% and 20% of extract per solvent using the Disc diffusion assay. Antimicrobial activity of Brassica rapa chenensis and A. altilis extracts were investigated at 0.025g/ml, 0.5g/ml and in some cases 0.1g/ml, using the Disc diffusion assays under asceptic conditions. Investigations were done against three pathogenic microorganisms: E. coli, S. aureus and Klebsiella pneumoniae using the Disc diffusion assay. The area of zone of inhibition was used as an indicator of the plant‘s antimicrobial properties. Larger the diameter of zone of inhibition, greater is the plant‘s antimicrobial activities. It is anticipated through the antimicrobial activity of plant extract, no area of growth will be induced around the disc. Bacteria colonies sensitive to the antimicrobial are inhibited at a distance from the disc whereas resistant strains grow up to the edge of the disc. Discs applied to the plates already streaked with bacteria and the fungus. A comparison of the effect of the various solvent type extracts against the three human pathogenic microorganisms at three different concentrations can be discussed. In general, there seem to be an increase in the plant‘s extract antimicrobial activity as the concentration of the extract is increased. For example, Solanum melongena C2H5OH extract induces area of zone of inhibition (AZOI) of 40.69, 43.33 and 45.7 mm2 against E.coli as the concentration of the plant extract increased from 5% to 20%. Likewise Moringa oleifera CH3CH2OH extract induces area of zone of inhibition (AZOI) of 37.69, 42.17 and 49.36 mm2 against Klebsiella pneumoniae at concentration of 5, 10 and 20% of extract respectively. However, there were exceptions to the above general increase in bacterial activity. For example, Solanum melogena n-C6H14 extract showed an increase in antimicrobial activity of 39.57 mm2 at 10% concentration against S.aureus, followed by a decrease of 19.86 mm2 at the 20% concentration. Moringa oleifera C2H5OH extract also showed a decreased in antimicrobial activity followed by an increase against K. pneumoniae. For example, against E.coli value of 33.35 mm2, 17.78 mm2 and 46.0 mm2 was obtained at the respective concentrations of 5, 10 and 20 % of extract. Of significance, there was a decrease in the area of zone of inhibition, AZOI for Moringa oleifera hexane extract against Klebsiella species at all three concentrations. Area of zone of inhibition of 42.17 mm2, 41.37 mm2 and 18.54 mm2 were obtained against K. pneumoniae at respective concentrations of 5, 10 and 20% of extract. The

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highest area of zone of inhibition, AZOI of 53.55 mm2 was induced by Solanum melogena C2H5OH extract against S. aureus at 20% concentration of extract. The smallest area of zone of inhibition of 15.04 mm2 was induced by Solanum melogena n-C6H14 extract against E. coli, where values of 15.04 mm2, 15.65 mm2 and 38.79 mm2 were registered at the respective concentration. The C2H5OH extract of either plant seems to be more antimicrobial than the n-C6H14 extract, suggesting greater localisation of plant natural products antimicrobial agents or the interactions of natural products via non covalent interactions to produce novel antimicrobial systems or assemblies. For example, Solanum melogena n-C6H14 extract induces area of zone of inhibition of 35.87 mm2, 39.57 mm2 and 19.86 mm2 against S. aureus. However, Solanum melogena CH3CH2OH extract induced area of zone of inhibition of 48.49 mm2, 46.9 mm2 and 53.53 mm2 against S. aureus at concentration of 5%, 10% and 20% concentration respectively.

Figure 4. Area of Zone of Inhibition (mm2) of plant extracts against E.coli at concentration of 5, 10 and 20%..

Graph 1, Figure 4, shows the area of AZOI (mm2) at 5%, 10%, & 20% concentrations of both plant extracts against colonies of E.coli. From the graph it can be observed that the nC6H14 extract of Moringa oleifera was more antibacterial at 20% concentration of extract. Values of 38.79 mm2 and 39.12 mm2 were recorded against E.coli respectively. Also, at the 20% concentration, Moringa oleifera C2H5OH extract was more antimicrobial than that of Solanum melogena. Values of 46.9 mm2 and 45.7 mm2 were registered respectively. Graph 2, Figure 5. shows the area of AZOI (mm2) at 5%, 10%, & 20% concentrations of both plant extracts against colonies of S.aureus. From the graph, the n-C6H14 extract of Moringa oleifera is more antimicrobial than that of Solanum melogena against S. aureus. Values of 44.51 mm2 and 19.86 mm2 were observed respectively. However, Solanum Melongena C2H5OH extract is more antimicrobial against S.aureus than Moringa’s C2H5OH extract at the 20% concentration. Values of 53.55 mm2 and 51.5 mm2 were observed respectively.

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Figure 5. Area of Zone of Inhibition (mm2) of plant extracts against S. aureus at concentration of 5, 10 and 20%.

Figure 6. Area of Zone of Inhibition (mm2) of plant extracts against Klepsiella species at concentration of 5, 10 and 20%.

Graph 3, Figure 6, shows the AZOI (mm2) at 5%, 10% and 20% concentrations of plant extract against colonies of Klebsiella pneumoniae. From the graph it can be observed that the n-C6H14 extract of Solanum melogena induces a higher area of zone of inhibition against Klebsiella pneumoniae compared with that of Moringa oleifera at the 20% concentration. Values of 40.24 cm2 and 18.54 cm2 were registered respectively. Likewise, C2H5OH extract of Solanum Melongena were less antimicrobial than that of Moringa oleifera at 20% concentration of plant extract. Area of ZOI registered were 44.15 mm2 and 49.36 mm2 respectively.

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Antimicrobial activity of Brassica rapa chenensis and A. altilis at a concentration of (0.025g/ml, 0.5g/ml and 0.1g/ml, were investigated using the Disc diffusion assays under asceptic conditions. It was found that the n-C6H12 extract of Brassica rapa chinensis was significantly more antimicrobial than that of A. altilis at both high and low concentration, Table 5.0 and Table 6.0. For example, the n-C6H12 extract of Brassica rapa chinensis is antimicrobial against all pathogens with the exception against E. coli and Bacillus subtilis at a concentration of 0.1g/ml. AZOI ranging from 34.92 to 119.34 mm2. Negligible AZOI was obtained against all pathogens at both concentrations for A. altilis. For A. altilis, only the nC6H14 extract at high concentration was antimicrobial against Bacillus subtilis. The AZOI being 89.2 mm2. The others show zero AZOI. These results are shown graphically in Figure 7. and Figure 8. Figure 13 shows the disc diffusion assay for the hexane extract of Artocarpus altilis against human pathogens.

Figure 7. Antimicrobial activity of Hexane extract of Brassica rapa chinensis at Low concentration.

Antimicrobial activity of the CH2Cl2 extract of A. altilis was conducted at a concentration of 0.01g/ml, 0.05g/ml and 0.1g/ml. For the CH2Cl2 extract at these three concentrations, the extract showed zero AZOI against all pathogens with the exception against Bacillus subtilis at a concentration of 0.01g/ml which showed AZOI of 59.0 mm2. At an higher concentration of 0.05g/ml, AZOI of 46.2 mm2 was induced against Bacillus subtilis. CH2Cl2 extract of A. altilis at a concentration of 0.1g/ml induces AZOI of 54.1 mm2 against S. aureus. Figure 9 shows the antimicrobial activity of the CH2Cl2 extract of Artocarpus altilis at various concentrations. The antimicrobial activity of the CH3CH2OH extract of Brassica rapa chinensis was investigated at a concentration of 0.01g/ml, 0.05g/ml and 0.1 g/ml with AZOI, ranging from 12.56 mm2 to 209.34 mm2. The highest AZOI of 209.34 mm2 was noted for the Brassica rapa chinensis extract against E.coli at a concentration of 0.01g/ml whereas the lowest of 12.56 mm2 was induced by Brassica rapa chinensis against Bacillus subtilis at a concentration of 0.01g/ml. Interestingly, the ethanol extract of A. altilis at a concentration of 0.1g/ml was microbial in nature as zero ZOI was induced. However, the CH3CH2OH extract of A. altilis at a low concentration induced a maximum AZOI of 94.99 mm2 against S. aureus and a minimum AZOI of 46.7 mm2 against Bacillus subtilis. Figure 10 and Figure 11 shows the antimicrobial profile of Brassica rapa chinensis and Artocarpus altilis at a concentration of 0.01g/ml, 0.05g/ml and 0.1g/ml respectively.

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Figure 8. Antimicrobial activity of Hexane Extract of Brassica rapa chinensis at high concentration.

Figure 9. Antimicrobial activity of CH2Cl2 extract of A. altilis at a concentration of 0.01g/ml, 0.05g/ml and 0.1 g/ml.

Figure 10. Antimicrobial activity of CH3CH2OH extract of Brassica rapa chinensis at a concentration of 0.01g/ml, 0.05g/ml and 0.1 g/ml..

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Figure 11. Antimicrobial activity of the CH3CH2OH extract of A. altilis at a concentration of 0.01 g/ml, 0.05g/ml and 0.1g/ml.

For the combined CH3CH2OH extract of A. altilis and Brassica rapa chinensis at a concentration of 0.1g/ml, significant AZOI was induced. These range from 67.8 mm2 to 168.9 mm2. AZOI of 67.8 mm2 and 168.9 mm2 were induced against Bacillus subtilis and C. albicans respectively. For the combined n-C6H14 extract of A. altilis and Brassica rapa chinensis at a concentration of 0.1 g/ml, zero AZOI were observed against E. coli, S.aureus and C. albicans. Only Bacillus subtilis showed antimicrobial susceptibility, with a registered AZOI of 73.40 mm2, Figure 12. Antimicrobial selectivity was observed for all of the extracts against human pathogens. For example, the n-C6H14 extract of Brassica rapa chinensis showed a high degree of inhibition against C. albicans (AZOI, 119.34 mm2) than against Bacillus subtilis (AZOI, 34.92 mm2) at a concentration of 0.01g/ml. Likewise the CH2Cl2 extract of A. altilis at a concentration of 0.01g/ml showed inhibition of 59.0 mm2 against Bacillus subtilis, but zero AZOI against E. coli, S. aureus and C. albicans. The CH2Cl2 extract of A. altilis at a concentration of 0.1g/ml registered a value of 54.1 mm2 against S.aureus but zero AZOI against E.coli, Bacillus subtilis and C. albicans. Further antimicrobial selectivity is seen for the CH3CH2OH extract of Brassica rapa chinensis at a concentration of 0.01g/ml against E. coli and Bacillus subtilis. For the former, AZOI of 209.37 mm2 is noted, whereas for the latter, AZOI of 12.56 mm2 was registered. Again for the CH3CH2OH extract, A. altilis at a concentration of 0.01g/ml showed antimicrobial selectivity against E.coli, S.aureus and Bacillus subtilis over C. albicans. For the latter, zero AZOI was observed whereas for the first three, AZOI, ranging from 46.7 mm2 to 95.00 mm2 were observed. Moringa oleifera n-C6H14 extract is more resistant than S. melogena extract against E.coli and S. aureus. Solanum melogena hexane extract is more resistant against Klebsiella pneumoniae compared to that of Moringa oleifera extract. For the CH3CH2OH extract, Moringa oleifera extract is more resistant against E. coli and Klebsiella pneumoniae. However, Solanum melogena extract is more resistant against S. aureus Thus, for Brassica rapa chinensis, extract at low concentration showed the solvent type extract selectivity: CH3CH2OH > n-C6H14. For A. altilis, at a low concentration, the solvent type extract showed the selectivity of CH3CH2OH > CH2Cl2 > n-C6H12. The n-C6H14 extract of Brassica rapa chinensis should be more selective for S.aureus, C. albicans infection whereas the CH3CH2OH extract of Brassica rapa chinensis should be more suited against

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E.coli and C. albicans infection. The CH3CH2OH extract of A. altilis at low concentration should be suited for S. aureus infection. Antimicrobial activity was also investigated for the positive control, tetracycline against the pathogens. It‘s found that the area of the zone of inhibition, AZOI in several instances is less than that induced by the n-C6H14 and CH3CH2OH extract of Solanum melongena, Moringa oleifera, Brassica rapa chinensis and Artocarpus altilis.

Figure 12. Antimicrobial activity of the combined ethanol and hexane extract of A. altilis and Brassica rapa chinensis at high concentration.

Figure 13. Disc diffusion assay of Artocarpus altilis hexane extract against human pathogens.

CONCLUSION From this study it can be concluded that n-C6H14 and CH3CH2OH extract of Solanum melogena, Moringa oleifera, Brassica rapa chinensis possess antibacterial activity as significant area of zone of inhibition, AZOI were observed. The area of ZOI ranging from 15.0 mm2 to 49.0 mm2 for the hexane extract of Solanum melogena and Moringa oleifera.

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The CH3CH2OH extracts showed more potent antimicrobial properties than the n-C6H14 extract with AZOI ranging from 18.0 mm2 to 53.55 mm2. For Brassica rapa chinensis and Artocarpus altilis, AZOI for the hexane extract range from 0.00 mm2 to 119.3 mm2. However, for the ethanol extract, AZOI ranges from 0.0 mm2 to 209.3 mm2. The combined hexane extract of Brassica rapa chinensis and Artocarpus altilis antimicrobial efficacy range from 0.0 mm2 to 73.4 mm2, whereas the combined ethanolic extract AZOI range from 67.8 mm2 to 169.0 mm2. The n-C6H14 and CH3CH2OH extract of Solanum melogena, Moringa oleifera, Brassica rapa chinensis and Artocarpus altilis also display antimicrobial selectivity, an important factor in preventing antimicrobial resistance. These fruits and vegetable extracts exhibit in some cases larger Area of Zone of Inhibition, AZOI compared to the reference antibiotic, Tetracycyline. Thus, these fruits and vegetables can be used as potent antimicrobial agents, in addition to their nutritional status (neutraceuticals).

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[8] [9] [10] [11] [12]

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Wilms, LR. Guide to Drugs in Canada. Third edition. Leo Paper Products. 2009. Smith, C.M., & Reynard, A.M. (1992). Textbook of Pharmacology. Third Edition. W.B.Saunders company; 96-1174. Macor JE. Annual reports in Medicinal Chemistry, sponsored by the Division of Medicinal Chemistry of the American Chemical Society, 43. Elsevier Inc. 2008; 3-497. Wood A. Topics in Drug design and discovery, Annual Reports in Medicinal Chemistry, Elsevier Inc. 41: 2008: 353-409. Bonner, J. Filling the Antibiotic Gap. Chemistry World, Royal Society of Chemistry, 2009, 6 (8): 16. Kelland, K. Antibiotic Resistance Poses Catastrophic Threat To Medicine, Huffington Post, 2013, 1-3. Westh, H; Zinn, CS; Rosdahl, VT; Sarisa Study Group, An international multicenter study of antimicrobial consumption and resistance in Staphylococcus aureus isolates from 15 hospitals in 14 countries. Microbial Drug Resistance. 2004, 10: 169-176. Shen, CC; Syu Wan-Jr; LiY; Shyh, LH; Chia, L; Gum, H; Sun, CM. Antimicrobial Diterpenes, Journal Natural Products, 2002, 65: 1857-1862. Fahey, JW; Zalcmann, AT; Talalay, P. The chemical diversity and distribution of glucosinolates and isothiocyanates amongst plants. Phytochemistry, 2001, 56: 5–51. Jagessar, RC; Mohamed, N. Antimicrobial activity of selected plants extracts from Guyana‘s flora. Journal of Pure and Applied Microbiology, 2010 4(2): 533-540. Jagessar, RC; Allen, R. Antimicrobial Potency of the Aqueous Extract of leaves of Terminalia catappa. Academic Research International, 2011, 13: 362-371. Jagessar, RC; Mars, A; Gomathigayam, S. Selective Antimicrobial properties of Leaf extract of Samanea Saman against Candida albicans, Staphylococcus aureus and Escherichia coli using several microbial techniques. Journal of American Science, 2011 7(3): 108-119. Jagessar, RC; Mars, A; Gomes, G. Leaf extract of Smilax schomburgkiana exhibit selective antimicrobial properties against pathogenic microorganisms. Life Science Journal, 2009, 6(1): 76-83.

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[14] Jagessar, RC; Mars, A; Gomes, G. Selective antimicrobial properties of Phylanthus acidus leaf extract against Candida albicans, Eschericia coli and Staphylococcus aureus using Disk diffusion, Well diffusion, Streak plate and a Dilution method. Nature and Science, 2008, 6(2): 24-38. [15] Jagessar, RC; Mohamed, A; Gomes, G. Antibacterial and antifungal activity of leaf extracts of Luffa operculata vs Peltophorum Pterocarpum against Candida albicans, Staphylococcus aureus and Escherichia coli. Nature and Science, 2007, 5(4): 81-93. [16] Jagessar, RC; Mohammed, A; Gomes, G. An evaluation of the antibacterial and antifungal activity of leaf extracts of Momordica Charantia against Candida albicans, Staphylococcus aureus and Eschericia Coli. Nature and Science, 2008, 6(1): 1-14. [17] Fahey, JW; Zalcmann, AT; Talalay, P. The chemical diversity and distribution of glucosinolates and isothiocyanates amongst plants. Phytochemistry, 2001, 56: 5–51. [18] Bennett, RN; Mellon, FA; Foidl, N; Pratt, JH; DuPont MS; Perkins L; Kroon PA; Profiling glucosinolates and phenolics in vegetative and reproductive tissues of the multipurpose trees Moringa oleifera L and Moringa stenopetala L. Journal of Agriculture and Food Chemistry 51: 3546-3553. [19] Makonnen, E; Hunde, A; Damecha, G. Hypoglycaemic effect of Moringa stenopetala aqueous extract in rabbits. Phytother Res. 1997, 11: 147–148. [20] Shen, G; Van Kiem, P; Cai, XF; Li, G, Dat, NT, Choi, YA, Lee, YM, Park, YK, Kim, YH. Solanoflavone, a new biflavonol glycoside from Solanum melongena: seeking for anti-inflammatory components, Archives Pharm Research. 2005, 28 (6): 657-659. [21] Sudheesh, S; Sandhya, C; Koshy, AS; Vijayalakshmi, NR. Antioxidants activity of Flavanoids from Solanum melongena, Phytotherapy research, 1999, 13(5): 393-396. [22] Liu X; Luo, J; Kong L. Phenylethyl cinnamides as potential alpha-glucosidase inhibitors. Natural product communications. 2011, 6 (6): 851-853. [23] Tiwari, ARS; Jadon, RS; Tiwari, P; Nayak S. Phytochemical Investigations of Crown of Solanum melongena fruit. International Journal of Phytomedicine. 2009. 1: 9–11. [24] Duke, JA; Ayensu ES. Medicinal Plants of China. Vols (1 & 2). Reference Publication., Inc. Algonac. Michigan, 1985. [25] Simona, IV; Alin, C; Teusdea, MC; Sonia, A; Socaci, CS. ―Glucosinolates Profile and Antioxidant Capacity of Romanian Brassica Vegetables Obtained by Organic and Conventional Agricultural Practices‖, Plant Foods for Human Nutrition, 2013, 68 (3), 313-321. [26] Rafatullah, S; Al-Yahya, M; Mossa, J; Galal, A; El-Tahir K. ―Preliminary Phytochemical and Hepatoprotective Studies on Turnip Brassica rapa L‖, International Journal of Pharmacology, 2006, 2 (6), 670-673. [27] Wu, Q; Cho, JG; Yoo, KH; Jeong, TS; Park JH; Kim, SY; Kang JH; Chung IS; Choi, MS; Lee, KT. ―A new phenanthrene derivative and two diarylheptanoids from the roots of Brassica rapa ssp. campestris inhibit the growth of cancer cell lines and LDLoxidation‖ Archives of Pharmacal Research. 2013. 36, (4), 423-429. [28] White, DA; Adams CD; Trotz UO. A guide to the Medicinal Plants of Coastal Guyana, Commonwealth Science Council, London, CSC Technical Publication series,1992, 225 (8): 111.

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[29] Siddesha, JM; Angaswamy, N; Vishwanath, BS. ―Phytochemical screening and evaluation of in vitro angiotensin-converting enzyme inhibitory activity of Artocarpus altilis leaf‖, Nat Prod Res. 2011, 25(20), 1931-40. [30] Khair, U; Khanama, S; Obab, S; Yanaseb, E; Murakamic, Y. ―Phenolic acids, flavonoids and total antioxidant capacity of selected leafy vegetables‖, Journal of Functional Foods. 2012. 4 (4), 979–987. [31] Huong, TT; Cuong, NX; Tram, H; Quang, TT; Duong, V; Nam, NH; Dat, NT; Huong, PT; Diep, CN; Kiem, PV; Minh CV. ―A new prenylated aurone from Artocarpus altilis”, J Asian Nat Prod Res. 2012. 14(9): 923-8. [32] Boonphong, S; Baramee, A; Kittakoop, P; Puangsombat, P., ―Antitubercular and Antiplasmodial Prenylated Flavones from the Roots of Artocarpus altilis‖, Chiang Mai Journal of Science, 2007, 34(3): 339-344. [33] Wang, Y; Deng, T; Lin, L; Pan, Y; Zheng, X. ―Bioassay‐guided isolation of antiatherosclerotic phytochemicals from Artocarpus altilis‖, Phytotherapy Research, 2006, 20, (12), 1052–1055. [34] Lan, WC; Tzeng, CW; Lin CC; Yen, FL; Ko, HH. ―Prenylated flavonoids from Artocarpus altilis: Antioxidant activities and inhibitory effects on melanin production‖, Phytochemistry, 2013, 89, 78-88. [35] Murray, PR; Baron, EJ; Pfaller, MA; Tenover, FC; Yolke RH: Manual of Clinical Microbiology, Mosby Year Book, London, 6th edition 1995. [36] Lorian, V; Antibiotics in Laboratory Medicine. 4th eds., Williams and Wilkins, Baltimore, London. 1996. [37] Sreenivasa, RP; Parekh, KS. Antibacterial activity of Indian seaweed extracts. Botanica Marina, 1981, 24: 577-582.

In: Fruit and Pomace Extracts Editor: Jason P. Owen

ISBN: 978-1-63482-497-2 © 2015 Nova Science Publishers, Inc.

Chapter 8

COCONUT WATER: AN ESSENTIAL HEALTH DRINK IN BOTH NATURAL AND FERMENTED FORMS Mansi Jayantikumar Limbad*, Noemi Gutierrez-Maddox and Nazimah Hamid School of Applied Sciences, Auckland University of Technology, Auckland, New Zealand

ABSTRACT Coconut water is the liquid endosperm fluid of the coconut fruit which contains high amounts of essential nutrients and minerals. This endosperm fluid is a widely consumed as a beverage in many parts of the world as it provides hydration along with increased nutritional, health and medicinal benefits. In addition to being used as a medium constituent, it also acts as a natural biocatalyst. One of the fermented products of coconut water, coconut water kefir, is made by fermenting coconut water with the kefir granules which contain essential lactic acid bacteria and yeast spp. known to have health benefits for a disease-free life. It has many applications in the food industry and functional food market. It is used as one of the important constituents in a variety of products or can be consumed ‗as-it-is‘. It is known to have no undesirable side effects and is said to improve digestion. This paper reviews the functional properties of coconut water, its applications in the food industry and recent advancements in this area.

INTRODUCTION Coconut (Cocos nucifera L.) is one of the important fruit trees in the world. From the various edible parts of coconut, coconut water is one of the main sources of nutrition in many tropical and subtropical countries (DebMandal & Mandal, 2011). This liquid endosperm is of cytoplasmic origin and is the product of cellularization, as a result of which the cavity within the coconut remains filled with the coconut water (Janick & Paull, 2008). It contains almost *

Corresponding author: E-mail address: [email protected].

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all the members of vitamin B group except B6 and B12, minerals, proteins, sugars, amino acids, magnesium, vitamin C, potassium and growth factors which reduce the risk of developing coronary heart disease, and aid in lowering blood pressure (Anurag & Rajamohan, 2003; Loki & Rajamohan, 2003; Massey, 2001; Sandhya & Rajamohan, 2006). This isotonic drink is very low in fat content and also contains optimum amounts of RNA phosphorous which plays an active role in transport of amino acids and respiratory metabolism in living cells (Chidambaram, Singaraja, Prasanna, Ganesan, & Sundararajan, 2013).

NUTRITIONAL INFORMATION The following is the nutritional composition of young coconut water as reported by USDA ((USDA)) and Arditti (Arditti, 2009): Chemical composition Energy value Water Dry Ash Protein Total lipid, fats Total dietary fibre Carbohydrates Total sugar Sucrose Fructose Glucose Mannitol Sorbitol Myo-inositol Scyllo-inositol Calcium Magnesium Phosphorus Iron Sodium Potassium Manganese Zinc Copper Chloride Selenium Sulfur Vitamin C, total ascorbic acid Thiamin (B1)sx Folate, total

Young coconut water 19 kcal 94.99 g/100g 5.01 g/100g 0.39 g/100g 0.72 g/100g 0.2 g/100g 1.1 g/100g 3.71 g/100g 2.61 mg/ml 9.18 mg/ml 5.25 mg/ml 7.25 mg/ml 0.8 mg/ml 15 mg/ml 0.01 mg/ml 0.05 mg/ml 24 mg/100g 30 mg/100g 37 mg/100g 0.29 mg/100g 105 mg/100g 312 mg/100g 0.142 mg/100g 0.1 mg/100g 0.04 mg/100g 183 mg/100g 0.001 mg/100g 24 mg/100g 2.4 mg/100g 0.03 mg/100g 0.03 mg/100g

Coconut Water Chemical composition Pyridoxine (B6) Folate, food Niacin (B3) Nicotinic acid (Niacin) Riboflavin (B2) Pantothenic acid (B5) Folate, Dietary Folate Equivalent (DFE) Biotin Folic acid Alanine γ-Aminobutyric acid β-Alanine Aspartic acid Cystine Arginine Asparagine and glutamine Glutamic acid Homoserine Glycine Lysine Leucine Isoleucine Histidine Methionine Ornithine Phenylalanine Proline Serine Tyrosine Tryptophan Threonine Valine Ethanolamine Pyridoline Malic acid Citric acid Shikimic and quinic acids Total lipids Total saturated fatty acids Total monounsaturated fatty acids Total polyunsaturated fatty acids Auxin Acid phosphatase Catalase Dehydrogenase

Young coconut water 0.032 mg/100g 0.003 mg/100g 0.08 mg/100g 0.64 mg/100g 0.057 mg/100g 0.52 mg/100g 3 (μg_DFE) 0.02 mg/100g 0.003 mg/100g 312 µg/ml 820 µg/ml 12 µg/ml 65 µg/ml 0.97-1.17 µg/ml 133 µg/ml ca. 60 µg/ml 240 µg/ml 5.2 µg/ml 13.9 µg/ml 150 µg/ml 22 µg/ml 18 µg/ml 0.017 g/100g 8 µg/ml 22 µg/ml 12 µg/ml 97 µg/ml 111 µg/ml 16 µg/ml 39 µg/ml 44 µg/ml 27 µg/ml 0.01 µmol/ml 0.39 mg/ml 34.31 meq/ml 0.37 meq/ml 0.57 meq/ml 0.2 g/100g 0.176 g/100g 0.008 g/100g 0.002 g/100g 0.07 mg/ml Present (units not given) Present (units not given) Present (units not given)

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Chemical composition Diastase Peroxidase RNA polymerase

Young coconut water Present (units not given) Present (units not given) Present (units not given)

The electrolyte concentration in coconut water generates an osmotic pressure similar to that of blood which helps in promoting health (Effiong, Ebong, Eyong, Uwah, & Ekong, 2010). Coconut water is also used in callus culture media as an important ingredient (Van Overbeek, Conklin, & Blakeslee, 1941). Coconut water has a significant impact on antiageing, anti-carcinogenic and anti-thrombotic effects due to the presence of a phytohormone (cytokinin) (Kende & Zeevaart, 1997; Rattan & Clark, 1994; Vermeulen et al., 2002). Micronutrients such as vitamins and inorganic ions help in the body‘s antioxidant system by aiding the removal of oxidizing species (reactive oxygen species) generated in the body due to hypermetabolism (Liu, Lin, Chen, Chen, & Lin, 2005). It is used as a ceremonial gift, as a traditional medicine and can also be processed into wine and vinegar (Prades, Dornier, Diop, & Pain, 2012). Thus, its use is not limited to being a refreshing drink but it is also used as one of the main ingredients in many food items such as bread, ice creams, biscuits and cakes (Chidambaram et al., 2013). In green coconut water, between pH 5.5 and 6.0, and at 25°C and 35°C, optimum activities of polyphenoloxydase (PPO) and peroxidase (POD) are observed. PPO is an enzyme that is found in chloroplasts, plastids and is also present in the cytoplasm of the ripened senescing plants. It helps the plant to resist microbial infections and extreme climatic conditions. The ratio of these enzymes (PPO/POD) in coconut water ranges from 0.2 to 16.7 and varies even within similar coconut varieties. This ratio depends on the stage of maturity at harvest, the variety and storage condition of the fruit, the cultivation conditions and on the mode of extraction of the coconut water. Young coconut water is able to synthesize more than one peptide which has antimicrobial activity and has novel properties and modes of action against human pathogenic bacteria (Mandal et al., 2009). Also, it is reported that coconut water contains (+)-catechin and (-)epicatechin, which have antimicrobial, anti-cancerous and antioxidant properties (Camargo Prado et al., 2015). It contains phytohormones such as auxins, cytokinin, kinetin, trans-zeatin, gibberellins, inorganic ions and vitamins, which play various roles in delaying ageing, reducing DNA damage by acting as anti-oxidants, and helping to cure neural diseases such as Alzheimer‘s. By being used as a potential drug, it has been suggested to have the ability to reduce the risk of cardiovascular disease and anaemia during pregnancy. Coconut water is often prescribed in cases of indigestion, burning pain during urination, dysuria, gastritis, burning pain of eyes or even expelling of the retained placenta (Prades et al., 2012). Overly Mature Coconut (OMC) water is lower in volume of nut water and is slightly salty in taste when compared to fresh young coconut water which is sweet and slightly sour in taste (Jackson, Gordon, Wizzard, McCook, & Rolle, 2004; Terdwongworakul, Chaiyapong, Jarimopas, & Meeklangsaen, 2009). OMC water has a high content of minerals such as sodium and potassium, and contains sugars such as sucrose and some proteins. These properties suggest that OMC water can be developed into a rehydration fluid product. In addition, the maturity of coconut water (due to changes in composition, physiochemical

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properties, sugar and salt content, and pH) influences the enzyme activity and enzyme inactivation kinetics. Very low thermal resistance is reported for both PPO and POD present in OMC water (Tan, Cheng, Bhat, Rusul, & Easa, 2014). Coconut water contains a large protein chain which binds easily to metal ions. A natural proteic solution of coconut water, in combination with appropriate metal ions, is used to synthesize a high quality nanosized powder (NiFe2O4) which exhibits size-dependent magnetic properties. This technique has been found to be an economical and efficient way to obtain nanosized nickel ferrite powder of a high quality (de Paiva, Graça, Monteiro, Macedo, & Valente, 2009). One of the most important uses of coconut water is in the preparation of a health drink, coconut water kefir. It is prepared by inoculating coconut water with kefir grains, which is then incubated for about 24-48 hours to allow appropriate fermentation. Detailed information on kefir is discussed below in order to understand the fermentation in depth.

KEFIR The name ‗kefir‘ has emerged from ‗keyif‘, which is a Turkish word meaning ‗good feeling‘ (Kabak & Dobson, 2011). Kefir is produced by the fermentation of kefir grains with milk giving rise to a viscous, acidic, slightly alcoholic and occasionally carbonated probiotic drink (Garrote, Abraham, Iacute, G., & De Antoni, 2001; Marshall, Cole, & Brooker, 1984; Yaman et al., 2010). Kefir grains, which are asymmetrically shaped and whitish to yellowish in colour, and consist of a mixture of various lactic acid bacteria and yeast species, are used for producing fermented food products (Beshkova, Simova, Simov, Frengova, & Spasov, 2002; Simova et al., 2002; Witthuhn, Schoeman, & Britz, 2005). The yeasts and lactic acid bacteria are embedded in a flexible, yet strong, polysaccharide matrix (consisting of galactose and glucose) which is often termed , ‗kefiran‘, the size of which varies over a large range of a few millimetres to a few centimetres (Garrrote et al., 2001; Yaman et al., 2010). The stability of kefir grains is reasonably good for several months if grown under specific conditions with appropriate sub-culturing (Simova et al., 2002). When kefir grains are added to fresh milk, the milk is fermented (Dobson, O'Sullivan, Cotter, Ross, & Hill, 2011; Witthuhn et al., 2005). Lactic acid, pyruvic acid, acetic acid, hippuric acid, butyric acid, propionic acid, diacetyl, and acetaldehyde are generated during fermentation, and these compounds are responsible for imparting the characteristic taste and aroma to kefir (Ahmed et al., 2013; Kesenkas, Dinkcedil, Seccedil, & Gouml, 2011; Kesenkas, Yerlikaya, & Ozer, 2013). Diacetyl, acetoin and acetaldehyde are also responsible for imparting aroma to kefir. Diacetyl is produced by Streptococcus lactis subsp. diacetlyactis and some Leuconostoc sp. (Cagindi, 2003). Fermentation can also be brought about by adding kefir grains to non-dairy products such as coconut water, soy milk, peanut milk, walnut milk, rice milk and cocoa-pulp beverage (Bensmira & Jiang, 2011; Cui, Chen, Wang, & Han, 2013; Liu, Chen, & Lin, 2005; Otles & Cagindi, 2003; Puerari, Magalhães, & Schwan, 2012). Kefir starter culture is also used in production of cheese and single cell protein by fermenting cheese whey under aerobic conditions (Filipcev, Šimurina, & Bodroza‐Solarov, 2007; Paraskevopoulou et al., 2003).

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Chemically, kefir composition is as reported below: (Liutkeviĉius & Šarkinas, 2004; Magalhães, Dias, et al., 2011; Magalhães, Pereira, Campos, Dragone, & Schwan, 2011; Wszolek, Tamime, Muir, & Barclay, 2001): Chemical composition Total solids Crude protein Carbohydrate Ash Moisture Fats

Percentage range (%) 10.6-14.9 2.9-6.4 3.8-4.7 0.7-1.1 86.3 0.03

Kefir is also prepared from commercial starters using bovine milk and it has been reported that the lactose concentration effectively decreased from 4.92%(w/v) to 4.02%(w/v) and the L(+)- lactic acid concentration increased to 0.76%(w/v) from 0.01%(w/v) after 24 hours of incubation. The acetic acid content increased from 2.10 to 2.73 mg/ml while the pH value was reported to be low as 4.24 in the first 24 hours after which it decreased gradually. The concentration of L(+)- lactic acid subsequently decreased while that of D(-)- lactic acid subsequently increased. These fermentation values depend on the type of starter culture used, the storage period and the medium used to grow the kefir (for example: the mammalian species from which the milk is derived, the coconut water, etc.), (García Fontán, Martínez, Franco, & Carballo, 2006; Magalhães, Pereira, et al., 2011; Öner, Karahan, & Çakmakçı, 2010). Recently, the structure of kefir has been studied in further detail, suggesting that the outer layer is composed of lactococci, yeast and lactobacilli. In contrast, the inner layer has a higher number of yeast cells compared to the outer layer and has longer lactobacilli. The ability of the kefir to auto-aggregate increases with increasing fermentation time (Wang et al., 2012). Kefir grains are thin, sheet-like structures which roll and scroll to form a mature grain-like structure. The smooth side consists of only short lactobacilli while the convoluted side has more yeasts than short lactobacilli. There is a zone of long, curved bacteria within the polysaccharide matrix which may play a role in forming kefiran or the polysaccharide matrix. Thus, the kefir structure is a product of folding and refolding of the flat sheet-like structures with an increase in the number of microorganisms and polysaccharides as folding occurs (Marshall et al., 1984). The charges on the microbial cell surface also play a role in autoaggregation, co-aggregation and microbial adhesion to a surface while contributing to survival in harsh conditions (Xie, Zhou, & Li, 2012). The following techniques have been used to identify different lactic acid bacteria and yeasts present in the kefir: Technique used to study kefir‘s microbial profile

Strains/type of isolates

References

Biochemical techniques

Saccharomyces sp., Kluyveromyces sp., Candida sp., Mycotorula sp., Torulaspora sp., Cryptococcus sp., Pichia sp. etc. L. acidophilus, B. bifidum, S. thermophiles, S. thermophiles, L. bulgaricus, Streptococcus spp., S. thermophiles, B. adolescentis, B. longum, B. lactis.

(Kolakowski & Ozimkiewicz, 2012)

PCR-based DGGE and species specific PCR

(Theunissen, Britz, Torriani, & Witthuhn, 2005)

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DNA sequencing of 16srRNA region Pulse Field gel electrophoresis (PFGE)

PCR DGGE, 16s rRNA sequencing for yeast

Strains/type of isolates

References

Lb. kefiranofaciens subsp. kefirgranum, Lb. kefiranofaciens subsp. kefiranofaciens, Lb. kefiri, Lb. parakefiri, Lactobacillus parabuchneri, Lactobacillus amilovorus, Lactobacillus crispatus and Lactobacillus buchneri.

(Leite et al., 2012)

Leuconostoc mesenteroides, Lactobacillus mali, Lactobacillus hordei, Leuconostoc mesenteroides, Enterococcus faecalis, Lactococcus lactis, Bifidobacterium psychraerophilum, Zygosaccharomyces fermentati, Saccharomyces cerevisiae, Dekkera bruxellensis, Pichia fermentans Lact. acidophilus complex, Lact. amylovorous, Lact. crispatus, Lact. galinarium, Lact. gasseri, and Lact. jonsonii Strains of Lact. acidophilus complex, including Lact. Delbrueckii subspecies (Lact. delbrueckii subsp. bulgaricus, Lact. Delbrueckii subsp. delbrueckii, Lact. delbrueckii subsp. lactis), Lact. plantarum, Lact. fermentum, Lact. rhamnosus, and Lact. sakei Kluyveromyces maxianus, Torulaspora delbrueckii, Saccharomyces cerevisiae, Candida kefir, Saccharomyces unisporus, Pichia fermentans, Kazachastania aerobia, Lachanceae meyersii, Yarrowia lipolytica, and Kazachstania unispora

(Hsieh, Wang, Chen, Huang, & Chen, 2012) (Kullen, SanozkyDawes, Crowell, & Klaenhammer, 2000) (Singh, Goswami, Singh, & Heller, 2009)

(Leite et al., 2012; Magalhães et al., 2010; Simova et al., 2002; Wang, Chen, Liu, Lin, & Chen, 2008)

Those kefir microorganisms which have been isolated have probiotic capabilities which have a positive effect on the body. Lactobacilli in the kefir have the ability to attach to the epithelial lining in the intestines and to auto-aggregate (Golowczyc et al., 2008). In addition, they are also known for their ability to produce certain bacteriocins and organic acids, which may protect the body from toxins, and, thus, have an antimicrobial effect (Sezer & Güven, 2009; Silva, Rodrigues, Xavier Filho, & Lima, 2009). It has been reported that kefir-derived polysaccharide also has an anti-tumor effect, anti-diabetic effect, anti-oxidative effect and plays a role in lowering blood pressure (Ahmed et al., 2011).

COCONUT WATER KEFIR A combination of young coconut water and the kefir granules gives rise to a healthy probiotic drink called coconut water kefir (Chatterjee, Bhattacharya, & Kandwal, 2011). It is made by adding kefir grains to the coconut water and allowing it to ferment for 24 to 48 hours (Gates & Schrecengost, 2013). It is high in nutritional value and is known to replenish the gut microflora for improved digestion.

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CONCLUSION Coconut water is a nutritious drink with numerous health benefits. This refreshing beverage has many potential biological applications for alleviating diseases such as cancer, and contains constituents to improve human health. The use of coconut water as a health drink should be encouraged so that more people consume such a product to lead a healthy life-style. Kefir consists of beneficial lactic acid bacteria, yeasts, acetic acid bacteria and others which aid in digestion and also give kefir its distinctive flavour. The proposed health benefits of kefir include being anti-cancerous, anti-tumoral, antibacterial and immunological, while there are also gastro-intestinal, anti-fungal and hypocholesterolaemic effects. Kefiran is used in the food industry as an edible biofilm and as a high quality health product with increased shelf-life and resistance to contamination. The probiotic properties of kefir and the health benefits of coconut water unite to form coconut water kefir. More research on coconut water kefir, its properties and its applications will provide an in-depth knowledge of this potentially useful health drink. It will also help in understanding its use to derive disease-curing drugs.

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In: Fruit and Pomace Extracts Editor: Jason P. Owen

ISBN: 978-1-63482-497-2 © 2015 Nova Science Publishers, Inc.

Chapter 9

EXTRACTION, CHARACTERIZATION AND POTENTIAL HEALTH BENEFITS OF BIOACTIVE COMPOUNDS FROM SELECTED CORNUS FRUITS Luminiţa David and Bianca Moldovan ―Babeş-Bolyai‖ University, Faculty of Chemistry and Chemical Engineering, Cluj-Napoca, Romania

ABSTRACT Cornus is a genus of the Cornaceae plant family, represented by about 30-60 species of woody plants commonly named Dogwoods, widely spread in Europe, Asia and North America. From about 2000 years ago, traditional Chinese medicine used different parts of plants belonging to Cornus genus for treatment of various diseases such as kidney and gastrointestinal disorders, diabetes, uterine bleeding and bladder incontinence. The fruits and the bark of Cornus species have been widely used for their analgesic, antiinflammatory, anti-malarial, anti-bacterial, anti-histamine, anti-allergic, anti-microbial, anti-parasitic, tonic, febrifuge and vulnerary properties as well as for their inhibitory effect on tumor cell proliferation. A high number of bioactive compounds have been identified in Cornus fruits, including ascorbic acid, phenolic compounds, anthocyanins, flavonoids, iridois, terpenoids, compounds that exert health effects especially by acting as potent antioxidants. This review will focus on the recent data reported on the extraction, characterization and biological activities of bioactive compounds isolated from fruits of selected Cornus plants in order to understand the high nutritional value of these fruits and their possible use as source of bioactive compounds for developing new pharmacological products.

Keywords: Cornus fruits, bioactive compounds, extraction, health benefits



Corresponding author: Luminiţa David, E-mail address: [email protected].

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1. INTRODUCTION Cornaceae, also known as the dogwood family, is a family of flowering trees, shrubs and subshrubs in the order Cornales, well known in the northern temperate areas for two genera: Cornus, the dogwoods and Nyssa, the tupelos. The genus Cornus consists of about 55 species, divided into four subgenera: Benthamidia, Chamaepericlymenum, Cornus and Swida and is widely distributed in the Northern hemisphere. These plants are often characterized by attractive flowers and fruits, hence are widely grown for ornamental purposes throughout Europe, North America and Asia. Most dogwood species have simple, opposite leaves, except Cornus alternifolia, the only Cornus spp. with alternate leaves [Vareed et al., 2006]. Dogwoods flowers are brilliant, colorful and attractive. Cornus florida, commonly known as flowering dogwood, is the most popular in U.S. gardens and landscaping, being recognized as the state flower of North Carolina and Virginia. Once pollination of flowers has occurred, all Cornus species produce one or two seeds drupes. The color of these drupes can range from white to yellow, red or dark bluish black. The drupes of 15 species are red while in the other species, they are blue or white. The bright color of the dogberries results from the presence of various anthocyanins, such as cyanidin and pelargonidin, pigments that impart the red color to some dogwood species fruits, or delphinidin and petunidin occurring in blue fruits [Eyde, 1988]. Yellow forms of the normally red dogwoods result from the presence of peonidin and petunidin, pigments typically found in fruit exocarp. The fruits of several species in the subgenera Cornus and Benthamidia are edible, though not all actually taste good or have much flavor. The drupes of the species in subgenus Swida are mostly toxic for humans though readily eaten by birds. The only dogwood commonly cultivated for its edible berries is the species Cornus mas (Cornelian cherry). Several cultivars from southeastern Europe have been selected for large and pulpy fruits and are grown for commercial purposes. The bright red cornelian cherries are used to produce jellies, jams and preserves, fermented into wine, or eaten raw. Cornus canadensis has been used as a minor food crop by some North American tribes but nowadays it lost its importance. The Cornus species are very rich in phenolic compounds, their leaves and fruits possess antioxidant, analgesic, antidiabetic, antimicrobial, antihistamine, anti-allergic and antiinflammatory properties [Vareed et al., 2006]. Due to these biological activities, they are widely used in traditional and modern medicine. In traditional medicine, Cornus officinalis fruits are used for their diuretic, tonic and analgesic activities in Korea, Japan and China [Kim et al., 1998]. Cornus officinalis (Japanese cornel) represents a major ingredient of many well known traditional Chinese herbal mixtures used to treat diabetes in Asian traditional medicine. They were also used in folk medicine for treatment of gastrointestinal disorders and various fever related diseases such as flu or malaria [Jayaprakasam et al., 2006; Wadl et al., 2014]. Recent studies reported the neuroprotective activity of some iridoid constituents from Cornus officinalis [Jeong et al., 2012] and antiproliferative effect of an aqueous extract prepared from these fruits against human mammary carcinoma cells [Telang et al., 2012]. The crude extract of Japanese cornelian cherries presents several pharmaceutical actions such as anti- arrhythmic, anti-inflammatory, hepatoprotective and anti-diabetic nephropathy [Kang et

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al., 2007; Zhao et al., 2010]. The dried ground bark of Cornus florida was used in the United States, during Civil War and World War II, as a quinine substitute in treating malaria [Hasegawa, 2007]. Later researches demonstrated that compounds isolated from bark of Cornus florida display antiplasmodial and antileishmanial activities [Graziose et al., 2012]. The fruits of Cornus kousa have been used in Korean traditional medicine as haemostatic and anti-diarrheic agents and their extract has been reported to possess immune-regulatory and cytotoxic effects [Kim et al., 2002; Lee et al., 2007a]. Cornelian cherry (Cornus mas L.) is known for its nutritional and health benefits. The bioactive compounds of Cornelian cherries such as vitamin C, pectins, phenolic acids, flavonoids, ursolic acid and iridoids are responsible for the health beneficial properties ascribed to these fruits [Seeram et al., 2002; Jayaprakasam et al., 2006; Pantelidis et al., 2007; Tural and Koca, 2008; Deng et al., 2013]. Antibacterial, anticancer, anti-inflammatory, antiobesity and antioxidant properties of Cornus mas anthocyanins have also been indicated [Seeram et al., 2002; Vareed et al., 2006]. The most phytochemically investigated Cornus species include: Tatarian dogwood (Cornus alba L.) - a large shrub or small tree, native to Siberia, Northern China and Korea. It‘s grown primarily for the winter display of bright red stems. The fruits are globular white bluish berries. Pagoda dogwood (Cornus alternifolia L.) - a flowering small tree growing up to 9 m tall, native to Eastern North America. The fruits are globular blue black drupes, eaten by birds and black bears. Silky dogwood (Cornus amomum L.) - a 5 m tall shrub, native to Eastern North America. The fruit is a small blue berry. Canadian dwarf cornel (Cornus canadensis L.) is a slow growing 10-20 cm tall subshrub, native to Eastern Asia and Northern America. The fruits are globose in shape, bright red drupes, containing one or two ovoid large seeds. They are edible with an apple like flavor. Giant dogwood (Cornus controversa L.) - an 18 m tall tree, native to China, Himalaya and Japan, is the largest and the fastest growing of the dogwoods. The fruits are globular bluish black berries, ripen from September to October. Flowering dogwood (Cornus florida L.) - a small tree growing up to 10 m high, native to Eastern North America is considered a short to medium-lived tree which can live up to 100 years on good sites. Cornus florida is one of the most beautiful ornamental trees in the world. The fruit is a cluster of up to 10 scarlet oblong oval berries which occasionally can be yellow and persist beyond January. There are valued fruits for birds and wildlife. Kousa dogwood (Cornus kousa L.) – an 8-12 m tall small tree, native to Asia, but also naturalized in the USA. The fruit is a globose pink to red 2-3 cm in diameter compound berry. It is edible, sometimes used for making wine. Cornelian cherry (Cornus mas L.) - a shrub or a 5 to 12 m tall tree, native to Central and South-Eastern Europe (from France to Ukraine) and Asia (from Turkey to China) produces one of the most valuable fruits within the Cornaceae. Cornelian cherry is a slow growing plant which can live up to 300 years [Wadl et al., 2014]. The shiny fruits are spherical or oval shaped one stone drupes which can be either yellow or deep red (almost black). Japanese Cornel (Cornus officinalis L.) - a shrub or a 4 to 10 m tall tree, very similar to Cornelian cherry, native to China, Japan and Korea. The edible but flavorless fruits are 2 cm spherical scarlet to purple drupes, ripen late in the fall.

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Common dogwood (Cornus sanguinea L.) - a medium to large deciduous shrub, growing 2-6 m tall is native to most of Europe and Western Asia. The fruit is a globose black berry, containing a single seed. Dwarf cornel (Cornus suecica L.) - a 20 cm tall subshrub is native to subarctic regions of Europe and Asia and to extreme North America. The edible fruit is a red berry.

2. BIOACTIVE COMPOUNDS FROM SELECTED CORNUS FRUITS 2.1. Polyphenols Phenolic compounds from fruits contribute to their quality, nutritional value, color, taste, aroma and flavor. They are also known to provide the beneficial health effects of many fruits. Phytophenols comprise a wide variety of compounds, divided into several classes: hydroxyacids, anthocyanins, flavonoids, lignans, and tannins. Determination of total phenolic content can be useful to express the biological and nutritive value of fruits. A wide variation in the total phenolic content of Cornus spp. fruits was observed. The total phenolic content (TPC) and composition of fruits depend on species, cultivars and environmental factors. TPC values of Cornelian cherries, estimated by Folin-Ciocalteu method, ranged from 25.9 to 74.83 mg gallic acid equivalents/g dry weight [Pantelidis et al., 2007; Yilmaz et al., 2009]. The total phenolic content of Cornus sanguinea fruits methanol extract was also determined, the obtained value being 88.56 mg gallic acid equivalents/g dry extract [Yousfbeyk et al., 2014].

2.1.1. Flavonoids The flavonoid family is the largest phytonutrients family of biosynthesized compounds that share some common chemical structural features and physicochemical properties, belonging to polyphenols. They are products of secondary metabolism of plants and more than 6000 unique flavonoids have been identified and described [Lehane and Saliba, 2008]. Flavonoids are characterized by benzo-γ-pyrone structure, the flavone skeleton, which consists of two phenyl rings and a heterocyclic pyrane ring. Flavonoids occur as aglycones, glycosides or methylated derivatives and can be divided into different subgroups, depending on their structure: flavones, flavanones, flavonols, flavanonols, isoflavones and flavan-3-ols, anthocyanidins and chalcones (Figure 1). When glycosides occur, usually the carbohydrate is rhamnose, glucose, glucorhamnose or arabinose [Midleton, 1984]. The isolation, identification and structural characterization of flavonoids from plant materials as single compounds or mixtures can be difficult due to the extended presence of isomeric forms of aglycones and their numerous patterns of glycosylation. Extraction is the main step for the isolation of flavonoids from plant materials. Mass spectrometry, ultraviolet and visible (UV-VIS) and infrared (IR) spectrophotommetry, usually applied for the characterization of organic compounds cannot provide enough information to elucidate all the structural features of flavonoids. Other analytical methods such as high performance liquid

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chromatography (HPLC) and nuclear magnetic resonance (NMR) are necessary for the unambiguous characterization of these compounds. 3' 2'

4'

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Figure 1. General structure of flavonoids.

2.1.1.1. Anthocyanins Anthocyanins are one of the major classes of naturally occurring dietary phenols, being the largest group of water-soluble pigments in plants. Anthocyanins are widely consumed in many edible plants [Mazza, 2000; Prodanov et al., 2005; Reyes and Cisneros-Zevallos, 2007]. The bright color of anthocyanins (orange, red, purple, blue), ensures a high potential of being used as natural colorants, as a healthy alternative to synthetic dyes. The fruits of the Cornus species are well known as a rich source of anthocyanins. These pigments are the most abundant bioactive compounds in the ripened fruits of these species [Seeram et al., 2002; Jayaprakasam et al., 2006; Vareed et al., 2006]. The total anthocyanin content varies from 0.28 mg/g fresh weight in Cornus kousa [Vareed et al., 2006], 2.23 mg/g in Cornus mas fruits [Pantelidis et al., 2007] to 16.67 mg/g in Cornus alternifolia [Vareed et al., 2006]. Anthocyanins contribute to the brilliant colors of these fruits. Anthocyanins are glycosides of glucose, galactose, rhamnose, arabinose or other monosaccharides of flavylium cation derivatives. Up to now, more than 500 different anthocyanins were reported [Castaneda-Ovando et al., 2009]. Their structure mainly differs by the number and the position of hydroxyl groups and the nature and number of bonded saccharides. The aglycons of anthocyanins, the anthocyanidins, are however limited to a few chemical structures. So far, only 23 anthocyanidins were identified of which only 6 are

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commonly present in higher plants: cyanidin, delphinidin, malvidin, peonidin, pelargonidin and petunidin (Figure 2) [Andersen and Jordheim, 2006]. R3' 3' 2'

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OH

4

OH Cyanidin: R3'= OH, R5'= H Delphinidin: R3'= OH, R5'= OH Malvidin: R3'= OCH3, R5'= OCH3 Peonidin: R3'= OCH3, R5'= H Pelargonidin: R3'= H, R5'=H Petudin: R3'= OH, R5'= OCH3

Figure 2. Structure of main anthocyanidins.

The color of anthocyanins depends essentially on the different structural forms in which they can be found, these structures being strongly influenced by the pH value. At pH values between 4 and 6, the typical value for fresh and processed fruits, an equilibrium between four structural forms: the red flavylium cation (I), the blue anhydrous quinoidal base (IV), the colorless carbinol pseudobase (II) and the yellow chalcone (III) occurs for all naturally anthocyanins (Figure 3). R3' OH O+

HO

R3' OH OR2

HO

R5'

OH R5'

OR2 OR1

I - H+

+ H+ O

OR1

+ H 2O

O

- H2O

R3'

R3'

OH O

R5'

OH HO

O

OH R5'

OR2 OR1

III

IV

OR2 R1 = H or glycosyl R2 = glycosyl R3', R5'= H, OH or OCH3

OR1

Figure 3. Chemical structures of anthocyanins at different pH values.

II

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The large variety of anthocyanins found in nature and their health promoting properties makes them a very complex and interesting group of bioactive compounds from fruits. Therefore, the food industry is interested in fruits with high content of anthocyanins among which Cornus fruits play an important role. The most common method for isolation of phenolic compounds found in fruits is the solvent extraction. Anthocyanins, like all polyphenols, are polar molecules thus polar solvents such as ethanol, methanol, water, acetone or mixtures of these are the most common solvents used in their extraction. The extraction can be conducted by grinding, drying, freeze-drying of fruits or only by soaking fresh fruits with subsequent solvent extraction [Kahkonen et al., 2001]. Among the most common methods are those which use acidic methanol (1% HCl) as extracting solvent at room temperature [Vareed et al., 2006]. Jayaprakasam et al. (2005) used acidified water (pH = 3) to isolate anthocyanins from Cornus officinalis and Cornus mas fruits, Moldovan et al. (2014) reported the extraction of pigments from frozen Cornelian cherries in acetone while Pantelidis et al. (2007) used air dried fruits (at 55°C) and 50% aqueous methanol as solvent. In the anthocyanins extraction from Cornus alba Sibirica the plant material was extracted with methanol containing 0.2% trifluoroacetic acid [Bjoroy et al., 2007]. To perform the extraction of anthocyanins from various Cornus mas genotypes, a Soxhlet extraction procedure with methanol was also reported [Yilmaz et al., 2009]. All the extraction methodologies are not selective for anthocyanins, implying the coextraction of non-anthocyanin substances, such as other flavonoids, sugars, proteins, organic acids, pectin [Castaneda-Ovando, 2009]. Consequently, new purification techniques in order to isolate the anthocyanin pigments from fruits of Cornus species are required. In this sense, a wide variety of techniques were applied, such as solid phase extraction (SPE), liquid-liquid extraction or more sophisticated chromatographic techniques like high performance liquid chromatography (HPLC). In order to remove chlorophylls, stilbenoids, less polar flavonoids and other non-polar compounds, the extract of Cornus alba fruits was partitioned with ethyl acetate [Bjoroy et al., 2007]. SPE is commonly carried out to purify the anthocyanins mixture obtained after extraction and partition. The extracts are usually fractionated by a C18 column, XAD Amberlite, Sephadex or hydroxylated polymethacrylic polymer resins (Toyopearl) in which the anthocyanins are strongly bounded through their hydroxyl groups [Seeram et al., 2002; Jayaprakasam et al., 2005; Bjoroy et al., 2007; Pawlowska et al., 2010]. The anthocyanin fraction is subsequently separated from other compounds by increasing the polarity of different solvents used for elution of the above mentioned columns. In order to separate the anthocyanin pigments, to elucidate their structure and to provide quantitative information, HPLC with UV-VIS or photodiode array (PDA) detectors is the most commonly used method. The difficulty of obtaining reference compounds and the spectral similarities of anthocyanin pigments represents a significant drawback of using these method therefore mass spectrometry (MS) and nuclear magnetic resonance (NMR) have became the preferred techniques for anthocyanin structure elucidation. The most encountered anthocyanins in Cornus spp. are the 3-glucosides and 3galactosides of cyanidin, pelargonidin and delphinidin. Other mono- and disaccharides attached in the aglycone 3-position have been also mentioned [Du and Francis, 1973a, b; Du

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et al., 1974a,b; Vareed et al., 2006; Bjoroy et al., 2007]. Slimenstad and Andersen (1998) identified an anthocyanin containing a very rare disaccharide: cyanidin-3-O-β-(2‖glucopyranosil-O-β-galactopyranoside) separated from the fruits of Cornus suecica (dwarf dogwood). Early studies demonstrated that Cornus mas fruits contain five anthocyanins: cyanidin-3galactoside, cyanidin-3-rhamnosylgalactoside, delphinidin-3-galactoside, pelargonidin-3galactoside and pelargonidin-3-rhamnosylgalactoside [Du and Francis, 1973a, b]. In a later work, Seeram et al. (2002) isolated and identified three pure anthocyanins from the fruits of Cornus mas. NMR and LC/ES-MS spectral data confirmed that the isolated pigments were delphinidin-3-O-β-galactoside, cyanidin-3-O-β-galactoside and pelargonidin-3-O-βgalactoside, in accordance with the result previously reported by Du and Francis [Du and Francis, 1973a]. Recent researches [Tural and Koca, 2008] indentified by HPLC with UVVIS detection three anthocyanins in Cornus mas berries: cyanidin-3-O-glucoside, cyanidin-3O-rutinoside and pelargonidin-3-O-glucoside. Pawlowska et al. (2010) reported the presence of other anthocyanins, identified by HPLC-PDA/UV and MS: cyanidin-3-O-galactoside, pelargonidin-3-O-glucoside and pelargonidin-3-O-rutinoside. The bluish white berries from Cornus alba Sibirica L. contain five anthocyanins, separated by HPLC and identified by NMR and LC-MS (ESI/TOF) as: delphinidin-3-Ogalactosyl-3‘,5‘-diglucoside as major pigment, delphinidin-3-O-galactosyl-3‘-glucoside, cyanidin-3-O-galactosyl-3‘-glucoside, delphinidin-3-O-galactoside and cyanidin-3-Ogalactoside [Bjoroy et al., 2007]. Anthocyanins with sugar moieties linked to both 3‘ and 5‘ positions have not been identified in other Cornaceae fruits. The first study of the anthocyanin content of Cornus officinalis and Cornus controversa was reported by Seeram et al. in 2002. The isolation, characterization and quantification of anthocyanins in these Cornus spp. fruits was achieved by HPLC, LC-ES/MS and NMR methods. The anthocyanins found in Cornus officinalis were identical to those isolated by the same authors in Cornus mas fruits: delphinidin-3-O-galactoside, cyanidin-3-O-galactoside and pelargonidin-3-O-galactoside but differ in their concentrations. The fruits of Cornus controversa were found to contain the same three anthocyanins as well as a number of unidentified pigments in their HPLC chromatogram [Seeram et al., 2002]. Vareed et al. (2006) found an unusually high concentration of anthocyanin compounds in these fruits. The anthocyanin profile reported by these authors is significantly different from that earlier reported by Seeram et al., 2002. The major anthocyanin in Cornus controversa fruits was found to be delphinidin-3-O-glucoside while also relative high amounts of delphinidin-3-O-rutinoside were identified. Negligible amount of cyanidin-3-Oglucoside when compared to delphinidin derivatives was also reported. The fruits of Cornus alternifolia showed similar anthocyanin profile as those of Cornus controversa [Vareed et al., 2006], the relative amounts of anthocyanins in these fruits were found in the following order: delphinidin-3-O-rutinoside > delphinidin-3-O-glucoside >> cyanidin-3-O-glucoside. The anthocyanin content in Cornus alternifolia fruits was several times higher than in other commonly consumed fruits.

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Cyanidin-3-O-galactoside was the major anthocyanin pigment identified in Cornus florida fruits [Vareed et al., 2006] which also contain cyanidin-3-O-glucoside in very small amounts. Table 1. Anthocyanins from Cornus species fruits Anthocyanin Cyanidin-3-galactoside

Cornus spp. C. mas

References Du and Francis, 1973a Seeram et al., 2002 Jayaprakasam et al., 2005 Jayaprakasam et al., 2006 Pawlowska et al., 2010

C. suecica

Slimestad and Andersen, 1998

C. alba C. officinalis

Bjoroy et al., 2007 Seeram et al., 2002

C. controversa C. florida

Seeram et al., 2002 Vareed et al., 2006

C. kousa

Vareed et al., 2006

C. mas

Tural and Koca, 2008 Capanoglu et al., 2011 Slimestad and Andersen, 1998 Vareed et al., 2006 Vareed et al., 2006 Vareed et al., 2006

OH OH

ClHO

O O

OH

O

HO

OH

HO

OH

Cyanidin-3-glucoside OH OH

ClHO

O O

OH

O OH

HO

OH

C. suecica C. controversa C. alternifolia C. florida C. kousa

Du et al., 1974a Vareed et al., 2006

C. mas

Tural and Koca, 2008 Capanoglu et al., 2011

C. alba

Bjoroy et al., 2007

HO

Cyanidin-3-rutinoside OH ClHO

OH O OH HO O

OH O

HO

O Me HO

O HO

OH

Cyanidin-3-galactosyl-3‘-glucoside HO HO

OH O

O

HO

OH

OH

ClO O OH

OH

O

HO HO

OH

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Table 1. (Continued) Anthocyanin

Cornus spp.

References

Cyanidin-3-(2-glucosylgalactoside)

C. suecica

Slimestad and Andersen, 1998

C. suecica

Slimestad and Andersen, 1998

C. mas

Du and Francis, 1973a

C. canadensis

Du et al., 1974b

C. mas

C. alba

Du and Francis, 1973a Seeram et al. 2002 Jayaprakasam et al., 2005 Jayaprakasam et al., 2006 Bjoroy et al., 2007

C. officinalis

Seeram et al., 2002

C. controversa

Seeram et al., 2002

C. controversa

Vareed et al., 2006

C. alternifolia C. kousa

Vareed et al., 2006 Du et al., 1974a

OH OH

ClHO

O O

OH

O

O

OH

HO

O HO

OH OH

OH OH

Cyanidin-3-(2-glucosylglucoside) OH OH

ClHO

O OH O

O

OH

O

OH

O

HO

HO

OH

OH OH

Cyanidin-3-robinobioside OH OH

ClHO

OH OH

O HO O

O OH

O Me HO

O HO

OH

Delphinidin-3-galactoside OH OH

ClHO

O

OH O

OH

O

HO

OH

HO

OH

Delphinidin-3-glucoside OH OH

-

Cl HO

O

OH O

OH

OH

O OH

HO HO

Extraction, Characterization and Potential Health Benefits … Anthocyanin Delphinidin-3- rutinoside OH ClHO

Cornus spp.

References

C. controversa

Vareed et al., 2006

C. alternifolia

Vareed et al., 2006

C. alba

Bjoroy et al., 2007

Cornus spp. C. alba

References Bjoroy et al., 2007

C. mas

Du and Francis, 1973b Seeram et al., 2002 Jayaprakasam et al., 2006

C. officinalis C. controversa

Seeram et al., 2002 Seeram et al., 2002

C. mas

Du and Francis, 1973b

C. canadensis

Du et al., 1974b

OH O OH OH HO O

OH O

HO

O Me HO

O HO

OH

Delphinidin-3- galactosyl-3‘glucoside HO HO

OH O

O

OH

OH

-

Cl HO

O

OH O

OH

O

HO

OH

HO

OH

Anthocyanin Delphinidin-3- galactosyl3‘,5‘-diglucoside HO HO

OH O

O

HO

OH

OH

ClO

O HO OH HO

O

OH

O OH

OH

OH O

OH HO

Pelargonidin-3-galactoside OH

-

Cl HO

O O

OH

O

HO

OH

HO

OH

Pelargonidin-3- robinobioside OH

ClHO

OH OH

O HO O

O

OH

O Me HO

O HO

OH

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168

Table 1. (Continued) Anthocyanin Pelargonidin-3-glucoside O O

C. mas

Pawlowska et al., 2010

OH

O OH

HO

OH

HO

Pelargonidin-3-rutinoside ClHO

C. kousa

References Tural and Koca, 2008 Pawlowska et al., 2010 Du et al., 1974a

OH

ClHO

Cornus spp. C. mas

OH O OH HO O

OH O

HO

O Me HO

O HO

OH

Cornus kousa fruits showed the lowest anthocyanin content among all Cornus spp. fruits studied so far. Du and co-workers [Du et al., 1974] revealed that Chinese dogwood, C. kousa fruits contain delphinidin-3-O-glucoside, cyanidin-3-O-glucoside and pelargonidin-3-Oglucoside, results not in accordance to the HPLC profile found by Vareed [Vareed et al., 2006], which only demonstrated the presence of cyanidin-3-O-glucoside (major pigment) and cyanidin-3-O-galactoside in negligible amount. Table 1 presents the anthocyanins found in Cornus spp. fruits. Two anthocyanins were identified by LC-MS in the fruit extract of Cornus amomum L.: a delphinidin-dipyranoside and a delphinidin-tripyranoside but their structure was not yet fully elucidated [Ma et al., 2010]. In recent years, various important biological activities, such as antioxidant, antimutagenic, anticancer, anti-inflammatory and antiobesity properties of anthocyanins have been reported. The methanol extract of C. alternifolia and C. controversa inhibited lipid peroxidation by 56% and 53% respectively, while delphinidin-3-O-glucoside and delphinidin3-O-rutinoside isolated from these fruits presented a higher inhibition of 71% and 68% respectively, at 5 time lower concentrations [Vareed et al., 2006]. Delphinidin-3-O-glucoside and delphinidin-3-O-rutinoside isolated from Cornus kousa fruits were evaluated for their inhibitory activities on tumor cell proliferation, lipid peroxidation and cyclooxygenase enzymes (COX), showing potent effects [Vareed et al., 2006]. The pure mixture of anthocyanins (cyanidin-3-O-galactoside, pelargonidin-3-Ogalactoside and delphinidin-3-O-galactoside) isolated from Cornus mas fruits possess a potent effect in amelioration of obesity and insulin resistance as well as reducing the body weight of high-fat-fed mice, independent of food intake. They also decreased the level of plasma cholesterol and liver lipids, reducing the risk of diabetes and obesity [Jayaprakasam et al., 2005; Jayaprakasam et al., 2006].

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2.1.1.2. Other Flavonoids Solid-liquid extraction is the most commonly used technique to isolate flavonoids from Cornus spp. fruits, the extraction solvents, the purification methods and the structural characterization techniques being the same as in the case of anthocyanins. Despite intensive investigation of anthocyanin compounds in Cornus spp., very little is known about other flavonoids from these fruits. After partition with n-hexane, ethyl acetate and n-butanol and chromatographic separation of the compounds from Cornus mas fruits extract, Pawlowska et al. (2010) evaluated the flavonoid profile of these. Using spectroscopic methods and HPLC-PDA-ESI-MS analysis, the authors identified 11 flavonoids of which 3 were anthocyanins. The quantitative analysis revealed a high flavonoid content 221.3 mg/10 g. Except one compound, which was identified as a dihydroflavonol (aromadendrin 7-Oglucoside), they were all O-flavonol glycosides of quercetin and kaempferol with sugar moieties such as glucose, xylose, rhamnose, rutinose and galactose. The major constituents of the flavonoid fraction were quercetin 3-O-glucuronide and kaempferol 3-galactoside. In contrast to results obtained by Pawlowska and co-workers, Sochor et al. found quercitrin (quercetin 3-O-rhamnoside) as the major poliphenol with flavonoid scaffold, followed by rutin (quercetin 3-O-rutinoside) in the fruits of eight investigated Cornelian cherry cultivars from Czech Republic [Sochor et al., 2014]. Cornus officinalis is another flavonoids rich Cornus spp. The ethanolic fruit extract of Cornus officinalis contains 5 flavonoids: kaempferol, kaempferide and quercetin (non glycosylated flavonoids) and two glycosyl derivatives of quercetin: isoquercitrin (3-Oglucoside) and hyperoside (3-O-galactoside) [Xie et al., 2012]. In a later work, six polyphenols with flavonoid backbone were identified, among which only one was an aglycone (quercetin), all others being glycosyl derivatives of kaempferol and quercetin [Ma et al., 2014]. OH

OH OH

HO

O

OH HO

O

OH OH

OH OH

O

OH

Quercetin

O

Myricetin OH

HO

O

OH HO

O

OH OH

O

Kaempferol

OH OH

O

Aromadendrin

Figure 4. Chemical structures of flavonoids (aglycones) from Cornus fruits.

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The fruits of Cornus kousa can also be considered a source of flavonoids. Four flavonoids were isolated and fully characterized by HPLC, NMR, MS and IR techniques. These compounds were: kaempferol, astragalin (kaempferol 3-O-glucoside), hyperoside and isoquercitrin [Lee et al., 2007b]. In contrast to these results, Vareed et al. (2007) identified only three flavonoids in the alcoholic extract of Cornus kousa fruits: kaempferin (kaempferol 3-O-rhamnoside), myricitrin (myricetin 3-O-rhamnoside) and astragalin. The chemical structures of flavonoids aglycones from Cornus spp. fruits are presented in Figure 4. The flavonoids isolated from Cornus kousa fruits have been reported to exhibit antioxidant activity and promising lipid peroxidation and cyclooxygenase enzyme inhibitory activity [Lee et al., 2007b; Vareed et al., 2007]. Astragalin presented cytotoxicity against human cancer cell lines [Yan et al., 2002]. Isoquercetin possess great anti-inflammatory and hepatoprotective activity [Puppala et al., 2007] and hyperin has significant anti-HIV activity [Lee et al., 2007b]. OH OH OH O

O

COOH

COOH COOH

HO

OH

OH

OH

OH

OH

Gallic acid

OH

Caffeic acid

HO

Chlorogenic acid OH

COOCH3 O

HO

OH

O OH

OH 3,5-Dihydroxy-2-(2-methoxy-2oxoethyl)phenyl 4-hydroxy-benzoate

Resveratrol

OH H3CO

OCH3 HO

HO

OH

OH

O

HO

OH OH

O

O

HO

O HO

OH

OH

Tachioside

Guaiacylglycerol-3-O-glucopyranoside

COOH O OH

p-Coumaric acid

Tyrosol

OH

OH

O

OH OH

HO

HO OH

HO

OH

O

OH 7-O-Galloyl-D-sedoheptulose

Figure 5. Chemical structures of phenolic compounds isolated from Cornus fruits.

Extraction, Characterization and Potential Health Benefits …

171

2.1.2. Other Phenolic Compounds The major polyphenolic compound identified in Cornelian cherries was chlorogenic acid, its content ranged from 2.63 mg/100 g fresh weight [Drkenda et al., 2014] to 135 mg/100 g fresh weight [Sochor et al., 2014]. Sochor and co-workers also identified gallic acid in much lower amounts (ranged from 11 to 32 mg/100 g fresh weight) compared to chlorogenic acid and traces of resveratrol in all investigated Cornus mas fruits from eight different cultivars. The fruits of Cornus officinalis, unlike Cornelian cherries, are reacher in gallic acid. The n-butanol extract of these fruits contains 60 mg gallic acid/ g dried extract, separated by highspeed counter-current chromatography [Tian et al., 2000]. Caffeic acid was also identified in these berries, in much lower amount: 0.08 mg/ g dried extract identified by NMR spectral data [Nawa et al., 2007] or even less (0.015 mg/ g dried extract) isolated by column chromatography by Ma and co-workers [2014]. p-Coumaric acid was isolated from the water extract of Cornus officinalis fruits by HPLC [Park et al., 2012]. The same study demonstrated the presence of other phenolics such as 3,5-dihydroxy-2-(2-methoxy-2-oxoethyl)-phenyl 4hydroxy-benzoate, tachioside and guaiacylglycerol-3-O-glucopyranoside [Ma et al., 2014]. Zhang isolated for the first time a low molecular weight polyphenol, 7-O-galloylsedoheptulose in the extract of Cornus officinalis fruits [Zhang et al., 1999]. The phytochemical investigation of Cornus amomum fruits first reported the presence of tyrosol, the major bioactive phenolic compound of olive oil, in a Cornus species and thus this compound may be regarded as a chemotaxonomic marker of Silky dogwood [Ma et al., 2010]. Figure 5 summarizes the chemical structures of main phenols isolated from Cornus spp. fruits. Biological studies reported the hepato- and renoprotective role of 7-O-galloyl-Dsedoheptulose on diabetes [Yamabe et al., 2009; Park et al., 2010]. 2.1.2.1. Lignans The lignans are a naturally occurring group of polyphenolic substances found in plants which are derived from phenylpropanoids, commonly encountered as dimers with complex dibenzylbutane skeleton in which the phenylpropane units are linked by the central C atom of their side chains. A few compounds of this class are trimers and tetramers. Most lignans found in plants occur freely and a small part can form glycosides with sugars. Lignans can be classified into 5 classes according their structural features: lignans, neolignans, norlignans, hybrid lignans and oligomeric lignans [Zhang et al., 2014]. Plants with high lignan content have been used in folk medicine due to their numerous pharmacological effects, nowadays lignans being an important class of compounds used to develop new synthetic derivatives with more potent medicinal actions [Gordaliza et al., 2004]. The isolation and characterization of the lignans from Cornus spp. fruits was mainly conducted by solvent extraction from the plant matrix. 80% aqueous methanol was used to extract the lignans from Cornus kousa L. fruits. After extraction, the obtained solution was partitioned with water, ethyl acetate and n-butyl alcohol and the ethyl acetate fraction was subjected to column chromatography [Lee et al., 2007a].Six lignan compounds were isolated and fully characterized by IR, EI-MS and NMR from the first time from this plant: pinoresinol, lariciresinol, balanophonin, erythro and threo guaiacylglycerol-β-coniferyl aldehyde ethers and a well known neolignan, dihydrodehydroconiferyl alcohol [Lee et al., 2007a].

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A new lignan glycoside was also identified in the methanol extract of fruits of Cornus kousa L.: cornuskoside A [Lee et al., 2008a]. This compound is a xylopyranoside of dihydrodehydroconiferyl alcohol and was isolated for the first time from a natural source in these fruits. The chemical structures of main lignans separated from Cornus spp. fruits are presented in Figure 5. The isolated lignans from Cornus kousa fruits were tested for their cytotoxic effects against two human carcinoma cell lines using the microculture tetrazolium (MTT) assay method [Lee et al., 2007a]. All investigated lignans from C. kousa fruits exhibited significant cytotoxic effects on colon and hepatocellular carcinoma cells, neolignans presenting the highest toxicity [Lee et al., 2007a]. Dihydrodehydroconiferyl alcohol and balanophonin showed potent cytotoxicity against human breast carcinoma, human cervix, human melanoma and human ovary carcinoma cell lines [Lee et al., 2008a]. Lariciresinol and pinoresinol isolated from methanolic extract of Cornus kousa fruits exhibited an efficient inhibitory activity against low density lipoprotein (LDL) oxidation [Lee et al., 2007c; Lee et al., 2010]. OH HO

OCH3

O

OH

O

O

H3OC OCH3 OCH3

O

Balanophonin

OH

Pinoresinol

OH

HO HO

O

OH OH

O

H3OC

O

O

OCH3

OCH3

OH

OH

OCH3

Cornuskoside A OH Lariciresinol OH

OCH3

HO O

HO

OCH3

OH

O

O OCH3 Dihydrodehydroconiferyl alcohol

Figure 6. Lignans isolated from Cornus kousa L. fruits.

OCH3

OH HO

Guaiacylglycerolconiferyl aldehyde ether

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173

2.1.2.2. Tannins Tannins are phenolic compounds widely distributed in plants, having a macromolecular structure with molecular weight ranging from 500 Da to 3000 Da. According to their chemical structure, tannins from vascular plants can be divided into two main classes: hydrolysable and condensed tannins. Hydrolysable tannins, including gallo- and ellagitannins, usually found in lower concentrations in plants, are biomolecules in which the hydroxyl groups of a central carbohydrate (usually glucose) are esterified with phenolic groups of gallic or ellagic acid. Condensed tannins, the most common type of tannins, have a variety of chemical structures, formed by the condensation of flavans. They are also called proanthocyanidins as by depolymerization they yield anthocyanidins [Hassanpour et al., 2011]. Cornelian cherry fruits are superior in tannin content to many other fruit species. Bijelic and co-workers investigated 18 Cornus mas L. genotypes from Voivodina province and reported values ranging from 560 mg to 1470 mg tannins / 100g fruits [Bijelic et al., 2011]. Comparable values of the tannin content are reported for Turkish Cornelian cherry genotypes by Ercisli et al. (2011) who found a mean value of 0.89% tannins in fruits. Tannins from Cornus officinalis fruits were separated and their structure was established for the first time by Okuda and co-workers [Okuda et al., 1984]. The authors isolated these compounds by extraction of the ripe fruits in 70% acetone followed by partition with diethyl ether and ethyl acetate. The compounds were purified by column chromatography and fully characterized using NMR spectroscopy and FAB-MS spectrometry. Seven new hydrolysable tannins were identified and named after Cornus fruits from which they were isolated: cornusiins A-G. They are monomeric, dimeric and trimeric ellagitannins (Figure 7) [Hatano et al., 1989a, b; Hatano et al., 1990]. Camptothins A and B, dimeric hydrolysable tannins, were also identified in the fruits of Cornus officinalis. Several other hydrolysable tannins were isolated, among which gemin D, isoterchebin, tellimagrandin I and II and oenothein. OH HO

O

O

OH

HO O

OH

O O

OH

OH

O

O

O O

O

O

O C

C

OH

OH

O OH

OH

HO

OH OH

HO HO

Figure 7. Chemical structure of cornusiin B.

O

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The dimeric hydrolysable tannin isolated from Cornus officinalis fruits, cornusiin A, showed a potent inhibitory effect on reverse transcriptase of avian myeloblastosis virus [Kakiuchi et al., 1985] and also antisarcoma activity [Myamoto et al., 1987]. Cornusiin B presented a high α-glucosidase inhibitory effect [Omar et al., 2012].

2.2. Terpenoids Terpenoids are a large class of naturally occurring organic compounds, derived from isoprene units, being probably the most wide-spread group of natural products, more than 23.000 terpenoids compounds being identified as now. Terpenoids can be described as modified terpenes where methyl groups are moved or removed or oxygen containing groups added. Terpenes are classified upon the number of isoprene units incorporated in their skeleton in: monoterpenes, sesquiterpenes, diterpenes, sesterpenes, triterpenes, carotenoids and rubbers [Wang et al., 2005].

2.2.1. Iridoids Iridoids represent a large class of secondary metabolites wide-spread in plants and in some animals. They are monoterpenoids biosynthesized from isoprene having a bicyclic β-cis fused cyclopentanopyrane ring system. Iridoids are responsible of many pharmaceutical activities of medicinal plants. Over 800 iridoids have been identified from plants and animals [Dinda et al., 2007a]. They are classified in four distinct groups: iridoid glycosides and aglicones (non-glycosidic iridoids), secoiridoids and bisiridoids [Suomi et al., 2000]. Solid-liquid extraction is the most used method for the separation of iridoids from the sample matrix. The most common solvents used for this purpose are alcohols, especially methanol and ethanol. Hot water can also be used as an environment friendly solvent for the extraction of these terpenoids compounds [Suomi et al., 2000]. Considering the potent biological activities of iridoids, the development of efficient purification methods is important for the pharmacological researches. The conventional methods used to separate and purify iridoid glycosides from Fructus Corni are column chromatography and preparative HPLC. Another efficient method is high-speed countercurrent chromatography [Liang et al., 2013]. Cornus is widely recognized as an iridoid rich genera. Morroniside was found to be the major irridoid in Cornus officinalis fruits. Du et al. (2008) reported a content ranging from 12.58 to 17.07 mg/g dry fruit. In contrast, other researchers identified cornuside as the major constituent of the total extract of Cornus officinalis fruits, representing 26.8% of the extract [Jiang et al., 2011]. Phytochemical researches during the past decades demonstrated that the main active components in Fructus Corni are the iridoids glycosides, including morroniside, sweroside and loganin as main compounds but also cornuside, swertiamarin, verbenalin, 7-O-methyl morroniside, 7-O-ethyl morroniside, 7-O-butyl morroniside. Also, an iridoid aglycone was identified as dehydromorroniaglycone [Cao et al., 2009]. Five iridoids glycosides were isolated from C. officinalis fruits by ultrasound assisted extraction in methanol, followed by successive partition of the extract with n-hexane, dichloromethane, ethyl acetate and n-butanol. The elucidation of the chemical profile of the extract was achieved by HPLC-DAD-ESI-MS, UV and NMR methods and the results

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indicated the presence of morroniside and loganin as major constituents as well as of 7-Obutyl morroniside and 7-O-methyl morroniside (R and S) [Jeong et al., 2012]. Recent researches led to isolation and characterization of two new iridoid glucosides: loganin derivatives logmalicids A and B as well as to the first report of loganic acid and secoxyloganin in Cornus officinalis fruits [Ma et al., 2014]. COOCH3

COOCH3

HO O

HO

HO

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O

OH OH

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HO Loganin

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O

O

O

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O

OH OH

O

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HO

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OH OH

O

O

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COOH OH HO

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H3C HO O O

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HO Loganic acid

HO

Figure 8. Chemical structures of some iridoids isolated from Cornus fruits.

The first report identifying the iridoid content of Cornus mas fruits mentioned loganic acid as the major monoterpenoid in Cornelian cherries with a concentration of 1.67 mg/g fresh fruit puree [West et al., 2012]. More bioactive iridoids of Cornus mas fruits were later isolated and identified using ultraperformance liquid chromatography (UPLC) coupled with PDA and ESI-TOF-MS.

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Loganin, sweroside and cornuside were identified by mass spectrommetry data [Deng et al., 2013]. Researches of iridoids from Cornus kousa fruits extract are limited, the only published study mentioning the presence of cornin [Vareed et al., 2007]. The chemical structures of main iridoids isolated from Cornus fruits are presented in Figure 8. Cornin, the iridoid glycoside isolated from Cornus kousa fruits, inhibited the proliferation of 5 human tumor cell lines: colon, breast, lung, central nervous system and stomach. It also showed promising lipid peroxidation and cyclooxygenase enzyme inhibitory activity [Vareed et al., 2007]. Investigation of the effect of loganin, an iridoid glycoside from Fructus Corni on hemorrhagic shock induced in rabbits revealed that this compound presents a potent effect on increasing blood pressure, indicating that loganin can be used as a new anti-shock drug [Cao et al., 2009]. The iridoid glycosides from Cornus officinalis fruits may act as advanced- glycation end products (AGE) inhibitory agents and subsequently prevent the progressive accumulation of extracellular matrix (ECM) in glomerular mesangium, being a potential agent in prevention and therapy of diabetic nephropathy. Loganin and morroniside inhibit oxidative stress and prevent renal deposition of laminin and fibronectin. In particular, morroniside has been proven effective in preventing diabetic angiophaty and nephropathy [Xu and Hao, 2004; Yokozawa, 2008; Ma et al., 2014]. Loganin, sweroside and morroniside, the major iridoid components in Cornus officinalis fruits, do not show a potent radical scavenging activity as compared to cornuside. Loganin and cornuside showed a significant inhibitory effect on melanogenesis in mouse melanoma cell line [Nawa et al., 2007]. Morroniside provide an efficient protection of neuroblastoma cells against hydroperoxyde induced cytotoxicity and protect rat brain from damage [Wang et al., 2009; Wang et al., 2010]. Loganin improves cognitive impairment caused by fimbria-fornix lesions [Zhao et al., 2010]. 7-O-butyl morroniside from Cornus officinalis fruits showed a neuroprotective activity against glutamate-induced neurotoxicity in HT22 hypocampal cells [Jeong et al., 2012]. Cornuside has been reported to have anti-inflammatory effect [Kang et al., 2007] and so does loganic acid too [Wei et al., 2013]. Strong antibacterial, antifungal and antispasmodic activities were demonstrated for loganin and sweroside [Dinda et al., 2007b]. Loganin, sweroside and cornuside isolated from Cornus mas fruits reduced the amount of DNA damages, suggesting potential antigenotoxic activity [Deng et al., 2013].

2.2.2. Triterpenoids Triterpenes are organic compounds consisting of six isoprene units. The pentacyclic triterpenes are classified into three groups: lupane, olenane and ursane. Like most triterpenoids, ursolic acid and its derivatives are constituents of numerous plants. Ursolic acid rarely occurs in plants without its isomers, oleanoic and betulinic acids, being among the main bioactive components from Cornus species fruits.

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Alcohol reflux extraction, ultrasound assisted extraction, Soxhlet extraction and supercritical fluid extraction (SPE) are the main techniques used to isolate triterpenoids from Cornus spp. fruits [Zhao et al., 2005]. Thin layer chromatography (TLC), HPLC and cyclodextrin-modified micellar electrokinetic chromatography (CD-MEKC) have also been applied for separation and determination of these three triterpenic acids in fruits [Zhang et al., 2005; Wang et al., 2008]. The isolated triterpenic compounds and their glycosides were identified by spectroscopic analysis: NMR, IR and MS. The content of ursolic acid in Cornus officinalis fruits ranged from 0.143% to 0.274%. Using reversed-phase HPLC, Wang and co-workers quantified ursolic acid as well as oleanolic acid in different parts of Cornus fruit. Their results suggested that the two triterpenic acids are mainly located in the exocarp. The reported values are 0.123% for ursolic acid and 0.085% oleanolic acid, expressed as mass fractions of dry powder of Cornus fruit pericarp [Wang et al., 2008]. The total triterpene acids (TTA) were isolated and quantified by ether extraction and HPLC and oleanolic acid and ursolic acid were identified together with other 7 unidentified compounds. The TTA content was 0.5% in dry weight of Fructus Corni [Qi et al., 2008]. In addition, β-sitosterol and daucosterol malate have been isolated from the fruits of this species [Xie et al., 2012]. The methanol extract of Cornus kousa fruits contains five triterpenoids isolated after partition with ethyl acetate, n-butanol and water by column chromatography. The five compounds were identified by EI-MS, UV-VIS, IR and NMR analysis to be ursolic acid, corosolic acid, taraxasterol, betulinic acid and betulinic aldehyde [Lee et al., 2009]. Ursolic aldehyde was also reported as an ursan-type triterpenoid from Cornus kousa fruits [Lee et al., 2010a]. Later phytochemical researches on the fruits of this plant revealed the presence of other triterpenoids in the extracts: lupeol, arjunolic acid, tormentic acid, asiatic acid and 19-αhydroasiatic acid [Lee et al., 2010b]. β-Sitosterol was also identified [Vareed et al., 2007]. The effort to isolate new bioactive compounds from the fruits of Cornus kousa Burg led to the identification and characterization by spectroscopic methods of two other steroids containing the conjugated ketone together with the allyl alcohol: 6-hydroxystigmast-4-en-3one (α and β isomers) [Lee et al., 2008b]. Triterpene glycosides were extracted from C. kousa berries purified by column chromatography and identified as tormetoside, niga-ichigoside F2 and arjunglucoside using NMR, IR and MS analysis [Jung et al., 2009]. Investigating the most abundant bioactive compounds from Cornus mas fruits, Jayaprakasam and co-workers reported the isolation of 2.2 g ursolic acid from 8 kg fruit pulp after successive extractions with n-hexane, ethyl acetate and methanol followed by column chromatography purification of the product [Jayaprakasam et al., 2006]. Figure 9 presents the chemical structures of some triterpenoids isolated from Cornus spp. fruits. Ursolic acid is considered one of the most promising chemopreventive agents for cancer, based on investigation of its antitumor activies [Kassi et al., 2007]. Kwon and co-workers tried to elucidate the apoptotic mechanism of ursolic acid from Corni Fructus in primary human malignant prostate tumor cells, proving that this compound inhibit prostate cancer cell growth [Kwon et al., 2010]. Ursolic acid, taraxasterol, betulinic acid and betulinic aldehyde showed a relative high inhibitory activity against human acyl-CoA: cholesterol acyltransferaze [Lee et al., 2009].

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Ursolic aldehyde isolated from Cornus kousa fruits was reported to possess an inhibitory activity against phosphatase of regenerating liver-3 (PRL-3), a key contributing factor in the development of metastatic properties of tumor cells [Lee et al., 2010a].

H H H

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H

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H H

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HO

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Taraxasterol

Sitosterol

H H HOOC

H

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H

O OH

OH

Daucosterol malate

Figure 9. Chemical structures of triterpenoids isolated from Cornus fruits.

Lupane triterpenoids: betulinic acid and betulinic aldehyde as well as ursolic acid exhibited a significant inhibitory activity against five human cancer cell lines: colon carcinoma (HCT-116), breast carcinoma (MCF-7), cervix carcinoma (HeLa), ovary carcinoma (SK-OV-3) and melanoma (SK-MEL-5), findings that suggest that methanol extract of Cornus kousa fruits and some of its isolated triterpenoids may be useful for cancer treatment [Lee et al., 2010b]. 6-Hydroxy-stigmast-4-en-3-ones α and β were also evaluated for their cytotoxic activity showing a potent action against three human cancer cell lines: colon carcinoma, melanoma and ovary carcinoma [Lee et al., 2008b]. Ursolic acid isolated from Cornus mas fruits has been demonstrated to improve some metabolic parameters related to high saturated fats diet and obesity. This bioactive

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triterpenoid showed a promising ability to ameliorate the obesity, to decrease the lipid accumulation in liver and to elevate the level of insulin in high fat fed mice [Jayaprakasam et al., 2006]. Recent researches demonstrated that ursolic acid from Cornus officinalis fruits ameliorate inflammatory diseases by regulating nuclear factor k light-chain enhancer of activated B cells (NF-kB) and mitogen-activated protein kinase (MAPK) pathways through the inhibition of lipopolysaccharide (LPS) binding on immune cells [Jang et al., 2014]. Total triterpenic acids isolated from Cornus officinalis fruits ameliorate diabetic cardiomyopathy in streptozocin-injected rats, by suppressing the endothelin reactive oxidative species pathway in the myocardium [Qi et al., 2008].

2.3. Other Organic Compounds Fresh cornelian cherry fruits are particularly rich in ascorbic acid, containing twice as much vitamin C as oranges [Seeram et al., 2002]. The vitamin C content is reported to be 16.4 to 38.5 mg/100 g [Brindza et al., 2009]. The fruits of Cornus mas are reported to contain 4.6% lipids in seed and 0.1-0.3% fats in the fleshy part of the fruit. Linoleic acid represents 67.3% in seed and 36.54% in pericarp from the total lipid content. Other fatty acids were also identified in the fruits, including lauric, myristic, pentadecenoic, palmitic, stearic, oleic, linoleic, linolenic, palmitoleic acids and other fatty acids [Brindza et al., 2009]. At least 15 aminoacids were identified in Cornelian cherries, e.g. aspartic acid, glutamic acid, serine, histidine, glycine, threonine and arginine [Brindza et al., 2009]. Cornelian cherries contain a special type of polysaccharide, calcium-pectate, which is able to reduce the level of low density lipoprotein cholesterol in human blood and also acts as diuretic [Bijelic et al., 2011].

2.4. Microcomponents Literature data report that Cornelian cherry juice is remarkable rich in various essential microelements. Copper, iron, phosphorus, zinc, manganese, potassium, sodium, calcium and magnesium are the main microelements identified by inductively coupled plasma atomic emission spectrometry (ICP-AES) in the ash of Cornus mas fruits mesocarp [Bijelic et al., 2011; Cindric et al., 2012; Deng et al., 2013]. The calcium level of the juice obtained from these fruits is 10 fold higher compared to other juices, e.g. apple and pear, reaching a value of 323 mg/L. The potassium concentration was also high (1639 mg/L) [Krosniak et al., 2010]. Mineral constituents are essential for normal development of humans. As essential elements are not synthesized in body they must be obtained from dietary sources. They play important roles in the metabolic functions. Calcium and phosphorus are essential for bone structure, potassium and sodium are involved in the functions of all organs, iron, copper and manganese are important for enzymatic functions. Cornelian cherry fruits, being rich in essential elements, might be considered as important nutritional supplements.

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3. ANTIOXIDANT ACTIVITY Cornus species fruits are significantly rich in phenolic compounds, anthocyanins, flavonoids, and ascorbic acid. Therefore, they could be considered as a valuable source of natural antioxidants. The evaluation of the antioxidant activity of their extracts demonstrated elevated levels, but also great variations among species and genotypes. The presence of these natural antioxidants provide protection against harmful free-radicals and therefore against cancer and heart diseases. In recent years, consumers have paid increasing attention to fruits with high antioxidant activity, such as elderberry, lingonberry, honeysuckle, medlar but also Cornelian cherries. Recently, a lot of studies have been published about the antioxidant activity of Cornus spp. fruits [Tural and Koca, 2008; Pantelidis et al., 2007; Popovic et al., 2012]. Due to the presence of different antioxidant compounds which may act through different mechanisms, no single method can be used to estimate the total antioxidant capacity of fruits extracts. Different antioxidant assays were used to assess the antioxidant activity, such as ferric reducing ability of plasma (FRAP), permanganate reducing antioxidant capacity (PRAC), 2,2-diphenylpicrylhydrazyl (DPPH) assay, β-carotene bleaching assay, deoxyribose method. Table 2. Some biological activities of Cornus spp. fruits Cornus spp. C. alternifolia C. controversa C. kousa

Biological activity Inhibition of lipid peroxidation Inhibition of lipid peroxidation as well as astringent and tonic effect Inhibitory activity on tumor cell proliferation, lipid peroxidation and cyclooxygenase enzymes (COX) Anticarcinogenic activity

C. mas

Inhibitory activity against low density lipoprotein (LDL) oxidation Anti-inflammatory, hepatoprotective and anti-HIV activity Inhibitory activity against human acyl-CoA: cholesterol acyltransferaze Inbititory of metastatic properties of tumor cells Treatment of diarrhea and gastrointestinal disorders

C. officinalis

Amelioration of obesity and insulin resistance, reduction of the body weight and decrease of the level of plasma cholesterol and liver lipids Antigenotoxic activity Hepatoprotective activity Diuretic effect Anti-inflammatory properties Cardioprotective effects Tonic, analgesic and diuretic activity

References Vareed et al., 2006 Vareed et al., 2006 Vareed et al., 2006 Vareed et al., 2007 Lee et al., 2007b Yan et al., 2002 Lee et al., 2007a Lee et al., 2008a Lee et al., 2008b Lee et al., 2010b Lee et al., 2007c Lee et al., 2010 Puppala et al., 2007 Lee et al., 2007b Lee et al., 2009 Lee et al., 2010a Celic et al., 2006 Jayaprakasam et al., 2005 Jayaprakasam et al., 2006 Deng et al., 2013 Alavian et al., 2014 Bijelic et al., 2011 Yilmaz et al., 2009 Eshaghi et al., 2012 Kean and Hwan, 1998

Extraction, Characterization and Potential Health Benefits … Cornus spp.

Biological activity Antitumoral activity Antiarrithmic, anti-shock, antineoplastic, antiinflammatory, hepatoprotective and antidiabetic effects Antibacterial activity Hepato- and renoprotective activity on diabetes Antiviral and antitumor activity; α-glucosidase inhibitory effect Prevention and therapy of diabetic nephropathy

Antibacterial, antifungal and antispasmodic activity

Amelioration of diabetic cardiomyopathy

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References Nawa et al., 2007 Telang et al., 2012 Kang et al., 2007 Cao et al., 2009 Zhao et al., 2010 Wu et al., 2008 Yamabe et al., 2009 Park et al., 2010 Kakiuchi et al., 1985 Myamoto et al., 1987 Omar et al., 2012 Xu and Hao, 2004 Yokozawa, 2008 Ma et al., 2014 Kang et al., 2007 Dinda et al., 2007b Wei et al., 2013 Jang et al., 2014 Qi et al., 2008

All investigated Cornelian cherry genotypes showed high antioxidant capacity evaluated by all mentioned antioxidant assays [Yilmaz et al., 2009; Popovic et al., 2012; Hassanpour et al., 2011]. The prooxidant activity was also assessed, Cornelian cherry fruits showing the lowest prooxidant activity among five investigated fruits [Pantelidis et al., 2007]. The ethanol extract of Cornus officinalis fruits acts as an efficient scavenger of hydroxyl radicals and protects human umbilical endothelial cells against peroxide induced apoptosis [Lee et al., 2006]. The Japanese cornel fruits extract have been reported to possess greater reducing power than vitamin E [Lim et al., 2011]. Table 2 summarizes the biological activity of some Cornus species fruits.

CONCLUSION The phytochemical investigations of Cornus species revealed that their fruits are rich in bioactive compounds such as polyphenols, anthocyanins, flavonoids, tannins, iridoids, triterpenoids, fatty acids, ascorbic acid and minerals. For many years they have been used in traditional and folk medicine to treat diabetes, liver and kidney diseases, gastrointestinal disorders, fever, pain and many others. Modern pharmaceutical studies indicated that Cornus spp. fruits exhibit therapeutic effects on diabetes, cancer, inflammatory diseases, cardiovascular disorders or obesity, especially due to their high antioxidant activity. They also have notable beneficial effects on hepatoprotection, hyperlipidemia, neuroprotection and inhibiton of bacteria and viruses. Due to their demonstrated health beneficial activities, the food and pharmaceutical industry should show an increasing interest for the plants belonging to Cornus species, these plants could be cultivated as alternate crops to yield fruits with high nutritional and therapeutic value and used to manufacture supplements and drugs with preventive and therapeutic uses.

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ACKNOWLEDGMENTS This work was supported by the Ministry of Education and Scientific Research, Romania [project no. 147/2011 PN-II-PT-PCCA-2011-3-1-0914].

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Jayaprakasam, B., Olson, LK, Schutzki, RE, Tai, MH, Nair, MG. Amelioration of obesity and glucose intolerance in high-fat-fed C57BL/6 mice by anthocyanins and ursolic acid in Cornelian cherry (Cornus mas). J. Agric. Food Chem., 2006, 54, 243-248 Jayaprakasam, B., Vareed, SK, Olson, NK, Nair, MG. Insulin secretion by bioactive anthocyanins and anthocyanidins present in fruits. J. Agric. Food Chem., 2005, 53, 28-31 Jeong, EJ, Kim, TB, Yang, H, Kang, SY, Kim, SY, Sung, SH, Kim, YC. Neuroprotective iridoid glycosides from Cornus officinalis fruits against glutamate-induced toxicity in HT22 hypocampal cells. Phytomedicine, 2012, 19, 317-321 Jiang, W-L, Zhang, S-M, Tang, X-X, Liu, H-Z.Protective roles of cornuside in acute myocardial ischemia and reperfusion injury in rats.Phytomedicine, 2011, 18, 266-271 Jung, L, Lee, D-Y, Cho, J-G, Ahn, E-M, Kim, S-Y, Baek, N-I.Isolation of triterpene glycosides from the fruit of Cornus kousa. J. Korean Soc. Appl. Biol. Chem., 2009, 52, 45-49 Kahkonen, MP, Hopia, AI, Heinonen, H. Berry phenolics and their antioxidant activity. J. Agric. Food Chem., 2001, 49, 4076-4082 Kakiuchi, N, Hattori, M, Namba, T, Nishizawa, M, Yamagishi, T, Okuda, T. Inhibitory Effect of Tannins on Reverse Transcriptase from RNA Tumor Virus. J. Nat. Prod., 1985, 48, 614-621 Kang, DG, Moon, MK, Li, AS, Kwon, TO, Kim, JS, Li, HS. Cornuside suppresses cytokineinduced pro-inflammatory and adhesion molecules in the human umbilical vein endothelial cells. Biol. Pharm. Bull., 2007, 30, 1796-1799 Kassi, E, Papoutzi, Z, Aligianis, S, Manoussakis, M, Moutsatsou, P. Ursolic acid, a naturally occurring triterpenoid, demonstrates anticancer activity on human prostate cancer cell line. J. Cancer Res. Clin. Oncol., 2007, 133, 493-500 Kean, KD, Hwan, KJ. A furan derivative from Cornus officinalis. Arch. Pharmacol. Res., 1998, 21, 787-789 Kim, DK, Kwak, JAH. A furan derivative from Cornus officinalis. Arch. Pharm. Res., 1998, 21, 787-789 Kim, JS, Oh, CH, Jeon, H, Lee, KS, Ma, SY.Immuno-regulatory property of fruit-extracts of Cornus kousa Burg. Kor. J. Med. Crop Sci., 2002, 10, 327-332 Krasniak, M, Gastol, M, Szalkowski, M, Zagrodzki, P, Derwisz, M. Cornelian cherry (Cornus mas L.) juices as a source of minerals in human diet. J. Toxicol. Environmen. Health A, 2010, 73, 17-18 Kwon, S-H, Park, H-Y, Kim, J-Y, Jeon, I-Y, Lee, M-K, Seo, K-I. Apoptotic action of ursolic acid isolated from Corni fructus in RC-58T/h/SA#4 primary human prostate cancer cells. Bioorg. Med. Chem. Lett., 2010, 20, 6435-6438 Lee, DY, Song, MC, Yoo, K-H, Bang, MH, Chung, IS, Kim, SH, Kim, DK, Kwon, BM, Jeon, TS, Park, MH, Baek, NI. Lignans from the fruit of Cornus kousa Burg.and their cytotoxic effects on human cancer cell lines. Arch. Pharm. Res., 2007a, 30, 402-407 Lee, DY, Lyu, H-N, Kwak, HY, Jung, L, Lee, YH, Kim, DK, Chung, IS, Kim, SH, Baek, NI. Isolation of flavonoids from the fruits of Cornus kousa Burg. J. Appl. Biol. Chem., 2007b, 50, 144-147 Lee, D-Y, Song, M-C, Kim, M-J, Jung, L-K, Jung, T-S, Ly, H, Baek, N-I.Isolation of inhibitory compound on LDL oxidation from the fruits of Cornuskousa Burg. Life Sci. Resources, Kyung He Univ., 2007c, 26, 34-37

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Lee, D-Y, Yoo, K-H, Chung, I-S, Kim, J-Y, Chung, D-K, Kim, D-K, Kim, S-H, Baek, N-I.A new lignin glycoside from the fruits of Cornus kousa Burg. Arch. Pharm. Res., 2008a, 31, 830-833 Lee, D-Y, Jung, L, Yoo, K-H, Song, M-C, Chung, I-S, Ahn, E-M, Kim, D-K, Baek, N-I. Cytotoxic sterols from the fruits of Cornus kousa Burg. J. Appl. Biol. Chem., 2008b, 51, 73-75 Lee, D-Y, Jung, L, Lyu, H-N, Jeong, T-S, Lee, Y-H, Baek, N-I.Triterpenoids from the fruits of Cornus kousa Burg.as human acyl-CoA: cholesterol acyltransferaze inhibitors. Food Sci. Biotechnol., 2009, 18, 223-227 Lee, D-Y, Lee, M-H, Jung, T-S, Kwon, B-M, Baek, N-I, Rho, Y-D. Triterpenoid and lignan from the fruits of Cornus kousa inhibit the activities of PRL-3 and LDL oxidation. J. Korean Soc. Appl. Biol. Chem., 2010a, 53, 97-100 Lee, D-Y, Jung, L, Park, J-H, Yoo, K-H, Chung, I-S, Baek, N-I.Cytotoxic triterpenoids from Cornus kousa fruits. Chem. Nat. Comp., 2010b, 46, 142-145 Lee, S-O, Kim, S-Y, Han, S-M, Kim, H-M, Ham, S-S, Kang, I-J.Corni fructus scavenges hydroxyl radicals and decreases oxidative stress in endothelial cells. J. Med. Food, 2006, 9, 594-598 Lehane, AM, Saliba, JK. Common dietary flavonods inhibit the growth of the intraerythrocytic malaria parasite. BMC Res. Notes, 2008, DOI:10.1186/1756-0500-1-26. Liang, J, He, J, Zhu, S, Zhao, W, Zhang, Y, Ito, Y, Sun, W. Preparative isolation and purification of iridoid glycosides from Fructus Corni by high-speed countercurrent chromatography. J. Liq. Chromatogr. Relat. Technol., 2013, 36, 983-999 Lim, SH, Choi, SH, Oh, YI, Kim, SJ. Anti-oxidative effects of flavonoids enriched Corni fructus extract and the mechanism. Afr. J. Pharmacol., 2011, 5, 506-514 Ma H, Li, L, Seeram, NP. Phenolics from Cornus amomun Mill.Fruit. Biochem. Syst. Ecol., 2010, 38, 1083-1084 Ma, W, Wang, KJ, Cheng, C-S, Yan, G, Lu, W-L, Ge, J-F, Cheng, Y-X, Li, N. Bioactive compounds from Cornus officinalis fruits and their effects on diabetic nephropathy. J. Ethnopharmacol., 2014, 153, 840-845 Mazza, G. Natural Food Colorants: Science and Technology. New York, NY, USA, Marcel Decker; 2000, 289–314 Midleton, E. The flavanoids. Trends Pharmacol. Sci., 1984, 5, 335-338 Moldovan, B, David, L. Influence of temperature and preserving agents on the stability of Cornelian cherries anthocyanins. Molecules, 2014, 19, 8177-8188 Myamoto, K, Kishi, N, Koshiura, R, Yoshida, T, Hatano, T, Okuda, T. Relatioship between the structures and antitumor activities of tannins. Chem. Pharm. Bull., 1987, 35, 814-822 Nawa, Y, Endo, J, Ohta, T. The inhibitory effect of the components of Cornusofficinalis on melanogenesis. J. Cosmet. Sci., 2007, 58, 505-517 Okuda, T, Hatano, T, Ogawa, N, Kira, R, Matsuda, M. Cornusiin A, a dimeric ellagitannin forming four tautomers and accompanying new tannins in Cornus officinalis. Chem. Pharm. Bull., 1984, 32, 4662-4665 Omar, R, Li, L, Yuan, T, Seeram, NP. α-Glucosidase inhibitory hydrolysable tannins from Eugenia jambolana seeds. J. Nat. Prod., 2012, 75, 1505-1509 Pantelidis, GE, Vasilakakis, M, Manganaris, GA, Diamantidis, G. Antioxidant capacity, phenol, anthocyanin and ascorbic acid contents in raspberries, blackberries, red currants, gooseberries and Cornelian cherries. Food Chem., 2007, 102, 777-783

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In: Fruit and Pomace Extracts Editor: Jason P. Owen

ISBN: 978-1-63482-497-2 © 2015 Nova Science Publishers, Inc.

Chapter 10

KUMQUAT (FORTUNELLA SPP.): BIOCHEMICAL COMPOSITION AND PROPHYLACTIC ACTIONS Theeshan Bahorun1, Darshini Narrain1, Piteesha Ramlagan2 and Chandra Tatsha Bholah2 1

ANDI Centre of Excellence for Biomedical and Biomaterials Research, University of Mauritius, Réduit, Republic of Mauritius 2 Department of Health Sciences, Faculty of Science and ANDI Centre of Excellence for Biomedical and Biomaterials Research, University of Mauritius, Réduit, Republic of Mauritius

ABSTRACT Natural plant products continue to be of increasing interest due to the wide range of health benefits they confer to humans. Citrus fruits have been extensively studied for their health-promoting potential and have been widely applied in the medical and food industry. Kumquat, a tropical fruit originally included in the genus Citrus has been classified a century ago in the genus Fortunella. The latter has so far been poorly studied compared to the genus Citrus, most probably due to its limited distribution and consumption. This chapter reviews selected interesting findings on phytochemical content of kumquats with emphasis on their prophylactic effects at biochemical and molecular levels.

Keywords: Kumquats, phytochemicals, nutrition, biochemistry, prophylaxis, molecular actions



Corresponding author: [email protected].

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CHAPTER HIGHLIGHTS  

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Kumquats are tropical fruits belonging to the genus Fortunella, a close relative of genus Citrus. They are rich in bioactive phytochemicals and nutrients that confer a wide range of prophylactic activities including anti-oxidant, anti-microbial, anti-inflammatory, antitumor, anti-diabetic, anti-obesity, anti-hypertensive activities and modulation neurodegenerative diseases. One characteristic feature of the Kumquat fruit is that it can be eaten whole, with the pulp and peel, both containing the health-promoting bioactive compounds. Kumquats are potential functional foods that can be included in normal diets and in nutritional programs for preventive and therapeutic treatments.

1. INTRODUCTION The interest in phytochemicals has grown exponentially due to the increasing evidence highlighting their prophylactic effects on human health. Phytochemicals are bioactive constituents of plant foods not identified as nutrients as they are not essential to life by themselves but confer important biological and curative properties. These phytochemicals are obtained through dietary sources like fruits, vegetables and natural beverages. Phytochemicals such as phenolic acids, flavonoids, carotenoids, stilbenes, tannins, lignans and essential oils can scavenge free radicals and quench Reactive Oxygen Species (ROS) and therefore provide effective means for preventing and treating free radical-mediated diseases. Reactive oxygen species, such as the superoxide radical (O2•−), hydrogen peroxide (H2O2), hypochlorous acid (HOCl) and the hydroxyl radical (HO•) have been widely suggested to play a determining role in the pathogenesis of several human diseases (Halliwell, 1996; Aruoma, 2003). ROS-induced oxidation can result in cell membrane disintegration, membrane protein damage and DNA mutation, which can further initiate or significantly increase the development of diseases including cancer (Li et al., 2013), diabetes (Etxeberria et al., 2012), neurodegenerative diseases (Ho et al., 2010), aging (Hensley and Floyd, 2002) and cardiovascular diseases (Hool, 2006). Plants represent an important and ubiquitous source of dietary ROS-quenching compounds. Citrus genus is a major source of phytochemicals including vitamins, minerals, dietary fibres, pectins and important classes and subclasses of phytophenolics that can be beneficial to health. It is one of the most important fruit tree crop in the world with an annual fruit production of over 102 million tons (Bahorun et al., 2012). It has been studied for its biochemical composition and potent biological properties. The Kumquat plant (Figure 1) is a perennial shrub belonging to the genus Fortunella, a citrus relative. Kumquats originate from South Asia, most likely in China and are commonly cultivated in the southern region of China (Wang et al., 2012). They are distributed throughout several other Asian countries including Japan, southern Pakistan and South Korea. Cultivation of kumquats is prominent in California, Florida and Texas and less prominent in South India, Australia, South Africa and Brazil and island states like Mauritius. Kumquat fruits have been called ―the little gold gems of the citrus family‖ and the plant is considered

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auspicious in China. Contrary to most Citrus, kumquats are eaten whole having a sweet and crunchy peel and slightly sour pulp. They have been cultivated since ancient times for ornamental, culinary and medical purposes. They are employed in the production of liqueurs, marmalades and sauces, and they can be candied or preserved whole in sugar syrup (Barreca et al. 2011). Fortunella species have been used in folk medicine in China and a number of studies have attempted to investigate the pharmacological properties of bioactive compounds in Kumquats (Kumamoto et al., 1985; Tan et al., 2014). They are considered a traditional medicine against cough, digestive problems, liver problems and hypercholesterolemia. They are also considered as a tonic and to have diuretic and laxative properties. In this chapter, we provide an insight on the phytochemical composition of the Kumquat fruit with particular emphasis on some of their prophylactic effects as evidenced by biochemical and molecular studies.

Figure 1. Kumquat (Fortunella margarita Nagami) plant.

2. TAXONOMIC CLASSIFICATION, SPECIES AND ANATOMY OF KUMQUATS Kumquats belong to the Rutaceae family and Fortunella genus (Table 1). They were originally classified in the genus Citrus but in 1915 were separated by Dr. Walter Swingle into another genus named Fortunella. Even though Citrus and Fortunella are closely related from a taxonomic point of view, they possess quite different flavonoid fingerprints (Berhow et al., 1998). The various Kumquats are distinguished as botanical species rather than cultivars (Morton, 1987). The kumquat tree is slow-growing, shrubby and reaches a height of 2 to 5 m tall. The fruit is oval-oblong or round in shape with a length of 1.6 to 5 cm (Figure 2). The Kumquat fruit comprises several parts (Figure 3): 1) The exocarp The exocarp of the kumquat fruit is made up of an outer layer called the flavedo (epicarp) and an inner layer called the albedo (mesocarp). The flavedo is golden-yellow to reddish-

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orange in color and comprises phenolic acids, flavonoids, terpenoids, pigments and essential oils (Koyasako and Bernhard, 1983). F. crassifolia peel contains terpenoid hydrocarbons (85.42%), alcohols (3.3%), ketones (1.8%), esters (1.72%) and aldehydes (0.18%) (Wang et al., 2012). 2) The endocarp The endocarp makes the fleshy part with usually 3 to 6 separate sections/segments. The juicy sacs inside the segments are called juice vesicles which are actually specialized hair cells (Sinha et al., 2012). F. japonica juice (unripe fruit) has been found to contain flavanone glycosides poncirin, dydimin, hesperidin, flavonol glycoside (rutin) and flavone glycosides like rhoifolin (Barreca et al., 2011). 3) The seeds The seeds are small, pointed and are at times absent in the fruit, for example in F. margarita Nordmann (Morton, 1987). Table 1. Taxonomic Classification, Botanical species and Common hybrids of Kumquats Taxonomic Classification Kingdom: Phylum: Class: Order: Family: Genus: Botanical Species F. hindsii

Plantae Tracheophyta Magnoliopsida Sapindales Rutaceae Fortunella Common Names Hong Kong or Golden Bean kumquat Marumi or round kumquat Meiwa or large round kumquat Nordmann Seedless kumquat Nagami, or oval kumquat Hong Kong or Golden Bean kumquat Marumi or round kumquat

F. japonica F. X crassifolia F. margarita Nordmann F. margaritaNagami F. hindsii F. japonica Common Hybrids Calamondin Citrangequat Limequat Mandarinquat Orangequat

= = = = =

Procimequat Sunquat Yuzuquat

= = =

Tangerine Citrange Key Lime Mandarin Satsuma Mandarin Limequat Lemon Yuzu

x x x x

Kumquat Kumquat Kumquat Kumquat

x

Kumquat

x x x

Kumquat Kumquat Kumquat

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Figure 2. Ripe and unripe Kumquat fruits.

Figure 3. Transverse section of a Kumquat fruit.

3. PHYTOCHEMICAL COMPOSITION OF KUMQUAT FRUITS 3.1. Nutrients Kumquats contain a large range of nutrients and phytochemicals, including vitamins, macro and micro elements carotenoids, essential oils and phytophenolics which are more extensively reviewed below. Major vitamins in kumquats are vitamin A, B1 (thiamin), B2 (riboflavin), B3 (niacin), B6 (pyridoxine), C and E (α-tocophecol) while calcium, magnesium, phosphorus, sodium, potassium, iron, zinc and selenium are the main macro and micro elements. According to the USDA National Nutrient Database (2012), the nutritional importance of Kumquats is conferred mostly by vitamin C (43.9 mg/100 g) which meets 73% of our recommended daily uptake, potassium (186 mg/100 g), calcium (62 mg/100 g) and magnesium (20 mg/100 g) contents. Vitamin C in kumquats seems to be more highly distributed in the pulp (> 500 µg/ml pulp juice) and flavedo (345 µg/g FW) as reported by Ramful and colleagues (2010a, 2011). The carotenoid composition of kumquats varies in peels and pulps. Peels contain mostly violaxanthin, β-citraurin, lutein, cryptochrome and βcryptoxanthin while pulps include additionally auroxanthin and luteoxanthin (Agόcs et al., 2007). β-carotene and β-cryptoxanthin have also been identified in Fortunella hindsii

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(Kanakapura and Pradeep, 2013). The essential oils of kumquat peel contain- much of the aroma of the fruit and is composed principally of limonene, which makes up around 93% of the total essential oil composition. Besides limonene and α-pinene (0.34%), both being monoterpenes, the oil is unusually rich in sesquiterpenes (0.38% total), such as αbergamotene (0.021%), caryophyllene (0.18%), α-humulene (0.07%) and α-murolene (0.06%), and these contribute to the spicy and woody flavor of the fruit (Koyasako and Bernhard, 1983). Wang and colleagues (2012) analysed essential oils from Fortunellacrassifolia peels and provided an order of occurrence as follows for major compounds: limonene>myrcene> camphene> α-selinene>α-pinene> 3,4-dimethyl styrene> βelemene.

3.2. Phytophenolics The prophylactic importance of kumquat seems to be ascribed to its richness in phenolics, notably flavonoids in addition to terpenoids and essential oils (Koyasako and Bernhard, 1983). In a study assessing the polyphenolic composition of citrus fruit pulps, Ramful et al. (2010a) reported that Kumquat pulps (Fortunella margarita, Variety Nagami) had the highest total phenols with amounts ranging between 1412 to 1694 µg/g FW (gallic acid equivalent). It further reported a flavonoid content between 300 and 400 µg/gFW (quercetin equivalent) in the pulps and a range of 1200-1500 µg/gFW (quercetin equivalent) in flavedo extracts. Flavanone, flavone and flavonol derivatives make up the majority of Kumquat flavonoids. Main flavanones identified in edible portions were naringin, hesperidin and neohesperidin; flavonols were mostly rutin, quercetin and kaempferol while diosmin and sinensetin were the flavone components (Wang et al., 2007). Dihydrochalcones are another class of flavonoids, identified in kumquat, characterized by the C6-C3-C6 basic backbone structure and regarded as the primary precursors and as vital intermediates in the synthesis of flavonoids (Gosch et al., 2009). Phloretin 3′,5′-di- C-glucoside (Figure 4), a dihydrochalcone, has been reported to be by far, the most abundant bioactive ingredient in Kumquat (Fortunellajaponica) (Barreca et al., 2011). In the same study, the analysis of the flavonoid composition of its crude juice, obtained from unripe and ripe fruits, highlighted for the first time the presence of thirteen additional compounds (C- and O-glycosyl flavonoids) among which acacetin 3,6-di-Cglucoside, vicenin-2, lucenin-2 4′-methyl ether, narirutin 4′-O-glucosideand apigenin 8-Cneohesperidoside were the main ones. Ogawa et al. (2001) found 3‘,5‘-di-C-betaglucopyranosylphloretin to be a flavonoid characteristic of the genus Fortunella. Other compounds such as neoeriocitrin and poncirin have also been characterized in Kumquat (Fortunella margarita) fruit extract (Tan et al., 2014). Besides flavonoids, kumquats are also rich in phenolic acids, more particularly cinnamic acids. The latter occurs naturally as free acids and esters. Cinnamic acid derivatives include boropinic acid and ferulic acid. In Fortunella japonica, boropinic acid was recorded as the most abundant phytochemical in the peels (Genovese et al., 2014). It is regarded as a novel compound which has anti-inflammatory and anti-bacterial properties, most specifically against Heliobacter pylori. Another compound of interest, 4′-geranyloxyferulic acid (GOFA) possesses valuable pharmacological properties as cancer chemopreventive, antiinflammatory, neuro-protective and anti-H.pylori agents. Chlorogenic acid seems to be one of

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the other most important phenolic acids in Kumquat as reported by Wang et al., 2007, who also quantified sinapic and p-coumaric acids in the edible portions.

Figure 4. Skeletal structure of Phloretin 3′,5′-di- C-glucoside.

Figure 5. Prophylactic effects of Kumquat phytochemicals.

4. MODULATORY EFFECTS OF KUMQUAT PHYTOCHEMICALS Various studies have highlighted the prophylactic effects of Kumquat phytochemicals: anti-oxidant, anti-microbial, anti-inflammatory, anti-tumor, anti-diabetic, anti-obesity, antihypertensive activities and modulation neurodegenerative diseases (Figure 5). Some of the outcomes of these investigations are discussed in the subsections that follow.

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4.1. Anti-Oxidant Activities There is currently no universal method to measure antioxidant capacities of plant samples accurately and consistently and in this respect antioxidant efficacy can only be predicted from data emanating from a multiplicity of assessing methods. Limited data exist on the antioxidant characterization of kumquat extracts. Few groups of researchers have worked on the estimation of their pharmacological properties. A survey of Mauritian citrus species showed that pulp extracts of F. margarita seem to be more potent than the flavedo extracts as evidenced by analyses from three antioxidant assays, notably Ferric Reducing Antioxidant Power (FRAP), Trolox Equivalent Antioxidant Capacity (TEAC) and hypochlorous acid scavenging assay (Ramful et al., 2011; Bahorun et al., 2012). The study was conducted on 11 citrus varieties and it was found that kumquat had the most potent antioxidant activities strongly linked to its phytophenolic content. It is widely suggested that phytophenolics exhibit a broad spectrum of biomedical functions and they can exert modulatory actions in cells by interacting with a wide range of molecular targets central to the cell signaling machinery. Many of these biological functions stem mostly from their free radical scavenging and antioxidant activity (Soobrattee et al., 2005). In a detailed study of the flavonoids of F. japonica, Barreca et al. (2011) reported that the juice showed remarkable antioxidant properties. This could be the outcome of the synergistic effects of flavonoid fractions containing the major component, phloretin 3′,5′-di- C-glucoside. The evaluation of radical scavenging ability of crude juices from unripe and ripe fruits showed a much higher antioxidant efficacy in ripe juices. The flavonoids from unripe fruits seem to be responsible for most of the antioxidant activity. However, in the ripe kumquat juice, flavonoids accounted for a limited amount of activity (Barreca et al., 2011), thereby speculating that other components of ripe kumquat juice may significantly influence the radical scavenging activity (Wang et al., 2007). Processing may also contribute extensively to variation of antioxidant propensities of kumquats. As such Lou et al. (2015), reported that when immature kumquat (F.japonica) was dried at both 110oC and 130oC for halfhour, total phenolics and flavonoids increased leading to a higher antioxidant activity. However, when dried at 130oC for a longer period(1.5 hours), the products underwent drastic browning and resulted in decreased flavonoid content. In the light of these data, kumquat could indeed be regarded as a significant addition to a diet rich in bioactive micronutrients that may help and enhance endogenous antioxidant protection (Barrecaet al., 2011).

4.2. Anti-Microbial Activities With evolving consumer trends and increasing antibiotic resistant pathogens, there is a pressing need to develop substitutes to chemical based bactericides. In this respect, essential oils which have been recognized for centuries for their antibacterial properties represent potent alternatives. Citrus essential oils not only lend themselves for this use but also are generally recognized as safe (GRAS) and have been found to be inhibitory both in direct oil and vapour form against a range of both Gram-positive and Gram-negative bacteria (Fisher and Phillips, 2008). The antimicrobial activity of kumquat oils is still debatable due to only few published reports. Wang et al., 2012, showed potent antimicrobial activity against both Gram-negative (E. coli and S. typhimurium) and Gram-positive (S. aureus, B. cereus, B.

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subtilis, L. bulgaricus and B. laterosporus) bacteria, together with a remarkable antifungal activity against C. albicans of essential oils isolated from FortunellacrassifoliaSwingle peels. They also showed in a food model of beef extract, the efficacy of essential oils to control viable bacteria counts. This investigation suggested that kumquat peel essential oils might be noteworthy as a natural food preservative against bacteria and fungus (Wang et al., 2012). The kumquat dihydrochalcones, phloretin and its glycosylated derivatives (phlorizin and phloretin 3‘,5‘-diC-glucoside) were shown to inhibit growth of Gram-positive and Gram-negative bacteria in Staphylococcus aureus, Listeria monocytogenes and methicillin-resistant Staphylococcus aureus, thereby confirming the data by Wang and colleagues (2012). Further analyses have evaluated the cytosolic activity of the molecules as being able to modify the use of biofuels and decreasing the ability of counteracting oxidative damage (Barreca et al., 2014).Moreover, the antimicrobial potential of Kumquat extracts is not only limited to antibacterialproperties. Essential oils from F.crassifolia show a strong antifungal ability against C.albicans (MIC 70 µg/ml). Rodov et al., (1992) showed that treatment of the flavedo of Kumquat (Fortunella margarita) by Ultraviolet (UV) illumination at 254 nm increases the antifungal activity by increasing the production of the phytoalexin scoparone.Phytoalexins are low-molecular antimicrobial substances of various chemical structures and they are elicited in plant tissues by either biotic (pathogen challenge) or abiotic (wounding, chemicals, irradiation, etc.) stresses (Bailey, 1982).

4.3. Anti-Inflammatory Activities Over the past decades, chronic inflammation has been regarded as a major risk factor for various diseases like diabetes, obesity, cardiovascular diseases and neurodegenerative diseases. This process can be triggered by cellular stress and dysfunction that are principally the effects of excessive calorie intake, elevated blood sugar levels and oxidative stress (Karin et al., 2006). Dietary phytochemicals have increasingly been shown to possess the ability to prevent and attenuate inflammatory responses through a variety of mechanisms (Pan et al., 2010). Studies investigating the pharmacological properties of kumquats phyto-constituents have shown promising results. During inflammation, pro-inflammatory cytokines lead to the formation of substantive amounts of nitric oxide (NO) by inducible nitric oxide synthase (iNOS). Flavonoids like flavones, the flavonols (isorhamnetin, kaempferol and quercetin) and the flavanone naringenin, present in citrus fruits, have inhibitory effects on iNOS protein and mRNA expression and also on NO production in a dose-dependent manner (Hämäläinen et al., 2007). These flavonoids, mostly present in kumquats, have been found to inhibit the activation of nuclear factor-B (NF-B), which is an important transcription factor for iNOS. Kaempferol and quercetin, present in edible fractions of kumquat also inhibited the activation of the signal transducer and activator of transcription 1 (STAT-1) which is another important transcription factor for iNOS. Hesperidin, present in Gannan kumquat peels, has shown to inhibit cytotoxicity and apoptosis induced by Aß25-35 and to prevent neurodegeneration (Luo et al., 2010). In traditional Chinese medicine, dried, whole immature and mature citrus fruits and peels, are widely used as remedies to stimulate appetite, aid digestion and improve menopausal syndromes (Ou, 1999). Dried whole kumquats have been used to cure inflammatory syndromes of the respiratory tract, such as coughing, hoarseness, and sore

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throats (Chiu and Chang, 1995). Heat treatment, to a temperature of ≤100 °C, has been shown to enhance the antioxidant activity of the peels without loss of anti-inflammatory flavonoid glucosides (Xuet al., 2007). Comparing various processing methods, Lin et al., (2008) demonstrated that heat treatment was efficacious to enhance NO-suppressing and peroxynitrite-intercepting activities of kumquat (Fortunella margarita Swingle) peel.

4.4. Management of Neurodegenerative Diseases Neurodegenerative diseases (ND) affect the brain, a vital organ in the body, involved in the control of all the involuntary functions and also in memory, cognition and emotion (Ho et al., 2010). The latter leads to a deterioration, often irreversible, of the intellectual and cognitive faculties (Iriti et al., 2010) usually associated with the progressive accumulation of misfolded proteins with the formation of toxic oligomers along with increasing oxidative damage and inflammation (Ricciarelli et al., 2007). Oxidative stress is considered as a key factor in the pathogenesis of neurodegenerative diseases. Dietary phytochemicals play a vital role in protecting neural cells from oxidative stress and neuroinflammation. To play their neuroprotective roles, dietary polyphenols should be able to cross the blood-brain barrier (BBB), which controls the entry of xenobiotics (drug, carcinogen or any foreign body) into the brain and the maintenance of the brain‘s microenvironment. The ability of polyphenols (for example, flavonoids) to penetrate through this barrier depends on the degree of their lipophilicity (Youdim et al., 2003). Less polar polyphenols or metabolites (i.e O-methylated derivatives) are more smoothly taken up by the brain as compared to more polar polyphenols and/or metabolites (i.e sulfated and glucuronidated derivatives). BBB penetration is also dependent on interactions of polyphenols with specific efflux transporters expressed in the BBB, such as the multi-drug resistance-associated proteins (MRPs) (Abbott et al., 2006). Dietary phytochemicals modulate neurogenerative disorders by either acting as antioxidants and/or by acting as signaling molecules. Kumquat phytochemicals (flavanones, dihydrochalcones, β carotene and cinnamic acids) exhibit a dual effect through indirect neuroprotection against oxidative stress and indirect protection through suppression of gliamediated inflammation (Wang et al., 2006) (Figure 6). Dietary intervention studies, in humans and animals with flavonoid-rich plant extracts (the same flavonoids as in kumquats), have highlighted their potential to influence cognition, memory and learning (Youdimet al., 2004; Wang et al., 2006). They exhibit protective effects against neuronal death in both oxidative stress-induced (Inanami et al., 1998) and β-amyloidinduced neuronal death models (Luo et al., 2002). The neuroprotective effects of flavonoids seem to be underpinned by their interaction with critical protein and lipid kinase signaling cascades in the brain, leading to an inhibition of apoptosis and to a promotion of neuronal survival and synaptic plasticity (Vauzour et al., 2013). Ferulic acid has been shown to be able to significantly protect against beta amyloid peptide toxicity by modulating oxidative stress, and by inducing the expression of protecting proteins in hippocampal cultures (Sultana et al., 2005). Carotenoids, in particular β carotene, can impact the hypothalamus (a brain region important for memory and regulation of metabolic activities like hunger, energy balance, body weight and insulin secretion). Long term β-carotene supplementation may have beneficial effects on cognitive functioning (Grodstein et al., 2007). Hesperidin (Figure 7) has gained considerable attention in the treatment of various oxidative stress mediated diseases

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such as neurodegenerative diseases, diabetes, cardiovascular diseases and certain types of cancers. Raza et al., (2011) showed that hesperidin treatment could reduce cerebral damage due to induced stroke in rat brain due to reduction of free radicals and associated neuroinflammation. In a study by Tamilselvam and colleagues (2013), it was postulated that hesperidin has a protective effect on human neuroblastoma SK-N-SH cells through its antioxidant and anti-apoptotic properties. Kumquat, therefore, represent a potential source of phytochemicals with neurodegenerative properties.

Figure 6. Kumquats are rich in flavonoids (flavanones and dihydrochalcones), carotenoids (beta carotene) and phenolic acids (cinnamic acids). These dietary polyphenols actually exhibit a dual effect through indirect neuroprotection against oxidative stress and indirect protection through suppression of glia-mediated inflammation.

Figure 7. Chemical structure of Hesperidin.

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4.5. Anti-Tumor Activities A large cohort of studies claims that citrus fruits with the bioactive flavonoids and limonoids represent promising agents in the area of cancer research. A likely explanation may be linked to the presence of over 60 types of flavonoids identified in citrus fruits (Robards et al., 1997), with wide structural diversity that may provide a rationale for the potential anticancer benefits through various action mechanisms. The preventive mechanisms of tumour protection by natural phytochemicals range from inhibition of genotoxic effects, increased antioxidant and anti-inflammatory activity, inhibition of proteases and cell proliferation, protection of intercellular communications to modulation of apoptosis and signal transduction pathways (Bahorun et al., 2012). Fruits generally containing phytophenolics like quercetin, kaempferol, luteolin, protocatechuic acid and phenolic acids such as gallic acid andchlorogenic acid are more potent against cancer (Fu et al., 2011). Kumquat with its cocktail of bioactive phytophenolics have potent anticarcinogenic propensity. Its peel, pulp and seed have been reported to have antiproliferative activities in a number of cancer cell lines (Li et al., 2013). Amongst its bioactive molecules is hesperidin. Apart from its anti-analgesic and anti-inflammatory properties, it has been considerably investigated for its anti-carcinogenic property. For instance, its action to modulate breast cancer markers has been highlighted in a number of studies (Nandakumar et al., 2011; Natarajan et al., 2011). Also, hesperidin was reported to induce apoptosis in colon cancer cells in a dose dependent manner. In the same study, it was reported to induce apoptosis through two signaling pathways: the down-regulation of BCL2, which is an anti-apoptotic regulator and the up-regulation of both BAX and CASP3 to promote apoptosis (Park et al., 2008). The prophylactic action of kumquat is not limited to ripe fruits. Barreca et al., (2011) has reported anti-cancer and anti-proliferative effects of rhoifolin. This flavone glycoside molecule chemically known as, apigenin 7 neohesperidoside, is present in unripe Kumquat fruits. Rhoifolin (Barreca et al., 2011) has been studied in its role as anti-carcinogenic and has revealed various different mechanisms by which it inhibits cell proliferation. It acts as a free radical scavenger and has been reported to modulate benzo(a)pyrene-induced mutagenesis. It also increases the glutathione concentration in skin and colon cancer cells to induce protection against oxidative stress. Its main protective function is through inhibition of ornithine decarboxylase, an enzyme which plays an important role in tumor promotion (Patel et al., 2007). In further support to anti-carcinogenic activities, carotenoids found in kumquat peels have the ability to impact multiple pathways in the carcinogenesis process (Figure 8), such as activation of the caspase cascade or activation of transcription factors. Carotenoids increase gap junctional communication by increasing expression of connexin43 (Cx43) promoter (Bertram, 2008). β-carotene found in kumquat peels, has shown to inhibit DNA damage from carcinogenic chemicals and together inhibit carcinogen activation (Sarkar et al., 1997). It reduces premenopausal breast cancer, particularly in smokers (Mignone et al., 2009).

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Figure 8. Carotenoids increase transcription of connexin43 (Cx43). This protein increases adherence of tumor cells through gap junction communication and hence reduces proliferation of malignant cells and limits their progress through the carcinogenesis process.

4.6. Anti-Diabetes and Anti-Obesity Activities Type II diabetes is a reactive oxygen species (ROS)-mediated pathology, with a worldwide prevalence estimated to double by 2030. A major effort has been launched to find therapeutic means to improve health conditions of diabetic and obese patients. Recent findings showed that supplemental natural antioxidants represent a potential strategy as adjunct therapy. Studies on citrus flavanones suggest that naringenin is able to reduce glucose uptake in the intestine and inhibits intestinal and renal Na+-glucose symporter (SGLT1) (Figure 9) (Li et al., 2006). Naringenin also activates AMPK (Adenosine monophosphateactivated protein kinase) in skeletal muscle in vitro leading to glucose uptake independent of insulin (Zygmunt et al., 2010). Additionally naringin, a glycoside of naringenin, has the ability to reduce the mRNA expression of phosphoenol pyruvate carboxykinase as well as glucose-6-phosphatase in the liver and both naringin and hesperidin significantly increase the

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glucokinase mRNA level (Jung et al., 2006) resulting in low glucose level. Furthermore, dietary phytochemicals including citrus phytophenolics such as, phenolic acids exhibit hypoglycemic activities through α-glucosidase inhibition. They are able to reduce breakdown of dietary carbohydrates into simple glucose molecules by inhibiting action of the membranebound α-glucosidase enzyme activity (Etxeberria et al., 2012) and thus reduce glucose uptake into the blood (Figure 9). Alpha-glucosidase inhibitors are available in the form of synthetic drugs, for example Acarbose and Miglitol. However, these drugs decrease the blood glucose levels on a short term basis only and are associated with side effects like flatulence, diarrhea and intestinal ulcers. Various phytochemicals, like phenolic acids (cinnamic acid and derivatives largely present in kumquats) (Adisakwattana et al., 2009) may be effective hypoglycaemic agents that have slight or no side effects and thus represent potential candidates for blood glucose up-regulation. At molecular level advanced glycated end products (AGEs) and their carbonyl derivatives highly contribute to the pathogenesis of diabetes by their interaction with specific receptors, known as RAGE (Receptor for Advanced Glycated End products), triggering, for instance, NF-B signaling pathway to induce the expression of pro-inflammatory mediators and elicit oxidative stress which exacerbate diabetic complications (Stern et al., 2002). Efforts are currently being geared towards the identification of useful AGE inhibitorsthatdelay or prevent glycation. It has been suggested that AGEs inhibitors from natural foods/dietary biofactors may reasonably serve as adjuvant. Studies on adipocytes treated with AGEs revealed a decrease in ROS production, carbonyl production and apoE secretions preventing oxidative stress when the cells were treated with polyphenolic rich citrus extracts (Ramful et al., 2010b). It has been reported that vitamin C, which is present in relatively high amount in citrus, including kumquats (Ramful et al., 2011), is regarded as an essential antioxidant in the plasma as it acts as a reducing agent and has the ability to chelate metal ions, to scavenge free radicals, to quench superoxide anions (·O2-) (Brewer, 2011) and also to inhibit lipid peroxidation (Vincent et al., 2004). The understanding of molecular changes underlying diabetes development offers the prospect of utilizing functional food supplements to augment therapies whichcan specifically target biochemical and signaling pathways. According to the World Health Organization, more than 1.4 billion adults were overweight [body mass index (BMI; in kg/m2) > 25] in 2008, of which 200 million and 300 million men and women, respectively, had a BMI > 30. Most importantly and alarming is the number of overweight children which has been estimated to be 42 million in 2013 (WHO, 2014) Overweight and obesity increase the risk of several serious chronic diseases including diabetes. The development of these complications is associated with enlargement of adipocytes that occur with abdominal obesity (Westphal, 2008) with an increase in the release of proinflammatory cytokines and a decrease in adiponectin secretion (Cannon, 2008). This leads to insulin resistance and thus type II diabetes (Sheng and Yang, 2008). Kumquat extracts may be a potential dietary supplement for the prevention and management of obesity and obesity-related metabolic disturbances.

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Figure 9. Diagrammatic representation of the glucose lowering effect of flavonoids and phenolic acids through inhibition of α-glucosidase enzyme activity in brush borders of small intestine.

In a study carried out by Tan et al., (2014), the preventive and therapeutic abilities of Fortunella margarita Swingle fruit extract (FME) on High-Fat Diet-Induced Obese C57BL/6 mice were investigated. In the preventive treatment, FME was found to control the body weight gain and the size of white adipocytes, to reduce the fasting blood glucose, serum total cholesterol, serum low density lipoprotein cholesterol levels as well as liver lipid contents in the high-fat diet-fed C57BL/6 mice. In the therapeutic treatment, FME decreased the serum triglyceride, serum total cholesterol, serum low density lipoprotein cholesterol, fasting blood glucose levels and liver lipid contents, improved glucose tolerance and insulin tolerance. Compared with the High-Fat Diet group, Kumquat extracts significantly increased the mRNA expression of PPAR-α and its target genes (Tan et al., 2014). PPARs (Peroxisome proliferator-activated receptors) are ligand-activated transcription factors belonging to the nuclear receptor family. They include PPA-α, PPA-β and PPA-γ and these control gene expression involved in lipid and glucose metabolism (Kersten et al., 2000).For example, PPA-α regulates genes involved in fatty acid uptake and oxidation (Aoyama et al., 1998). Tan and colleagues (2014) concluded that Fortunella margarita Swingle fruit extract had a modulatory effect on obese mice partly through regulation of PPAR-α signaling pathway. Furthermore it has been reported that quercetin, a flavone present in kumquats, inhibits the differentiation of pre-adipocytes to adipocytes in vitro through the activation of the AMPK signal pathway in addition to decreasing the activation of PPARγ and C/EBPα (CCAAT/Enhancer Binding Protein-α) - the key regulator of adipogenesis. Quercetin also reduces the viability of adipocytes as a result of increased apoptosis of preadipocytes by the activation of caspase-3. This programmed cell death mediator cleaves PARP that eventually promotes apoptosis and down regulates bcl-2 (a protein that regulates cytochrome c- an apoptotic mediator).

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Quercetin-induced apoptosis of mature adipocytes occurs most probably through the modulation of the ERKs (Extracellular signal-regulated kinases) pathway, which is important in cell survival, differentiation and proliferation (Ahn et al., 2008, Yang et al., 2008).

4.7. Anti-Hypertensive Activities Hypertension is a chronic condition where arterial walls are constricted leading to elevated blood pressure, usually ≥ 140/90 mmHg. Hypertensivity is approximately twice as frequent in patients with diabetes compared with patients without the disease (Sowers et al., 2001). Excess of insulin in the blood changes the retention rates of Na+ and Ca2+ therefore altering the main components of the blood pressure regulators such as the vascular reactivity increase in cardiac output and peripheral resistance (González-Castejón and RodriguezCasado, 2011). Kumquats are a rich source of potassium and flavanones (for example, hesperitin) and these phytochemicals lower blood pressure through chronic increase in production of nitric oxide by activation of endothelial nitric oxide synthase in the vascular endothelium (Galleano et al., 2010). Other mechanisms such as inhibitory effect on Angiotensin I-converting enzyme (ACE) could also be responsible for the blood lowering effects of flavanones. Angiotensin I-converting enzyme is an important enzyme involved in maintaining vascular tension. It converts angiotensin I to angiotensin II, a potent vasoconstrictor and stimulator of aldosterone secretion by the adrenal gland (Skeggs and Khan, 1956). Inhibition of ACE is considered a useful therapeutic approach in the treatment of high blood pressure in both diabetic and non-diabetic patients (Erdos and Skidgel, 1987). Animal and clinical studies have indicated the potential of specific phenolic phytochemicals in hypertension management with direct absorption into the blood (Suda et al., 2003; Kwon et al., 2006). A clinical study carried out by Egert et al. (2009) reported that quercetin leads to a decrease in the systolic blood pressure in an entire study group and more particularly in subjects between 25-65 years together with a decrease in the concentration of plasma atherogenic oxidized Low Density Lipoprotein (LDH) (‗bad‘ cholesterol‘), therefore providing protection against cardiovascular disease. However, quercetin also decreased the serum High Density Lipoprotein (HDL) concentration, but the LDL: HDL ratio was unaltered.

CONCLUSION In light of research findings on kumquat so far, it is clear that the fruit represents a potential candidate for possible inclusion in nutrition programs to manage health and diseases due to its arsenal of prophylactic ingredients. This tropical fruit still warrants further well designed observational epidemiological studies, structure-activity relationship analyses, bioavailabity investigations and above all well planned clinical trials which remain the ultimate means to measure efficacy in human subjects. To materialize this concept, we need to move much further in research and evaluate individual needs and recommendations of functional foods for an individual genotype. In this respect, nutrigenomics and application of

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personalized nutrition programs will have instrumental roles. The resulting evidence will pave the way towards development of effective functional foods, with Kumquats being a significant addition to a diet rich in bioactive nutrients and phytophenolics.

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In: Fruit and Pomace Extracts Editor: Jason P. Owen

ISBN: 978-1-63482-497-2 © 2015 Nova Science Publishers, Inc.

Chapter 11

ALOE VERA EXTRACTS: FROM TRADITIONAL USES TO MODERN MEDICINE Taukoorah Urmeela and Mahomoodally Mohamad Fawzi Department of Health Sciences, Faculty of Science, University of Mauritius, Réduit, Mauritius

ABSTRACT Aloe vera, one of nature‘s most curative medicinal plants, has been traditionally used as alternative treatment against a plethora of human ailments in various countries like China, India, and Egypt, amongst others. Its therapeutic attributes have been well investigated and proven by numerous in vitro, in vivo and clinical studies. Native to North Africa, this succulent plant has been shown to be beneficial in the treatment and management of a wide range of conditions including skin disorders, constipation, noninsulin dependent diabetes mellitus, cardiovascular disorders, cancer and even AIDS. During the past recent years, the commercialisation of crude Aloe vera extracts and/or formulated products has experienced a boom in the pharmaceutical, food, cosmetic and the wellness industries. The beneficial effects of Aloe vera can be attributed to the panoply of phytonutrients and phytochemicals including non-nutritive constituents like phenolic compounds present in the plant. This chapter attempts to give an updated overview of the therapeutic uses of Aloe vera extracts and related formulation in the treatment and manage of human diseases.

1.0. INTRODUCTION Aloe vera is one of nature‘s most sacred gifts bestowed with the tremendous potential to prove itself as ‗Green medicine‘. Use of this tropical succulent plant has been referred to in ancient text like Ayurvedic medicine textbooks (Khare, 2004) and has acquired an esteemed position in the human society since time immemorial. Indeed, there are reports that Alexander the Great used Aloe vera to treat his wounded soldiers and Cleopatra used it for skin care 

E-mail address: [email protected]; [email protected].

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(Ajabnoor, 1990 cited Akinyele et al., 2007, p.559). The therapeutic effects of Aloe vera have urged recurrent myths about its properties that have persisted from the fourth century BC throughout world history (Reynolds and Dweck, 2004 cited Mahomoodally, 2014). Native to Northern Africa, this plant has been in existence for over 2000 years (Akinyele and Odiyi, 2007). Aloe vera forms part of the Liliaceae (Tribe Aloineae) family which are characterised by perennial succulent plants, often arboreal, bearing rosettes of leaves at the end of juicy green branches (Hepper, 1968). The leaves are fleshy or succulent, stiff, spotted, lance- shaped with smooth surfaces, sharp apices and spiny edges. When broken or injured, they release gluey exudates. The name Aloe is derived from the Arabic word Alloeh which means shining bitter substances (Ajabnoor, 1990). As from February 2013, there are about 550 species of the genus Aloe that have been accepted by the World Checklist of Selected Plant families. All various species of Aloe have similar constituents but Aloe vera remains the most popular one due to the fact that it propagates itself more readily than the other species making it more easily available for use (Anselm, 2004). Native to Northen Africa, Aloe vera has later been propagated in other tropical countries e.g. Mexico, Venezuela and India. United States of America started cultivating Aloe vera in the late 1970s. This plant has the ability to close its stomata to completely avoid water loss and thus can survive long periods of droughts. It can grow up to about 3 feet in height (Davis et al., 2000 cited, Akinyele et al., 2007) and matures in about 4-5 years. Under appropriate climatic conditions, it can live up to 25 years. The plant grows best in tropical climates (Akinyele and Odiyi, 2007). Besides being ornamental, Aloe vera has also been extensively used for millennia all across the world by several cultures in traditional medicine: Greece, Egypt, India, Japan, China and Mauritius. The sap and gel of the succulent leaves are used to treat numerous conditions including burns, rashes, insect bites, wounds, acne, cancer, intestinal ulcer and its effect against the Human Immunodeficiency virus has also been proved (Anselm, 2004 cited Akinyele et al., 2007, pp. 559). Parenchymatous gel from Aloe vera leaves are also extensively used in health drinks, topical creams, toiletries and cosmetics (Atherton, 1998 cited Vijayalakshmi et al., 2012, p.542). Without any doubt, there has been a boom in the commercialisation of Aloe vera during the past recent years. Products made of or containing Aloe vera gel can be found ubiquitously. Consequently, various kinds of natural-based industries have a share in the Aloe vera market, most notably the cosmetic, food, beverage and dietary supplement industries. The major constituents of Aloe vera gel can be classified into five groups namely phenolics, saccharides, vitamins, enzymes and low molecular weight substances (Choi and Chung, 2003 cited Ray et al., 2013, p. 712). Aloe vera gel has an assortment of pharmacological properties which encompasses anti-viral, anti-bacterial, laxative, protection against radiation, antioxidant, anti-inflammation, anti-cancer, anti-diabetic, anti-allergic, and immuno-stimulation amongst others (Rodriguez et al., 2010; Ray et al., 2012 cited Ray et al., 2013, p.712). Recently, the topical use of Aloe vera gel in cosmetics and skin care products has been emphasised due to its demonstrated moisturising and wound-healing effects. It has been designated as a treatment for dry skin (Gediya et al., 2011). Studies have shown that freezedried Aloe vera extract is a natural effective ingredient for ameliorating skin hydration, possibly through a humectant mechanism and thus can be used in moisturising cosmetic formulations and also as a complement in the treatment of dry skin (Dal‘Belo et al., 2006). Moreover, bactericidal attributes of Aloe vera relieve itching related to scabies (Oyelami et

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al., 2009). It is also known to help slow down the appearance of wrinkles and actively repair the damaged skin cells that cause the visible signs of aging (Mohammadirad et al., 2013). Scientific studies have also proven the efficacy of Aloe vera in the management of psoriasis (Choonhakarn et al., 2010), UV induced erythema (Reuter et al., 2008), as well as acne vulgaris (Hajheydari et al., 2013). Apart from skin disorders, Aloe vera gel can also be also applied on superficial or partial thickness burns to fasten healing process and reduce pain (Shahzad and Ahmed, 2013). It helps in soothing skin injuries affected by burning, skin irritations, cuts and insect bites. Faster wound closure has also been demonstrated in rats treated with isolated and characterised Aloe vera polysaccharides (Oryan et al., 2014). Aloe vera further reduces inflammation by downregulating pro-inflammatory cytokine production in activated human macrophages and thus interfering with the cytokine overproduction during early sepsis or in chronic inflammatory or autoimmune disease, thereby ameliorating the outcome and quality of life of patients (Budai and Varga et al., 2013). Aloe vera polysaccharides have also been speculated to enhance immunity and exert antioxidant effects in oral ulcer animal models (Yu et al., 2009). Furthermore, Aloe vera has shown its potential in the management of diabetes mellitus (DM). In this age of increased number of diabetics, Aloe vera gel can prove to be an inexpensive and reliable source of treatment. Clinical trials have shown that in obese individuals with prediabetes or early untreated DM, Aloe vera gel complex reduced body weight, body fat mass, and insulin resistance (Choi et al., 2013). Abo-Youssef and Messiha (2013) also proved the antidiabetic effect of Aloe vera leaf pulp extract in vivo and in vitro as compared to glimiperide. Another important detail is that Aloe vera contains anthraquinones, namely: aloesin, aloe-emodin and barbaloin, that exert chemo-preventive effect through modulating antioxidant and detoxification enzyme activity levels, which are one of the indicators of tumorigenesis (El-Shemy et al., 2010) and can thus be used as a cancer treatment.

2.0. CONSTITUENTS OF ALOE VERA Aloe vera contains 75 potentially active constituents: vitamins, enzymes, minerals, sugars, lignin, saponins, salicylic acids and amino acids (Surjushe et al., 2008).

2.1. Sugars Aloe vera contains monosaccharides (glucose and fructose) and polysaccharides: (glucomannans/polymannose). Glucose and mannose are responsible for the mucilage consistency of the gel. It also contains alprogen which is a glycoprotein with anti-allergic properties and C-glucosyl chromone, a novel anti-inflammatory compound (Surjushe et al., 2008).

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2.2. Vitamins Aloe vera contains vitamins A (beta-carotene), C and E, B12, folic acid, and choline among which vitamin A, C and E act as antioxidants.

2.3. Minerals Calcium, chromium, copper, selenium, magnesium, manganese, potassium, sodium and zinc are present in Aloe vera. Magnesium lactate is responsible for the anti- allergic attribute of Aloe vera (Javed and Rahman, 2014). Most of the minerals are essential for the proper functioning of various enzyme systems in different metabolic pathways while others act as antioxidants (Surjushe et al., 2008).

2.4. Enzymes The enzymes present in Aloe vera include aliiase, alkaline phosphatase, amylase, bradykinase, carboxypeptidase, catalase, cellulase, lipase, and peroxidise. Bradykinase prevents inflammation while applied topically on the skin while the others participate in metabolism of fats and sugars (Malik Itrat et al., 2013; Javed and Rahman, 2014).

2.5. Anthraquinones Aloe vera contains phenolic compounds which basically confer laxative effects. Aloin and emodin act as analgesics, antibacterials and antivirals (Surjushe et al., 2008).

2.6. Fatty acids and steroids Cholesterol, campesterol, β-sisosterol and lupeol present in Aloe vera anti- inflammatory relief while lupeol also possesses antiseptic and analgesic properties (Surjushe et al.,2008).

2.7. Hormones Auxins and gibberellins are hormones found in Aloe vera and confer anti- inflammatory effects which help in wound healing (Surjushe et al., 2008).

2.8. Others Aloe vera contains a myriad of other substances among which feature salicylic acid and saponins (summarised in Table 1). Salicylic acid possesses anti-inflammatory and

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antibacterial properties (Surjushe et al., 2008) and is well reputed in the treatment of acne. Saponins are soapy and have cleansing and antiseptic properties (Surjushe et al., 2008). Table 1. chemical constituents of Aloe vera (Malik Itrat et al., 2013; Javed and Rahman, 2014) Name of constituent Acemannans

Aloetic acid Cinnamic acid Chrysophanic acid Salicylic acid

Alo-emodin Aloin or Barbaloin Isobarbaloin Alprogen

Vitamins Enzymes

Minerals

Functions and mode of action (where applicable) Deals with damaging processes by acting as immune stimulant mainly via stimulation of production of T-lymphocytes and macrophages from thymus and β- cells of pancreas. It has germicidal, bactericidal and antifungal actions. Additionally, it coats and permeates the surfaces of the gastrointestinal tract allowing for easy expulsion of toxins and faster absorption of nutritive factors. The specific properties are not fully known but it seems to act as a natural antibiotic. It procures antiseptic, germicidal, anaesthetic and analgesic effects. Moreover, it has a strong detergent action due to molecular similarity with saponins. It is a good purifying agent with strong laxative and diuretic effects. It also stimulates bile secretion and has fungicidal action. This acid confers antiseptic, antibacterial and anti- inflammatory actions. It has been used in pharmaceutical industry as analgesics, anti-rheumatics and even in acne treatment. Salicylic acid work through various routes, cyclooxygenase (COX) activity inhibition or adenosine monophosphate activated protein kinase (AMPK) activation, etc. This compound is present in the yellow exudates found in lining of Aloe vera leaf and has bactericidal and laxative properties. It has purging, detoxifying and markedly antibiotic properties. Acts as a natural antibiotic. It procures anti-allergic properties by inhibiting histamine and release of leukotriene by multiple signals and Ca(2þ) blocking influx inhibition and antigen–antibody reactions. It is rich in all vitamins except vitamin D. Vitamins A, C, E and B12 act as antioxidants. Carboxypeptidase relieves pain, swelling and is anti-inflammatory. Bradykinase reduces inflammation and consequently reduces pain via vasodialtion. Other enzymes digest dead tissues in wounds. Lipases and proteases aid in digestion. They are required in the body for the proper functioning of various enzyme systems in different metabolic pathways.

3.0. THERAPEUTIC USES OF ALOE VERA As mentioned, Aloe vera has been used extensively in traditional medicine by different cultures all over the world. Even in Mauritius, people have not been indifferent to the therapeutic attributes of Aloe vera. Some of the numerous conditions treated with Aloe vera are as follows:

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3.1. Skin Problems Preliminary evidence suggests that Aloe vera may improve symptoms of certain skin conditions such as:

3.1.1. Eczema Eczema is a chronic inflammatory skin disorder that affects 20% of the population in developed nations, and frequently manifests in early childhood. The term ‗eczema‘ is used to refer to a wide range of relentless skin conditions: from dryness to recurring skin rashes characterised by edema, itching, crusting, flaking, blistering, cracking, oozing or bleeding (Andrew and Craig, 2001 cited Ahamed et al., 2012). Skin discolouration can occur in some areas due to healed injuries. Scratching a healing lesion may lead to scarring or propagation of the rash. The defined aetiology and pathogenesis of eczema are not yet fully understood, but a multifaceted interaction between genetic and environmental factors has been implicated in the predisposition and development of the disease. Food allergy plays a pathogenic role in some eczema patients, mainly infants and children with severe eczema (Kelly and Hourihane, 2011). There are limited studies on the efficacy of Aloe vera in treating eczema. A doubleblind, randomized, placebo-controlled prospective clinical trial was carried in 44 adult patients with seborrheic dermatitis. The results indicated that Aloe vera crude extract emulsion is effective in the therapy of patients with seborrheic dermatitis (Vardy et al., 1999). 3.1.2. Psoriasis Psoriasis is an immune-mediated disease that affects the skin. It is normally a lifelong condition. Till now, no cure exists. This condition is characterised by scaly, reddened patches, papules and plaques that are itchy (Menter et al., 2008). There are various types of psoriasis, 5 being more common: 1) plaque, 2) guttate, 3) inverse, 4) pustular and 5) erythrodermic. The most familiar one is plaque psoriasis characterised by red and white hues of scaly patches on the epidermis. The skin accumulates rapidly at these sites giving it a silvery- white appearance (Menter et al., 2008). These are more frequent at elbows and knees but can affect any other area on the body. Even fingernails and toenails are affected (referred to as psoriatic nail dystrophy). Psoriasis can also affect the joints leading to psoriatic arthritis. One study showed a major favourable effect of Aloe vera extract 0.5% in hydrophilic cream compared to hydrophilic cream alone in reducing psoriatic plaques and inflammation (Syed et al., 1996). Another study compared Aloe vera cream containing 70% mucilage to 0.1% triamcinolone acetonide cream over the course of 8 weeks and found it to be equally effective (Choonhakarn et al., 2010). Yet, further research is needed to conclude the efficacy of Aloe vera to treat Psoriasis. 3.1.3. UV Induced Erythema UV induced erythema refers to the skin becoming red due to hyperemia of the capillaries in the lower layers of the skin induced by solar radiation (commonly called sunburns). In severe cases, blistering and peeling of the skin can occur. One randomized, double-blind, placebo-controlled trial (Reuter et al., 2008) compared the anti-inflammatory effect of 97.5% pure Aloe vera gel to 1% hydrocortisone and a placebo gel and concluded that if Aloe vera gel is applied under an occlusive bandage for 2 days following UV exposure, inflammation is

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reduced considerably when compared to placebo gel or 1% hydrocortisone in placebo gel, but was however less effective than 1% hydrocortisone cream. The authors suggest that Aloe vera gel might be useful for the treatment of inflammatory skin conditions and can thus protect the skin from solar radiation. Exact role is not known, but following the administration of Aloe vera gel, an antioxidant protein, metallothionein, is generated in the skin, which scavenges hydroxyl radicals and prevents suppression of superoxide dismutase and glutathione peroxidase in the skin (Byeon et al., 1998). It reduces the production and release of skin keratinocyte-derived immunosuppressive cytokines such as interleukin-10 (IL-10) and hence prevents UV-induced suppression of delayed type hypersensitivity (Byeon et al., 1998). More research is needed in this area.

3.1.4. Acne Acne vulgaris is a skin disease, characterized by areas of skin with seborrhea (scaly red skin), comedones, papules, nodules, pimples, and possibly scarring (Adityan, Kumari and Thappa, 2009). This occurs mainly in adolescents but can persist through adult years as well. Acne affects mostly the face, neck and back. Acne occurs due to blockages in the follicles. Consequently, hyperkeratinisation takes place resulting in the formation of a plug of keratin and sebum, also termed as a microcomedo, (Benner & Sammons, 2013). During adrenarche, there is enlargement of sebaceous glands and increased production of sebum due to the increased production of androgen (DHEA-S). The microcomedo enlarges and forms an open comedo, commonly called blackhead, or a closed comedo. Comedones are the result of sebaceous glands becoming clogged with sebum and dead skin cells (Benner and Sammons, 2013). This leads to naturally occurring Propionibacterium acne bacterium to cause inflammation resulting in papules, infected pustules or nodules. This in turn can lead to redness and hyperpigmentation (Simpson and Cunlife, 2004). One study demonstrated that the combination tretinoin/Aloe vera gel was well tolerated and significantly more effective than tretinoin cream in the treatment of mild to moderate acne vulgaris (Hajheydari et al., 2013).

3.2. Wound Healing and Treatment of Burns Aloe vera gel is generally applied topically on first degree burns and wounds. It is believed to soothe pain and lead to quicker healing. In a study, twenty-seven patients with partial thickness burn wound were treated with Aloe vera gel compared with vaseline gauze. The results revealed that the Aloe vera gel treated lesion healed faster than the vaseline gauze area. This study showed the effectiveness of Aloe vera gel on a partial thickness burn wound (Visuthikosol et al., 1995). A more recent study (Shahzad and Ahmed, 2013) assessed the efficacy of Aloe vera gel compared with 1% silver sulfadiazine cream as a burn dressing for the treatment of superficial and partial thickness burns. The results were quite noteworthy in the sense that patients treated with Aloe vera gel showed remarkably earlier healing of burn wounds than those patients treated with 1% silver sulfadiazine (SSD). All the patients of Aloe vera group were relieved of pain earlier than those patients who were treated with SSD. This leads to the conclusion that thermal burns patients dressed with Aloe vera gel showed advantage compared to those dressed with SSD regarding early wound epithelialization, earlier pain relief and cost-effectiveness. Polysaccharides, particularly

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mannose-containing polysaccharides, cellulose, and pectic polysaccharides, comprise the major part of Aloe vera gel. Acetylated glucomannan is primarily responsible for the gel‘s mucilaginous properties (Hamman, 2008) and has been found in vitro and in animal studies to modulate immune function (through macrophage activation and cytokine production) and accelerate wound healing (Ulbricht et al., 2008). Veracylglucan B and veracylglucan C, two maloyl glucans isolated from Aloe vera gel, have been demonstrated in vitro to have potent anti-inflammatory effects, although their effects on cell proliferation appear antagonistic (Esua and Rauwald, 2006). One study isolated and characterised Aloe vera polysaccharides (Oryan et al., 2014). In the study, open wounds of rats were treated on a daily basis with different doses of Aloe vera polysaccharides for 30 days. The results showed an enhanced wound closure in treated rats and demonstrated that Aloe vera polysaccharides, at transcriptional level, regulate MMP-3 and TIMP-2 gene expression during the dermal wound repair. Consequently, it may influence the granulation tissue formation and wound closure by increased production of extracellular matrix constituents including glycosaminoglycans and collagen. Moreover, among the non-polysaccharide gel constituents, salicylic acid and other antiprostaglandin compounds may contribute to the local anti-inflammatory activity of Aloe vera via the inhibition of cyclooxygenase pathway (Ulbricht et al. 2008). Another study demonstrated that Aloe vera downregulates pro-inflammatory cytokine production in activated human macrophages and thus interfering with the cytokine overproduction during early sepsis or in chronic inflammatory or autoimmune disease may ameliorate the outcome and quality of life of patients (Budai and Varga et al., 2013). As a result, Aloe vera could be a new therapeutic tool to target Nlrp3 inflammasome-mediated cytokine production. Further, Aloe vera contains lupeol, salicylic acid, urea nitrogen, cinnamonic acid, phenols and sulphur which act as antiseptic agents which all confer inhibitory action against fungi, bacteria and viruses (Surjushe et al., 2008).

3.3. Treatment of Diabetes Mellitus Non- insulin dependent diabetes mellitus (NIDDM) is the most common form of the disease, and accounts for more than 90% of diabetes patients. It is characterised by insulin resistance in peripheral tissues leading to compensatory hyperinsulinemia, followed by β-cell failure, which eventually leads to prandial and later to overt fasting hyperglycemia (Defronzo et al., 1992). The number of people diagnosed with NIDDM is increasing at an alarming rate in western societies; driven by a drastic rise in the occurrence of obesity and sedentary lifestyles (Kwanghee et al., 2009). NIDDM is a progressive disease with related complications of retinopathy, nephropathy, neuropathy, and atherosclerosis (Marcovecchio et al. 2005). Therefore, maintaining a near-normal blood glucose level is the primary goal of diabetic patients. Due to its strongly bitter taste, numerous Mauritians and worldwide citizens take Aloe vera orally in order to maintain a normal blood glucose level. The Aloe vera leaf is peeled and gel paste gulped directly or blended with some water and a whole glass of the resulting blend is consumed everyday in the morning. Yongchaiyudha et al. (1996) conducted clinical trials to evaluate the potential antidiabetic activity of Aloe vera. In the trial, one tablespoon of Aloe vera juice was given to diabetic patients twice a day for at least 2 weeks. The study observed that the blood sugar and triglyceride levels of these patients fell, suggesting the potential of Aloe vera as an antidiabetic agent. Another study showed that in

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obese individuals with prediabetes or early untreated DM, Aloe vera gel complex reduced body weight, body fat mass, and insulin resistance (Choi et al., 2013). Abo-Youssef and Messiha (2013) studied the antidiabetic effect of Aloe vera leaf pulp extract in vivo and in vitro as compared to glimiperide. The results showed that the serum levels of malondialdehyde (MDA) and superoxide dismutase (SOD) were significantly decreased whereas the level of blood glutathione (GSH) was considerably increased by the treatment of Aloe vera in diabetic mice as compared to controls. As for the in vitro study, both Aloe vera (10 μl/l) and glimiperide (10 μmol/l) remarkably increased both basal and stimulated insulin secretion from isolated islets of pancreas. These findings show a promising antidiabetic effect of Aloe vera for further clinical trials regarding pharmaceutical use of Aloe vera extract for treating type II diabetes. The evaluation of the presence of hypoglycaemic activity in the alcoholic extract of Aloe vera gel demonstrated that Aloe vera extract maintained the glucose homeostasis by controlling the carbohydrate metabolizing enzymes (Rajasekaran et al., 2004). Tanaka et al. (2006) identified five phytosterols from Aloe vera that can act as antidiabetic agents namely lophenol, 24-methyl-lophenol, 24-ethyl-lophenol, cycloartanol, and 24methylene-cycloartanol.

3.4. Hypertension Hypertension also known as arterial hypertension or elevated blood pressure, is a chronic medical condition in which the blood pressure in the arteries is always high. Blood pressure refers to the ratio of maximum pressure during systole (contraction of heart) to minimum pressure during diastole (relaxation of heart). Normal blood pressure at rest is within the range of 100–140 mmHg systolic and 60–90 mmHg diastolic (bottom reading). High blood pressure is prevalent if ration is at or above 140/90 mmHg. Hypertension puts strain on the heart, increasing the possibility of hypertensive heart disease, coronary artery disease, strokes, and aneurysms of the arteries amongst others. In traditional medicine, it is believed that consuming the gel of Aloe vera can lower blood pressure. However, scientific information is sparse. In a double-blind, placebo-controlled, crossover study, healthy volunteers above 18 years of age received either 1200 mg of oral Aloe vera powder or matching placebo (Shah et al., 2010). Electrocardiographic variables, systolic blood pressure and diastolic blood pressure were evaluated. The study concluded that a single dose of oral Aloe vera had no effect on blood pressure of young healthy volunteers (Shah et al., 2010). No recent investigation concerning the link between Aloe vera and hypertension has been carried out in hypertensive patients.

3.5. Cardiovascular Diseases Cardiovascular diseases have been reported as one of the major cause of deaths globally. It is characterised by atheroma formation in the arteries due to accumulation of lipids. The risk of atheroma formation increases with elevated plasma cholesterol and obesity. The ethnopharmacological use of Aloe vera to lower blood cholesterol has been reported (Mootoosamy and Mahomoodally, 2014). Kumar et al., (2013) tested the effects of Lactobacillus rhamnosus GG and Aloe vera gel on lipid profiles in rats with induced

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hypercholesterolemia and concluded that the combination of both of these compounds may have a therapeutic potential to decrease cholesterol levels and the risk of cardiovascular diseases. The effects of lophenol and cycloartanol, minor phytosterols of Aloe vera gel, in obese animal model of type II diabetes, Zucker diabetic fatty (ZDF) rats were examined (Misawa et al., 2008). Consecutive treatment of phytosterols suppressed the hyperglycemia, and random blood glucose levels after 35 days of treatment were 39.6 and 37.2% lower than the control. These observations suggest that Aloe vera-derived phytosterols could reduce visceral fat accumulation, and would be useful for the improvement of hyperlipidemia and hyperglycemia which normally increase the risk of cardiovascular problems. These results were further exploited by Nomaguchi et al., (2011) who confirmed that Aloe phytosterols, lophenol and cycloartanol, activate PPAR transcription in vitro. Furthermore, quantitative gene expression analysis in DIO mice in the same study suggested that Aloe phytosterols improve fatty acid metabolism in the liver. All these findings support the use of Aloe vera in the treatment of cardiovascular diseases.

3.6. Cancer Cancer refers to a group of diseases involving abnormal cell growth resulting in formation of tumours. Current treatments involve chemotherapy, radiation, and targeted therapies (Anand et al., 2008) which are accompanied ny numerous side effects. Therefore, plant based formulations, such as Aloe vera, used by ancient healers represent a potential source for pharmacological exploitation in order to come up with a solution for cancer. In mice previously implanted with murine sarcoma cells, acemannan enhances the production and release of interleukin-1 (IL-1) and tumor necrosis factor from macrophages (Peng et al., 1991). Consequently, this initiated an immune attack that resulted in necrosis and regression of the cancerous cells. Aloe vera gel also contains several low-molecular-weight compounds with the ability of inhibiting the discharge of reactive oxygen free radicals from activated human neutrophils (Hart et al., 1990). Alprogen in Aloe vera inhibit calcium influx into mast cells, thereby inhibiting the antigen-antibody-mediated release of histamine and leukotriene from mast cells (Ro et al., 2000). In a more recent study, rats were orally fed with Aloe vera polysaccharides. Rats in control group were orally fed the same volume of saline. The results showed that Aloe vera polysaccharides enhanced immunity activity and exerted antioxidant effects compared with vehicle controls (Yu et al., 2009). The antiviral and antitumour activity can be due to indirect or direct effects. Indirect effect is due to stimulation of the immune system and direct effect is due to anthraquinones. The anthraquinone aloin inactivates various enveloped viruses such as Herpes simplex and Varicella zoster. (Sydiskis et al., 1991). Studies have also shown that a polysaccharide inhibits the binding of benzopyrene to primary rat hepatocytes, thereby preventing the formation of potentially cancer-initiating benzopyrene-DNA adducts. An induction of glutathione S-transferase and an inhibition of the tumor-promoting effects of phorbol myristic acetate has also been reported which suggest a possible benefit of using Aloe vera gel in cancer chemoprevention (Kim et al., 1999). Another study extracted three anthraquinones, namely: aloesin, aloe-emodin and barbaloin, from Aloe vera leaves and the data suggested that these may exert their chemo-preventive effect through modulating antioxidant and detoxification enzyme activity levels, as they are one of the indicators of tumorigenesis (El-Shemy et al., 2010).

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3.7. Constipation Constipation refers to the symptom of painful defecation and also the frequency of passing stool is less than three times per week. Severe constipation includes obstipation (failure to pass stools or gas) and fecal impaction, which can progress to bowel obstruction and become life-threatening (Milford, 2011). Constipation often occurs due to lack of fibre and fluid in the diet. There are other non- nutritional factors contributing to this condition namely: stress, illness, lack of exercises and drugs. Constipation can lead to more serious problems like haemorrhoids and cancer of the colon. One remedy adopted by Mauritians is the intake of Aloe vera together with the pulp and gel every morning. The laxative effects are due to the yellowish latex found in the sap of Aloe vera. However, it can cause painful cramping and is not recommended. Other gentler, herbal laxatives from the same plant family as Aloe (such as cascara and senna) are generally recommended first (University of Maryland medical, 2011). In vitro and in vivo studies in rats demonstrated that aloe-emodin-9-anthrones reduce the absorption of water from the intestinal lumen by inhibiting the activity of Na+, K+-adenosine triphosphatase (ATPase) and stimulate water secretion by increasing the paracellular permeability across the colonic mucosa (Ishii, Tanizawa and Takino, 1990). The net result is a reduction in water absorption and the formation of softer stools (Boudreau and Beland, 2006). Aloe-emodin has been suggested to have antiangiogenic properties; it has been demonstrated to be a potent inhibitor of urokinase secretion and tubule formation of endothelial cells, both key events in angiogenesis (Cárdenas, Quesada, and Medina, 2006).

3.8. In Cosmetics Cosmetics are care substances used to enhance one‘s appearance. In the U.S., as per Food and Drug Administration (FDA), which regulates cosmetics, defines cosmetics as "intended to be applied to the human body for cleansing, beautifying, promoting attractiveness, or altering the appearance without affecting the body's structure or functions." These products include soap, facial cleansers, facial and body scrubs, creams, face masks, astringents, etc. Many Mauritian females apply Aloe vera gel paste as face mask everyday in the morning or at least once a week. This is believed to lighten skin tone, moisturise and prevent aging of the skin. Apart from being used in its natural form, there are various popular skin care products that contain Aloe vera extract as an ingredient. One study evaluated the effects of cosmetic formulations containing different concentrations of Aloe vera. It showed that freezedried Aloe vera extract is a natural effective ingredient for ameliorating skin hydration, possibly through a humectant mechanism and thus can be used in moisturising cosmetic formulations and also as a complement in the treatment of dry skin (Dal‘Belo et al., 2006). Mucopolysaccharides in Aloe vera gel help in binding moisture into the skin (Surjushe et al., 2008). Aloe vera also stimulates fibroblast which in turn stimulates production of collagen and elastin fibers (Surjushe et al., 2008). Consequently, skin is more elastic and less wrinkled. Moreoever, it exhibits cohesive effects on the superficial flaking epidermal cells by sticking them together, resulting in softening of the skin. Amino acids present in Aloe vera as well

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participate in the softening of hardened skin cells (Surjushe et al., 2008). Zinc present acts as an astringent to tighten pores. Another study showed that Aloe vera gel gloves improved the skin integrity, decreases appearance of wrinkles and reduces erythrema (West et al., 2003).

CONCLUSION Aloe vera remains one of the most commonly exploited plant extract in the pharmaceutical, cosmetic and the wellness industry for its plethora of biological properties. The beneficial effects of Aloe vera in skin disorders, wound healing, diabetes mellitus, cardiovascular diseases, cancer and in cosmeticeutical products have been well documented. However, its use in the treatment of hypertension is still inconclusive. The authenticity and dosage of Aloe vera in cosmetic products and food supplements remain a challenging issue. Further research geared towards Aloe vera formulation and its use as a functional food should be carried out.

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Vijayalakshmi, D., Dh, Apani, R., Jayaveni, S., Jithendra, P., Rose, C., M and Al, A. 2012. In vitro anti inflammatory activity of Aloe vera by down regulation of MMP-9 in peripheral blood mononuclear cells. Journal of Ethnopharmacology, 141 (1), pp. 542--546. Visuthikosol, V., Chowchuen, B., Sukwanarat, Y., Sriurairatana, S., Boonpucknavig, V. and Others. 1995. Effect of aloe vera gel to healing of burn wound a clinical and histologic study. Journal of the Medical Association of Thailand- Chotmaihet thangphaet, 78 (8), pp. 403--409. West, D. P. and Zhu, Y. F. 2003. Evaluation of aloe vera gel gloves in the treatment of dry skin associated with occupational exposure. American Journal of Infection Control, 31 (1), pp. 40--42. Yongchaiyudha, S., Rungpitarangsi, V., Bunyapraphatsara, N. and Chokechaijaroenporn, O. 1996. Antidiabetic activity of Aloe vera L. juice. I. Clinical trial in new cases of diabetes mellitus. Phytomedicine, 3 (3), pp. 241--243. Yu, Z., Jin, C., Xin, M. and Jianmin, H. 2009. Effect of Aloe vera polysaccharides on immunity and antioxidant activities in oral ulcer animal models. Carbohydrate polymers, 75 (2), pp. 307--311.

In: Fruit and Pomace Extracts Editor: Jason P. Owen

ISBN: 978-1-63482-497-2 © 2015 Nova Science Publishers, Inc.

Chapter 12

ELDERBERRIES EXTRACTS: BIOLOGIC EFFECTS, APPLICATIONS FOR THERAPY: A REVIEW Mihaela Mirela Bratu and Ticuta Negreanu-Pirjol ―Ovidius‖ University Constanta, Faculty of Pharmacy, Aleea Universitatii no.1, Campus Corp B, Constanta, Romania

ABSTRACT As many berries, the fruits of Sambucus nigra (L.) contain large amounts of flavonoids with different structures, mostly anthocyanins (mainly cyanidin-3-glucoside and cyanidin-3-sambubioside) and small quanities of flavonols and flavonol ester. Flavonoids are a broad class of low-molecular-weight secondary metabolites encompassing more than 10,000 scaffolds, and are commonly found in leaves, seeds, bark and flowers. Their role in plants is to afford protection against ultraviolet radiation, pathogens and herbivore animals. Due to their activity as safe and potent antioxidants, they are considered as important nutraceuticals. Due to the content in anthocyanins, elderberries have an attractive bright purple color, which make elderberry anthocyanins extracts valuable foodstuff colorants but also therapeutic agents. There are many studies showing the biologic effects of certain elderberries extracts, such as: in vitro and in vivo antioxidant activities, anti-inflammatory properties, stimulant of cell division. Some of them offers contradictory information. There are also reports concerning attempts to formulate and develop new pharmaceutical/nutraceutical products. This chapter tries to join together the information concerning the main therapeutic effects of elderberries extracts as they are presented in the recent publications. Also, it presents some attempts to apply the elderberries extracts in pharmacy as active principles.

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1. SHORT HISTORY ABOUT APPLICATIONS OF ELDERBERRIES IN FOLK MEDICINE The elder, Sambucus nigra (L.), is a flowering small tree, mainly distributed in temperate and subtropical regions such as: Central and Southern Europe, Asia, North America. It is a common plant in Europe, being related to different old magical traditions and ethnoiatric uses. After many study concerning the phylogenetic relationship of Sambucus genus [1, 2] it was concluded that it belongs to the Adoxaceae instead of the Caprifoliaceae family. This botanical family belongs to Dipsacales order, Magnoliopsida class, Asteridae subclass. The European cultures used the elder for longtime for magical purposes, to keep the evil spirits away [3]. Romanian tradition consider the elder (Sambucus nigra) and the dwarf elder (Sambucus ebulus) as a couple (man and wife) with magical power for rain calling ceremonies and evil protection. The collection of their berries were done only after accomplishing a magical ceremony [4]. All parts of the plant have medicinal uses in many traditions all over Europe. Stembark, leaves, flowers, fruits, and root extracts are used to treat upper respiratory cold infections, fever but also stomach ache, constipation, diarrhea [5, 6, 7]. The flowers are said to have diaphoretic, anti-catarrhal, expectorant, diuretic and topical anti-inflammatory actions [7]. Leaves and inner bark have also been used for their purgative, emetic, diuretic, laxative, topical emollient, expectorant, and diaphoretic action [7]. The Austrian traditional medicine uses the elderberries prepared as tea, jelly, juice or syrup to cure viral infections, fever, flu, colds, respiratory tract, mouth, gastrointestinal tract problems but also skin diseases [8]. Other recent studies isolated from elderberry (S. nigra L.) bark a lectin with an exclusive specificity toward the Neu5Ac(a2,6)Gal/GalNAc disaccharide [9, 10]., which demonstrate insecticidal potency [11]. Inspired from tradition, the modern production of elderberries extracts and dietary supplements is growing year by year. Meanwhile, the scientific facts confirm the therapeutic activities of elderberries and elderberries extracts.

2. ACTIVE COMPOUNDS IN ELDERBERRIES AND ELDERBERRIES EXTRACTS Fresh elderberries contains large amounts of anthocyanins, the main constituents have been identified as cyanidin-3-glucoside (65.7% of total anthocyanins) and cyanidin-3sambubioside (32.4% of total anthocyanins) [12] in addition to small amounts of other types of anthocyanins, flavonols and flavonol ester [13]. The dried seeds contain a lectin (0.1%), identified as Sambucus nigra agglutinin III (SNA-III, synonym SNA-IVf) (Assessment report on Sambucus nigra L., fructus EMA/HMPC/44208/2012). Another lectin, SNA-Vf (synonym nigrin f) has been found in fresh fruits [9]. The fruits contain about 0.01% essential oil which include 34 identified components [13]. As other ingredients elderberries contain vitamins and minerals in small amounts and glucides (pectin and up to 7.5% glucose and fructose) [13].

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The leaves and the unripe berries of Sambucus nigra contain toxic constituents such as cyanogenic glycosides (sambunigrin) and should be avoided in elderberry preparation [13, 14].

Figure 1. Sambunigrin.

The main components that may contribute to pharmacological activity of elderberries are the polyphenols, especially anthocyanins, which have been proved as powerful antioxidants. On that reason, the elderberries extracts producers are interested in preserving as much as possible the content and the structure of those compounds.

Figure 2. Cyanidin-3 glucoside (chrysanthemin).

Figure 3. Cyanidin-3-sambubioside.

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Due to their supposed content in some toxic compounds (a cyanogenic glycoside which is destroyed by heat) elderberries are predominately used processed. Despite the cyanogenic glycosides, the anthocyanins are quite stable at high temperatures; therefore cultivars with higher concentrations in anthocyanins and other polyphenols are particularly chosen for commercial growing. Verbenic et al., [15] measured the content in anthocyanins, quercetin and sugars in the fruit of two cultivars and three elderberry selections. Their results show that the leading European cultivar is ‗Haschberg‘ has significantly higher amounts of total anthocyanins given to other cultivars/selections but lower concentrations of secondary metabolites than some selections [15] From their results one can see that the cultivar with the highest amounts of total anthocyanins is ‗Rubini‘; which also contains high amounts of quercetins, especially quercetin 3-rutinoside, a compound with important antioxidant activity and therapeutic applications. Given to other berries, elderberries are major sources of anthocyanins, as reveals the study of Veberic et al., [16]. Their study determine the anthocyanin composition in species of Ribes, Rubus, Vaccinium, Fragaria, Crataegus, Morus, Sorbus, Sambucus and Aronia genus. The highest total anthocyanin content was determined in dark colored fruit of cultivated elderberry and bilberry whereas light-colored dog rose and Chinese hawthorn fruit had the lowest anthocyanin content. The composition of anthocyanidin glycosides do not differ between the fruit of wild growing and cultivated species, but their contents are generally different. Seabra et al., [17] performed fractionated high pressure extractions from dry and in natura elderberry pomace in order to obtain anthocyanin rich extracts. Experiments were carried out using CO2 supercritical fluid extraction followed by enhanced solvent extraction (ESE) with CO2/ethanol–water mixtures (1–100%, v/v), to obtain anthocyanin rich fractions in the second step, at 313 K and ~20 MPa. Higher extract yields, anthocyanin contents and antioxidant activities occurred by the presence of water, both in the raw material and in the solvent mixture. The CO2 dissolved in the ESE solvent mixture favored either anthocyanin contents or antioxidant activities, which were not directly related. ESE has several additional advantages over conventional solvent extraction, such as the possibility of extract fractionation and a higher extraction flexibility, which is offered by the possibility of modifying solvent dissolution capability just by changing operational conditions as dissolved CO2, and temperature and pressure, which can also be explored. The presented work protocols for anthocyanins extraction use solvents and techniques considered as ―acceptable‖ and ―generally regarded as safe‖ in the food and pharmaceutical industries [17]. Duymus et al., [18] prepared elderberries extracts using different extraction phases and different protocols, in order to establish an optimal method for polypenolic compound extraction. The extraction phases used were: water, 70% ethanol, 70% acetone and methanol at room temperature, HCl (0.1%) in methanol by using an ultrasonic bath at room temperature, as well as water at 100°C (infusion). All these extracts were subsequently evaporated to dryness at 35°C. According to the author‘s data, the highest yields of extraction were obtained when it was used the acidified methanol, 70% ethanol and methanol as extraction phases.

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Other authors studied the effects of extraction time, temperature and pH on the anthocyanins during the extraction form vegetable products [19].The results showed that the stability of anthocyanin-based extracts depended on pH and temperature, heating time, and anthocyanin sources. Elevated pH levels cause anthocyanin degradations which affect both color and intensity. The company BerryPharma AG produces an elderberry extract commercially known as Rubini. This extract is standardized by HPLC and is always produced from the same ―Haschberg‖ variety of Sambucus nigra L. fruits, which is grown under cultivation in the Steiermark region of Austria. The elderberry-to-extract ratio of the product is 18:1. The juice of pressed elderberries is filtrated by a specific filter system which separates the different sizes of the molecules due to their active agents. Subsequently, the resulting flavonoidconcentrated liquid is either used for the Rubini. The extract is concentrated and standardized using membrane filtration to achieve a minimum anthocyanin concentration of 3.2%. The concentration of anthocyanins is achieved using a mechanical filtration procedure in which semipermeable membranes separate substances according to their different molecular sizes. In the product it is not add any preservatives or sugar [20]. A product used as dietary supplement prepared from elderberries is also Sambucol®. The product was developed in Europe in 1991 as a result of 20 years of research, and, as a natural product, has been marketed in Europe since 1998 for the treatment of colds and flu [21]. Its formulation and extraction method is producer‘s property.

3. MEASUREMENTS OF ANTIOXIDANT ACTIVITY IN ELDERBERRIES EXTRACTS Antioxidants are considered as possible protection agents reducing oxidative damage of human body. Natural compounds occur in all parts of plant, may be beneficial in reducing oxidative stress-induced diseases. The phytochemicals in plant tissues responsible for the antioxidant capacity can largely to be attributed to the phenols, anthocyanins carotenoids, vitamins, dietary gluthatione and endogenous metabolites. Antioxidants derived from fruits and vegetables have been shown to function as free radical scavengers, peroxide decomposers, enzyme inhibitors or synergists. The vegetable consumption has been associated with the increased cancer rates, low incidence of heart diseases, pollutants detoxification, reducing of blood pressure and inflammation [22]. One of the most important sources of antioxidants among dietary plants is small red fruits. These plants represent a source of natural antioxidants that might serve as leads for the development of novel drugs. Several studies both in vitro [23 – 26] and in vivo [27, 28] emphasized that elderberries extracts have a potent antioxidant effect, property due by the presence of numerous phytochemicals, such as organic acids, flavonols glycosides and anthocyanins, importants for the beneficial health effects, with referring to their ability to protect against colon cancer, influenza A and B virus and Helicobacter pylori infections [29]. Recently, extracts of both European, black or common elderberry (Sambucus nigra L.) and American elderberry (Sambucus canadensis L.) demonstrated that the purple-black fruits of elderberries (Sambucus spp. L.) are one of the richest sources of anthocyanins and phenolic compounds among small fruits and have strong antioxidant capacity [30 - 33]. Among

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common small fruits, elderberries are perhaps most comparable to blackberries and black raspberries [34, 35]. Due to the chemical diversity of antioxidant compounds present in natural samples, it is unrealistic to separate each antioxidant component and study it individually. In addition, levels of single antioxidants do not necessarily reflect their total antioxidant capacity because of the possible synergistic interactions among the antioxidant compounds in a food mixture, for that Barros et al., 2011, recommend to evaluated the antioxidant properties of the entire extracts obtained from the plant [36]. As suggested in the literature [33], earlier studies of small fruit phytonutrient contents reported high correlations among total phenols, anthocyanin values and antioxidant capacities, as determined by oxygen radical absorbance capacity (ORAC) and FRAP assays. Using the ORAC method, showed that especially the American elderberry species Sambucus canadensis L. had a much higher potential than cranberry and blueberry [37]. Anton et al., 2013, [29] reported the presence in Sambucus nigra L. (black elder) berries crude-extract, as primary sources of antioxidant compounds, the total flavonoids (38,36 mg/100g FW) as quercetin-3-O-glucoside and quercetin-3-O-rutinoside, phenolic acid as pcoumaric acid (0,55 mg/100g FW), total flavonol aglycons (2,60 mg/100g FW) as quercetin and kaempferol, total anthocyanins (272.87 mg/100g FW) as cyanidin-3-O- sambubioside-5glucoside, cyanidin-3,5-O-diglucosid, cyanidin-3-O-sambubioside, cyanidin-3-O-glucoside and total anthocyanindins (121.87 mg/100g FW) as cyanidin-3-O-glucoside, pelargonidin, cyanidin. The authors used the DPPH assay based on the reduction of the DPPH radical in the presence of hydrogen donating antioxidant, to emphasize that these phytochemical components, are responsible for the higher antioxidant capacity of elderberries (63,26 % DPPH radical scavenging activity) than for gooseberries, black gooseberries, black and red currants [29]. Two methods are frequency used to determine total antioxidant capacity of elderberries fruits, the ferric reducing antioxidant power (FRAP) method according to Benzie and Strain [38] and the DPPH radical scavenging capacity using the method of Brand-Williams et al. [39]. Because antioxidant capacity estimates are likely to vary with techniques, the author‘s recommend to use of more than one antioxidant assay for the determination of antioxidant power of small fruit samples [40]. Dawidowicz A.L. et al., 2006 [23], estimated the antioxidant properties of alcoholic extracts from berries of Sambucus nigra L. by means of DPPH and β-carotene/linoleic acid methods [39, 41] and considered in relation to the extraction temperature (in the range 20°– 200°C) and to the level of flavonoids most representative for this species. The phenolic compounds from elderberries extracts act as antioxidants neutralizing the activities of free radicals and inhibiting the co-oxidation reactions of linoleic acid and β-carotene. According the authors, the antioxidant properties of Sambucus nigra extracts are mainly connected with the presence of flavonols and anthocyanins. Rutin is the most representative flavonol of this species. Isoquercitrin and astragaline, also belonging to flavonols, exist in Sambucus nigra in smaller amounts. Anthocyanins occurring in the plant are cyanidin-3-sambubioside and cyanidin-3-glucoside (large amounts), and cyanidin-3-sambubioside-5-glucoside and cyanidin-3,5-diglucoside (smaller amounts) [23]. In the case of the DPPH assay, at 100°C, the higher concentrations of flavonols and anthocyanins present in the extract, emphasize their higher antioxidative ability, probably the hydrolysis of flavonols being responsible for the difference between the antioxidant properties and the amount of flavonols in berries extract

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obtained at higher temperature [23]. Cejpek et al., 2009, [25] proposed for determination the antioxidants of fresh juice pressed from elder fruits, the electrochemical activity (EA) expressed in g of l-ascorbic acid equivalents (AAE) per kg. The most important polyphenols found in elderberry fruits were cyanidin 3-sambubioside and cyanidin 3-glucoside, which comprised 51% and 40% EA of anthocyanins, respective. The major part of the juice EA was originated from catechins and phenolic acids such as chlorogenic acid; rutin and other flavonols provided 8,1% of the total EA. The expected antioxidant capacity associated with electrochemical activity was compared with a free-radical scavenging capacity. Using DPPH assay, elderberry juice presented 0.3% antiradical activity of 1-ascorbic acid [25]. Barros et al., 2011, [36] developed a new portable and enable rapid in field analysis of electrochemical techniques applicable to non-transparent samples, such as cyclic voltammetry and differential pulse voltammetry to provide a further insight into redox processes within plant extracts. These techniques have been tested and developed as an alternative and/or complementary tool for the evaluation of antioxidant power, respectively the content in vitamins (ascorbic acid) and pigments (β-carotene) phenolics and flavonoids of the Sambucus nigra L. The most important antioxidants found in elderberry were phenolic compounds (92.7 mg GAE/g DW), flavonoids (26,2 mg CE/g DW) and ascorbic acid in fresh fruits of S. nigra (1730 μg/g DW) and particularly elderberry wine has been found to contain higher concentrations of phenolics than red wine (1753 mg GAE/L). Also the anticarcinogenic and antioxidative effect of elderberry juice has also been attributed to the high content of anthocyanins and other flavonoid. Due to the presence of several electroactive species, more easily oxidisable in the extact, the cyclic voltammograms (CV) showed a stronger antioxidant activity as measured by DPPH, reducing power, β-carotene bleaching and inhibition of lipid peroxidation using thiobarbituric acid-reactive substances (TBARS) methods. Also, in order to quantify the electrochemical antioxidant activity of samples the authors used the differential pulse voltammogram (DPV) and compared the current density of all oxidation peaks (peak height) with that of ascorbic acid (AA), in terms of equivalents of ascorbic acid. The capability for the sample to act as oxidative protector arises from the existence of easily oxidized species (low oxidation potential) and their amount, as well as from the presence of other less oxidisable species, providing that the substance to be protected has a higher oxidation potential, expressed by the sum of AA equivalents as total electrochemical antioxidant power (TEAP) [36]. The literature data confirmed that elder fruits can serve as a good source of antioxidant principles as polyphenols in human diet and all the studied spectrum of black elder products can be regarded as good candidates for nutraceutical formulations.

4. BIOLOGIC EFFECTS AND THERAPEUTIC APPLICATIONS OF ELDERBERRIES EXTRACTS The scientific literature many studies are in agreement with the antiviral effects of elderberries extracts. The anti influenza efficacy of elderberry syrup Sambucol® has been investigated by Zakay-Rones et al., [42, 43]. In a placebo-controlled, double-blind clinical study during an outbreak of influenza B/Panama [42]. The authors observed that the treatment with elderberry syrup Sambucol® in

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a complete cure had as effects the achieving of the influenza symptoms within 2 – 3 days in nearly 90% of the elderberry-treated group compared with at least 6 days in the placebo group. The syrup formulation contained 38% of the standardized extract plus small amounts of raspberry extract, glucose, citric acid and honey. In a second study, Zakay-Rones et al., [43], investigated the efficacy and safety of a standardized elderberry extract (Sambucol®) for treating influenza A and B infections. In their experiment, sixty patients (aged 18 – 54 years) suffering from influenza-like symptoms for 48 h or less were enrolled in a randomized, double-blind, placebo-controlled study during the influenza season of 1999 – 2000 in Norway. Patients received 15 ml of elderberry or placebo syrup four times a day for 5 days, and recorded their symptoms using a visual analogue scale. The treatment efficacy was evaluated by assessing the symptoms and overall wellbeing (global evaluation). The symptoms assessed were: aches and pains, degree of coughing, frequency of coughing, quality of sleep, mucus discharge in the respiratory tract and nasal congestion. These symptoms were assessed at baseline to investigate if the two groups were clinically comparable at the start of the study. A positive correlation was found between the amount of unused medication and the information provided by each patient on the number of days the preparation was used. Symptoms were relieved on average 4 days earlier and use of rescue medication was significantly less in those patients receiving elderberry extract compared with placebo. None of the patients reported any adverse reactions related to the medication. The author‘s conclusion is that elderberry extract seems to offer an efficient, safe and cost-effective treatment for influenza. The results of study show that elderberry syrup Sambucol® is also effective against influenza A virus infections. Both studies show that the duration of the illness can be reduced by 3 – 4 days with elderberry syrup compared with placebo. A possible mechanism of action of elderberry extract in the treatment of influenza is that the flavonoids stimulate the immune system by enhancing production of cytokines by monocytes [44]. In addition, elderberry has been shown to inhibit the haemagglutination of the influenza virus and thus prevent the adhesion of the virus to the cell receptors [42]. An in vitro experiment [45] describes the antiviral effects and mechanisms against H1N1 influenza virus of an anthocyanin elderberries extract. The extract was prepared from wild crafted elderberries which were extracted using supercritical CO2 at 60 °C and 300 bar for 2 hours, followed by two extractions using ethanol:water (100 mL, 4:1, v/v) ethanol for 2 h each. The combined extracted slurry was filtered through Fisherbrand P4 filter paper and centrifuged at 537 x g for 20 min. The supernatant was vacuum distilled to remove ethanol, and the final solution concentration was about 35 mg/mL. In this optimized extract two antiinfluenza flavonoids were identified: 5,7-dihydroxy-4-oxo-2-(3,4,5trihydroxyphenyl)chroman-3-yl-3,4,5 trihydroxycyclohexanecarboxylate and 5,7,30,40-tetraO-methylquercetin. The molecular mode-of-action of these flavonoids was determined by demonstrating their direct binding to H1N1 virus particles resulting in the inability of the H1N1 viruses to enter host cells, effectively preventing H1N1 infection in vitro. This modeof-action was further verified using synthesized 5,7,30,40-tetra-O-methylquercetin and racemic dihydromyricetin which bind to H1N1 virions and, when bound, blocked H1N1 infection in vitro. Elderberries extracts are also active against human pathogenic bacteria as well as against influenza viruses A and B [46]. The quoted study used in the trials as elderberries extract the company BerryPharma AG‘s product commercially known as Rubini. Strains of S. pyogenes,

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group C and G Streptococci, and B. catarrhalis were directly isolated from patient samples and growth in standardized culture media in the presence of elderberry liquid extract added in various amounts. Also, the infected cell cultures with different influenza virus strains (the human HPAIV isolate A/Thailand/KAN-1/2004 (KAN-1, H5N1) and the human strain B/Massachusetts/71 (B/Mass)). It was shown that the standardized elderberry liquid extract possesses antimicrobial activity against both Gram-positive bacteria of Streptococcus pyogenes and group C and G Streptococci, and the Gram-negative bacterium Branhamella catarrhalis in liquid cultures. The liquid extract also displays an inhibitory effect on the propagation of human pathogenic influenza viruses. Polyphenolic elderberries extracts exhibit also inhibition of the infectious bronchitis virus at an early point during replication [47]. In this study the authors used an elderberries extract in 80% ethanol. This extract shows no-cytotoxic effects but inhibited viral replication, reducing viral titers by four to six orders of magnitude in a dose-dependent manner. The authors suppose that polyphenols are the source of this inhibition, as plants with high polyphenol concentrations often have antiviral properties. Sambucus nigra extract has been shown to inactivate two enveloped viruses, in the case of infectious bronchitis virus by compromising its membrane directly. The membranes of these two viruses are chemically distinct, with infectious bronchitis virus membranes being derived from the endoplasmic reticulum Golgi intermediate compartment, while influenza membranes are derived from the plasma membrane. These results suggest that S. nigra extract may have broad anti-viral effects against other enveloped viruses. Waknine-Grinberg et al. [48] studied the immunomodulatory effect of standardised elderberry extract on leishmanial and malarial infections. A nontoxic dose of a standardised elderberry extract was examined in murine models of leishmaniasis and malaria. The elderberry extract causes a shift in the immune response, as demonstrated in human monocyte cultures, to Th1 (inflammation-associated) responses. Treatment of leishmania-infected mice with standardised elderberry extract delayed the development of the disease. As there was no direct in vitro anti-leishmanial effect, the observed partial protection in vivo is most likely related to immune modulation. Although increased Th1 responses are associated with protection from leishmaniasis, they are considered to be the main immunopathological processes leading to cerebral malaria. Administration of standardised elderberry extract to mice prior to and following infection with Plasmodium berghei ANKA increased the incidence of cerebral malaria, while administration of standardised elderberry extract after infection had no effect on the disease. The results indicate how an inflammatory-like response may alleviate or exacerbate clinical symptoms of disease and hint at the importance of administration timing. The overall effect of depends on the ongoing immune response and the Th1/Th2 balance determined by both host and parasite defense mechanisms. Elderberry extracts have been tested for immunomodulatory activity on monocytes from healthy individuals [44]. An increase in their cytokine production was observed in vitro following stimulation. The production of inflammatory cytokines was tested using blood derived monocytes from 12 healthy human donors in vitro. Elderberry extracts and lipopolysaccharid (as a positive control for monocyte activation) were added to the monocytes and incubated. The results show an increase in secretion of proinflammatory cytokines (tumour necrosis factor-alpha and interleukins IL-1β, IL6, and IL-8), and the stimulatory activity was dose dependent. Standardized elderberry extract showed the highest cytokine stimulation.

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An experiment performed on human subjects evaluate the influence of the standardized black elderberry concentrate (Rubini) upon blood parameters related to the oxidative stress [49]. The subjects (men and women) were given 1.5 g extract daily for 10 days. Before and after the treatment the following parameters were determined: erythrocyte superoxide dismutase, catalase, plasmatic magnesium and plasma reducing power (FRAP assay). The superoxide dismutase and catalase activities showed significantly increasing after treatment in all group members. This changes in the enzymes activities suggest that anthocyanins from the elderberry extract act as molecular inducers of the self-defense mechanisms of the organism focused against hyper reactive oxygen radicals. Modern therapeutic formulations include elderberry extracts as antiiflamatory agents. An original topic product based on an elderberry extract (550-660 mg% anthocyanins) and zinc salts included in collagen membrane or collagen sponge was evaluate in vitro [50]. . The new products show a good regenerating capacity measured as growth dynamics of cells in cultures. Silver nanoparticles using black elderberry fruit extracts were synthesized and their antiinflammatory effects were evaluate [51]. The anthocyanins from elderberries were extracted with a solvent mixture (acetone-water 4:1) at room temeperature. In order to obtain a silver nanomaterial a silver salt solution was mixed and boiled with 16.6 mL fruit extract (total anthocyanin content 24 × 10−3mM). The synthesized nanoparticles presented a promising anti-inflammatory effect, investigated both in vitro and in vivo. In vitro, the anti-inflammatory effect was demonstrated by the decrease of cytokines production and by maintaining their low level after UVB irradiation. In vivo, the pre-administration of silver nanoparticles decreased the level of cytokines in the paw tissues and also presented long-term protective effect. The local treatment of psoriasis vulgaris skin lesions confirmed the good anti-inflammatory effect of silver nanoparticles, which proved to be even better than that of hydrocortisone.

5. TOXICITY The presence of small amounts of HCN developed from sambunigrin decomposition in fruit seeds is the only concern of elderberry preparations. As it was mentioned, this toxic substance is removed by heat treatment or cooking of berries/juice since it is volatile and evaporates. Non-clinical data indicate no signals of toxicological concern when the preparations are based on cooked or heat treated products. Recent studies concerning the potential of five berries (bilberry, blueberry, cranberry, elderberry, and raspberry ketones) to inhibit uridine diphospho-glucuronosyl transferase and therefore to cause clinically significant interactions with drug in current medication revealed that these five berries are unlikely to cause clinically significant herb–drug interactions mediated via inhibition of uridine diphospho-glucuronosyl transferase enzymes involved in drug metabolism. [52]. In vitro studies regarding the genotoxicity of Rubini extract were done using Allium cepa as a test organism [53]. The results lead to the conclusion that the elderberry extract have mutagenic activity, but only in very high concentrations.

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[16] Veberic R., Slatnar Ana, Bizjak J., Stampar F., Mikulic-Petkovsek Maja. (2015) Anthocyanin composition of different wild and cultivated berry species. LWT - Food Science and Technology 60 509-517 [17] Seabra Inês J.,. Braga Mara E. M, Batista Maria T. P., de Sousa H. C. (2010) Fractioned High Pressure Extraction of Anthocyaninsfrom Elderberry (Sambucus nigra L.) Pomace, Food Bioprocess Technol. 3:674–683 [18] Duymus H. G., Göger F., Hüsnü Can Baser K. (2014) In vitro antioxidant properties and anthocyanin compositions of elderberry extracts. Food Chemistry 155 112–119 [19] Ekici Lutfiye, Simsek Zeynep, Ozturk I., Sagdic O., Yetim H. (2014) Effects of Temperature, Time, and pH on the Stability of Anthocyanin Extracts: Prediction of Total Anthocyanin Content Using Nonlinear Models Food Anal. Methods 7:1328–1336 [20] http://www.rubini.com/en/rubini-elderberry/production-method.html [21] http://www.sambucolusa.com/store/aboutsambucol [22] Shiow Y. Wang, Hsin-Shan Lint (2000) Antioxidant activity in fruits and leaves of blackberry, raspberry and strawberry varies with cultivar and developmental stage, J. Agric. Food Chem. 48, 140-146 [23] Dawidowicz A.L., Wianowska D., Baraniak B. (2006) The antioxidant properties of alcoholic extracts from Sambucus nigra L. (antioxidant properties of extracts). LWT 39 308–315 [24] Kahkonen M.P., Hopia A.I., Heinonen M. (2001) Berry phenolics and their antioxidant activity. J. Agric. Food Chem. 49: 4076–4082 [25] Cejpek K., Malouskova I., Konecny M., Velisek J. (2009) Antioxidant Activity in Variously Prepared Elderberr y Foods and Supplements. Czech J. Food Sci, 27, Special Issue, 45-48 [26] Akbulut M., Ercisli S., Tosun M. (2009) Physico-chemical characteristics of some wild grown European elderberry (Sambucus nigra L.) genotypes. Phcog. Mag. 20:320–3 [27] Debasis Bagchi, Sashwati Roy, Viren Patel, Guanglong He, Savita Khanna, Navdeep Ojha, Christina Phillips, Sumona Ghosh, Manashi Bagchi and Chandan K. Sen. (2006) Safety and whole-body antioxidant potential of a novel anthocyanin-rich formulation of edible berries. Molecular and Cellular Biochemistry 281: 197–209 [28] Wu, X., Gu, L., Prior, R. L., McKay, S. (2004) Characterization of anthocyanins and proanthocyanidins in some Cultivars of Ribes, Aronia, and Sambucus and their antioxidant capacity. J. Agric. Food Chem. 52, 7846–7856 [29] Anton, A.M., Pintea A.M., Rugină D.O., Sconţa Z.M., Hanganu, D., Vlase L., Benedec D. (2013) Preliminary studies on the chemical characterization and antioxidant capacity of polyphenols from Sambucus sp. Dig. J. Nanomater Bios. Vol. 8, 3, 973 – 980 [30] Lee J., Finn C.E. (2007) Anthocyanins and other polyphenolics in American elderberry (Sambucus canadensis) and European elderberry (Sambucus nigra) cultivars. J. Sci. Food Agric. 87:2665–75 [31] Velioglu Y.S., Mazza G., Gao L., Oomah B.D. (1998) Antioxidant activity and total phenolics in selected fruits, vegetables, and grain products. J. Agric. Food Chem. 46: 4113–7 [32] Wang H., Cao G., Prior R.L. (1996) Total antioxidant capacity of fruits. J. Agric. Food Chem 44:701–5

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[33] Özgen M., Scheerens J.C., Reese R.N., Miller R.A. (2010) Total phenolic, anthocyanin contents and antioxidant capacity of selected elderberry (Sambucus canadensis L.) accessions. Pharmacogn. Mag., 6 (23): 198–203 [34] Halvorsen B.L., Carlsen M.H., Phillips K.M., Bohn S.K., Holte K., Jacobs Jr D.R. et al. (2006) Content of redox-active compounds (i.e., antioxidants) in foods consumed in the United States. Am. J. Clin. Nutr. 84:95–135 [35] Koca I, Karadeniz B. (2009) Antioxidant properties of blackberry and blueberry fruits grown in the Black Sea Region of Turkey. Sci. Hort. 121:447–50 [36] Barros L., Cabrita L.,Vilas Boas M., Carvalho A.M., Ferreira I.C.F.R. (2011) Chemical, biochemical and electrochemical assays to evaluate phytochemicals and antioxidant activity of wild plants. Food Chemistry 127 1600–1608 [37] Wu X., Beecher G.R., Holden J.M., Haytowitz D.B., Gebhardt S.E., Prior R .L. (2004) Lipophilic and hydrophilic antioxidant capacities of common foods in the U.S. Journal of Agricultural and Food Chemistry, 52: 4026–4037 [38] Benzie I.F., Strain J.J. (1996) The ferric reducing ability of plasma (FRAP) as a measure of ―antioxidant power―: The FRAP assay. Anal Biochem. 239:70–6 [39] Brand-Williams W., Cuvelier M.E., Berset C. (1995) Use of free radical method to evaluate antioxidant activity. Lebensm Wiss Technol. 28:25–30 [40] Özgen M., Reese R.N., Tulio A.Z., Miller A.R., Scheerens J.C. (2006) Modified 2,2Azino-bis-3-ethylbenzothiazoline-6-sulfonic Acid (ABTS) method to measure antioxidant capacity of selected small fruits and comparison to ferric reducing antioxidant power (FRAP) and 2,2′-diphenyl-1-picrylhydrazyl (DPPH) methods. J. Agric. Food Chem. 54:1151–7 [41] Tag, M. S., Miller, E. E., Pratt, D. E. (1984) China seeds as a source of natural lipid antioxidants. Journal of the American Oil Chemists’ Society 61, 928–931 [42] Zakay-Rones Z, Varsano N, Zlotnik M, Manor O, Regev L, Schlesinger M, et al: (1995) Inhibition of several strains of influenza virus in vitro and reduction of symptoms by an elderberry extract (Sambucus nigra L.) during an outbreak of influenza B Panama. J. Altern Complement Med; 1: 361 – 369. [43] Zakay-Rones Z, Thom E, Wollan T, Wadstein J (2004) Randomized Study of the Efficacy and Safety of Oral Elderberry Extract in the Treatment of Influenza A and B Virus Infections J. Int. Med. Res. 32, 132–140 [44] Barak V, Halperin T, Kalickman I. (2001) The effect of Sambucol, a black elderberrybased natural product, on the production of human cytokines: I. Inflammatory cytokines. Eur Cytokine Netw; 12: 290 – 296 [45] Roschek B. Jr, Fink R. C., McMichael M. D., Li Dan, Alberte R. S. (2009) Elderberry flavonoids bind to and prevent H1N1 infection in vitro Phytochemistry 70 1255–1261 [46] Krawitz C., Abu Mraheil M., Stein M., Imirzalioglu C., Domann E., Pleschka S., Hain T. (2011) Inhibitory activity of a standardized elderberry liquid extract against clinically-relevant human respiratory bacterial pathogens and influenza A and B viruses Complementary and Alternative Medicine, 11:16 [47] Chen Christie, Zuckerman D. M, Brantley Susanna, Sharpe M., Childress K., Hoiczyk E., Pendleton Amanda R (2014) Sambucus nigra extracts inhibit infectious bronchitis virus at an early point during replication BMC Veterinary Research, 10:24

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[48] Waknine-Grinberg JH, El-On J, Barak V, Barenholz Y, Golenser J (2009) The immunomodulatory effect of Sambucol on leishmanial and malarial infections Planta Med. May;75(6):581-586 [49] Bratu Mihaela Mirela, Porta S., Balaban D. P., Roncea F., Negreanu-Pirjol Ticuta, Belc M. C., Petcu L. C. (2008) Influence of an anthocyanin rich extract upon blood parameters related to oxidative stress. Archives of the Balkan Medical Union vol. 43, 4, 256-259 [50] Bratu Mihaela Mirela, Roncea F., Moldovan Lucia, Craciunescu Oana, Negreanu-Pirjol Ticuta (2009) In vitro evaluation of new topical preparation based on polyphenols, zinc and a matrix animal components Archives of the Balkan Medical Union vol. 44, 1, 3134 [51] David Luminita, Moldovan Bianca, Vulcu Adriana, Olenic Liliana, Perde-Schrepler Maria, Fischer-Fodor Eva, Florea A., Crisan Maria, Chiorean Ioana, Clichici Simona, Filip Gabriela Adriana (2014) Green synthesis, characterization and anti-inflammatory activity of silver nanoparticles using European black elderberry fruits extract Colloids and Surfaces B: Biointerfaces 122 767–777 [52] Choi E. J., Park J. B., Yoon K. D., Bae S. K. (2014) Evaluation of the in vitro/in vivo potential of five berries (bilberry, blueberry, cranberry, elderberry, and raspberry ketones) commonly used as herbal supplements to inhibit uridine diphosphoglucuronosyl transferase.. Food and Chemical Toxicology 72 13–19 [53] Bratu Mihaela Mirela, Doroftei Elena, Negreanu-Pirjol Ticuta, Hostina Corina, Porta S. (2012) Determination of Antioxidant Activity and Toxicity of Sambucus nigra Fruit Extract Using Alternative Methods Food Technol. Biotechnol. 50 (2) 177–182

In: Fruit and Pomace Extracts Editor: Jason P. Owen

ISBN: 978-1-63482-497-2 © 2015 Nova Science Publishers, Inc.

Chapter 13

TUMOR CELL GROWTH ACTIVITY OF FRUIT AND POMACE EXTRACTS Dragana Četojević-Simin Oncology Institute of Vojvodina, Faculty of Medicine, University of Novi Sad, Serbia

ABSTRACT Fruit and fruit waste by-products that are usually obtained after industrial processing should be regarded as a potential nutraceutical resource capable of offering low-cost, nutritional and health promoting dietary supplements. They can contain significant amounts of carotenoids, phenolics, flavonoids, anthocyanins and other bioactive phytochemicals that can modulate cell proliferation, oxidative reactions in cellular systems and exert excellent anti-oxidative, anti-microbial, anti-proliferative and proapoptotic activities. Fruit and fruit pomace extracts of different genotypes of tomato, pepper, raspberry, bilberry and rosehip exerted pronounced and selective tumor cell growth inhibition effects in cervix, breast and colon tumor cells. They also demonstrated favorable non-tumor/tumor cell growth ratios and increased apoptosis/necrosis ratios. These are the qualities that favor their use as healthy food and promote the development of dietary supplements on their basis. Anti-tumor activity of different fruit species, genotypes and their waste by-products was compared and discussed with regard to different extraction procedures and bioactive phytochemicals content.

INTRODUCTION Fruits are well-known sources of health-promoting compounds that comprise vitamins, minerals, and a range of different polyphenolic antioxidants, such as flavonoids and tannins (Beekwilder et al., 2005). It has been reported that small fruit crops have high contents of antioxidant compounds such as ascorbic acid, β-carotene, glutathione, α-tocopherol, 

Date of Birth: November 12th 1970, Education: B.Sc., M.Sc., Ph.D., Specialist in Medical Genetics, Address: Dr Goldmana 4, 21204 Sr. Kamenica, Serbia, e-mail: [email protected].

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anthocyanins and other phenolics. Fruits of Rosa canina L. (Rosaceae) - rosehips, are best known for their high content of vitamin C and are widely used as food. Berries are important dietary sources of fibre and micronutrients which are essential to health. They also contain a vast number of other phytochemicals for which there are no known deficiency conditions but which may have marked bioactivities and potential health benefit (Baettie et al., 2005). It has been shown that the potent antioxidant activity of these fruits is based on the high phenolic value (Rouanet et al., 2010). Berry fruits are renowned for their high concentration of bioactive compounds such as anthocyanins, flavonols, catechins, hydroxybenzoic acids etc. with demonstrated antioxidant, antimicrobial, anti-inflammatory, vasodilatory, antiproliferative and anticancer activities (Bobinaite et al., 2012) and thus considered as a top class of healthy food (Jimenez-Garcia et al., 2012). Bao et al. (2008) reported that bilberry consumption triggers genetic signalling in disease prevention and promotes human health due to biomedical activities on conditions such cardiovascular disorders, advancing age-induced oxidative stress, inflammatory responses, and diverse degenerative diseases. Raspberry (Rubus idaeus L.) is known as a rich source of dietary antioxidants such as phenolic acids (ellagic acid and its conjugates, ellagitannins - lambertianin C and sanguiin H6), flavonoids (flavan-3-ols and their oligomers, quercetin) and anthocyanins (cyanidin-3sophoroside, cyanidin-3-(2-glucosylrutinoside), cyanidin-3-glucoside, pelargonidin-3sophoroside, cyanidin-3-rutinoside, pelargonidin-3-(2-glucosylrutinoside), pelargonidin-3glucoside, pelargonidin-3-rutinoside) (Bobinaite et al., 2012). Besides phenolic compounds, raspberries contain vitamin C, dietary fibers, α-tocopherol, tocotrienol, calcium, potassium, magnesium, carotenoids, linoleic acid (Lee et al., 2012). Among antioxidant properties, raspberries have also shown other beneficial bioactivities including anti-inflammatory, antiproliferative (in human liver, breast, colon, and prostate cancer cells), anti-neurodegenerative, anti-viral, and anti-bacterial activities (Bobinaite et al., 2012). Tomatoes are one of the most widely used and versatile fruit crops. They are consumed fresh and processed into a wide range of manufactured products (De Sousa et al., 2008). Epidemiologic studies suggest that consumption of tomato and tomato-based products reduces the risk of chronic diseases such as cardiovascular disease and cancer (Willcox et al., 2003). In particular, intake of tomato and tomato-based products has been relatively consistently associated with a lower risk of cancers of the prostate, lung and stomach (Yang et al., 2013). This protective action is attributed to antioxidant components like carotenoids (in particular, lycopene and -carotene), ascorbic acid, flavonoids and tocopherols and synergistic interactions among them (Raffo et al., 2006). Lycopene is the major carotenoid present in tomatoes, accounting for >80% of the total tomato carotenoids in fully red-ripe fruits (Lenucci et al., 2006). Tomatoes also contain moderate amounts of - and -carotene and lutein (George et al., 2004). The skin and seed fractions of tomatoes have been found to be a rich source of antioxidant compounds (Toor & Savage, 2005). Thus, removal of skin and seeds of tomato during processing results in a significant loss of these antioxidants and their potential health benefits (Ćetković et al., 2012). Peppers are a good source of antioxidant bioactive compounds which are intestinally bioaccessible, particularly extractable polyphenols, β-carotene and zeaxanthin, vitamins C and E (Hervert-Hernández et al., 2010). According to Halvorsen et al. (2006) paprika is among the top 50 foods with the highest antioxidant content higher than in blueberries or red wine.

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By-products of fruit and vegetable processing represent a major disposal problem, but they are promising resources of compounds which may be used due to their favourable technological, nutritional and health promoting properties (Ćetković et al., 2008) as functional foods, nutraceuticals or cosmeceuticals, for the increase of the stability of foods by preventing lipid peroxidation, protection from oxidative damage in living systems by scavenging oxygen free radicals (Makris et al., 2007) or as "non-chemical" ingredients in pharmaceutical and cosmetic industry (Peschel et al., 2006). Multi-endpoint bioassays that are based on whole cell response of human cell lines are powerful indicators of metabolic, biochemical, and genetic alterations that arise under the influence of evaluated extracts (Ĉetojević-Simin et al. 2012). Taking into account renowned biological activity of fruits and substantial potential of their industrial fruit-processing byproducts, in this study raspberry and tomato pomace, as well as raspberry, bilberry, rosehip and paprika fruit extracts were used to determine: (1) cell growth activity in a panel of human tumor and non-tumor cell lines, (2) non-tumor/tumor IC50 ratios, (3) mechanism of induced cell death i.e. apoptosis and necrosis and (4) apoptotic increase.

EXPERIMENTAL Chemicals All chemicals and standards were purchased from Sigma Chemical Co. (St Louis, MO, USA), apart from ascorbic acid that was purchased from POCH Spólka Akcyjna (Gliwice, Poland).

Fruit and Pomace Origin and Extraction All fruits and pomaces were obtained from the producers or localities renowned for the highest quality of fruits. Two raspberry (R. idaeus L.) cultivars (Meeker and Willamette) were obtained from ″Alfa RS″, Lipolist, Serbia. Samples of the raspberry were stored at 20 °C prior to analysis. Raspberry pomace from both cultivars was obtained after juice separation. Samples of the raspberry pomaces (70 g) were extracted two times, for 60 min (560 ml) and 30 min (280 ml), at room temperature, using a homogenizer (Ultraturax, DIAX 900, Heidolph Instruments GmbH, Kelheim, Germany). The extraction was performed with 80% methanol aqueous solution containing 0.05% acetic acid (Ĉetojević-Simin et al. 2015). Fresh undamaged berries of raspberry cultivar Meeker were frozen and stored at -20ºC until use. For the freeze-drying process, the raspberry samples were frozen at -40ºC for 2h in a Martin Crist Alpha 2-4 (Osterode, Germany) lyophilizator. The main drying process was performed at p = 0.01 mbar and temperatures from -40 ºC to 20 ºC for 59.5h. The final drying step was 5.5h at p=0.005 mbar and temperature from 20 ºC to 30 ºC. Samples (20 g) were extracted at room temperature using a high performance homogenizer (Heidolph, Silent Crusher M, Kelheim, Germany). The extraction was performed on a laboratory shaker (200 rpm, Heidolph Unimax 1010, Kelheim, Germany), two times with different amounts of 80% methanol aqueous solution containing 0.05% acetic acid: 160 ml in 60 min and 80 ml for 30

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min at room temperature (Vulić et al. 2014). The obtained extracts were combined and evaporated to dryness under reduced pressure and lyophilised (Alpha 2-4 LSC Martin Christ, Osterode, Germany). Dried and ground rosehip (R. canina L.) and bilberry (Vaccinium myrtillus L.) fruits were obtained from the Research Institute for Medicinal Herbs (Panĉevo, Serbia). The rosehip has been grown in the area of Rtanj mountain (Serbia) (Tumbas Šaponjac et al. 2015) and bilberry was grown in the area of Kopaonik mountain (Serbia) (Tumbas et al. 2012). Both fruits were extracted using the same protocol. Sample of dried fruit was passed through a 0.36 mm sieve and 20 g was macerated with 500 ml of 80% acetone at room temperature during 24 h. The macerat was filtered (Whatman No.4) and the maceration was repeated once more. The two macerates were mixed and the obtained extract was evaporated to dryness under reduced pressure. In order to separate vitamin C from the phenolic antioxidants and to remove the organic acids, residual sugars, amino acids, proteins and other hydrophilic compounds as well as to exchange solvents, a clean-up by solid phase extraction (SPE) with a vacuum manifold processor (system spe-12G; J.T. Baker, Deventer, Holland) was performed according to Rigo et al. (2000) with Chromabond C-18 (1000 mg, J.T. Baker, Deventer, Holland). Extract was re-dissolved with 0.5 M H2SO4, filtered through 0.45 mm pore size membrane filters (Millipore, Bedford, MA) and slowly loaded on the Chromabond C-18 previously conditioned with 2 ml of methanol followed by 5 ml of 5 mM H2SO4. The polar substances were removed with 2 ml of 5 mM H2SO4 and this fraction, together with unretained substances in loading eluent, was marked as Fr1. The phenolic compounds were eluted with 2 ml of methanol followed by 5 ml of distilled water and this solution was considered as purified dried bilberry extract. Further, extraction and fractionation of neutral and acidic phenolics was conducted according to Chen et al. (2001). Chromabond C-18 cartridge was preconditioned for neutral flavonoids by sequentially passing 8 ml of methanol and 4 ml of distilled deionized water adjusted to pH 7.0. For acidic phenolics, cartridges were preconditioned by passing 4 ml of 0.01 M HCl instead of distilled deionized water. The purified extract was adjusted to pH 7.0 with diluted NaOH solution, loaded onto the neutral fractionating Chromabond C-18 and washed with 10 ml of pH 7.0 distilled and deionized water. The effluent portion was adjusted to pH 2.0 with 2.0 M HCl, passed through the preconditioned acidic column, and washed with 5 ml of 0.01 M HCl. The adsorbed fractions were eluted with 12 ml of methanol. Neutral fraction was labelled as Fr2 and fraction containing acidic phenolics as Fr3. The obtained three fractions were evaporated using a rotary evaporator until dryness, at 35 °C with a water bath under reduced pressure. Tomato genotypes (Baĉka, Knjaz, Novosadski niski, O2, Rutgers and Saint Pierre) grown in the fields of the Institute of Field and Vegetable Crops, Novi Sad, Serbia were taken for experimentation. Tomatoes (1 kg) of each genotype were washed and cut in four pieces and tomato juice was prepared using the juice processor Neo, SK-400. Fresh tomato waste was dried in vacuum-dryer (Alpha 2-4 LSC Martin Christ, Osterode, Germany). Samples of dried tomato waste (10 g) were (1) treated with hexane to remove non-polar compounds, then extracted with ethanol at room temperature (Stajĉić et al. 2015) and (2) extracted with hexane at room temperature, using a high performance homogenizer, Heidolph DIAX 900 (Heidolph Instruments GmbH, Kelheim, Germany). The extraction was performed three times with (1) different amounts of 80% ethanol: 160 ml in 30 min, 80 ml in 30 min, 80 ml in 15 min at room temperature and (2) with 160 ml hexane for 10 min at room temperature

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(Ćetković et al., 2012). The total extraction time was (1) 75 min and (2) 30 min. The extracts were combined and evaporated to dryness under reduced pressure. Ground paprika, ″Aleva N.K.″ variety was obtained from the Aleva a.d. company from Novi Kneževac. Soxhlet oleoresin (SX) of paprika was obtained using technical grade hexane. Ground pepper was placed into the thimble in the middle portion of the Soxhlet apparatus, the solvent was then added and the process was continued until complete discoloration of sample was achieved (Tepić et al., 2009). The solvent was evaporated from extract under vacuum. Supercritical fluid oleoresins (SF) were obtained with commercial carbon-dioxide (Tehno-gas, Novi Sad, Serbia) using a laboratory scale high-pressure extraction plant (NOVA-Swiss, Effretikon, Switzerland) at 40°C and pressures of 20 (SF20), 30 (SF30) and 40 (SF40) MPa, with the carbon-dioxide flow rate of 3.59 g min-1 (Tumbas Šaponjac et al. 2014), as previously described (Tepić et al., 2009).

Extracts and Standards Extracts were re-dissolved in DMSO to obtain 500 mg/ml stock solution for the evaluation of cell growth and cell death activity and were investigated in the concentration range from 0.005-2.5 mg/ml. Standards were diluted in DMSO, apart from ascorbic acid that were diluted in 9 mg/ml NaCl and sterilized using 0.22 m syringe filters (Sartorius, Germany). Standard solutions were investigated in the concentration range from 0.002-0.5 mg/ml.

Cell Lines Cell growth activity was evaluated in vitro in human cell lines: HeLa (cervix epitheloid carcinoma, ECACC No. 93021013), MCF7 (breast adenocarcinoma, ECACC No. 86012803), HT-29 (colon adenocarcinoma, ECACC No 91072201) and MRC-5 (human fetal lung, ECACC 84101801). Cell lines were grown in DMEM medium with 45 mg/ml glucose, supplemented with 10% heat inactivated fetal calf serum (FCS), 100 IU ml–1 of penicillin and 100 μg ml–1 of streptomycin. Cells were cultured in 25 ml flasks (Corning, New York, USA) at 37 C in the atmosphere of 5% CO2 and high humidity, and sub-cultured twice a week. A single cell suspension was obtained using 0.1% trypsin with 0.04% EDTA.

Cell Growth Activity The cell lines were harvested and plated into 96-well microtiter plates (Sarstedt, Newton, USA) at seeding density of 3–5x103 cells/well, in a volume of 199 or 180 µL, and preincubated in complete medium supplemented with 5% FCS, at 37 C for 24 h. Serial two-fold dilutions of extracts and standards in DMSO or 9 mg/ml NaCl (1 L or 20 L) were added in 199 or 180 L of medium to achieve the required final concentrations. Equal volumes of solvents were added in control wells. Concentration of DMSO in cell culture was ≤5 L/ml.

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After the addition of dilutions microplates were incubated at 37 C for 48 h. The cell growth was evaluated by the colorimetric sulphorhodamine B (SRB) assay of Skehan et al. (1990), modified by Ĉetojevic-Simin et al. (2009). Color development was measured using Multiscan Ascent (Labsystems; Helsinki, Finland) photometer at 540 nm against 620 nm as background. The effect on cell growth was calculated as 100 × (AT/AC) (%), where AT is the absorbance of the test sample and AC of the control. The concentration–cell growth (dose effect) curves were drawn for each treatment and EC50 values (concentration that inhibit cell growth by 50%) were determined using OriginPro 8 SRO (OriginLab Corporation, Northampton, USA). Non-tumor/tumor EC50 ratios (NT/T) were calculated for extracts and standards using EC50 values obtained in non-tumor cell line and in respective tumor cell line, values above 1 corresponding to favorable ratios and values below 1 corresponding to nonfavorable ratios. The results of cell growth activity were obtained in two independent experiments, each performed in quadruplicate (n=8).

Cell Death Detection Apoptosis and necrosis were detected using the Cell Death Detection ELISAPlus kit, Roche (Version 11.0), a photometric enzyme-immunoassay for the qualitative and quantitative in vitro determination of cytoplasmic histone associated DNA fragments after induced cell death. Assay is based on a quantitative sandwich enzyme immunoassay principle using mouse monoclonal antibodies directed against DNA and histones that allows specific determination of mono- and oligonucleosomes in the cytoplasmatic fraction of the cell. Cell death detection experiments were performed based on the results of cell growth experiments, using the EC50 < 100 g/ml criterion i.e. experiments were performed in all cell lines using both CB and TF pomace extracts. Extract concentrations were 8-fold multiplied EC50 concentrations (calculated based on a 48 h exposition time in cell growth experiments) due to the shorter exposition time in cell death experiments (2 h) and depending on the extract and the cell line (Ĉetojević-Simin et al. 2015). Briefly, 1 x 104 cells were seeded in a 96-well microplate and preincubated for 24 h. Extracts and negative control (solvent) were added and incubated for 2 h. At this point plate was centrifuged, cell culture supernatants pooled for each treatment and used for the evaluation of necrosis. Cells were than lysed, centrifuged, lysates pooled and evaluated for apoptosis. The cell culture supernatant (for the evaluation of necrosis) and cell lysis fraction (for the evaluation of apoptosis) both containing the cytoplasmic histone-associated DNA fragments as well as positive control were reacted with the anti-histone antibodies labeled with biotin, and anti-DNA antibodies coupled to peroxidase and incubated for 2 h. Microplate was washed, substrate of the peroxidase was added and color development was measured using Multiscan Ascent (Labsystems; Helsinki, Finland) photometer at 405 nm against 492 nm as background. Background value was subtracted from the average and enrichment factors (EF) both for apoptosis and necrosis we calculated as AT/AC, where AT is absorbance of the treatment and AC of the negative control. Apoptosis/necrosis ratios (A/N) were calculated for each treatment and negative control using absorbance values. Apoptotic increase (AI) was obtained by dividing A/N of the treatment with A/N of negative control

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(Table 4), values above 1 corresponding to favorable ratios and values below 1 corresponding to non-favorable ratios. In cell death experiments the results for treatments and negative control were obtained from pooled quadruplicates (n=4). Using these pooled quadruplicates specific enrichment of mono- and oligonucleosomes (apoptosis or necrosis) was evaluated in duplicate (n=2) according to manufacturer‘s recommendation.

RESULTS The highest antiproliferative activity was observed by raspberry pomace extracts (IC50=40-70 µg/ml) and by dried rosehip fruit and dried bilberry fruit extracts (IC50=80-190 µg/ml) towards cervix (HeLa) and breast (MCF7) carcinoma cells (Table 1). Both raspberry pomace extract (Meeker and Willamete) increased apoptosis in cervix carcinoma (HeLa) (AI=2.8-3.1), while Meeker cultivar also increased apoptosis in breast adenocarcinoma cells (MCF7) (AI=3.1) (Table 1). Raspberry fruit extract of the same cultivar (Meeker) obtained after freeze drying and using the same extraction procedure (80% methanol with 0.05% acetic acid) demonstrated moderate antitumor activity (IC50=400-720 µg/ml) towards all cell lines. Tomato pomace extracts obtained using hexane extraction demonstrated moderate activity towards cervix (HeLa) and breast (MCF7) carcinoma cells (IC50=510-680 µg/ml) but this activity was higher compared to extracts obtained by ethanol extraction (IC50=13.7-23.7 mg/ml). The antitumor activity of dried pepper fruit was low towards all cell lines (IC50=14.2-45 mg/ml). The highest antiproliferative activity towards the most resilient, colon adenocarcinoma cell line (HT-29) was observed by raspberry pomace extracts (IC50=160-190 µg/ml) and dried billberry fruit extracts (IC50=200-300 µg/ml). Non-tumor/tumor cell growth ratios (NT/T) that were calculated for all extracts and cell lines were low for the majority of examined extracts (NT/T 1) (Table 1). Favorable, high ratios were obtained in all cell lines for raspberry pomaces and fruit (NT/T=1.39-3.25), and they were highest in breast adenocarcinoma cells (MCF7) (NT/T=2.50-3.25). The highest antiproliferative activity of standard was observed by synthetic antioxidant butylated hydroxytoluene (BHT), reaching IC50 values in the range from 1.09-6.1 µg/ml in all investigated cell lines (Table 2). Very high and nonselective antiproliferative activity was also recorded for kaempferol (IC50=2.84-14.08 µg/ml), ellagic acid (IC50=2.47-25 µg/ml), butylated hydroxyanisole (BHA) (IC50=7.62-10.94 µg/ml), -carotene (IC50=10.65-21.08 µg/ml), quercetin (IC50=11.7438.91µg/ml), gallic acid (IC50=2.33-169.74 µg/ml), naringenin (IC50=9.93-102.43 µg/ml), myricetin (IC50=17.57-92.57 µg/ml), caffeic acid (IC50=19.73-274.93 µg/ml), ascorbic acid (IC50=26.69-398.60 µg/ml) and morin (IC50=54.06-216.16 µg/ml). High and selective antiproliferative activity was demonstrated by cinnamic acid in colon carcinoma cell line (HT-29) (IC50=8.74 µg/ml) and by gentisic acid in cervix carcinoma cell line (HeLa) (IC50500

1 2.07 1.17 1.13 1.18 2.87 8.40 1 0.92 1 1

500 348.20±16.70 456.46±30.73 11.10±2.96 159.71±41.66 33.93±2.49 68.70±10.83 500 12.21±0.60 500 >500

1 1.13 0.68 0.29 0.40 1.48 1.21 1 0.96 1 1

500 500 500 14.083.15 216.1654.48 92.573.62 102.4318.75 500 38.91±5.89 500 >500

1 0.79 0.62 0.23 0.30 0.54 0.81 1 0.30 1 1

500 393.16±8.97 311.22±62.76 3.20±0.11 64.01±2.68 50.39±3.94 83.40±13.59 500 11.74±2.98 500 >500

Simin et al. 2013 Simin et al. 2013 Simin et al. 2013

19.73±4.93  250 >500 2.47±0.40 >500 2.33±0.30 31.25 144.71±21.70 194.35±31.95  250 103.44±13.98 500 94.69±38.48

1.73 1 0.21 1.17 0.41 8.22 1 1.91 1.45 1 3.44 1 5.28

75.02±10.42 203.99±29.11 164.75±21.92 11.10±0.73 114.35±16.46 6.59±0.27 500 284.91±42.43 313.96±51.26 185.80±9.10 299.12±86.99 500 298.84±35.26

0.46 1.22 0.65 0.26 1.77 2.91 0.06 0.97 0.90 1.34 1.19 1 1.67

274.937.91 221.97±6.93 8.74±19.22  25 271.79±24.60 169.7413.03 152.5127.10 242.470.78 500 165.42±35.66 376.3787.89 482.1417.18 338.0563.47

0.12 1.13 12.22 0.12 0.74 0.11 0.20 1.14 0.56 1.51 0.95 1.04 1.48

34.16±1.77  250 106.80±16.86 2.89±0.91 202.69±15.43 19.16±6.12 31.25 276.19±49.19 281.43±29.35  250 355.93±82.73 500 500

Simin et al. 2013 Ĉetojević-Simin et al. 2015 Simin et al. 2013 Ĉetojević-Simin et al. 2015 -

7.87±0.056 1.11±0.14 241.13±62.18

1.39 2.71 14.74

8.23±0.40 1.09±0.28 248.61±11.12

1.33 2.76 1.58

7.620.66 6.102.33 275.5122.17

1.43 0.49 1.43

10.94±2.26 3.01±0.49 393.56±45.78

Stajĉić et al. 2015 -

26.69±5.07 13.83±10.87

14.93 1.25

32.93±1.84 10.65±1.71

12.10 1.63

98.3635.64 21.085.23

4.05 0.82

398.60±24.96 17.33±1.31

Ĉetojević-Simin et al. 2015 Tumbas Šaponjac et al. 2014

NT/T- non-tumor/tumor IC50 ratio.

References

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High NT/T ratios were recorded also for naringenin, galic acid, vanillic acid, sinapic acid and myricetin (NT/T=2.87-8.40) in cervix carcinoma (HeLa); ascorbic acid, gallic acid and butylated hydroxytoluene (BHT) (NT/T=2.76-12.10) in breast adenocarcinoma (MCF7); cinnamic and ascorbic acid (NT/T=4.05-12.22) in colon adenocarcinoma cell line (HT-29). Rutin, phloridzin, quercitrin, apigenin 7-O-glucoside and syringic acid did not show any activity in the investigated concentration range (0.005-500 µg/ml). Aniproliferative activity of all other standards was moderate to low. All extracts contained considerable amount of phenolic antioxidants and exhibited good antioxidant properties by effectively scavenging hydroxyl, superoxide anion and DPPH radicals. Majority of investigated extract (apart from ethanolic tomato pomace and paprika fruit extracts) were evaluated for their DPPH radical-scavenging activity, with EC50 values in the narrow range of concentrations - from 0.042 mg/ml (Willamette raspberry pomace and rosehip fruit, Fr 2) (Ĉetojević-Simin et al. 2015; Tumbas et al. 2012) to 0.25 mg/ml (freeze dried raspberry fruit; Vulić et al. 2014). The high anti-tumor activity was demonstrated by rosehip and billberry extracts that contained high amounts of ascorbic acid (Fr 1), quercetin, kaempferol and myricetin (Frs 2), elagic, caffeic and gallic acid (Fr 3, rosehip) and caffeic, ellagic and gallic acid (Fr 3, billberry) (Tumbas et al. 2012; Tumbas Šaponjac et al. 2015). Total phenolic, flavonoid and anthocyanin contents of the raspberry cultivar Meeker were 1.2-30 fold higher in pomace (Ĉetojević-Simin et al. 2015) than in freeze dried fruit (Vulić et al. 2014). This could explain almost 10-fold higher antitumor activity of the pomace compared to fruit (Table 1).

CONCLUSION The highest antiproliferative activity was observed by raspberry pomace extracts (IC50=40-70 µg/ml) and by dried rosehip fruit and dried bilberry fruit extracts (IC50=80-190 µg/ml) towards cervix and breast carcinoma cells. Raspberry pomace extracts increased apoptosis 3-fold in cervix carcinoma (Meeker and Willamete) and breast adenocarcinoma cells (Meeker).The highest antiproliferative activity towards the most resilient, colon

adenocarcinoma cell line was observed by raspberry pomace extracts and dried billberry fruit extracts. Non-tumor/tumor cell growth ratios were low for the majority of examined extracts. These ratios were favorable and high in all cell lines for raspberry pomaces and fruit, the highest in breast adenocarcinoma cells. Considering the fact that contemporary anti-tumor therapy relies on the higher citotoxicity of selective drugs towards tumor tissue than healthy tissue high non-tumor/tumor ratios of raspberry fruit and pomaces encourage their use in the health prevention and suggest their use in the production of nutraceuticals and food supplements. The highest antiproliferative activity of standard was observed by synthetic antioxidant BHT in all investigated cell lines. Very high and nonselective antiproliferative activity was also recorded for kaempferol, ellagic acid, BHA, -carotene, quercetin, gallic acid, naringenin, myricetin, caffeic acid, ascorbic acid and morin. High and selective antiproliferative activity was demonstrated by cinnamic acid in colon carcinoma cell line and by gentisic acid in cervix carcinoma cell line. The most favorable non-tumor/tumor cell growth ratios were obtained by ascorbic acid (14.93) and synthetic antioxidant trolox (14.74)

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in cervix carcinoma cell line. High non-tumor/tumor ratios were recorded also for naringenin, galic acid, vanillic acid, sinapic acid and myricetin (2.87-8.40) in cervix carcinoma; ascorbic acid, gallic acid and BHT (2.76-12.10) in breast adenocarcinoma; cinnamic and ascorbic acid (4.05-12.22) in colon adenocarcinoma cell line. Rutin, phloridzin, quercitrin, apigenin 7-Oglucoside and syringic acid did not show any activity in the investigated concentration range (0.005-500 µg/ml). Aniproliferative activity of all other standards was moderate to low. The high anti-tumor activity was demonstrated by rosehip and billberry extracts that contained high amounts of ascorbic acid (Fr 1), quercetin, kaempferol and myricetin (Frs 2), elagic, caffeic and gallic acid (Fr 3, rosehip) and caffeic, ellagic and gallic acid (Fr 3, billberry). Total phenolic, flavonoid and anthocyanin contents of the raspberry cultivar Meeker were 1.2-30fold higher in pomace than in freeze dried fruit, probably due to their higher concentrations in pomace. This could explain almost 10fold higher antitumor activity of the pomace compared to fruit. Considering the superb anti-tumor activity of investigated fruits, pomaces and standards it is evident that some emphasized flavonoids, phenolic acids, provitamins and vitamins in higher amounts significantly contribute to this activity. But all other investigated standards that were characterized as moderate or even low in antitumor activity most certainly contribute synergistically when in the complex mixtures with other bioactives that are habitually found in fruits and pomaces.

ACKNOWLEDGMENTS This research is part of project TR31044 financially supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia.

The Author declares no conflict of interest.

REFERENCES Bao, L., Yao, X.-S., Tsi, D., Yau, C.-C., Chia, C.eS., Nagai, H., et al. (2008). Journal of Agricultural and Food Chemistrye. Journal of Agriculture and Food Chemistry, 56, 420425. Beattie, J., Crozier, A., & Duthie, G. (2005). Potential health benefits of berries. Current Nutrition & Food Science, 1, 71-86. Beekwilder, J., Jonker, H., Meesters, P., Hall, R. D., van der Meer, I. M. and Ric de Vos, C. H.: Antioxidants in raspberry: On-line analysis links antioxidant activity to a diversity of individual metabolites. J. Agric. Food Chem. 53 (2005) 3313-3320. Bobinaite R, Viškelis P & Rimantas Venskutonis PR (2012) Variation of total phenolics, anthocyanins, ellagic acid and radical scavenging capacity in various raspberry (Rubus spp.) cultivars. Food Chemistry, 132, 1495–1501.

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Chen, H., Zuo, Y., & Deng, Y. (2001). Separation and determination of flavonoids and other phenolic compounds in cranberry juice by high-performance liquid chromatography. Journal of Chromatography, 913, 387-395. Ĉetojevic-Simin D, Svirĉev Z & Baltić V (2009) In vitro cytotoxicity of Cyanobacteria from water systems of Serbia. Journal of Balkan Union of Oncology, 14(2), 289-294. Ĉetojević-Simin D, Velićanski A, Cvetković D, Markov S, Ćetković G, Tumbas Šaponjac V, Vulić J, Ĉanadanović-Brunet J, Djilas S. (2015) Bioactivity of Meeker and Willamette raspberry (Rubus idaeus L.) pomace extracts. Food Chemistry 166: 407-413. Ĉetojević-Simin D, Velićanski A, Cvetković D, Markov S, MrĊanović J, Bogdanović V, Šolajić S. (2012) Bioactivity of Lemon Balm Kombucha. Food Bioprocess Technol 5 (5), 1756-1765. Ćetković , G., Ĉanadanović-Brunet, J., Djilas, S., Savatovic´ , S., Mandic´ , A., & Tumbas, V. (2008). Assessment of polyphenolic content and in vitro antiradical characteristics of apple pomace. Food Chemistry, 109, 340–347. Ćetković G, Savatović S, Ĉanadanović-Brunet J, Djilas S, Vulić J, Mandić A, ĈetojevićSimin D. (2012) Valorisation of phenolic composition, antioxidant and antiproliferative properties of tomato waste. Food Chemistry 133 (3), 938-945. De Sousa, A. S., Borges, S. V., Magalhăes, N. F., Ricardo, H. V., & Azevedo, A. D. (2008). Spray-dried tomato powder: Reconstitution properties and colour. Brazilian Archives of Biology and Technology, 51(4), 807–814. George, B., Kaur, C., Khurdiya, D. S., & Kapoor, H. C. (2004). Antioxidants in tomato (Lycopersium esculentum) as a function of genotype. Food Chemistry, 84, 45–51. B.L. Halvorsen, M.H. Carlsen, K.M. Phillips, S.K. Bøhn, K. Holte, D.R.Jr. Jacobs, R. Blomhoff, Am. J. Clin. Nutr. 84, 95 (2006) D. Hervert-Hernández, S.G. Sáyago-Ayerdi, I. Goñi, J. Agric. Food Chem. 58, 3399 (2010) Jimenez Garcia SN, Guevara-Gonzalez RG, Miranda-Lopez R, Feregrino-Perez AA, TorresPacheco I & Vazquez-Cruz MA (2012) Functional properties and quality characteristics of bioactive compounds in berries: Biochemistry, biotechnology, and genomics. Food Research International, http://dx.doi.org/10.1016/j.foodres.2012.11.004 Lenucci, M. S., Cadinu, D., Taurino, M., Piro, G., & Dalessandro, G. (2006). Antioxidant composition in cherry and high-pigment tomato cultivars. Journal of Agricultural and Food Chemistry, 54, 2606–2613. Makris, D. P., Boskou, G., & Andrikopoulos, N. K. (2007). Polyphenolic content and in vitro antioxidant characteristics of wine industry and other agri-food solid waste extracts. Journal of Food Composition and Analysis, 20, 125–132. Peschel, W., Sanchez-Rabaneda, F., Diekmann, W., Plescher, A., Gartzěa, I., Jim nez, D., Lamuela-Raventós, R., Buxaderas, S., & Codina, C. (2006). An industrial approach in the search of natural antioxidants from vegetable and fruit wastes. Food Chemistry, 97, 137– 150. Raffo, A., La Malfa, G., Fogliano, V., Maiani, G., & Quaglia, G. (2006). Seasonal variations in antioxidant components of cherry tomatoes (Lycopersicon esculentum cv. Naomi F1). Journal of Food Composition and Analysis, 19, 11-19. Rigo, A., Vianello, F., & Clementi, G. (2000). Contribution of proanthocyanidins to the peroxy radical scavenging capacity of some Italian red wines. Journal of Agricultural and Food Chemistry, 48, 1996-2002.

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Rouanet, J.-M., Décordé, K., Del Rio, D., Auger, C., Borges, G., Cristol, J.-P., et al. (2010). Berry juices, teas, antioxidants and the prevention of atherosclerosis in hamsters. Food Chemistry, 118, 266-271. Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica D, Warren JT, Bokesch H, Kenney S & Boyd MR (1990) New colorimetric cytotoxicity assay for anticancer-drug screening. Journal of National Cancer Institute, 82, 1107–1112. Tumbas Šaponjac, V., Ĉanadanović-Brunet, J., Ćetković, G., Djilas, S., Ĉetojević-Simin, D. (2015) Dried bilberry (Vaccinium myrtillus L.) extract fractions as antioxidants and cancer cell growth inhibitors. LWT - Food Science and Technology. ISSN: 00236438. DOI: 10.1016/j.lwt.2014.04.021 Tumbas Šaponjac, V., Ĉetojević-Simin, D., Ćetković, G., Ĉanadanović-Brunet, J., Djilas, S., Mandić, A., Tepić, A. (2014) Effect of extraction conditions of paprika oleoresins on their free radical scavenging and anticancer activity. Central European Journal of Chemistry, 12 (3), 377-385. Natasa Simin, Dejan Orcic, Dragana Cetojevic-Simin, Neda Mimica-Dukic, Goran Anackov, Ivana Beara, Dragana Mitic-Culafic, Biljana Bozin (2013) Phenolic profile, antioxidant, anti-inflammatory and cytotoxic activities of small yellow onion (Allium flavum L. subsp. flavum, Alliaceae). LWT-Food Science and Technology 54, 139-146. Stajĉić S, Ćetković G, Ĉanadanović-Brunet J, Djilas S, Mandić A, Ĉetojević-Simin D. (2015) Tomato waste: carotenoids content, antioxidant and cell growth activities. Food Chemistry 172: 225-232. Tepić A., Z. Zeković, S. Kravić, A. Mandić, CyTA – Journal of Food 7(2), 95 (2009). Toor, R. K., & Savage, G. P. (2005). Antioxidant activity in different fractions of tomatoes. Food Research International, 38, 487–494. Tumbas V, Ĉanadanović-Brunet J, Ćetković G, Djilas S, Ĉetojević-Simin D. (2014) Dried bilberry (Vaccinium myrtillus L.) extract fractions as antioxidants and cancer cell growth inhibitors. LWT-Food Science and Technology. DOI: http://dx.doi.org/ 10.1016/j.lwt.2014.04.021 Tumbas V, Ĉanadanović-Brunet J, Ĉetojević-Simin D, Ćetković G, Djilas S, Gille L. (2012) Effect of Rose hip (Rosa canina L.) phytochemicals on stable free radicals and human cancer cells. Journal of the Science of Food and Agriculture 92 (6), 1273- 1281. Vulić J. J., Velićanski S.A., Ĉetojević-Simin D.D., Tumbas Šaponjac V.V., Djilas M.S., Cvetković D.D, Markov L.S. (2014) Antioxidant, antiproliferative and antimicrobial activity of freeze-dried raspberry. Acta Periodica Technologica, 45,99-116. Willcox, J. K., Catignani, G. L., & Lazarus, S. (2003). Tomato and cardiovascular health. Critical Reviews in Food Science and Nutrition, 43, 1-18. Yang, T., Yang, X., Wang, X., Wang, Y., & Song, Z. (2013). The role of tomato products and lycopene in the prevention of gastric cancer: A meta-analysis of epidemiologic studies. Medical Hypotheses, 80, 383–388.

In: Fruit and Pomace Extracts Editor: Jason P. Owen

ISBN: 978-1-63482-497-2 © 2015 Nova Science Publishers, Inc.

Chapter 14

INFLUENCE OF TWO MATURATION STAGES AND THREE IRRIGATION REGIMES ON FATTY ACID COMPOSITION OF CV. ARBEQUINA PRODUCED UNDER TUNISIAN GROWING CONDITIONS Faten Brahmi1, Chehab Hechmi2, Imed Chraief1 and Mohamed Hammami1 Laboratory of Biochemistry, UR ‗‗Human Nutrition and Metabolic Disorder‖ Faculty of Medicine, Monastir, Tunisia 2 Institute of Olivier of Sousse, Sousse, Tunisia

1

ABSTRACT The effects of three-irrigation managements (50% evapotranspiration [ETc], 75% ETc and 100% ETc) and two-maturation degrees (maturation I and maturation II) on the fatty acid composition of fruits from olive grown in Tunisian conditions were evaluated. At maturation grade I, at the highest level of water supplied to the variety arbequina of olive produced under Tunisian growing conditions, a statistically significant decrease of oleic acid percentage (from 66.71 to 64.73%) and an increase of gadoleic and linolenic acids levels (from 0.6 to 1.53% and from 0.84 to 1.1% respectively) were observed. At the second maturation stage, an inverse trend of the fatty acids composition at the different water managements was noted for the linolenic acid. Hence, when the percentage of palmitoleic acid increased (from 2.42 to 3.16%) the percentage of oleic acid decreased (from 64.94 to 63.35%) as the amount of water supplied to the olive tree increased. These results could be due the fact that the levels of saturated, polyunsaturated, monounsaturated fatty acids and oleic to linoleic acid ratio may have undergone some changes during ripening and also to the three different amounts of water supplied to the olive tree. Therefore, we noticed that, the oleic linoleic acid ratio in the second stage of maturation increased proportionally with water managements and proportionally with maturation.



E-mail addresses: [email protected], [email protected].

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Keywords: Introduced cultivars, irrigation, Fatty acids

1. INTRODUCTION The olive tree is a crop mainly located in the Mediterranean basin, which cultivation has recently been extended outside of its traditional environment in new producer countries such as Australia, as it is a crop of great economic and social relevance. Almost all these new orchards employ different irrigation techniques in order to accelerate the rate of growth of the olive trees, diminish the characteristic alternate bearing pattern of this crop, and increase fruit yields per hectare and consequently the oil production [1]. Olive trees exhibit great adaptability to adverse soil conditions and are traditionally grown under rain feeding. Nevertheless, in Tunisia, like many Mediterranean countries, most of the newly planted olive orchards are grown under irrigation and intensive conditions, which allows the affection of plant survival and productivity, all of which affect VOO composition and the demand of the consumer. Then, to apply a successful irrigation programme to olive trees, it is of critical importance to have knowledge of their physiological responses and sensitivity as well as oil composition to different irrigation strategies at different stages of their growth cycle. Therefore, the chemical and organoleptic characteristics of olive oil depend on several factors [2]. According to Aparicio and Luna (2002) [3], these factors are clustered into four main groups: environmental (soil, climate), cultivation (ripeness, harvesting), technological (fruit storage, extraction procedure), and agronomic factors (fertilisation, irrigation). Among these factors irrigation is a major determinant of olive oil quality [4]. Among these factors irrigation is a major determinant of olive oil quality [4]. High quality olive oil cannot be obtained from fruit that have suffered a severe water stress [4]. As water supplies decrease and better quality water supplies are reserved for more sensitive crops to drought conditions than olive tree [5].In fact, studies have shown that irrigation can increase olive production [6-7,1] thereby increasing total fruit yield and oil production per tree [6].Therefore, studies differ regarding their overall performance to applied water. Chemical and sensory characteristics, however, allow distinguishes clearly between virgin olive oils from irrigated and non-irrigated olive trees [8-10]. Physical, biochemical, and physiological changes which occur during fruit development imply that intracellular variations play an important role in the distribution of different metabolites in the cells [11]. For years food analysts and plant physiologists have been interested in the effects of maturation on the chemical components in the industrial parts of fruits because of their impact in the market quality of some industrial products made with petroselinic acid derivatives. Lipid components in fruits, though occurring in minor amounts, are presumed to contribute to the development of characteristic aromas and flavours during ripening as they are considered as precursors for various volatile odorous principles of fruits [12]. Supran (1978) [13] reported that lipids contribute to the industrial and nutritional value as well as characteristic aromas and flavours. In this chapter, we evaluated the effects of three-irrigation regimes and two-maturation degrees on the fatty acid composition of fruits from olive grown in Tunisian conditions.

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2. MATERIALS AND METHODS 2.1. Plant Material and Growth Conditions Olive fruits were collected from arbequina olive (Olea europaea L.) cultivar planted in the experimental farm of Elkef in north- western of Tunisia (Latitude: 36‘‘ 18‘ N; Longitude: 09‘‘ 07‘ E; Altitude: 500 m above sea level). Elkef region is characterized by a mean annual rainfall of 450 mm, concentrated mainly from autumn to spring and an average evapotranspiration (ETc) of 1500 mm. The warmer months are July/August and the coldest are December/January with a mean annual temperature varied from 7.8°C to 28.5°C. In this olive orchard (20 ha), water was delivered three times per week at a rate of 4 h j-1 using a localized irrigation system with four drip nozzles of 4 l h-1 each per tree (two per side), set in a line along the rows at a distance of 0.5 around the trunk with a unit flow of 8 l h-1 (total flow per tree was 16 l h-1). Arbequina olive oil cultivar, was tested in a factorial combination with three irrigation levels [three plots of 36 m2 (6m x 6 m) (+ three trees per treatement, three plot of 3x 36m2) each were designed for each irrigation]: stressed (T1), moderated (T2) and well irrigation (T3) receiving a seasonal water irrigation amount equivalent to 50, 75 and 100% ETc [14]. The calculation procedures used by this model are based on the Penman–Monteith– FAO method [15] with a single estimated crop coefficient (Kc = 0.6) and a coverage coefficient (Kr = 0.5) [16] where ETc = Kr . Kc . ET0. The soil was silty (18%) with alkaline pH (8.10) and consisted of 33% calcium carbonate, 1.20% organic matter, 0.65‰ N2, 255 mg kg-1 K2O, and 6 mg kg-1 P2O6. The experimental plot was grown intensively at a planting density of 286 plants/ha and a tree spacing of 6 m x 6 m with olive oil trees of 5-year-old after planting.

2.2. Total Lipid Extraction and Fatty Acids Methylation Triplicate sub-samples of 0.5 g were extracted using the modified method of (Bligh & Dyer, 1959) [17]. Thus, fruit samples were kept in boiling water for 10 min to inactivate lipase (Douce, 1964)29 and then ground manually using a mortar and pestle. A chloroform/methanol (Analytical Reagent, LabScan, Ltd., Dublin, Ireland) mixture (1:1, v/v) was used for total lipid extraction. After washing with water and centrifugation at 3000×g for 10 min, the organic layer containing total lipids was recovered and dried under a nitrogen stream. Total fatty acids (TFA) were converted into their methyl esters using sodium methoxide solution (Sigma, Aldrich) according to the method described by (Cecchi et al., 1985) [18]. Methyl heptadecanoate (C17:0) was used as an internal standard. Those fatty acids methyl esters (FAMEs) obtained were subsequently analyzed.

2.3. Gas Chromatography The fatty acid methyl esters were analyzed on a HP 5890 gas chromatograph (Agilent Palo Alto, CA, USA) equipped with a flame ionization detector (FID). The esters were separated on a RT-2560 capillary column (100 m length, 0.25 mm i.d., 0.20 mm film

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thickness). The oven temperature was kept at 170°C for 2 min, followed by a 3°C/min ramp to 240°C and finally held there for an additional 15 min period. Nitrogen was used as carrier gas at a flow rate of 1.2 ml/min. The injector and detector temperatures were maintained at 225°C. A comparison of the retention times of the FAMEs with those of co-injected authentic standards (Analytical Reagent, LabScan, Ltd., Dublin, Ireland) was made to facilitate identification.

2.4. Statistical Analyses All extractions and determinations were conducted in triplicate. Data is expressed as mean±S.D. The means were compared by using the one-way analysis of variance (ANOVA) followed by Duncan‘s multiple range tests. The differences between individual means were deemed to be significant at p < 0.05. A principal component analysis (PCA) was performed in order to discriminate between different maturity stages and irrigation regime on the basis of their fatty acids composition.

3. RESULTS AND DISCUSSION The influence of both maturation index and irrigation regimes on fatty acid composition are reported in the Table 3.The results showed that major fatty acids for three irrigation regimes and two maturation stage were oleic, palmitic and linoleic acids. Furtheremore, the differences were statistically significant among three watering levels treatments studied of the arbequina especially in stearic and palmitoleic acids .At the two stage of maturation, the percentage of saturated fatty acids; palmitic, arachidic, behenic and lignoceric acids were not influenced by the kind of irrigation management (Table 1). Therefore, we noted that drip irrigation in the first stage of maturation decreased the SFA proportions. However, at maturation grade I, at the highest level of water supplied to the olive tree, a statistically significant increase of margaric acid percentage (from 0.13 to 0.30%) was observed. At the second maturation stage, an inverse trend of the fatty acids composition at the different water managements was noted. Hence, the percentage of margaric acid decreased (from 0.25 to 0.13%) as the amount of water supplied to the olive tree increased (Table 1).Another saturated fatty acids is the palmitic, behenic and lignoceric acids its content was barely affected by irrigation regimes. The percentage of behenic acid (C16:0) decreased from 0.52% on the first stage of maturation to 0.08% when fruit was fully ripened (Table 1). The irrigation regime and the two maturation stage not affected significantly the saturated fatty acid ratio that influences the organoleptic characteristics of the oil because oil with a high content of saturated fatty acids could influence the organoleptic characteristics of the oil. This gives rise to the defect defined as a ―fatty sensation‖ [9,19]. Therefore; the results showed that oleic acid decreased significantly (64.94%, 64.32% and 63.35%, respectively) in the fruits from the second maturation stage (T1, T2 and T3). Consequently, elicit significant changes in the oleic acid proportion. In fact, Current biochemical evidence indicates that, in olive and other plant species, the polyunsaturated fatty acids are produced by the consecutive desaturation of oleic acid. For the samples analyzed, fruits from the second maturation stage (T1, T2 and T3) had

Influence of Two Maturation Stages and Three Irrigation Regimes …

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higher contents of palmitoleic acid (2.42%, 2.79% and 3.16%, respectively); whereas the first maturation stage, showed a significantly higher content of oleic acid (Table 1). However, at full maturity, there was a negligible level of euricic acid detected. Quantitative gadoleic acid accumulation began from the third watering levels treatments studied of the arbequina (Table 1). As for arachidic acid, its rate of accumulation was lower during fruit ripening and irrigation regime. The ratio of monounsaturated fatty acids, decreased significantly (from 69.72% to 68.18%) during fruit ripening, except a higher amount detected at T2 in the second maturation stage (69.30%). Thus, the important variation was noted for linoleic acid. In fact; during fruit ripening, linoleic acid increased significantly (p < 0.05) from 11.37% on the first grade of maturation to 13.05% when fruit was fully ripened (Table 1). It was also noticed in maturation grade I, a statistically significant increase of linolenic acid percentage (from 0.84 to 1.10 %). At the second maturation stage, an inverse trend of the linolenic acid composition at the different water managements was noted (Table 1).Thus, at the last stages of maturity, the linolenic acid had the highest percentages. Gomez-Rico et al. (2007) [4] and Salas et al. (1997) [20] found that irrigation induces an increase in palmitic and linoleic acids and a decrease in oleic and linolenic acid content in virgin olive oil. Moreover, Patumi et al. (1999) [21] found that the fatty acid composition of different Italian varieties was affected mainly by cultivar and not by irrigation practices. Consequently, the major variation consisted in an increase of the polyunsaturated fatty acids and reached a highest amount (15.33%) in the second stage of maturation. This is not in agreement with the results obtained by Lakshminarayana et al. (1981) [22]. Jameison and Reid (1969) [23] and Peiretti et al. (2004) [24] also reported variations in the proportions of PUFA during the growth cycle of borage. Interest in the PUFA, as health-promoting nutrients, has expanded dramatically in recent years. A rapidly growing literature illustrates the benefits of PUFA in alleviating cardiovascular, inflammatory, heart diseases, atherosclerosis, autoimmune disorder, diabetes and other diseases [25-26]. The major variation consisted in a decrease of the monounsaturated fatty acids in favor of an increase in polyunsaturated ones. These results could be due the fact that the levels of saturated, polyunsaturated, monounsaturated fatty acids may have undergone some changes during ripening and also to the three different amounts of water supplied to the olive tree. However, the changes of oleic to linoleic acid ratio at the first stage of maturation, are very slight and do not have any nutritional relevance. Our results are in good agreement with those of Gomez-Rico et al. (2006, 2007) [8,4]. With regard to the effect produced in the fatty acid composition by the irrigation regime and maturation stage, we noticed that, the MUFA/PUFA decrease when 100% of water was used (from 5.71 to 4.54 %).As for the ratio of saturated fatty acids to unsaturated fatty acids, its rate of accumulation was almost stable during the first stage of ripening. However, at the second stage of maturation, this latter showed no significantly variation. The multivariate PCA was used to classify samples from the cultivar planted under three irrigation regimes and two maturation stages. The PCA multivariate technique, based on the values of fatty acids composition generated a score and loading plot (Figure 1a,b). In fact, along the first-dimension PC1 (accounting for 40.48% of the total variance), Arbequina 50%, at the first stage of maturation and Arbequina 75%Etc, at the second stage of maturation were discriminated, whereas along the second dimension (accounting for 22.88% of variance), Arbequina T2MI, T3MI, T1MII and T3MII, were differentiated.

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Table 1. Fatty acid composition (%) of fruits from Arbequina variety growing in the same area under three levels of irrigation and two maturation stage Fatty acid

Watering level 50% Etc 75% Etc 100% Etc 50% Etc 75% Etc 100%Etc MaturationI MaturationII Palmitic acid 14.54a 14.10a 14.72a 14.70a 14.68a 14.95a bc ab a abc bc Margaric acid 0.13 0.27 0.30 0.25 0.15 0.13c bc a ab abc cd Stearic acid 1.73 1.94 1.78 1.77 1.58 1.51d a a a a a Arachidic acid 0.86 0.98 0.60 1.14 0.79 0.83a a a a a a Behenic acid 0.52 0.34 0.21 0.24 0.14 0.08a a a a a a Lignoceric acid 0.25 0.37 0.34 0.19 0.11 0.52a SFA 18.06a 18.02a 17.97a 18.32a 17.48a 18.04a c d c bc ab Palmitoleic acid 2.05 1.57 2.28 2.42 2.79 3.16a b b ab b a Margaroleic acid 0.30 0.43 0.50 0.42 0.73 0.34b a a ab ab ab Oleic acid 66.71 66.24 64.73 64.94 64.32 63.35b a a a a a Gadoleic acid 0.60 0.92 1.53 0.71 1.35 1.21a b a b b b Euricic acid 0.04 0.66 0.11 0.17 0.10 0.10b a a ab ab ab MUFA 69.72 69.84 69.16 68.68 69.30 68.18b Linoleic acid 11.37c 11.27c 11.75bc 12.01bc 12.47ab 13.05a bc a ab abc c Linolenic acid 0.84 1.17 1.10 0.98 0.74 0.71c b b b ab ab PUFA 12 .21 12.44 12.85 12.99 13.21 15.33a a a a a ab MUFA/PUFA 5.71 5.61 5.38 5.32 5.24 4.54b a a ab ab bc O/L ratio 5.87 5.87 5.50 5.45 5.15 4.85c a a a a a UFA/SFA 4.54 4.56 4.56 4.46 4.72 4.54a T1: 50% Etc, flow of 8 l/tree h_1 (1540 l tree) being 440 m3 ha-1, T2: 75% Etc, flow of 12 l/tree h-1 (2300 l tree) being 660 m3 ha-1, T3: 100% Etc, flow of 16 L/tree h-1 (3070 l tree) being 880 m3 ha1. SFA, saturated fatty acid; UFA, unsaturated fatty acid; MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid; O/L ratio, Oleic/linoleic ratio; UFA/SFA, ratio of saturated fatty acids to unsaturated fatty acids.

Comparisons between the two PCA plots indicated that the variables palmitoleic acid, linoleic acid, polyunsaturated fatty acids, gadoleic acid, lignoceric acid, arachidic acid, linolenic acid, stearic acid, margaric acid, behenic acid and erucic acid were mainly responsible for discrimination of the Arbequina under the two treatments 75% and 100%Etc, in the first stage of maturation and Arbequina treated with 50% and 100%Etc, in the second stage of maturation, whereas the ratio of oleic to linoleic acid, ratio of monounsaturated fatty acids to polyunsaturated fatty acids, oleic acid and monounsaturated fatty acids were major contributors to the separation of Arbequina 50%, at the first stage of maturation. The variables margaroleic acid, palmitic acid and the ratio of saturated fatty acids to unsaturated fatty acids differentiated the Arbequina 75%Etc, at the second stage of maturation. The data obtained by the multivariate analysis confirm those obtained previously, showing that each sample behaves differently with respect to irrigation treatments and maturation stage. Similar investigations based on chemometric analysis showing that the genetic factor (olive cultivar) is effective in discriminating of virgin olive oil [27-28].

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Figure 1. Score plot (a) and loading plot (b) of principal component analysis applied to the data set of fatty acid composition of fruit from the olive obtained from arbequina cultivar as affected by the different irrigation regimes and two maturation stages. T1, 50% Etc; T2, 75% Etc; T3, 100% Etc; MI, Maturation I; MII, Maturation II ; C16:0, palmitic acid; C16:1, palmitoleic acid; C17:0, margaric acid; C17:1, margaroleic acid; C18:0, stearic acid; C18:1, oleic acid; C18:2, linoleic acid; C18:3, linolenic acid; C20:0, arachidic acid; C20:1, gadoleic acid; C22:0, behenic acid, C22:1, erucic acid and C24:0, lignoceric acid; SFA, saturated fatty acids; UFA, unsaturated fatty acid; UFA/SFA, ratio of saturated fatty acids to unsaturated fatty acids, MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; O ⁄ L, Oleic ⁄ Linoleic ratio.

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CONCLUSION The highest oil content was reached at full fruit ripeness for the polyunsaturated fatty acids. Percentages of fatty acids varied significantly among the two stages of maturity and the three irrigations regimes which indicate potential for selection of industrial and nutritional fatty acid profiles. Then, the multivariate analyses imply that fatty acids were influenced by the irrigation regimes and the maturation stages.

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INDEX A A. altilis, x, 121, 125, 127, 132, 133, 134, 137, 138, 139, 140 Abraham, 149, 153 accessions, 239 accounting, ix, 70, 107, 109, 116, 242, 259 acetaldehyde, 149 acetic acid, 61, 92, 149, 150, 152, 243, 247 acetone, 23, 55, 163, 173, 230, 236, 244 acetylcholine, x, 108, 114 acidic, 71, 149, 163, 244 acidity, 34, 83 acne, 212, 213, 215, 217, 222, 223 acne vulgaris, 213, 217, 222, 223 active compound, 95, 239 activity level, 213, 220 adaptability, 256 additives, vii, viii, 1, 2, 6, 21, 24, 53, 65, 70 adenine, 114 adenocarcinoma, 245, 247, 250, 251 adenosine, 215, 221 adhesion, x, 60, 87, 104, 108, 114, 150, 184, 234 adipocyte, 116 adiponectin, 202 adolescents, 217, 224 adrenal gland, 204 adults, 104, 202 advancements, xi, 145 adverse effects, 9 adverse soil conditions, 256 aerobic bacteria, 12 aetiology, 216 aflatoxin, 114 Africa, 70, 113, 212, 223 agar, 127, 128, 187 age, 213, 219, 222, 242 aggregation, 55, 59, 150, 208 agonist, 115

AIDS, xii, 211 alcohols, 54, 174, 192 aldehydes, 15, 20, 21, 54, 192 aldosterone, 204 algae, 54 alkaloids, ix, 107, 124 allergy, 216 aloe, 213, 220, 221, 223, 225, 226 alters, 42 aluminium, 35 amine(s), 9, 11, 20, 21, 24, 25, 54, 57, 63 amino, 11, 21, 146, 213, 244 amino acid(s), 146, 213, 244 ammonium, 63 amplitude, 111 amylase, 206, 214 anaerobic digestion, 49 analgesic, xi, 157, 158, 180, 200, 214, 215 androgen, 217 angiogenesis, 221 angiotensin II, 204 anomalities, 31 ANOVA, 258 anthocyanin(s), xi, xii, 17, 22, 62, 65, 71, 96, 99, 108, 110, 157, 158, 159, 160, 161, 162, 163, 164, 165, 168, 169, 180, 181, 182, 183, 184, 185, 186, 188, 209, 227, 228, 229, 230, 231, 232, 234, 236, 237, 238, 239, 240, 241, 242, 250, 251 antibiotic(s), ix, 107, 122, 128, 141, 196, 215 antibody, 215, 220, 225 anti-cancer, 91, 125, 148, 152, 200, 212 anticancer activity, 123, 125, 184, 253 anticarcinogenic, viii, 69, 71, 200, 233 antidiabetic, viii, 69, 71, 158, 181, 213, 218 antigen, 215, 220, 225 anti-inflammatory, viii, ix, xi, xii, 69, 77, 107, 113, 114, 115, 116, 123, 125, 142, 157, 158, 168, 170, 176, 181, 190, 194, 195, 198, 200, 213, 214, 215, 216, 218, 225, 227, 228, 236, 237, 240, 242, 253

266

Index

antimicrobial, vii, viii, x, xi, 1, 4, 10, 14, 18, 26, 53, 59, 60, 62, 65, 71, 74, 75, 78, 99, 108, 113, 116, 121, 122, 127, 128, 129, 134, 135, 136, 137, 139, 141, 142, 148, 151, 154, 158, 196, 197, 205, 206, 235, 242, 253 antioxidative activity, 27 antioxidative potential, 124 antipyretic, 123 antitumor, 177, 181, 185, 187, 247, 250, 251 antiviral, ix, 108, 113, 220, 233, 234, 235 apoptosis, x, xiii, 77, 108, 114, 115, 181, 197, 198, 200, 203, 204, 207, 208, 209, 210, 241, 243, 246, 247, 250 appetite, 197 apple pomace, ix, 2, 3, 25, 26, 82, 88, 91, 92, 102, 252 apples, 26 aptitude, 263 aquifers, 31 Argentina, 51, 70 arginine, 179 aromatic rings, 71, 108 ARS, 142, 205 arterial hypertension, 219 arteries, 219 Artocarpus altilis, x, xi, 121, 122, 124, 125, 126, 127, 128, 129, 130, 133, 137, 140, 141, 143 aryl hydrocarbon receptor, 186 ascorbic acid, xi, 11, 62, 65, 146, 157, 179, 180, 181, 185, 233, 241, 242, 243, 245, 247, 250, 251 Asia, xi, 21, 113, 157, 158, 159, 160, 207, 228 Asian countries, 190 aspartic acid, 179 assessment, 59, 66, 78, 117, 186 asthma, 86, 104, 124 astringent, 180, 222 atherosclerosis, 115, 218, 253, 259 atmosphere, 8, 12, 26, 34, 50, 62, 66, 67, 73, 245 atmospheric pressure, 72 atomic emission spectrometry, 179 Austria, 231, 237 authentication, 127 authenticity, 222 autoimmune disease, 213, 218 autooxidation, 6, 17 auxins, 148 avian, 174 awareness, 70

B Bacillus subtilis, 18, 131, 132, 133, 137, 139

bacteria, xi, 6, 7, 8, 14, 54, 62, 67, 70, 74, 75, 82, 91, 122, 128, 134, 145, 148, 149, 150, 152, 154, 155, 181, 196, 218, 234 bacterial colonies, 128 bacterial pathogens, 239 bacterial strains, 74, 128 bactericides, 196 bacteriocins, 151 bacterium, 217, 235 Bangladesh, 222 base, 162 basement membrane, x, 108, 115 BBB, 198 beef, vii, 1, 5, 6, 9, 10, 11, 13, 14, 15, 16, 17, 20, 21, 23, 24, 25, 26, 27, 28, 197 beet pulp, ix, 82, 88, 104, 105 beneficial effect, viii, xii, 6, 58, 60, 69, 84, 181, 198, 211, 222 benefits, ix, xi, 2, 17, 26, 48, 54, 62, 84, 85, 107, 145, 152, 157, 159, 186, 189, 200, 242, 251, 259, 263 benzene, 71, 125 benzo(a)pyrene, 200 beta-carotene, 214 beverages, 108, 116, 153, 154, 155, 190 bilberry, xii, 230, 236, 240, 241, 242, 243, 244, 247, 250, 253 bile, 215 bioactive compounds, ix, xi, 1, 17, 64, 81, 85, 96, 97, 107, 111, 112, 157, 159, 161, 163, 177, 181, 190, 191, 210, 242, 252 bioassay, 125 bioavailability, 65, 72, 109 biochemistry, 65, 182, 189 bioconversion, 119 bioflavonoids, 5 biogas, viii, 69 biological activity(s), vii, xi, 74, 84, 112, 157, 158, 168, 174, 180, 181, 188, 243 biologically active compounds, 76, 116 biomarkers, 182 biomass, 40, 49, 154 biomaterials, 111 biomedical applications, 82, 91, 102 biomolecules, 173 biopolymers, 96 biosynthesis, 64, 102, 103, 156 biotechnology, 112, 252 biotic, 197 biotin, 246 birds, 158, 159 black tea, 94 bladder incontinence, xi, 157

Index bleaching, 180, 233 bleeding, xi, 157, 216 blood, x, 85, 108, 114, 125, 146, 148, 151, 155, 176, 179, 197, 198, 202, 203, 204, 205, 210, 218, 219, 222, 225, 231, 235, 236, 240 blood pressure, x, 108, 114, 125, 146, 151, 155, 176, 204, 219, 225, 231 blood-brain barrier, 198, 210 body fat, 213, 219 body mass index (BMI), 202 body weight, 114, 115, 168, 180, 198, 203, 213, 219 bonds, 96 bone, 115, 179 bone marrow, 115 Bosnia, 183 bowel, 221 bowel obstruction, 221 brain, 176, 198, 205 Brassica rapa chinensis, x, xi, 121, 122, 124, 127, 128, 129, 130, 131, 132, 133, 134, 137, 138, 139, 140 Brazil, viii, 14, 77, 81, 82, 83, 86, 98, 107, 190 breakdown, 202 breast cancer, 125, 186, 200, 208 breast carcinoma, 172, 178, 250 bronchitis, 124, 235, 239 bulk density, vii, 29, 41, 42, 45, 46, 47, 51 burn, 40, 217, 225, 226 by-products, vii, viii, xii, 1, 2, 4, 5, 6, 8, 17, 18, 25, 29, 49, 50, 53, 54, 55, 58, 60, 61, 66, 70, 78, 81, 99, 109, 117, 241, 243

C C. albicans, x, 74, 121, 122, 128, 131, 132, 133, 139, 197 Ca2+, 204 Cairo, 222 calcium, 36, 90, 123, 179, 193, 220, 242, 257 calcium carbonate, 257 calorie, 197 cancer, xii, 84, 98, 114, 124, 142, 152, 170, 177, 178, 180, 181, 184, 190, 194, 200, 205, 207, 208, 211, 212, 213, 220, 221, 222, 231, 242, 253 cancer cells, 114, 253 cancerous cells, 220 candidates, 202, 233 capillary, 109, 186, 257 carbohydrate(s), 2, 19, 54, 72, 83, 84, 85, 87, 110, 124, 160, 173, 202, 206, 219 carbon, 34, 35, 40, 72, 77, 154, 183, 223, 245 carbon dioxide, 72, 77, 223 carbon tetrachloride, 154, 183

267

carboxyl, 89, 90 carboxylic acid(s), 71, 247 carcinogen, 198, 200 carcinogenesis, 114, 200, 201 carcinoma, 125, 158, 172, 178, 208, 245, 247, 250 cardiac diseases, ix, 108, 114 cardiac dysfunction, x, 108 cardiac output, 204 cardiomyopathy, 179, 181 cardiovascular disease(s), 84, 99, 113, 148, 190, 197, 199, 204, 206, 209, 220, 222, 242 cardiovascular disorders, xii, 181, 211, 242 carotene, 72, 83, 123, 180, 193, 198, 199, 200, 206, 209, 232, 241, 242, 247, 250 carotenoids, vii, viii, xii, 1, 4, 53, 96, 103, 113, 123, 174, 190, 193, 199, 200, 208, 231, 241, 242, 253 cascades, 198 cascara, 221 catalysis, 111 catechin, viii, 14, 57, 69, 125, 148, 207 cation, 161, 162 C-C, 41 cell culture, 114, 115, 186, 235, 245, 246 cell death, 116, 203, 243, 245, 246, 247 cell differentiation, 116 cell division, xii, 227 cell line(s), 125, 142, 170, 172, 176, 178, 184, 200, 208, 243, 245, 246, 247, 250 cell membranes, 93 cell signaling, 196 cell surface, 150 cellulose, 40, 50, 87, 88, 110, 113, 218 Central Europe, 187, 253 central nervous system, 176 ceramic, vii, viii, 29, 30, 31, 32, 35, 36, 40, 42, 46, 48, 49, 50, 51, 95 ceramic materials, vii, 29, 32, 51 cervix, xii, 172, 178, 241, 245, 247, 250 challenges, 76 cheese, 149, 154 chemical characteristics, 238 chemical properties, 87, 187 chemical structures, 116, 125, 161, 170, 171, 172, 173, 176, 177, 197 chemoprevention, 73, 208, 220 chemopreventive agent(s), ix, 107, 114, 177 chemotherapy, 220 chicken, vii, 1, 3, 4, 5, 6, 8, 9, 10, 13, 14, 15, 18, 19, 21, 24, 26, 27, 28, 96 childhood, 216 children, 15, 22, 202, 216, 224 Chile, 70

268

Index

China, xii, 70, 142, 158, 159, 187, 190, 211, 212, 239 Chinese medicine, xi, 157, 197, 208 chitosan, 104 chloroform, 23, 257 cholera, 124 cholesterol, x, 21, 85, 86, 91, 108, 115, 124, 155, 168, 177, 179, 180, 185, 203, 204, 219 choline, 214 chromatographic technique, 163 chromatography, 63, 161, 171, 173, 174, 177, 185, 186, 188 chromium, 214 chronic diseases, ix, 65, 107, 202, 242 cigarette smoke, 186 citizens, 218 citrus albedo, ix, 82, 88 Civil War, 159 classes, 22, 26, 71, 108, 160, 161, 171, 173, 190 clay minerals, 36, 39 cleavage, 113 climate(s), 30, 71, 77, 83, 208, 212, 256 climate change, 30 clinical symptoms, 86, 235 clinical trials, 204, 218 closure, 213, 218 CO2, viii, 40, 69, 72, 73, 75, 78, 230, 234, 245 cocoa, 149, 155 coconut water, xi, 145, 146, 148, 149, 150, 151, 152, 153, 154, 155 coconut water kefir, xi, 145, 149, 151, 152 Code of Federal Regulations, 84, 98, 99 coffee, 12, 73 cognition, 198 cognitive function, 198, 206 cognitive impairment, 176 coke, 35 colitis, 183 collagen, 115, 218, 221, 236 collateral, 86 Colombia, 99 colon, ix, xii, 82, 84, 91, 107, 114, 172, 176, 178, 200, 208, 221, 231, 241, 242, 245, 247, 250 colon cancer, 84, 200, 208, 231 colon carcinogenesis, ix, 107, 114 color, xii, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17, 19, 20, 21, 22, 23, 24, 25, 26, 57, 58, 59, 61, 62, 86, 158, 160, 161, 162, 192, 227, 231, 246 combined effect, 8 combustion, vii, 30, 34, 39, 40, 41, 47 commercial, viii, 3, 4, 12, 13, 14, 54, 61, 81, 82, 86, 105, 109, 117, 122, 150, 153, 158, 182, 186, 230, 245

communication, 200, 201 compaction, 41, 42 compatibility, 82, 97 compilation, 117 complement, 212, 221 complications, x, 108, 115, 202, 209, 218, 224 composites, 110 compression, vii, 29, 32, 41, 42, 43, 45, 46, 47, 111 computer, 95 condensation, 173 conductance, 94 conductivity, vii, 29, 34, 35, 46, 47, 50, 51 conflict, 251 conflict of interest, 251 congress, 67, 76, 263 conjugated dienes, 58, 124 connective tissue, 115 conservation, 30, 95, 96 conserving, 46 constipation, xii, 211, 221, 228 constituents, ix, xi, xii, 4, 40, 73, 75, 86, 107, 145, 152, 158, 169, 175, 176, 179, 182, 187, 190, 197, 211, 212, 213, 215, 218, 223, 228, 229, 262, 263 construction, 30, 31, 41, 48, 49 consumers, 2, 5, 11, 14, 30, 54, 58, 64, 70, 84, 180 consumption, xii, 12, 35, 85, 97, 115, 141, 155, 189, 231, 242 contact time, 84, 85 contamination, 152 control group, 59, 61, 62, 114, 220 cooking, 3, 4, 5, 6, 13, 16, 17, 20, 22, 24, 25, 55, 59, 236 copper, 179, 214 Cornaceae plant, xi, 157 Cornus, vi, xi, 157, 158, 159, 160, 161, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188 coronary artery disease, 219 coronary heart disease, 84, 99, 146 correlation(s), 47, 51, 56, 232 cortex, 126 cosmetic(s), ix, xii, 71, 72, 75, 76, 82, 91, 211, 212, 221, 222, 223, 243 cost, xii, 31, 54, 72, 112, 122, 217, 234, 241 cough, 86, 104, 122, 191 coughing, 197, 234 cracks, 32, 40 critical value, viii, 69 crop(s), viii, 54, 69, 104, 122, 158, 181, 190, 241, 242, 256, 257, 262 crown, 124 crystalline, 34, 36, 90

Index crystals, 123 cultivars, 105, 158, 160, 169, 171, 191, 230, 238, 243, 251, 252, 256, 263 cultivation, viii, 69, 70, 75, 83, 148, 156, 231, 256 cultivation conditions, 75, 148 culture, 74, 128, 148, 149, 150, 153, 154, 235, 246 culture media, 148, 235 cure, 148, 197, 216, 228, 234 curing process, 7 CV, 143, 233 cycles, 97 cyclooxygenase, 168, 170, 176, 180, 215, 218 cytochrome, 203 cytokines, 197, 202, 217, 234, 235, 236, 239 cytoplasm, 148 cytotoxicity, 114, 125, 170, 172, 176, 187, 197, 252, 253 Czech Republic, 169, 186

D data set, 261 deaths, 219 decay, 65 decomposition, 39, 40, 41, 112, 236 defecation, 221 defects, 32, 40, 43 defense mechanisms, 235, 236 deficiency, 242 deficit, 3 degradation, 56, 63, 72, 83, 92, 99, 110, 120 degradation process, 72 dehydration, 92, 93 Dekkera bruxellensis, 150 denaturation, 94 density values, 47 deoxyribose, 180 Department of Agriculture, 152 depolymerization, 173 deposition, 176 depth, 111, 128, 149, 152 derivatives, 110, 160, 161, 164, 169, 171, 175, 176, 183, 194, 197, 198, 202, 205, 256 dermatitis, 216 dermatology, 225 destruction, 96, 111 detectable, 14 detection, 116, 117, 118, 164, 246 detergents, 122 detoxification, 213, 220, 231 developed nations, 216 deviation, 130

269

diabetes, ix, xi, 84, 85, 86, 108, 113, 114, 115, 124, 125, 157, 158, 168, 171, 181, 187, 190, 197, 199, 201, 202, 204, 205, 207, 209, 213, 218, 220, 222, 224, 225, 226, 259 diabetic nephropathy, 158, 176, 181, 185 diabetic patients, 103, 204, 218, 223 diarrhea, 180, 202, 228 diastole, 219 diastolic blood pressure, 219 dienes, 58, 76 diet, 3, 77, 84, 91, 102, 178, 184, 196, 203, 205, 209, 221, 224, 233 dietary fiber, vii, viii, ix, 1, 3, 4, 5, 6, 8, 19, 25, 26, 28, 53, 54, 55, 58, 59, 66, 81, 83, 84, 85, 86, 101, 102, 242 diffusion, 91, 93, 94, 96, 101, 103, 110, 111, 128, 134, 137, 140, 142 digestibility, 49, 70, 72, 77 digestion, xi, 34, 84, 145, 151, 152, 182, 197, 215 digestive enzymes, 84 discrimination, 155, 260 discs, x, 121, 128 diseases, ix, xi, xii, 107, 113, 114, 116, 122, 148, 152, 157, 158, 181, 190, 197, 198, 204, 211, 219, 220, 231, 242, 259 disorder, 216, 259 distillation, 70, 110 distilled water, 17, 59, 112, 127, 128, 244 distribution, xii, 56, 119, 141, 142, 154, 156, 183, 189, 205, 256 diuretic, 158, 179, 180, 191, 215, 228 diversity, 141, 142, 154, 200, 232, 251 DNA, 148, 151, 154, 176, 190, 200, 209, 220, 246 DNA damage, 148, 176, 200 DNA sequencing, 151 docosahexaenoic acid, 64 Dogwoods, xi, 157, 158, 183 DOI, 65, 182, 185, 186, 253 donors, 235 dopamine, 77 dosage, 13, 222 down-regulation, 200 drought, 256 drug delivery, 82, 91 drug interaction, 236 drug metabolism, 236 drug resistance, 198 drugs, 122, 152, 181, 187, 202, 221, 231, 237, 250 dry matter, ix, 19, 21, 70, 82, 87, 88 drying, 6, 7, 36, 40, 92, 127, 163, 243, 247 DTA curve, 40 durability, 42, 43 dyes, 161

270

Index

dysuria, 124, 148

E E.coli, x, xi, 121, 122, 128, 130, 131, 134, 135, 137, 139, 140 Eastern Europe, 159 ECM, 176 ecology, 30, 153 economic growth, 30 eczema, 216, 223 edema, 115, 216 effluent(s), 47, 182, 187, 244 effluents, viii, 30, 32, 47 efflux transporters, 198 Egypt, xii, 211, 212 eicosapentaenoic acid, 64 elaboration, 36, 86, 97 elastin, 221 elbows, 216 elderberries, xii, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236 electric current, 95 electric field, ix, 82, 83, 93, 94, 95, 99, 101, 103, 104 electrical conductivity, 34, 93 electrodes, 95 electrolyte, 148 electromagnetic, 83 electron, 27, 35, 75 electrophoresis, 151 electroporation, 94 elucidation, 163, 174, 223 e-mail, 29, 107, 241 emission, 30 emotion, 198 employment, 31 endocrine, 115 endosperm, xi, 145 endosperm fluid, xi, 145 endothelial cells, x, 108, 114, 181, 184, 185, 221 endothelial dysfunction, 114 endothelium, x, 108, 114, 204 endothermic, 39, 40 energy, viii, ix, 31, 35, 46, 48, 50, 69, 82, 85, 93, 95, 97, 110, 111, 112, 198 energy consumption, 31 energy efficiency, 93 energy supply, 95 enlargement, 202, 217 environment, 30, 31, 48, 54, 82, 85, 93, 174, 256 environmental factors, 160, 216 environmental impact, viii, 30, 31, 47, 70, 72 environmental issues, ix, 30, 81

environmental protection, 30 enzyme(s), ix, 2, 3, 96, 98, 107, 112, 113, 114, 116, 120, 125, 143, 148, 149, 168, 170, 176, 180, 187, 200, 202, 203, 204, 205, 206, 207, 208, 209, 212, 213, 214, 215, 219, 220, 223, 231, 236, 246, 263 enzyme immunoassay, 246 enzyme inhibitors, 231 epicarp, viii, 81, 82, 85, 191 epicatechin, viii, 14, 69, 74, 76, 148 epidemiologic, 253 epidemiologic studies, 253 epidermis, 216 equilibrium, 162 equipment, 95, 97, 105 Eschericia coli, xi, 122, 142 ESI, 164, 169, 174, 175 essential fatty acids, 64 ester, xii, 88, 89, 102, 110, 123, 209, 227, 228 estrogen, 186 ethanol, 22, 23, 55, 57, 60, 62, 70, 73, 74, 75, 78, 79, 111, 115, 125, 134, 137, 140, 141, 163, 174, 181, 230, 234, 235, 244, 247 ethers, 171 ethyl acetate, 23, 125, 163, 169, 171, 173, 174, 177 Europe, xi, 70, 83, 157, 158, 160, 228, 231 European Community, 70 European Union, 30, 62 evaporation, 31, 40, 47 evapotranspiration, xiii, 255, 257, 263 evidence, 65, 72, 84, 91, 114, 125, 190, 205, 216, 225, 258 evil, 228 evolution, 7, 12, 50, 56, 262 exothermic peaks, 40 expectorant, 228 experimental condition, 64 experimental design, 59, 101 exploitation, 220 exposure, 11, 92, 93, 94, 216, 222, 226 expulsion, 215 extracellular matrix, 176, 218 extrusion, vii, 29, 32, 34, 41, 42, 43, 45, 46, 47, 50

F fabrication, 33 factories, 47 families, 212 fasting, 203, 218 fat, 1, 3, 4, 5, 6, 8, 9, 11, 14, 15, 16, 17, 19, 20, 22, 24, 28, 82, 91, 102, 146, 168, 184, 203, 209, 220, 224

271

Index fatty acids, xiii, 54, 63, 64, 72, 82, 84, 91, 98, 179, 181, 255, 257, 258, 259, 260, 261, 262, 263 FDA, 95, 104, 221 fecal impaction, 221 fermentation, 7, 54, 70, 71, 77, 93, 110, 112, 149, 150, 153 ferrite, 36, 149 fever, 158, 181, 228 fiber(s), ix, 2, 3, 5, 7, 8, 15, 19, 24, 25, 28, 58, 59, 71, 81, 82, 83, 84, 85, 87, 88, 90, 91, 99, 102, 103, 105, 221 fiber content, 3, 5, 58, 88 fibroblasts, 155 fibrosis, x, 108, 114 fillers, 5 film thickness, 258 films, 99 filters, 244, 245 filtration, 112, 231 fingerprints, 191 Finland, 118, 246 fish, viii, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 76, 77 fish oil, 63, 64, 65, 66, 76 flame, 257 flammability, 23 flatulence, 202 flavanones, ix, 107, 108, 110, 116, 118, 125, 160, 194, 198, 199, 201, 204 flavonol, xii, 22, 63, 71, 169, 192, 194, 227, 228, 232 flavor, 3, 4, 5, 8, 9, 11, 13, 14, 15, 16, 20, 22, 54, 58, 59, 60, 61, 62, 158, 159, 160, 194 flavour, 152, 263 flexibility, 230 flight, 117 flocculation, 123 flora, 122, 141 flour, 64, 104, 119 flowers, xii, 123, 158, 227, 228 fluid, xi, 72, 75, 78, 79, 97, 112, 118, 145, 148, 177, 221, 230, 245 fluid extract, 72, 78, 79, 112, 177, 230 fluorescence, 34, 56 folic acid, 214 follicles, 217 food additive, 56 Food and Drug Administration, 84, 95, 221 food industry, vii, viii, xi, 4, 6, 8, 53, 84, 85, 87, 92, 95, 97, 118, 145, 152, 163, 189 food intake, x, 108, 115, 168 food production, 186 food products, ix, 2, 6, 64, 85, 87, 107, 149 food safety, 104

food spoilage, 2, 5, 14 force, 16 formation, 5, 6, 7, 8, 9, 10, 11, 12, 17, 20, 21, 22, 24, 25, 40, 45, 55, 57, 58, 61, 63, 76, 115, 152, 197, 198, 217, 218, 219, 220, 221 formula, 88 fragments, 246 France, 70, 159 free radicals, 71, 190, 199, 202, 220, 232, 243, 253 freezing, 14, 60, 62 freshwater, viii, 53, 54 fructose, 213, 228 fruit-processing, vii, viii, 53, 54, 243 functional analysis, 104 functional food, xi, 25, 54, 58, 70, 74, 79, 85, 90, 145, 190, 202, 204, 208, 222, 243 fungi, 5, 74, 119, 128, 218 fungus, 74, 122, 134, 197 furan, 184

G gastritis, 148 gastrointestinal disorders, xi, 157, 158, 180, 181 gastrointestinal tract, 215, 228 gel, 3, 26, 88, 90, 103, 104, 151, 212, 213, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226 gel formation, 3, 88 gelling agent, ix, 81, 82, 90, 91 gene expression, 203, 218, 220 genes, 203, 224 genetic alteration, 243 genetic diversity, 187 genetics, 64 genital herpes, 225 genomics, 252 genotype, ix, 108, 204, 244, 252 genus, viii, xi, 81, 82, 109, 123, 157, 158, 189, 190, 191, 194, 208, 212, 228, 230 geographical origin, 206 Germany, 70, 156, 243, 244, 245 germination, 70 glia, 198, 199, 210 glucose, 85, 91, 98, 109, 113, 115, 149, 160, 161, 169, 173, 184, 201, 203, 207, 210, 213, 218, 220, 222, 228, 234, 245 glucose tolerance, 203 glucoside, xii, 109, 113, 117, 164, 165, 166, 167, 168, 169, 170, 183, 194, 195, 196, 197, 227, 228, 229, 232, 242, 249, 250, 251 glucosinolates, 123, 124, 141, 142 glutamate, 176, 184 glutamic acid, 179

272

Index

glutamine, 147 glutathione, 56, 114, 115, 200, 217, 219, 220, 241 glycerol, 116 glycine, 179 glycosaminoglycans, 218 glycoside, 109, 110, 112, 123, 142, 172, 176, 185, 187, 188, 192, 200, 201, 230 glycosylation, 109, 117, 160 granules, xi, 145, 151 grape, viii, 1, 9, 10, 11, 12, 13, 14, 24, 25, 26, 27, 28, 53, 54, 55, 56, 57, 58, 59, 61, 62, 64, 65, 66, 67, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 99 grape pomace, viii, 9, 11, 14, 24, 25, 28, 54, 55, 56, 57, 65, 66, 67, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 grape seeds, viii, 54, 69, 77, 78 graph, 135, 136 GRAS, 196 Greece, 109, 117, 212 growth, ix, xii, 5, 7, 9, 18, 19, 23, 59, 70, 74, 87, 108, 115, 124, 128, 134, 142, 146, 154, 155, 177, 185, 197, 220, 225, 235, 236, 241, 243, 245, 246, 247, 248, 249, 250, 253, 256, 259, 263 growth dynamics, 236 growth factor, 115, 146 guidelines, 31, 224 Guyana, 121, 122, 127, 141, 142

H hair, 192 hair cells, 192 hardness, 3, 4, 6, 21 harvesting, 88, 256 healing, 125, 212, 213, 216, 217, 226 health, vii, xi, xii, 2, 17, 53, 54, 62, 66, 70, 84, 91, 101, 102, 105, 145, 148, 149, 152, 153, 157, 159, 160, 163, 181, 186, 189, 190, 201, 204, 207, 212, 225, 231, 241, 242, 243, 250, 251, 253, 259, 263 health benefits, xi, 2, 17, 54, 62, 84, 145, 152, 157, 159, 186, 189, 242, 251, 263 health care, 105 health condition, 201 health effects, vii, xi, 91, 157, 160, 231 health promotion, 153 heart disease, 180, 219, 231, 259 heat loss, 46 heat transfer, 102 height, 191, 212, 233 Helicobacter pylori, 231 heme, 57 hemicellulose, 40, 50, 87 hemisphere, 158

hemoglobin, 65 hepatocellular carcinoma, 172 hepatocytes, 220 hepatotoxicity, 182 herbal medicine, 207 hesperetin, ix, 107, 108, 109, 112, 113, 114, 115, 116, 120 hexane, 134, 137, 139, 140, 169, 174, 177, 244, 245, 247 high blood pressure, 204 high fat, 179 histamine, xi, 54, 157, 215, 220 histidine, 179 histone(s), 246 history, 100, 212 HIV, 170, 180 HM, 77 homeostasis, 219 Hong Kong, 192 hormone(s), x, 108, 113, 114, 115, 214 host, 234, 235 House, 153 human body, 221, 231 human health, viii, ix, 30, 65, 67, 69, 83, 107, 152, 190, 206, 242 human neutrophils, 220, 223 human subjects, 204, 236 humidity, 10, 24, 73, 245 hybrid, 171 hydration, xi, 145, 212, 221 hydrocarbons, 20, 21, 192 hydrocortisone, 216, 236 hydrogen, x, 34, 35, 71, 75, 90, 96, 108, 114, 187, 190, 232 hydrogen peroxide, x, 108, 114, 187, 190 hydrolysis, 49, 91, 92, 112, 113, 120, 206, 232 hydroperoxides, 56, 58, 60, 61, 124 hydrophobicity, 73 hydroxyl, 71, 75, 112, 161, 163, 173, 181, 185, 190, 217, 250 hydroxyl groups, 71, 112, 161, 163, 173 hygiene, 60 hypercholesterolemia, 86, 191, 220 hyperemia, 216 hyperglycemia, 218, 220, 224 hyperinsulinemia, 218 hyperlipidemia, 181, 220 hypersensitivity, 217 hypertension, ix, 108, 114, 125, 204, 207, 209, 219, 222 hypertrophy, x, 108, 114 hypotensive, 114, 123 hypothalamus, 198

273

Index hypothesis, 94

I ICAM, 114 ID, 182 ideal, 85 identification, 70, 78, 124, 154, 155, 160, 177, 182, 202, 258 IL-8, 235 illumination, 197 immersion, 35 immune function, 218 immune modulation, 235 immune response, 235 immune system, 220, 234 immunity, 78, 207, 213, 220, 226 immunomodulator, 224 immunomodulatory, 235, 240 improvements, 93 in vitro, xii, 49, 57, 60, 76, 77, 110, 113, 115, 116, 119, 120, 122, 127, 128, 134, 143, 201, 203, 210, 211, 213, 218, 219, 220, 222, 223, 227, 231, 234, 235, 236, 237, 239, 240, 245, 246, 252 in vivo, ix, xii, 77, 107, 113, 114, 116, 119, 120, 211, 213, 219, 221, 222, 227, 231, 235, 236, 240 incidence, 114, 231, 235 India, xii, 153, 190, 211, 212 individuals, 213, 219, 235 induction, x, 57, 108, 114, 210, 220 induction period, 57 industrial processing, xii, 241 industrial wastes, 31, 109 industry(s), vii, viii, xi, 2, 7, 8, 14, 21, 28, 29, 30, 31, 32, 35, 46, 47, 49, 50, 54, 70, 71, 75, 76, 78, 82, 91, 95, 104, 109, 113, 118, 122, 145, 181, 211, 212, 215, 222, 230, 243, 252 infants, 216 infection, 139, 234, 235, 239 inflammasome, 218, 222 inflammation, 197, 198, 199, 207, 210, 212, 213, 214, 215, 216, 217, 231, 235 inflammatory disease, 179, 181 inflammatory responses, 197, 242 influenza, 231, 233, 234, 235, 239 influenza virus, 234, 239 ingestion, 84, 97 ingredients, viii, 2, 3, 5, 14, 15, 16, 27, 54, 57, 74, 81, 82, 85, 103, 148, 186, 204, 208, 228, 243 inhibition, ix, x, xi, xii, 18, 22, 27, 56, 57, 58, 60, 63, 74, 108, 115, 121, 122, 125, 128, 131, 134, 135, 136, 139, 140, 168, 179, 198, 200, 202, 203, 206, 210, 215, 218, 220, 233, 235, 236, 241

inhibitor, 22, 75, 186, 221 initiation, ix, 107, 114 injury(s), 77, 154, 184, 213, 216 inoculum, 128 inositol, 146 insulation, 46, 47 insulin, 98, 115, 116, 168, 179, 180, 198, 201, 202, 203, 204, 209, 213, 218, 224 insulin dependent diabetes, 218 insulin resistance, 115, 168, 180, 202, 209, 213, 218 integrity, 222 interphase, 76 intervention, 198, 223 intestine, 201 intracellular calcium, x, 108, 115 investment(s), 31, 76, 97 ion channels, 207 ionization, 257 ions, 94, 148, 149 Iowa, 209 Iran, 70 Ireland, 257, 258 iron, 18, 57, 179, 193 irradiation, 5, 6, 10, 13, 27, 111, 197 irrigation, xiii, 255, 256, 257, 258, 259, 260, 261, 262, 263 ischemia, 187, 207 Islam, 222 isoflavonoids, 108 isolation, 49, 91, 122, 124, 125, 143, 160, 163, 164, 171, 175, 177, 185, 223 isomers, 176, 177 isoprene, 174, 176 Israel, 262 issues, 30 Italy, 1, 50, 53, 69, 70, 262, 263

J Japan, 95, 158, 159, 190, 209, 212 joints, 216

K K+, 221 kaempferol, 169, 170, 186, 194, 197, 200, 206, 232, 247, 250, 251 keratin, 217 keratinocyte, 217 ketones, 54, 192, 236, 240 kidney, xi, 157, 181, 187 kinetics, 149, 155, 186

274

Index

Klebsiella pneumoniae, xi, 122, 130, 131, 134, 136, 139 knees, 216 Korea, 158, 159 kumquats, xii, 189, 190, 193, 194, 196, 197, 198, 202, 203, 209

Luo, 142, 197, 198, 207, 208 lutein, 193, 242 lycopene, 96, 242, 253 lymphatic system, 113 lymphocytes, 215 lysis, 246

L

M

lactic acid, xi, 6, 7, 8, 12, 62, 145, 149, 150, 152, 155 Lactobacillus, 62, 150, 154, 155, 156, 219, 224 lactose, 150 Latin America, 84 laxatives, 221 LC-MS, 164, 168 LDL, 124, 125, 142, 172, 180, 184, 185, 204 leaching, 94 lead, 7, 31, 42, 113, 122, 152, 197, 216, 217, 221, 236 leakage, x, 108, 115 learning, 198 leishmaniasis, 235 lesions, 115, 176, 236 life quality, 30 ligand, 203 light, 9, 57, 59, 61, 72, 74, 129, 179, 196, 204, 230 lignans, 71, 160, 171, 172, 188, 190 lignin, 40, 50, 70, 87, 185, 213 linoleic acid, xiii, 72, 232, 242, 255, 258, 259, 260, 261 linolenic acid, xiii, 54, 255, 259, 260, 261 lipid metabolism, 155, 207 lipid oxidation, 2, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 55, 56, 57, 58, 59, 60, 61, 63, 64, 65, 66, 67, 71 lipid peroxidation, 64, 114, 168, 170, 176, 180, 202, 208, 233, 243 lipid peroxides, 57 lipids, 21, 23, 56, 63, 66, 83, 86, 147, 168, 179, 180, 219, 256, 257, 263 lipolysis, 116 lipophilic compounds, viii, 69 liquid chromatography, 116, 117, 161, 163, 175, 183, 252 liquid phase, 45 liquids, 72, 100 Listeria monocytogenes, 9, 18, 26, 60, 197 liver, 10, 12, 27, 124, 154, 168, 178, 179, 180, 181, 191, 201, 203, 209, 220, 242 low-density lipoprotein, 206 LTD, 237 lumen, 84, 221 lung cancer, 77

machinery, 196 macromolecules, 91 macronutrients, 84 macrophages, ix, 107, 114, 115, 183, 206, 213, 215, 218, 220, 222 magnesium, 123, 146, 179, 193, 214, 236, 242 magnetic properties, 149 magnitude, 93, 235 Maillard reaction, 95 majority, 15, 30, 111, 194, 247, 250 malaria, 158, 185, 235 malignant cells, 201 man, 117, 228 management, x, xii, 71, 108, 115, 202, 204, 205, 207, 211, 213, 224, 225, 258, 262 Mandarin, 118, 192 manganese, 179, 214 manipulation, 4, 72 manufacturing, 35, 39, 47, 102 marine fish, 54 Mars, 141, 142 Maryland, 104, 221, 225 mass, 34, 40, 85, 91, 92, 95, 96, 109, 116, 117, 156, 163, 176, 177, 213, 219, 224 mass loss, 40 mass spectrometry, 117, 163 mast cells, 220, 225 materials, viii, 30, 31, 32, 35, 36, 40, 45, 46, 48, 71, 72, 73, 81, 82, 91, 94, 95, 99, 102, 110, 112, 113, 127, 160 matrix, 49, 72, 104, 110, 112, 149, 150, 171, 174, 240 matrixes, 187 matter, ix, 35, 40, 41, 72, 75, 82, 88, 93 Mauritius, 189, 190, 211, 212, 215, 224 MCP, 114 MCP-1, 114 measurement(s), 12, 20, 59, 61, 94, 102, 225 meat, vii, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 54, 91, 96 mechanical properties, 46, 58 media, 30, 153, 187 medical, xi, 189, 191, 219, 221, 225

275

Index medication, 234, 236 medicine, 17, 122, 125, 148, 158, 171, 181, 187, 191, 211, 212, 215, 219, 228, 237 Mediterranean, vii, 20, 25, 29, 30, 31, 47, 48, 60, 83, 256 Mediterranean countries, 30, 47, 256 melanin, 143 melanoma, 172, 176, 178 mellitus, 85, 213, 218, 224 membrane permeability, 94 membranes, 23, 94, 96, 99, 231, 235 memory, 188, 198 mesocarp, viii, 81, 82, 85, 86, 179, 191 meta-analysis, 253 Metabolic, 223, 224, 255 metabolic disorder(s), 209 metabolic disturbances, 202 metabolic pathways, 214, 215 metabolism, ix, 2, 98, 100, 107, 115, 124, 146, 203, 214, 220, 223 metabolites, ix, xii, 71, 96, 99, 107, 111, 112, 114, 118, 174, 183, 198, 227, 230, 231, 251, 256 metabolized, 112 metabolizing, 205, 219 metal ion(s), 149, 202 metals, 58 meter, 34 methanol, 23, 75, 110, 111, 112, 125, 160, 163, 168, 171, 172, 174, 177, 178, 230, 243, 244, 247, 257 methodology, 33, 76, 92, 101, 102 methyl group(s), 89, 174 Mexico, 83, 212 Miami, 208 mice, x, 108, 114, 168, 179, 183, 184, 186, 203, 205, 207, 209, 219, 220, 222, 224, 235 microbial communities, 154 micronutrients, 196, 242 microorganism(s), 14, 54, 74, 75, 96, 100, 112, 122, 128, 134, 141, 150, 151, 155, 156 microsatellites, 187 microscope, 35 microstructure(s), vii, 29, 32, 35, 41, 45, 47 microwave heating, 83, 99, 101 microwaves, 111, 112 middle lamella, 87 migration, 61 Ministry of Education, 182, 251 mitochondria, 207 mitogen, 179 mixing, vii, 29, 32, 34, 41, 42, 43, 44, 45, 46, 47, 50 MMP, 218, 226 MMP-3, 218 MMP-9, 226

model system, 11, 26 modelling, 49 models, 55, 61, 62, 198, 213, 226, 235 moderate activity, 74, 247 modifications, 87, 108 moisture, 3, 4, 7, 8, 10, 14, 16, 17, 32, 34, 50, 86, 93, 221 moisture content, 3, 10, 16, 34 mold, 6, 7, 34 molecular structure, 87, 99 molecular weight, 75, 95, 171, 173, 212 molecules, vii, viii, 53, 76, 96, 102, 111, 163, 184, 197, 198, 200, 202, 231 monocyte chemoattractant protein, x, 108, 114 monomers, 56, 89 monoterpenoids, 174 monounsaturated fatty acids, xiii, 147, 255, 259, 260, 261 Moon, 184 Moringa oleifera, x, xi, 121, 122, 123, 127, 128, 129, 130, 131, 134, 135, 136, 139, 140, 142 morphology, 222 mortality, 99, 102, 224 moulding, 32 MR, 186, 253 mRNA, x, 108, 114, 116, 197, 201, 203, 207 mucosa, 221 mucus, 234 multivariate analysis, 260 muscles, 15, 16, 57, 58, 61 mutagenesis, 200 mutation, 190 mycotoxins, ix, 107 myocardial infarction, 152 myocardial ischemia, 184 myocardium, 179 myoglobin, 8

N Na+, 201, 204, 207, 221 Na2SO4, 127 NaCl, 6, 9, 10, 245 nanoparticles, 236, 240 National Academy of Sciences, 208 national product, 30 National Research Council, 1, 53 natural compound, 12, 64, 72, 73 natural food, vii, viii, 1, 2, 27, 53, 64, 65, 101, 197, 202 natural resources, 30 necrosis, xiii, 220, 235, 241, 243, 246, 247 negative effects, 7, 31

276

Index

neolignans, 171, 172 nephropathy, 176, 218 neuralgia, 124 neuroblastoma, 176, 187, 199 neurodegeneration, 197, 210 neurodegenerative diseases, 190, 195, 197, 198, 199, 207, 209 neuroinflammation, x, 108, 115, 198, 199, 208 neurons, 77, 209 neuropathy, 218 neuroprotection, 181, 198, 199, 210 neurotoxicity, 77, 176, 209 neutral, ix, 35, 81, 87, 89, 244 New Zealand, 145 niacin, 193 nickel, 149 nicotinamide, 114 Nigeria, 222 nitrates, 5 nitric oxide, 114, 125, 197, 204, 206 nitric oxide synthase, 197, 204 nitrite, 5, 6, 7, 8, 21, 28 nitrogen, 8, 34, 61, 62, 218, 257 nitrosamines, 5 NMR, 123, 161, 163, 164, 170, 171, 173, 174, 177 nodules, 217 non-communicable diseases, ix, 108, 114 non-insulin dependent diabetes, xii, 211 non-polar, 163, 244 normal development, 179 North Africa, xii, 83, 211 North America, xi, 83, 153, 157, 158, 159, 160, 228 Norway, 234 nuclear magnetic resonance, 161, 163 nutraceutical, xii, 22, 64, 116, 118, 227, 233, 241 nutrients, xi, 93, 95, 145, 190, 193, 205, 259 nutrition, 54, 77, 97, 145, 189, 204, 209 nutritional status, 141

O obesity, x, 60, 108, 113, 115, 168, 178, 180, 181, 184, 190, 195, 197, 202, 205, 206, 218, 219, 224 oedema, x, 108 OH, 74, 75 oil, vii, 4, 7, 8, 11, 28, 29, 30, 31, 32, 35, 39, 47, 48, 49, 50, 59, 60, 61, 63, 64, 66, 72, 76, 77, 86, 104, 113, 119, 194, 196, 228, 237, 256, 258, 262, 263 oil production, vii, 29, 256, 262 oil samples, 64 oleic acid, xiii, 72, 75, 255, 258, 260, 261 oligomers, 55, 56, 57, 198, 242

olive oil, vii, viii, 29, 30, 31, 32, 34, 35, 36, 41, 47, 48, 49, 50, 61, 66, 171, 256, 257, 259, 260, 262, 263 olive oil wastewater, vii, 29, 32, 35, 36, 41 olive wastewater, vii, 29, 32, 41 operations, 93 opportunities, 4, 118 organ(s), 179, 198 organic compounds, 108, 160, 174, 176 organic matter, vii, 30, 35, 39, 40, 41, 42, 47, 50, 257 organic soils, 50 organic solvents, viii, 69, 72, 78 organism, 128, 236 ornithine, 200 osmotic pressure, 148 overproduction, 213, 218 overweight, 202, 206 ox, 4 oxalate, 123 oxidation, 5, 6, 7, 9, 10, 12, 13, 14, 16, 17, 20, 21, 22, 23, 25, 54, 56, 57, 58, 59, 61, 63, 64, 66, 76, 124, 142, 172, 180, 184, 185, 190, 203, 232 oxidation products, 12, 20, 56, 57, 58 oxidative damage, 197, 198, 231, 243 oxidative reaction, xii, 241 oxidative spoilage, vii, 1, 53 oxidative stress, x, 108, 115, 176, 185, 186, 187, 197, 198, 199, 200, 202, 208, 209, 210, 231, 236, 240, 242 oxygen, 8, 13, 27, 34, 40, 70, 71, 108, 174, 190, 206, 207, 232, 243 oysters, 96

P Pacific, 113, 153, 207 pain, 148, 181, 213, 215, 217 Pakistan, 24, 152, 155, 190, 225 Panama, 233, 239 pancreas, 215, 219 parallel, 21 parasite(s), ix, 107, 113, 185, 235 partition, 11, 23, 163, 169, 173, 174, 177 Passiflora edulis Sims f. flavicarpa Degener, viii, 81 Passiflora L., viii, 81, 82 Passifloraceae, viii, 81, 82, 83 passion fruit, viii, 81, 82, 83, 84, 85, 86, 87, 88, 91, 92, 93, 97, 98, 101, 102, 103, 104, 105 passion fruit pomace, viii, 81, 83, 86, 88, 91, 92 pathogenesis, 190, 198, 202, 216 pathogens, x, xii, 5, 9, 10, 14, 60, 85, 87, 121, 124, 128, 131, 137, 139, 140, 196, 227 pathology, 201

Index pathways, 179, 200 PCA, 258, 259, 260 PCR, 150, 151, 154, 155 peat, 50 pectin, ix, 2, 4, 81, 82, 83, 85, 88, 89, 90, 91, 92, 93, 98, 99, 100, 101, 102, 103, 104, 105, 110, 163, 228 penicillin, 122, 245 peptide(s), 148, 154, 198, 209 periodontal, 152 peripheral blood, 226 peripheral blood mononuclear cell, 226 permeability, 93, 94, 96, 101, 210, 221 permeation, 91 permittivity, 94 peroxidation, 187 peroxide, 12, 56, 57, 181, 231 peroxynitrite, 198, 207 Peru, 83 petroleum, 77 pH, 3, 4, 6, 7, 8, 10, 11, 14, 17, 22, 34, 35, 57, 61, 62, 83, 88, 90, 91, 92, 100, 148, 149, 150, 162, 163, 231, 238, 244, 257 pharmaceutical, ix, xii, 71, 74, 76, 82, 91, 109, 113, 158, 174, 181, 187, 211, 215, 219, 222, 227, 230, 243 pharmacological research, 174 pharmacology, 77 pharmacotherapy, 225 phenol, 65, 76, 185, 188 phenolic compounds, vii, viii, ix, xi, xii, 1, 7, 14, 19, 22, 23, 53, 54, 56, 65, 76, 85, 86, 107, 109, 110, 112, 116, 119, 124, 125, 157, 158, 163, 170, 173, 180, 210, 211, 214, 231, 232, 242, 244, 252 phenotype, 206 phosphate(s), 15, 16, 26, 40, 114 phosphorous, 146 phosphorus, 179, 193 physical and mechanical properties, 59 physical phenomena, 111 physical properties, 50, 51, 62, 67 physicochemical characteristics, ix, 82, 83, 91, 93 physicochemical properties, 24, 27, 83, 155, 160 Physiological, 209 physiology, 104 phytochemicals, xii, 54, 71, 123, 143, 189, 190, 193, 195, 197, 198, 199, 200, 202, 204, 206, 211, 231, 239, 241, 242, 253 phytosterols, 219, 220, 224 placebo, 86, 206, 216, 219, 225, 233, 234 placenta, 148 plant growth, ix, 92, 107 plant phenolics, ix, 107, 110, 116

277

plants, ix, x, xi, xii, 21, 48, 64, 71, 81, 85, 86, 87, 88, 89, 98, 102, 107, 109, 112, 113, 116, 119, 120, 121, 124, 129, 141, 142, 148, 157, 158, 160, 161, 162, 171, 173, 174, 176, 181, 183, 211, 212, 225, 227, 231, 235, 237, 239, 257 plaque, 216, 223 plasma membrane, 207, 235 plasma proteins, 3, 25 plasticity, vii, 29, 32, 34, 36 platinum, 95 PM, 262 pneumonia, xi, 122, 128 Poland, 156, 243 polar, 19, 73, 76, 111, 163, 198, 244 polarity, x, 73, 75, 110, 111, 112, 121, 127, 134, 163 pollination, 158 pollutants, 118, 231 pollution, 30, 35, 48 polycyclic aromatic hydrocarbon, 118 polymer(s), 71, 88, 89, 98, 104, 163, 226 polymerase, 148 polymerization, 56, 57 polyphenols, 7, 11, 19, 24, 35, 56, 57, 58, 63, 65, 66, 70, 71, 73, 76, 77, 78, 84, 118, 160, 163, 169, 181, 198, 199, 206, 229, 230, 233, 235, 237, 238, 240, 242 polysaccharide(s), ix, 81, 87, 95, 104, 120, 149, 150, 151, 179, 213, 218, 220, 223, 226 Polysaccharides, 217 polyunsaturated fat, 53, 76, 147, 258, 260, 261, 262 polyunsaturated fatty acids, 53, 76, 147, 258, 260, 261, 262 pomace extracts, vii, viii, xii, 1, 2, 11, 14, 53, 54, 57, 74, 75, 78, 241, 246, 247, 248, 250, 252 pomegranate, viii, 1, 17, 18, 19, 24, 25, 26, 27, 28, 53, 54, 60, 61, 62, 64, 65, 67 ponds, 31, 47 pools, 48 population, 84, 156, 182, 216 porosity, vii, 30, 34, 40, 42, 43, 44, 45, 46, 47 Portugal, 237 positive correlation, 234 positive interactions, 75 potassium, 4, 8, 34, 40, 146, 148, 179, 193, 204, 214, 242 potato, 3, 25, 102, 104, 209 poultry, 12, 14, 15, 22, 26, 27 pregnancy, 148 preparation, 58, 85, 149, 209, 229, 234, 240 preservation, 23, 56, 65, 98, 100, 105 preservative, vii, 1, 5, 6, 9, 10, 11, 12, 18, 61, 197 prevention, ix, 66, 73, 84, 107, 112, 115, 116, 153, 176, 187, 202, 242, 250, 253

278

Index

principal component analysis, 258, 261 principles, xii, 31, 118, 223, 227, 233, 256 probe, 35 probiotic(s), 85, 103, 149, 151, 152, 153, 155, 156 process innovation, 119 procyanidin B1, viii, 69 producers, 30, 70, 76, 229, 243 profitability, 2 pro-inflammatory, 184, 197, 202, 213, 218 project, 182, 251 proliferation, xi, xii, 31, 114, 157, 168, 176, 180, 187, 200, 201, 204, 207, 218, 241 promoter, ix, 107, 114, 200 propagation, 111, 216, 235 prophylactic, xii, 189, 190, 191, 194, 195, 200, 204, 205, 208 prophylaxis, 189 prosperity, 30 prostate cancer, 177, 184, 242 protection, xii, 10, 12, 14, 17, 19, 60, 66, 77, 176, 180, 196, 198, 199, 200, 204, 210, 212, 227, 228, 231, 235, 243 protein oxidation, 18, 19, 20, 22, 26, 206 proteinase, 3 proteins, 3, 55, 57, 59, 63, 72, 83, 84, 95, 98, 100, 110, 115, 124, 146, 148, 163, 198, 244 proteolytic enzyme, 70 Pseudomonas aeruginosa, 74 psoriasis, 213, 216, 223, 224, 236 psoriatic arthritis, 216, 224 pulp, viii, 3, 4, 17, 19, 28, 54, 81, 82, 84, 86, 88, 91, 99, 104, 105, 149, 155, 177, 190, 191, 193, 196, 200, 205, 213, 219, 221 purification, 2, 83, 91, 109, 163, 169, 174, 177, 185, 263 purity, 11, 92, 105 PVC, 11 pyridoxine, 193

Q quality of life, 213, 218 quantification, 164 quartz, 36, 39 quercetin, 21, 22, 23, 76, 123, 169, 194, 197, 200, 203, 204, 205, 206, 210, 230, 232, 242, 247, 250, 251

R race, 234 radiation, xii, 212, 216, 220, 222, 227

radicals, 108, 180, 181, 185, 217, 236, 250 rainfall, 257 ramp, 34, 258 rancid, 9, 11, 57 rash, 216 raspberry, xii, 63, 64, 65, 234, 236, 238, 240, 241, 243, 247, 250, 251, 252, 253 raw materials, viii, 30, 32, 36, 50, 51, 69, 86 RCD, 51 RE, 184, 186, 187 reaction time, 64 reactions, 40, 41, 54, 215, 225, 232, 234 reactive oxygen, 148, 201, 220, 236 reactivity, 7, 204 reading, 219 receptors, 202, 203, 234 recommendations, 204 recovery, 49, 70, 71, 76, 99, 109, 110, 113, 115, 118 recycling, 47 red wine, 54, 70, 233, 242, 252 regression, 220 regulations, 76 rehydration, 148 relaxation, 219 relevance, 256, 259 relief, 214, 217 repair, 213, 218 replication, 235, 239 requirements, 31, 35, 48 researchers, viii, 2, 30, 31, 73, 81, 88, 89, 96, 114, 174, 196 residues, viii, 2, 14, 28, 51, 64, 71, 81, 82, 87, 89, 109, 110, 112, 119 resins, 163 resistance, 83, 93, 94, 95, 122, 141, 152, 204, 219 resolution, 35 resources, 30, 31, 186, 243 response, x, 71, 75, 92, 101, 102, 108, 114, 117, 235, 243 resveratrol, viii, 69, 71, 76, 78, 171, 210 retail, 96 retention rate, 204 reticulum, 235 retinopathy, 218 reverse transcriptase, 174 RH, 143, 183 riboflavin, 193 ribosome, 237 rice husk, 50 rings, 108, 160 risk, viii, 5, 81, 84, 99, 146, 148, 168, 197, 202, 206, 208, 219, 242 RNA, 146, 148, 184

Index Romania, 157, 182, 227 room temperature, 32, 34, 39, 163, 230, 243, 244 root(s), 60, 123, 124, 125, 142, 228 routes, 215 Royal Society, 141 rubbers, 174

S S.aureus, x, 121, 122, 130, 131, 134, 135, 139 safety, vii, viii, 1, 2, 7, 20, 53, 54, 65, 66, 97, 105, 234 Salmonella, 9, 18 salts, 8, 40, 236 Sartorius, 245 saturated fat, 84, 147, 178, 258, 259, 260, 261 saturated fatty acids, 147, 258, 259, 260, 261 savings, 112 scabies, 212, 224 scanning electron microscopy, 93 scavengers, 231 science, 119 scientific publications, ix, 82, 83, 91, 93 SDS-PAGE, 61 sea level, 257 seafood, 58, 65, 66 seborrheic dermatitis, 216, 225 sebum, 217 secondary metabolism, 160 secretion, x, 108, 115, 184, 198, 202, 204, 215, 219, 221, 235 sedentary lifestyle, 218 sedimentation, 123 seed, 8, 9, 10, 11, 12, 13, 14, 17, 18, 24, 25, 26, 27, 55, 59, 60, 61, 62, 63, 64, 65, 67, 70, 71, 72, 74, 77, 99, 104, 117, 123, 160, 179, 183, 200, 242 seeding, 245 selectivity, viii, 69, 94, 110, 122, 139, 141 selenium, 193, 214 SEM micrographs, 42, 44, 45, 47 sensation, 258 sensitivity, 18, 114, 256 sepsis, 213, 218 sequencing, 151 Serbia, 182, 241, 243, 244, 245, 251, 252 serine, 179 serum, 91, 182, 203, 204, 219, 245 services, 30 shape, viii, 45, 81, 95, 159, 191 shear, 16, 24, 111 shelf life, vii, viii, 2, 5, 6, 8, 9, 10, 13, 18, 19, 24, 26, 28, 53, 54, 57, 61, 62, 63, 64, 65 shock, 176, 181

279

shortness of breath, 104 showing, xii, 30, 47, 57, 64, 84, 112, 168, 178, 181, 227, 260 shrubs, 158 Siberia, 159 side chain, 89, 104, 171 side effects, xi, 122, 145, 202, 220 signal transduction, 200 signaling pathway, 200, 202, 203, 205 signalling, 207, 242 signals, 215, 236 signs, 213 silver, 57, 65, 217, 225, 236, 240 sintering, 41 SiO2, 36, 37 skeletal muscle, 201 skeleton, 160, 171, 174 skin, xii, 10, 12, 22, 84, 125, 127, 200, 211, 212, 213, 214, 216, 217, 221, 222, 223, 226, 228, 236, 242 skin diseases, 228 Slovakia, 182 sludge, 49 small intestine, 82, 91, 115, 203 SNP, 114 society, 211 sodium, 8, 10, 14, 15, 21, 28, 114, 148, 179, 193, 214, 257 Solanum melongena, x, xi, 121, 122, 124, 127, 130, 134, 142 solid phase, 163, 244 solid state, 110 solid waste, 31, 48, 49, 50, 54, 252 solidification, 40 solubility, 10, 58, 73, 78, 110, 112 solution, 19, 28, 31, 32, 47, 48, 56, 63, 91, 103, 127, 149, 171, 220, 222, 234, 236, 243, 244, 245, 257 solvents, x, 23, 72, 73, 110, 111, 121, 127, 163, 169, 174, 230, 244, 245 South Africa, 70, 155, 190 South America, 113, 121 South Asia, 190 South Korea, 190 soybeans, 112 soymilk, 154 SP, 107, 188 Spain, 29, 30, 31, 32, 70 specifications, 87 spectral techniques, 123 spectroscopic techniques, 123 spectroscopy, 173 sponge, 236

280

Index

stability, 3, 4, 6, 7, 8, 9, 10, 12, 13, 17, 19, 20, 21, 24, 26, 28, 58, 60, 61, 64, 66, 149, 185, 231, 243, 262 stabilization, 2, 3 Staphyloccocus aureus, xi, 122 starch, 3, 25, 88, 98 state(s), x, 32, 4, 44, 101, 108, 110, 112, 127, 158, 190 steel, 95, 109 sterile, 128 steroids, 124, 177, 214 sterols, 185 stimulant, xii, 124, 215, 227 stimulation, 116, 212, 215, 220, 235 stock, 245 stomach, 176, 228, 242 stomata, 212 storage, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 22, 24, 25, 26, 28, 31, 48, 54, 55, 56, 57, 58, 59, 61, 62, 65, 66, 67, 74, 77, 87, 88, 99, 148, 150, 256 Streptozotocin, x, 108, 115 stress, x, 58, 75, 108, 197, 198, 202, 221, 256 stroke, 155, 199, 208 structural changes, 54 structure, ix, 26, 62, 75, 81, 83, 87, 88, 89, 90, 96, 100, 102, 110, 123, 125, 150, 154, 160, 161, 163, 168, 173, 179, 194, 195, 199, 204, 221, 223, 229, 237 style, 5, 7, 152 styrene, 109, 194 subgroups, 108, 160 substitutes, 196 substitution, 43, 47 substrate, 112, 246 sucrose, 4, 148 sugar beet, 101, 104, 105 sulfur, 34 sulfuric acid, 92 sulphur, 218 Sun, 117, 119, 141, 185, 187, 224 supercritical fluids, viii, 69, 72, 73, 77, 78 supplementation, ix, 85, 107, 114, 198, 206 suppression, 198, 199, 210, 217 surface properties, 105 survival, 150, 188, 198, 204, 256 susceptibility, xi, 23, 111, 122, 128, 139 suspensions, 105 sustainability, 30 sustainable development, 31 swelling, 59, 215 Switzerland, 207, 245 symptoms, 86, 216, 234, 239

synaptic plasticity, 198 synergistic effect, 196 synthesis, ix, 107, 115, 116, 194, 240 systolic blood pressure, 204, 206, 219

T Taiwan, 206, 208, 210 tannins, 4, 17, 19, 110, 124, 160, 173, 181, 183, 185, 190, 241 target, 73, 112, 202, 203, 218, 224 techniques, 32, 41, 65, 76, 110, 112, 141, 150, 163, 169, 170, 177, 223, 230, 232, 233, 256 technology(s), viii, ix, 2, 31, 50, 69, 73, 82, 83, 89, 93, 94, 95, 96, 97, 103, 104, 105, 110, 111, 112, 116, 118 temperature, viii, ix, 3, 34, 39, 40, 50, 51, 59, 61, 69, 72, 82, 83, 90, 91, 92, 94, 95, 97, 101, 104, 110, 185, 198, 230, 231, 232, 243, 244, 257, 258 tension(s), 40, 204 terpenes, 174 textbook(s), 211, 225 textural character, 58 texture, 3, 4, 6, 7, 13, 15, 17, 20, 21, 25, 59, 60, 62, 86, 87, 97 TGA, 34, 39, 40 Thailand, 226, 235 therapeutic agents, xii, 209, 227 therapeutic effects, xii, 181, 212, 227 therapeutic use, xii, 181, 211 therapy, 113, 176, 181, 201, 216, 250 thermal analysis, 34, 50, 99 thermal conductivity, vii, 29, 35, 46, 47, 51 thermal decomposition, 40 thermal degradation, ix, 40, 82, 95 thermal properties, 30 thermal resistance, 149 thermal stability, 22 thiamin, 193 thickening agents, 2 threonine, 179 thymus, 215 TIMP, 218 tissue, 20, 21, 24, 82, 87, 91, 93, 94, 101, 102, 110, 114, 218, 250 tissue engineering, 82, 91 TNF, 115, 116 TNF-α, 115, 116 tobacco, 105, 237 tocopherols, 1, 64, 72, 242 tonic, xi, 157, 158, 180, 191 total cholesterol, 21, 203 toxic effect, 114

281

Index toxicity, viii, 23, 69, 153, 172, 184, 187, 198 traditions, 228 traits, 10 transcription, 115, 116, 197, 200, 201, 203, 220 transcription factors, 116, 200, 203 transducer, 95, 197 transformation, 39, 186 transforming growth factor, 115, 187 transition metal, 108 transmission, 35 transport, 146 trial, 35, 58, 206, 216, 218, 223, 225, 226 trifluoroacetic acid, 163 triggers, 242 trypsin, 245 tumor(s), xi, xii, 78, 113, 114, 151, 157, 168, 176, 177, 178, 180, 187, 190, 195, 200, 201, 220, 241, 243, 246, 247, 248, 249, 250, 251 tumor cells, xii, 177, 178, 180, 201, 241 tumor necrosis factor, 220 tumorigenesis, 213, 220 tumours, 220 turbulence, 111 Turkey, 14, 15, 67, 70, 153, 156, 159, 186, 239 type 2 diabetes, 84, 209 tyramine, 63, 124 Tyrosine, 147

vapor, 72 variables, 117, 219, 260 variations, 86, 154, 180, 252, 256, 259 varieties, 14, 21, 28, 74, 76, 77, 83, 148, 196, 259 vascular system, 114 vasodilation, x, 108, 114 vegetables, 24, 84, 87, 91, 95, 96, 98, 99, 100, 108, 116, 117, 122, 124, 141, 143, 190, 231, 238 vein, 114, 184 Venezuela, 212 vessels, x, 108, 115 viral infection, 228 virus infection, 234 virus replication, ix, 108 viruses, 181, 218, 220, 225, 234, 235, 239 viscosity, 45, 85 vitamin A, 83, 123, 193, 214 vitamin B6, 123 vitamin C, 1, 4, 19, 83, 99, 103, 123, 146, 159, 179, 193, 202, 208, 242, 244 Vitamin C, 83, 146, 193 vitamin D, 215 vitamin E, 12, 56, 181 vitamins, vii, viii, 1, 53, 73, 83, 93, 95, 148, 190, 193, 212, 213, 214, 215, 228, 231, 233, 241, 242, 251

W U Ukraine, 159 ulcer, 212, 213, 226 ultrasound, 96, 98, 105, 109, 110, 111, 112, 118, 119, 174, 177 United, 70, 84, 96, 118, 152, 159, 187, 208, 212, 239 United States, 70, 84, 96, 118, 152, 159, 187, 208, 212, 239 urban, 49 urea, 218 urokinase, 221 USA, 24, 159, 185, 205, 209, 243, 245, 246, 257 USDA, 146, 152, 193, 205, 209 uterine bleeding, xi, 157 UV, 75, 160, 163, 164, 174, 177, 197, 213, 216, 225 UV radiation, 75 UVB irradiation, 236

V vacuum, 8, 12, 13, 14, 15, 16, 27, 28, 63, 234, 244, 245 valorization, viii, 31, 47, 48, 81, 82

Washington, 98, 100, 263 waste, vii, viii, xii, 2, 29, 30, 31, 32, 34, 35, 39, 40, 41, 42, 43, 44, 47, 48, 49, 50, 69, 70, 76, 82, 85, 103, 109, 241, 244, 252, 253, 262 waste management, 30, 76 waste water, 48, 49, 109, 262 wastewater, vii, 29, 31, 32, 34, 35, 36, 39, 41, 42, 47, 48, 49, 50, 262 water absorption, vii, 29, 42, 45, 47, 221 water purification, 123 water supplies, 256 water vapor, 40 weight loss, 39, 40, 41, 85 wellness, xii, 211, 222 wells, 245 wheeze, 104 wheezing, 86 wildlife, 159 wine making process, viii, 69 wine production, viii, 69, 70, 71 workers, 168, 169, 171, 173, 177 World Health Organization (WHO), 202, 210 World War I, 159 worldwide, vii, viii, 1, 53, 69, 76, 109, 201, 218

282

Index

worms, ix, 108, 113 wound healing, 82, 91, 214, 218, 222

yield, ix, 3, 4, 10, 12, 16, 17, 24, 32, 55, 59, 74, 75, 82, 83, 89, 91, 92, 93, 96, 105, 111, 112, 117, 129, 134, 173, 181, 256, 262

X Z X-ray analysis, 88 XRD, 36, 37

zinc, 179, 193, 214, 236, 240 ZnO, 37

Y yeast, xi, 6, 7, 14, 145, 149, 150, 151