Fruit and Pomace Extracts

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
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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
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CONTENTS
Preface vii
Chapter 1 Fruit and Pomace Extracts: Applications to Improve the Safety
and Quality of Meat Products 1
P. G. Peiretti and F. Gai
Chapter 2 Valorization of Liquid Effluents from Olive Oil Extraction Activity
in the Production of Ceramic Bricks: Influence of
Conformation Process 29
D. Eliche-Quesada and F. A. Corpas-Iglesias
Chapter 3 Fruit and Pomace Extracts: Applications to Improve the Safety
and Quality of Fish Products 53
F. Gai and P. G. Peiretti
Chapter 4 Supercritical Fluid Extraction of Pharmaceutic Compounds from
Waste Materials Derived from Vinification Processes 69
Cleofe Palocci and Laura Chronopoulou
Chapter 5 Passion Fruit Pomace Powder: Potential Applications of
Emerging Technologies for Extraction of Pectin 81
Cibele Freitas de Oliveira and Poliana Deyse Gurak
Chapter 6 Hesperetin: Simple Natural Compound with Multiple
Biological Activity 107
José Valdo Madeira Junior, Vânia Mayumi Nakajima,
Fabiano Jares Contesini, Camilo Barroso Teixeira,
Juliana Alves Macedo and Gabriela Alves Macedo
Chapter 7 A Review of the Antimicrobial Activity of Various Solvent Type
Extracts from SOME Fruits and Edible Plants 121
R. C. Jagessar, N. Ramchartar and O. Spencer
Chapter 8 Coconut Water: An Essential Health Drink in Both Natural
and Fermented Forms 145
Mansi Jayantikumar Limbad, Noemi Gutierrez-Maddox
and Nazimah Hamid
vi Contents
Chapter 9 Extraction, Characterization and Potential Health Benefits

of Bioactive Compounds from Selected Cornus Fruits 157
Luminiţa David and Bianca Moldovan
Chapter 10 Kumquat (Fortunella Spp.): Biochemical Composition and
Prophylactic Actions 189
Theeshan Bahorun, Darshini Narrain, Piteesha Ramlagan
and Chandra Tatsha Bholah
Chapter 11 Aloe Vera Extracts: From Traditional Uses to Modern Medicine 211
Taukoorah Urmeela and Mahomoodally Mohamad Fawzi
Chapter 12 Elderberries Extracts: Biologic Effects, Applications for Therapy:
A Review 227
Mihaela Mirela Bratu and Ticuta Negreanu-Pirjol
Chapter 13 Tumor Cell Growth Activity of Fruit and Pomace Extracts 241
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 255
Faten Brahmi, Chehab Hechmi, Imed Chraief
and Mohamed Hammami
Index 265
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 by-
products 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.
viii Jason P. Owen
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, trans-
resveratrol 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
Preface ix
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 (15-
20% 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
x Jason P. Owen
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
Preface xi
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-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.
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, 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.
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
xii Jason P. Owen
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 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 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
Preface xiii
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.
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 ISBN: 978-1-63482-497-2
Editor: Jason P. Owen © 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: francesco.gai@ispa.cnr.it
2 P. G. Peiretti and F. Gai
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 González-
Aguilar, 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 (Viuda-
Martos 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
Fruit and Pomace Extracts 3
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 non-
meat 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 (freeze-
dried 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 Type of ingredient Impact on product Reference

Pork meat batters Apple fiber Behave as an inert filler & Fernández-Martín et
Increase hardness al., 2000
Mutton nuggets Apple pomace Reduce hardness, texture, flavor & Huda et al., 2014
overall acceptability scores
Pork meat Apple powder Improve gel hardness & may Lantto et al., 2006
homogenate contain suitable
enzyme activities for food protein
stabilization
Raw pork sausages Apple puree Together with plum, decrease fat & Nuñez de Gonzalez et
increased moisture al., 2008a
Low fat chicken Apple pulp Increase dietary fiber content, Verma et al., 2010
nuggets redness,
yellowness & chroma index
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
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 physico-
chemical, 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 (González-
Aguilar 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
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 (Viuda-
Martos 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 by-
products (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

Ground beef meat Citrus by-products Increase nutritive value, preserve Abd El-Khalek &
& extend shelf life Zahran, 2013
Dry-cured Raw & cooked lemon Decrease nitrite levels & delay Alesón-Carbonell et al.,
sausages albedo oxidation development 2003, 2004
Beef burger Lemon albedo Improve cooking properties & Alesón-Carbonell et al.,
increase fiber content 2005
Raw ground goat Kinnow rind powder Antioxidant effect Devatkal & Naveena,
meat 2010
Goat meat patties Kinnow rind powder Antioxidant effect Devatkal et al., 2010
Swedish-style Orange & lemon Control rancidity & off-flavor Fernández-López et al.,
meatballs extracts development 2005
Dry-cured Orange dietary fiber Decrease residual nitrite levels Fernández-López et al.,
sausages 2007
Dry-fermented Orange dietary fiber Decrease residual nitrite levels & Fernández-López et al.,
sausages favor micrococcus growth 2008
Fresh ground Citrus extract Slight preservative effect Mexis et al., 2012
chicken
Dry-cured sausage Lemon albedo & Reduce nitrite levels, thus Viuda-Martos et al.,
& Bologna orange dietary fiber avoiding the formation of 2009
sausage nitrosamines & nitrosamides
Bologna sausage Orange dietary fiber Improve shelf life of meat Viuda-Martos et al.,
products 2010a,b
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 by-
products 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 by-
products 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 (Alesón-
Carbonell et al., 2004). These authors concluded that the addition of lemon albedo to dry-
cured 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
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 radical-
scavenging 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 water-
soluble 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.
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
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

Cooked ground beef Grape seed extract Improve oxidative stability & Ahn et al., 2002
reduce warmed-over flavor
development
Cooked ground beef Grape seed extract Positive effect on microbial Ahn et al., 2007
growth, color change & lipid
oxidation
Raw beef patties Grape seed extract Increase shelf life Bañón et al., 2007
Ground chicken thigh Grape seed extract Inhibit TBARS formation & Brannan, 2008
meat mitigate the prooxidative effects
of NaCl
Ground chicken thigh Grape seed extract Inhibit intensity of musty & Brannan, 2009
& breast rancid odor, & rancid flavor
Cooked pork patties Grape seed extract Decrease lipid oxidation Carpenter et al., 2007
Pork burger Red grape pomace Increase color stability & Garrido et al., 2011
extract acceptability & decrease lipid
oxidation
Fried beef patties Grape seed extract Inhibit formation of heterocyclic Gibis & Weiss, 2012
amines
Pre-cooked, frozen, Grape seed extract Protect against oxidation & Kulkarni et al., 2011
re-heated beef retain fresh odor & flavor longer
sausage
Ground dark turkey Grape seed extract Inhibit development of TBARS Lau & King, 2003
meat
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
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

Pork patties Grape extract Increase the quality & extend the Lorenzo et al., 2014
shelf-life
Cooked turkey breast Grape seed extract Improve oxidative stability Mielnik et al., 2006
meat during heat treatment & storage
Cooked pork patties Grape skin Provide partial protection against Nissen et al., 2004
lipid oxidation
Pig liver pâté Grape seed extract Reduce the oxidative Pateiro et al., 2014
deterioration of lipid
Irradiated & non- Grape seed extract Prevent & minimize major Rababah et al., 2005
irradiated chicken sensory changes during
breast meat irradiation
Irradiated & non- Grape seed extract Decrease the amount of TBARS, Rababah et al., 2006
irradiated chicken hexanal
breast meat & pentanal values
Baladi Goat Meats Grape seed extract Minimize lipid oxidation Rababah et al., 2011
Cooked beef & pork Grape seed extract Reduce oxidative rancidity & Rojas & Brewer, 2007
patties improve shelf life
Raw beef & pork Grape seed extract Provide minimal protection Rojas & Brewer, 2008
patties against oxidation
Beef patties Grape pomace Inhibit some foodborne Sağdıç et al., 2011
extract pathogens
Raw & cooked Grape seed & peel Prevent lipid oxidation & alter Selani et al., 2011
chicken meat extracts color of cooked meat
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
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-3,8-
dimethylimidazo[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 pre-
cooked, 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.
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:
Coffee<Tea<Grape skin< Rosemary. In particular, rosemary extract at a sensorily acceptable
level was found to yield efficient protection, as demonstrated both by sensory evaluation and
chemical analyses, and thus displayed potential for maintaining sensory eating quality in
processed pork products. Other plant extracts tested, including grape skin, were found to
provide less and insufficient protection to obtain an acceptable product for consumption.
Pateiro et al., (2014) studied the effect of adding grape seed, tea, and chestnut extracts as
natural antioxidants on the physicochemical and oxidative stability of refrigerated stored pig
pâtés at 0, 4, 8 and 24 weeks of refrigerated storage. They compared this effect with that of
the synthetic antioxidant BHT. Antioxidant extract and storage period affected color
parameters. In particular, grape seed extract addition showed less total color difference, while
with the other extracts color changes could not be linked to oxidative processes or at least
could be affected by initial extract color. Pig liver pâté with grape seed and tea extracts
showed lower total color difference between 0 and 24 weeks. Furthermore, they found that
samples added with natural antioxidants showed a decrease both in TBARS index and in the
evolution of peroxide index, marking an improvement over the results obtained with BHT, so
they concluded that it would be advisable to replace the synthetic antioxidant with these
natural extracts.
Rababah et al., (2005) evaluated the effectiveness of commercial grape seed extract (3000
ppm), green tea extract (3000 ppm), and a combination of the two (6000 ppm) on the sensory
evaluation of irradiated chicken breasts (at a dosage of 3.0 kGy) using instrumental,
descriptive, and consumer tests. The results showed that infusing plant extracts into skinless,
boneless chicken breast meats could be an effective technique for minimizing undesirable
changes in sensory properties during irradiation, which increases texture attributes, though
does not affect sensory flavor attributes apart from giving the meat a brothy flavor. These
authors concluded that the infusion of chicken meat with green tea extract is an effective
method for enhancing the sensory changes caused by irradiation, while the addition of grape
seed extract increased the darkness and redness of the meat samples. Rababah et al., (2006)
also investigated the effect of irradiation on volatile compounds and TBARS contents in raw
and cooked non-irradiated and irradiated chicken breast meat infused with green tea and
grape seed extracts and stored at 5°C for 12 d. They found that irradiation increased hexanal
and TBARS values both of meat infused with plant extracts and controls, while cooking the
samples increased the amounts of TBARS and volatiles. They concluded that though
irradiation increased lipid oxidation, infusion of chicken meat with plant extracts could
reduce the lipid oxidation caused by irradiation because the addition of plant extracts
decreased the amount of TBARS as well as hexanal and pentanal values in comparison with
un-infused meat. Finally, Rababah et al., (2011) evaluated the effect of commercial grape
seed or green tea in combination with synthetic tert methyl-butylhydroquinone (TBHQ) at
different concentrations on lipid oxidation and the redness of goat meats stored at 5°C for 3,
6, and 9 days. They found that the infusion of goat meat with TBHQ and these plant extracts
is an effective method for minimizing lipid oxidation caused by storage; in particular, TBHQ
was the most effective antioxidant at retarding lipid oxidation in goat meat, while grape seed
and green tea extracts/combinations at a higher level (6000 ppm) were more effective than at
a lower level (3000 ppm).
Rojas & Brewer (2007) determined the effect of grape seed extract (0.01% and 0.02%),
oleoresin rosemary (0.02%) and water-soluble oregano extract (0.02%) mixed with salt (2%)
on oxidative and color stability in cooked beef and pork patties stored at 4°C for 2, 4, 6, and 8
d. The higher grape seed extract concentration resulted in the best antioxidant activity in both
beef and pork and did not affect instrumental color measures of redness, yellowness, or color
intensity, and appeared to reduce visual green discoloration in beef patties. Therefore, grape
seed extract at 0.02% has the potential to reduce oxidative rancidity and improve shelf life in
refrigerated cooked beef and pork patties. Rojas & Brewer (2008) also studied the effect of
grape seed extract, oleoresin rosemary and water-soluble oregano extract mixed with salt on
the oxidative and color stability of raw beef and pork patties, vacuum packaged and stored
frozen at -18°C for 1, 2, 3 and 4 months. Lipid oxidation was assessed using TBARS and
descriptive sensory evaluation and varied little between different extracts, while minimal
oxidation occurred, probably because the product had not been precooked; therefore little, if
any, lipid oxidation would have been initiated, and the product was vacuum packaged, which
excluded oxygen, thus limiting the progress of any oxidative rancidity that may have been
initiated. Moreover, the authors stated that the concentrations of polyphenolics in these
extracts varied substantially from those used in other studies. Grape seed extract addition
provided small degrees of protection against oxidation in both meat species and did not alter
the sensory perception of oxidation, redness, yellowness or color intensity.
Sağdıç et al., (2011) compared the antimicrobial effects against food spoilage
microorganisms, yeast and moulds and lipolytic bacteria and against foodborne pathogens,
coliform bacteria and Enterobacteriaceae of grape pomace ethanolic extracts obtained from 5
different grape varieties grown in Turkey (Emir, Gamay, Kalecik Karası, Narince, and
Öküzgözü). The extracts were concentrated, incorporated into beef patties at 0%, 1%, 2%,
5%, and 10% concentrations and stored in the refrigerator (4°C) for 2, 12, 24, and 48 h.
During the storage period, microorganism numbers generally decreased in proportion to
extract concentration and all the microorganisms tested were inhibited by the extract
concentration of 10%. Furthermore, foodborne pathogens and spoilage microorganisms were
also inhibited by 5% of Emir (white grape cultivar), Gamay (red grape cultivar), and Kalecik
Karası (red grape cultivar) varieties in beef patties. The authors concluded that environmental
and agricultural factors can also influence the composition of grape pomace extracts and this
should be taken into account when classifying grape cultivar extracts in respect of their
antimicrobial effectiveness.
Selani et al., (2011) studied the effect of grape seed and peel extracts obtained from two
different grape varieties (Isabel and Niagara) grown in Brazil on pH, lipid oxidation, color
and sensory properties of raw and cooked processed chicken meat stored at −18°C for nine
months. Neither extracts altered the pH values of raw and cooked samples or the color of raw
samples, but they led to alterations in the color of the cooked product (darkening and lower
intensity of red and yellow color). In the sensory evaluation, only the Niagara variety
interfered with the natural chicken meat flavor and odor. These two grape residue extracts,
that showed considerable amounts of total phenolic compounds, containing the flavonoids
catechin and epicatechin as major compounds, were effective in inhibiting the lipid oxidation
of meat, with results comparable to a synthetic antioxidant (0.01% BHT) or a commercial
mixture of sodium erythorbate, citric acid and sugar. These authors stated that the use of
residues from the wine industry as natural antioxidants, combined with the use of vacuum
packaging and storage under freezing temperatures, may be considered an effective method
for retarding lipid oxidation in both raw and cooked processed chicken meat.
PLUM
Various dried plum puree ingredients have been promoted to aid the retention of juices in
precooked meat and poultry products (Table 4) and have been reported to function as
antioxidants, antimicrobials, fat replacers, and flavorings (Karakaya et al., 2011). In fact,
plum (Prunus domestica L.) contains a known humectant, sorbitol, which has the potential to
alleviate the ―dry‖ mouth-feel in low-fat meat because it naturally binds moisture (Lee &
Ahn, 2005). These authors evaluated the effects of plum extract (1%, 2% and 3%) on the
quality characteristics of vacuum-packaged, irradiated ready-to-eat turkey breast rolls at 0 and
7 days of storage. They found that the addition of plum extract had no detectable effect on the
proximate composition, but decreased lightness value and increased redness and yellowness
values of this ready-to-eat product due to the original color of plum extract. However, the
color of sample with 3% plum extract was dark and might not be appealing to consumers. The
juiciness of turkey breast rolls was increased by plum extract, while texture was not
influenced. The authors recommended the addition of 3% or higher of plum extract with the
aim of improving mouth-feel and antioxidant effect in irradiated turkey breast rolls.
Table 4. Recent articles about meat and meat products with plum by-product

Turkey breast rolls Plum extract Increase juiciness, control lipid Lee & Ahn, 2005
oxidation & production of
aldehydes
Precooked pork Dried plum puree Increase sweetness Leheska et al., 2006
sausage patties
Chicken breast Dried plum products Suitable substitute for alkaline Jarvis et al., 2012
fillets marinated phosphates as a marinade
Raw pork sausages Dried plum puree Suppress lipid oxidation Nuñez de Gonzalez et al.,
2008a
Boneless beef roasts Fresh & dried plum Reduce lipid oxidation & Nuñez de Gonzalez et al.,
juice concentrate, warmed-over flavor 2008b
spray dried plum
powder
Boneless ham Fresh & dried plum No differences Nuñez de Gonzalez et al.,
muscles juice concentrate, 2009
spray dried plum
powder
Low fat beef patties Plum puree Increase juiciness & texture Yıldız-Turp & Serdaroğlu,
scores 2010
Leheska et al., (2006) evaluated the phenolic content and sensory attributes of precooked
pork sausage patties enhanced with 5% or 10% dried plum puree in comparison with 5% or
10% blueberry puree. Results indicate that adding dried plum puree to precooked pork
breakfast sausage increased total phenolics. Trained sensory panel evaluations showed that
dried plum puree treatments were significantly sweeter than all other treatments, in fact, as
the fruit amount increased, sweetness scores also increased with the dried plum puree
treatments being sweeter than the blueberry puree treatments. On the consumer panel, 70% of
children said they would eat the dried plum puree sausages again, while 90% said they would
like to eat the blueberry puree sausage again. Therefore, the concentration of dried plum
puree may need to be reduced to increase the number of children who would choose to eat the
sausage again.
In order to address the growing demand for more natural poultry products, Jarvis et al.,
(2012) determined physical and sensory attributes of vacuum-marinated boneless breast meat
containing various dried plum ingredients (0.06% dried plum fiber, 0.06% dried plum
powder, 1.1% and 2.2% plum juice concentrate, 0.06% and 0.22% fiber/powder mix), as
compared to the traditional marinade with 0.06% and 0.45% sodium tripolyphosphate
(STPP). The combination of plum powder and plum fiber marinade was found to have similar
sensory characteristics when compared to STPP in boneless/skinless chicken breast fillets for
a majority of the attributes measured. Plum concentrate at 1.1% produced an equivalent
marinade pick-up as compared to STPP, and drip loss was comparable to STPP for both
concentrations of plum concentrate. Cooking loss for the STPP treatment was similar to the
plum fiber/powder mix at 0.22% and the 1.1% plum concentrate. No differences were
observed in thaw loss for any treatment as compared to STPP. The authors concluded that a
blend of plum fiber and powder or a plum concentrate could be a suitable substitute for the
alkaline phosphates commonly used in chicken breast meat marinades.
Phenolic compounds in dried plums appear to be the main contributors to their
antioxidant capacity. Therefore, this may be a useful natural ingredient for retarding lipid
oxidation in raw ground or precooked pork sausage that routinely contains higher levels of fat
than other processed meat products. Nuñez de Gonzalez et al., (2008a) determined the
antioxidant properties of dried plum purees (3% or 6% dried plum puree and 3% or 6% dried
plum and apple puree) in both raw and precooked pork sausages stored either refrigerated or
frozen and compared the results with those obtained with raw pork sausages without
antioxidant or with addition of 0.02% BHA/BHT. All treatments decreased fat and increased
moisture content in raw sausages but only 6% dried plum puree reduced cooking yields.
Inclusion of 6% dried plum puree decreased internal redness in raw sausage. Treatment with
6% dried plum puree was even more effective than BHA/BHT at retarding oxidative
rancidity, with lower TBARS values in pork sausages, while the result of the treatment with
3% dried plum puree was similar to those obtained with synthetic antioxidants. A trained-
panel sensory evaluation showed that dried plum puree decreased salt and bitter tastes,
enhanced sweet taste, and masked cooked pork/brothy, cooked pork fat, spicy/peppery, and
sage flavors. Therefore, they concluded that inclusion of 3% dried plum puree was effective
as a natural antioxidant for suppressing lipid oxidation in precooked pork sausage patties.
Nuñez de Gonzalez et al., (2008b) determined the chemical, physical, and sensory attributes
of brine-injected, cooked roast beef (Semimembranosus + Adductor) containing varying
levels of fresh and dried plum ingredients (2.5 or 5% fresh plum juice concentrate, 2.5 or 5%
dried plum juice concentrate, or 2.5 or 5% spray dried plum powder) and subjected to
refrigerated storage over a 10 wk period. All plum ingredients reduced TBARS values and
had minimal effects on tenderness, sensory characteristics, color and appearance. Small
changes in TBARS values, purge, color values, and some sensory properties were found
during storage. These results indicate that 2.5% dried plum juice concentrate or fresh plum
juice concentrate could be incorporated into precooked beef roasts to reduce lipid oxidation
and, potentially, warmed-over flavor. Nuñez de Gonzalez et al., (2009) investigated whether
the inclusion of different levels of plum juice concentrates (fresh, dried or spray dried at 2.5%
or 5%) could improve quality, retain cured color during refrigerated storage and retard lipid
oxidation in sliced vacuum-packaged, brine-injected boneless ham muscles. Hams were
cooked, vacuum-packaged, stored at <4 °C and evaluated at 2-week intervals over 10 weeks.
All plum ingredient treatments increased the percentage of cooking loss in cured hams, but
did not affect vacuum-package purge or sliced vacuum-package purge at 21-days post
storage. Hams with dried plum juice concentrate were slightly darker in color, having more
off-color. All treatments increased redness when measured by the colorimeter, but the dried
plum juice concentrate was much more intense in color and atypical of traditional cured pork
when compared to fresh plum juice concentrate, spray dried plum powder and the control
without plum ingredient. TBARS values were not affected by any treatment and the inclusion
of plum ingredients at the 5% level slightly increased shear force values. Sweet taste
increased and salty taste was reduced by the inclusion of 5% plum ingredients. Based on these
results, the injection of fresh/dried plum ingredients into cured ham at 2.5% or 5% may not be
acceptable due to reductions in product yield and changes in product color.
Yıldız-Turp & Serdaroğlu (2010) determined the effects of using different amounts of
plum puree (5%, 10% or 15%) on some properties of low fat beef patties. Plum puree was
used as an extender in beef patties. Moisture content decreased with increasing concentration
of plum puree. Increasing amounts of plum puree decreased beef patty pH. The highest
cooking yield and moisture retention were found in 5% plum puree samples. Diameter
reduction increased and thickness reduction decreased with increasing amounts of plum
puree. The addition of plum puree to the formulation significantly affected the color of
samples. TBARS values of control samples were higher than in plum puree-added samples at
the end of the storage period. Higher plum puree concentrations in the formulations led to
increased juiciness and texture scores. The results indicated that 5% or 10% plum puree can
be used as an extender in low-fat beef patties.
POMEGRANATE
Pomegranate (Punica granatum L.) is an important source of bioactive compounds and
has been used in folk medicine for many centuries (Karakaya et al., 2011). Pomegranate fruit
contains many different kinds of polyphenolic antioxidants such as punicalagins,
anthocyanins, catechins, ellagic tannins, gallic acid, and ellagic acid (Gil et al., 2000). It is
important to note that while processing pomegranate into juice, the pulp and rind are
discarded and this by-product can provide protection against lipid oxidation in high-fat meat
products with added health benefits and increased consumer appeal. Use of pomegranate by-
products as a source of natural antioxidant in processed meat products had been investigated
and their antioxidant effect had been demonstrated (Table 5).
Devatkal et al., (2010) evaluated the anti-oxidant effect of extracts of pomegranate rind
powder and pomegranate seed powder in cooked goat meat patties. They evaluated 1,1-
diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, total phenolics content, and
the effect of these extracts on TBARS values, instrumental color, and sensory attributes
during meat storage at 4°C. These authors found that addition of 10 ml extract derived from
the extraction of 10 g of these powders in 100 ml of boiled distilled water could protect
cooked goat meat patties against lipid oxidation during refrigerated storage. In fact, TBARS
values were significantly lower in pomegranate rind powder, followed by pomegranate seed
powder as compared to control. Sensory evaluation indicated no significant differences
among patties. They concluded that extracts of these powders could be successfully added to
meat to function as antioxidants without affecting sensory attributes. Similarly, Devatkal &
Naveena (2010) studied the effects of salt and pomegranate fruit by-product powders (rind
powder and seed powder) on color and oxidative stability of raw ground goat meat stored at
4°C. They found that the addition of salt resulted in a reduction in redness scores. During
storage, lightness increased in control and unchanged in treated samples, while redness scores
declined. Salt addition accelerated TBARS formation, while adding pomegranate by-products
counteracted this effect. Percentage reduction in TBARS values was highest in pomegranate
seed powder (443%) followed by pomegranate rind powder (227%). These authors confirmed
that these powders have the potential to be used as natural antioxidants to minimize
autooxidation and salt-induced lipid oxidation in ground meat.
Table 5. Recent articles about meat and meat products with pomegranate by-product

Goat meat patties Pomegranate rind Antioxidant effect Devatkal et al., 2010
powder & seed
powder
Raw ground goat Pomegranate rind Antioxidant effect Devatkal & Naveena,
meat powder & seed 2010
powder
Meat paté Pomegranate peel Inhibit Listeria monocytogenes Hayrapetyan et al., 2012
extract
Chicken meat Pomegranate peel Enhance shelf life & control Kanatt et al., 2010
products extract oxidative rancidity
Cooked chicken Pomegranate juice & Protect against oxidative Naveena et al., 2008a
patties rind powder extract rancidity
Freshly minced Pomegranate rind Inhibit lipid oxidation Naveena et al., 2008b
chicken meat powder extract
Hen breast muscle Pomegranate fruit Reduce protein oxidation & Vaithiyanathan et al.,
juice inhibit microbial growth 2011
Hayrapetyan et al., (2012) evaluated the potential of different pomegranate extracts (dried
pomegranate seeds, pink pomegranate peels, and red pomegranate peels) to be used as a
natural preservative and an antilisterial agent in ready-to-eat products and meat patés. They
showed the inhibitory effect of pomegranate extract against all five tested species, in the
following order of increasing sensitivity: Listeria monocytogenes, Bacillus cereus, Bacillus
subtilis, Escherichia coli and Staphylococcus aureus. The Minimal Bactericidal
Concentration found for the pomegranate extract was 24.7 mg/ml and it was tested against L.
monocytogenes in meat paté at different temperatures. These authors also tested two pure
components (gallic and ellagic acids) commonly found in pomegranate by-products, but they
did not show considerable inhibition of L. monocytogenes. Conversely, the results of this
work indicated that the pomegranate extract could serve as a good natural bifunctional
additive, working both as an antimicrobial and antioxidant compound in meat products.
The antimicrobial and antioxidant potential of pomegranate seed and peel extract was
also investigated by Kanatt et al., (2010) in chicken products. They found that pomegranate
seed extract did not have any significant activity, while pomegranate peel extract showed
excellent antioxidant activity. The latter also had good reducing power and iron chelation
capacity and showed good antimicrobial activity against Bacillus cereus and Staphylococcus
aureus having a minimum inhibitory concentration of 0.01%. Pseudomonas could be
inhibited at a higher concentration of 0.1%, while it was ineffective against Salmonella
typhimurium and Escherichia coli. Pomegranate peel extract added to popular chicken meat
products was effective in controlling oxidative rancidity and enhanced its shelf life by 2–3
weeks during chilled storage.
The efficacy of pomegranate juice and pomegranate rind powder extract as an alternative
to the most commonly used synthetic antioxidants such as BHT (10 mg/100 g meat) in
cooked chicken patties during refrigerated storage was observed by Naveena et al., (2008a).
These authors grilled patties for 20 min and stored them under aerobic conditions at 4 °C for
15 days. They found that pomegranate juice or rind powder extract at a level of 10 mg
equivalent phenolics/100 g meat would be sufficient to protect chicken patties against
oxidative rancidity for longer than BHT. The addition of pomegranate juice or rind powder
extract did not affect any of the sensory attributes of the meat.
Naveena et al., (2008b) compared the effects of adding various levels of pomegranate
rind powder extract (5, 10, 15 and 20 mg equivalent phenolics/100 g meat) and vitamin C (50
mg/100 g meat) on color and lipid stability in cooked chicken patties during chilled storage.
Firstly, they determined the radical-scavenging activity, reducing power and phenolic
composition of this by-product that exhibited significantly higher reducing power and DPPH
radical-scavenging activity. Addition of pomegranate rind powder extract to chicken patties
did not affect any of the sensory attributes, reduced lightness values compared with control
and vitamin C patties, increased total phenolic content (as tannic acid equivalent) and reduced
TBARS values in treated patties. Addition of pomegranate rind powder extract at a level of 10
mg equivalent phenolics ⁄ 100 g meat would be sufficient to protect chicken patties against
oxidative rancidity for longer than vitamin C. These authors concluded that this pomegranate
by-product has substantial amounts of phenolic compounds which can easily inhibit lipid
oxidation in cooked chicken patties.
Vaithiyanathan et al., (2011) studied the effect of dipping chicken meat in 0.02%
pomegranate fruit juice phenolics solution on shelf life under refrigerated storage at 4 °C for
28 days. Increasing the level of pomegranate fruit juice phenolics increased scavenging
activity (DPPH). In pomegranate fruit juice phenolics-treated samples, TBARS values were
lower and total sulfhydryl and protein-bound sulfhydryl content values were higher than in
control samples. Microbial quality evaluation showed that psychrotrophic and aerobic counts
were higher in control samples. The authors concluded that this natural antioxidant reduced
protein oxidation and inhibited microbial growth in spent hen breast muscle and the treated
samples were sensorily acceptable for up to 12 days of refrigerated storage.
OTHER FRUITS AND BERRIES

The antioxidant activity of other fruit and berry extracts containing phenolic compounds
against lipid oxidation has been investigated in various meat products (Table 6).
One of the dietary fibers with the highest polyphenol content is the pulp of the Carob
(Ceratonia siliqua L.) fruit contains sweet carbohydrate (40–50% dry matter) as well as
dietary fiber, tannins, and polyphenols. Bastida et al., (2009) studied the addition of Carob
fruit in the form of non-purified (Liposterine) or purified (Exxenterol) extracts at functional
doses (3 g/100 g) in cooked meat systems and compared with the effects of α-tocopherol,
during both chilling and frozen storage. These authors evaluated meat lipid alteration as
TBARS and polar material-related triglyceride compounds. They concluded that these
extracts can be successfully used to reduce fat alteration in cooked pork meat; indeed,
TBARS levels resulted lower in samples containing Carob fruit extracts and α-tocopherol
than in the control sample under chilled storage, while the changes in polar material were
proportionally smaller after six months‘ frozen storage than after chilled storage, with
Exxenterol displaying the highest antioxidant protection.
Table 6. Recent articles about meat and meat products with fruit and berry extracts

Frankfurters Dog rose & strawberry Inhibit lipid oxidation Armenteros et al.,
tree berries 2013
Cooked pork meat Carob fruit extracts Reduce fat alteration Bastida et al., 2009
Raw & cooked Tart cherry tissue Prevent the formation of Britt et al., 1998
ground beef patties oxidation products & reduce
& fried patties the formation of aromatic
amines
Cooked pork Bearberry extract Decrease lipid oxidation Carpenter et al., 2007
patties
Porcine burger Mediterranean berries Rosa canina improve the Ganhão et al., 2010
patties extracts oxidative stability, color &
texture
Porcine burger Mediterranean berries Inhibit lipid oxidation & protect Ganhão et al., 2013
patties extracts PUFA during cooking &
storage
Raw pork patties Black currant extract Inhibit lipid & protein Jia et al., 2012
oxidation
Chicken breast Prunus mume extract Decrease volatile aldehydes & Jo et al., 2006
meat hydrocarbons
Mechanically Cranberry powder Inhibit lipid oxidation processes Lee et al., 2006
separated turkey &
cooked ground
pork
Precooked pork Blueberry puree Increase tenderness, Leheska et al., 2006
sausage patties cohesiveness, & flavor
Mechanically Cranberry press cake Inhibit lipid oxidation Raghavan &
separated turkey extract Richards, 2006 &
2007
Cooked pork Cloudberry extract Stabilize hexanal production Rey et al., 2005
patties
The efficiency of extracts from the following Mediterranean berries: strawberry tree
(Arbutus unedo L.), common hawthorn (Crataegus monogyna L.), dog rose (Rosa canina L.),
and elm-leaf blackberry (Rubus ulmifolius Schott) to inhibit lipid oxidation in raw, cooked,
and cooked and chilled porcine burger patties were investigated. Ganhão et al., (2013)
showed that extracts from these berries are promising antioxidants which could enhance the
nutritional, safety and sensory properties of meat products. They used the following as
indicators of lipid oxidation: modification of the FA profile during cooking and chilling,
quantitative measurements of TBARS, and lipid-derived volatiles. PUFA gradually decreased
during cooking and subsequent storage of cooked burger patties, with this decrease being
significantly greater in control patties than in those with added berry extracts. Accordingly,
the control patties showed significantly higher TBARS numbers and counts of lipid-derived
volatiles in all treatments when compared to the berry-added counterparts. These wild
Mediterranean fruits added to burger patties (3% of total weight) also displayed intense
antioxidant activity against protein oxidation, that might play a major role in the nutritional
and sensory quality of meat products (Ganhão et al., 2010). The addition of elm-leaf
blackberry would affect the color displayed by burger patties, whereas the addition of
common hawthorn would supply some negative textural properties to these products. Dog
rose berries improve the oxidative stability, color and texture of cooked burger patties with no
apparent drawbacks.
Armenteros et al., (2013) studied the effect of adding phenolic-rich extracts from dog
rose (Rosa canina L.) and strawberry tree (Arbutus unedo L.) berries in frankfurters
elaborated with or without the addition of sodium ascorbate and nitrite. They found that fruit
phenolics and additives inhibited lipid oxidation: indeed, TBARS increased only during
chilled storage of control frankfurters, while the amount of protein carbonyls increased in all
treatments except in frankfurters treated with strawberry tree extract and additives. Textural
characteristics such as springiness, cohesiveness, hardness, and gumminess suffered a
deterioration only in control frankfurters, while the addition of substances with proven
antioxidant activity reduced the discoloration process that occurred during chilling. These
authors concluded that the combination of the additives typically employed in the meat
industry together with these fruit extracts is a successful strategy to enhance the oxidative
stability of frankfurters without modifying their texture and color properties.
The fruit of Prunus mume has been used as a traditional drug and healthy food in Asia
and the antioxidant properties of their methanolic extract were determined in chicken breast
meat systems by Jo et al., (2006). They showed TBARS values at day 3 decreased by about
45% compared to the control, while the amounts of volatile aldehydes and hydrocarbons
decreased significantly when using this extract.
The effects of adding 11.5% tissue from two varieties of tart cherries (Prunus cerasus
Montmorency and P. cerasus Balaton) on the oxidation of lipids in raw and cooked ground
beef patties and on the formation of heterocyclic aromatic amines in the fried patties were
investigated by Britt et al., (1998). They found that TBARS values for raw and cooked
ground beef patties containing cherry tissue were significantly lower than those for the
control meat product. The addition of cherry tissue also affected cholesterol oxidation;
indeed, after 4 days of refrigerated storage, cholesterol oxides represented 2.0 and 1.7% of the
total cholesterol in patties containing Montmorency and Balaton cherry tissue, respectively,
while they were 5.2% of the total cholesterol content in the control. Tart cherry tissue also
effectively reduced the formation of heterocyclic aromatic amines in cooked beef patties to
levels normally formed in patties fried at lower temperatures. The concentrations of the
principal amine (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine) in cooked muscle foods,
were reduced by 93 and 87% by Montmorency and Balaton cherry tissue, respectively.
Rey et al., (2005) investigated the antioxidant activity of cloudberry (Rubus
chamaemorus) extracts (100 and 500 mg/kg) on TBARS development and hexanal content in
cooked pork patties. These authors performed tests in parallel for comparison using pure
quercetin, rutin, caffeic acid and the extracts of two plants: beetroot (Beta vulgaris) and
willow herb (Epilobium angustifolium). Cloudberry extract, rich in total phenolics (containing
16.2 mg of gallic acid equivalents/g dry matter), at a concentration of 500 mg/kg, was a
potent antioxidant for stabilizing hexanal production of cooked pork patties after 6 days of
refrigerated storage and comparable to that observed for pure quercetin. while the least potent
antioxidant on stabilizing oxidation was pure rutin, while caffeic acid showed intermediate
activity. Beetroot and willow herb extract stabilized hexanal production of cooked pork
patties after 6 days of refrigerated storage to an extent comparable to that observed for pure
quercetin and cloudberry extract. At a concentration of 100 mg/kg, willow herb and beetroot
extracts showed lower antioxidant activity on TBARS and hexanal contents, similar to that
observed for caffeic acid over 3 days, while cloudberry extract was still as potent as
quercetin.
Other rich sources of phenolic compounds such as anthocyanins and flavonols are
berries.
Carpenter et al., (2007) investigated the effect of bearberry (Arctostaphylos uva-ursi) (0–
1000 μg/g muscle), also known as Uva Ursi, on lipid oxidation, color, pH, microbial status
and sensorial properties of cooked pork patties. Bearberry extract addition had decreased lipid
oxidation in raw pork patties at days 9 and 12 of storage, relative to the controls. The
antioxidant activity of bearberry was also observed in cooked pork patties, thus demonstrating
its thermal stability. The sensory properties of cooked pork patties, mesophilic plate counts
and pork pH were unaffected by bearberry extract addition.
Jia et al., (2012) determined the optimal extracting condition (40% ethanol in water for 2
h) of frozen crude blackcurrant (Ribes nigrum L.) by determining the extract's anthocyanin
content, reducing and radical-scavenging ability. Additionally, they evaluated the inhibitory
effect of this extract (5, 10 or 20 g/kg) on lipid and protein oxidation and color deterioration
in raw pork patties during chilled storage. These tests confirmed blackcurrant extract to be a
highly effective antioxidant in patties, because it stabilized the red color of the meat and
inhibited oxidation during refrigerated storage. The findings demonstrated strong potential for
blackcurrant extracted with 40% ethanol in water as a natural antioxidant source for
preserving meat and meat-product quality.
Leheska et al., (2006) evaluated the phenolic content and sensory attributes of precooked
pork breakfast sausage patties enhanced with blueberry (Vaccinium angustifolium) puree (5%
or 10%). They compared the results with a control without fruit and two treatments with dried
plum puree (5% or 10%). They found that the addition of blueberry puree and dried plum
puree increased total phenolics in the cooked sausage by an average of 36%, that may be
nutritionally beneficial while also having consumer appeal. The blueberry puree treatments
were lower in phenolics than the dried plum puree treatments, but the blueberry treatments
were more acceptable to children than the plum ones. Comparisons of fruit type, percentage
of fruit added, and fruit treatments versus control were all significant for tenderness,
cohesiveness, and pork sausage flavor, but were not significant for other attributes.
Cranberry (Vaccinium macrocarpon) juice powder is a stronger inhibitor of lipid
oxidation than cranberry press cake, which is derived from juice production and is a highly
underutilized by-product containing seeds and skin with many nutraceutical compounds, such
as phenolic acids, flavonols, anthocyanins, and proanthocyanidins (Lee et al., 2006).
Cranberry press cake could be used as an antioxidant in meat and poultry products (Lee et al.,
2006; Raghavan & Richards, 2006, 2007). Lee et al., (2006) examined the potential of the
different polyphenolic classes found in cranberries to inhibit lipid oxidation in mechanically-
separated turkey and cooked ground pork. They demonstrated that in cooked pork (30% fat
before cooking), crude cranberry extract exhibited 51% inhibition on TBARS formation in
samples held for 9 days at 2°C. Furthermore, treatment with cranberry juice powder at 0.32%
showed equal inhibition of lipid oxidation to rosemary extract used at 0.04% in mechanically-
separated turkey samples held for 14 days at 2°C. These authors suggested that flavonol
aglycones were the most effective at inhibiting lipid oxidation compared to the other classes
of polyphenolics. In particular, quercetin, a non-glycosylated flavonol present in cranberry
powder, inhibited lipid oxidation in meat at low concentrations.
Because of the susceptibility of muscle membrane lipids to oxidation, the ability of

quercetin in the cranberry extracts to partition between the aqueous and the membrane phases
was studied by Raghavan & Richards (2006). Cranberry press cake (extracted using five
different solvent mixtures) was compared with extracts from cranberry juice powder,
prepared using chloroform:methanol (1:1), for their ability to inhibit lipid oxidation in
mechanically-separated turkey. The amount of quercetin in the extracts and the amount of
quercetin that partitioned into the membranes and, consequently, the effectiveness of the
extracts to inhibit lipid oxidation followed the same order: juice powder extract > ethyl
acetate extract of press cake ≥ ethanol extract of press cake. The total phenolic content of the
extracts decreased in the following order: ethyl acetate extract of press cake > ethanol extract
of press cake ≥ juice powder extract. These authors stated that the ability of cranberry extracts
to inhibit lipid oxidation depends on the type and amount of specific constituent phenolic
compounds rather than the total amount of phenolic components. They concluded that a
proper choice of solvents and extraction method, which would increase the amount of
quercetin in the press cake extracts, might increase their antioxidant potential and hence their
economic value. Raghavan & Richards (2007) compared the ability of extracts from
cranberry press cake using solvent extraction (water, ethanol and acetone) and microwave-
assisted solvent extraction to inhibit lipid oxidation in mechanically-separated turkey. In
terms of choice of solvent, based on their flammability and toxicity, they found that the most
effective extracts were obtained by microwave-assisted solvent extraction with 100% ethanol
or simple solvent extraction with 100% acetone, while water extracts were least effective at
inhibiting lipid oxidation in mechanically-separated turkey.
ACKNOWLEDGMENTS
The author would like to thank Dr. Anthony Green for the English revision of the paper.
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Chapter 2
VALORIZATION OF LIQUID EFFLUENTS FROM OLIVE

OIL EXTRACTION ACTIVITY IN THE PRODUCTION OF
CERAMIC BRICKS: INFLUENCE OF
CONFORMATION PROCESS
D. Eliche-Quesada*1,2 and F. A. Corpas-Iglesias2

1
Department of Chemical, Environmental, and Materials Engineering,
Higher Polytechnic School of Jaén, University of Jaén, Jaén, Spain
2
Department of Chemical, Environmental, and Materials Engineering.
Higher Polytechnic School of Linares. University of Jaén, Jaén, Spain
ABSTRACT
Olive oil production industry is characterized by relevant amounts of by-products
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
*
Corresponding author: Tel.:+34953211861; Fax:+34953212141; e-mail address: deliche@ujaen.es.
30 D. Eliche-Quesada and F. A. Corpas-Iglesias
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.
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.
Keywords: Wastewaters olive oil industry, clay brick, organic wastes, forming process,
physical, mechanical and thermal properties
1. INTRODUCTION
Developed countries started to realise from the 1970s that economic prosperity was based
especially on the intensive use of finite natural resources and that, therefore, in addition to the
economic and social issues, a third aspect was neglected: the environment. Thus arises the
concept of "development sustainable" in 1987 as the "development that ensures the needs of
the present without compromising the ability of future generations to meet their own needs".
Intuitively, a sustainable activity is one that can be kept. Thus, "ecology", "sustainability",
"natural materials", "emission control", "waste minimization", are increasingly used in
ordinary language concepts, which reveals the great interest for consumers, industry and
researchers in the vast majority of productive sectors.
The ability to ensure the present necessities without compromising future generations is
an issue that has been gaining great importance. Particular care has been taken to match the
economic growth with environmental protection, managing resources according to a
sustainable perspective in order to ensure a better life quality. Among the main concerns
related to environmental issues by companies and organizations, are the development and
production of new products in order to avoid negative impact on the natural environment [1].
There is a growing volume of waste generated at global level, and increasingly less the
ability of the planet to assimilate them. Neglect or inadequate waste management produces
significant impacts on media receivers, and they can cause pollution in the water, on the
ground, in the air, contribute to climate change and affect ecosystems and human health. On
the other hand, when the waste is managed properly they can become resources that
contribute to the saving of raw materials and ensure economic sustainability, with a positive
effect on the conservation of natural resources and ecosystems.
The generation of waste in Spain, in 2010, was of some 137 million tons, showing a
downward trend compared to previous years [2]. Regarding the sectorial source of waste,
construction and mining were the two sectors that generated a higher volume, followed by
those generated in households and in the sectors industrial and services.
The olive oil industry is very important in Mediterranean countries. Although there are
more than 40 producers of olive oil, over 75% of world production is concentrated in
European countries. Within the European Union, Spain is the main world producer, it
produces more than half of the oil [3]. More than 80 % of the national production is
concentrated in Andalusia, a region in the South of Spain, especially in the provinces of Jaen
and Cordoba, what generates 20% of world production of olive oil [4].
Valorization of Liquid Effluents from Olive Oil Extraction Activity … 31
Olive oil is nowadays produced by either two or three-phase extraction systems. The
three phase method in which a solid fraction (wet pomace or alperujo), a liquid residue
(―alpechin‖), and the olive oil are obtained. Two phase method in which a solid fraction, wet
pomace and a liquid one, the olive oil, are obtained. The main advantage of the two-phase
system is the reduction of the olive mill wastewater amounts compared to that originated from
the three phase technology, in which 0.6-1.31 water/kg olives are used at the centrifugation
step, by eliminating the production of alpechin thus avoiding its consequences environmental
[5]. Therefore, the two-phase process still generates some wastewater coming basically from
washing of the olive fruits, before entering the extraction process, and the olive oil, in the late
centrifugation wash. Wastewater is a dark-coloured liquid containing many dissolved and
suspended substances, therefore presenting high-polluting charge. Wastewater is typically
sent to evaporation in storage ponds due to its low investment and, however, it needs large
areas and produces several problems such as bad odour, infiltration and insect proliferation
[6]. The production of olive oil, throughout the Mediterranean area, generates about 30
million tons of wastewater yearly [7]. Therefore, this activity generates huge quantities of
wastes that may have a great impact on land and water environment because of their high
phytotoxicity. Several studies have proven the negative effects of these wastes on soil
microbial populations [8], on aquatic ecosystems [9] and even in air medium [10]. In
addition, it has been estimated that every ton of processed olives in the two-phase extraction
process produced 100-120 m3 of olive washing (OW) and 200 m3 of olive oil washing
(OOW), obtaining 200 Kg of olive oil [1]. As the production of olive oil reached 618200 tons
during 2012/13 campaign in Spain [11], the production of waste is considerable, and so
important is to consider its valorization. There is a need for guidelines to manage these wastes
through technologies that minimise their environmental impact and lead to a sustainable use
of resources.
Due to the large production of wastewater, would be desirable to find appropriate
solutions for disposal and/or reuse of the waste compatible with the environment and more
sustainable from an economic point of view [12]. An alternative to the environmental
problems caused by storage ponds, then breaks, overflows, and other anomalities can cause
damage to the soil, aquifers, etc., can be its utilization in industrial production processes. The
building industry has called upon scientists to develop more sustainable, low cost, lightweight
construction materials. Rapid ways for implementing principles of sustainable development
have been emerging within the construction, as well as a trend to achieve building with
minimal energy consumption. The employment of ceramic products manufactured from waste
in building is an option that fits perfectly with the principles of sustainable development,
since it implies a value solution that allows the reuse of materials that today are considered to
be waste and, on the other hand, can give to the ceramic material better performance.
Recently, some researchers [13-18] have reviewed the use of various wastes in brick
production to achieve lightweight materials that are environmentally friendly while respecting
the requirements of the relevant standards. Clay-based materials show a natural forgiveness
towards the incorporation of a variety of materials, including potentially hazardous industrial
wastes or sub-products. Traditional clay-based materials are heterogeneous products that can
accommodate different wastes or sub-products without modification of its production process
or the final product properties [19-21]. Several authors have studied the valorization of solid
waste fraction, alperujo [22-28] and olive mill wastewater from three-phase extraction system
[29-32] as raw materials in clay bricks, but there are few studies of the use of two phase olive
mill wastewater for building materials production [33, 34].
On the other hand, the properties of ceramic materials are closely related to several
factors, among them, the process of forming, it conditions the microstructure and properties
of processed materials. The regional ceramics industry is characterized by the use of
techniques of pressing and extrusion forming of parts made, this being last technical of higher
frequency.
Taking into account the high amounts of wastewater produced by the process of
extraction of olive oil, and their chemical composition, in this study, 22 wt % of olive
wastewater (OW) or olive oil wastewater (OOW) was used instead of fresh water (FW) on the
stage of compression or extrusion molding. The technological properties of the ceramic bricks
with waste were compared with those with added fresh water. In addition, the results obtained
by extrusion and compression will be compared, in order to evaluate whether the waste bricks
are attractive for an industrial implementation. The use of these effluents used to mix clays
when preparing paste for bricks could provide a solution to an environmental problem.
2. EXPERIMENTAL
2.1. Preparation of the Samples
Clay was supplied by a clay quarry located in Bailen, Jaen (Spain) and was obtained by
mixing three types of raw clay in equal parts: red, yellow and black clay. Clay was crushed
and ground to yield a powder with a particle size suitable to pass through a 150 m sieve. The
waste, olive wastewater and olive oil wastewater were supplied by a local olive oil extraction
plant and used directly without any prior pretreatment. The ceramic paste for the extrusion
was prepared by adding fresh water (FW) or residue resulting from olive oil extraction (OW
or OOW) to the clay in a mixer. The amount of added water in the mixer depends on clay
plasticity and on its consistency while performing the extrusion. In the present work 22 wt %
of FW, OW or OOW was added to the clay. The same value as used at industrial scale for this
kind of clay mixture. Extrusion was carried out in a laboratory Venco extruder. Extruded test
pieces were dried at room temperature for about 24 h, and then heated in an oven at 110 ºC
until constant weight for at least 24 h.
To study the influence of the conformation process, specimens also formed by
compression in a semi-dry state. The same amount of water, OW or OOW as in the process of
forming by extrusion (22 wt %) was added to the samples to obtain comparative results. The
amount of waste is high to obtain adequate plasticity and absence of defects, mainly cracks,
during the semi-dry compression moulding stage under 19.6 MPa of pressure, using a
uniaxial laboratory-type pressing Mega KCK-30 A. So, the OW and OWW samples were
formed after 24 hours in order to remove some of the moisture. Samples were fired in a
laboratory furnace at a rate of 3 ºC/min up to 950 ºC for 4h. Samples were then cooled to
room temperature by natural convection inside the furnace. The shaped samples were
designated as x-C-FW for the bricks with fresh water, x-C-OW for the mixture with olive
wastewater and x-C-OOW for the mixture with olive oil wastewater, being x the forming
process; x=E: extrusion or x=C: compression. In Figure 1 can be observed a scheme a

sequential diagram of the methodology used.
Figure 1. Scheme a sequential diagram of the methodology used for the fabrication of extruded and
compressed bricks.
2.2. Characterisation of Brick Raw Materials
The acidity of the OW and OWW was determined with Crison pH meter Basic 20. The
electrical conductivity was electrometrically measured using a Crison Basic 30 EC
conductivity meter. The determination of the organic content of dry residue was performed
according to ASTM D-2974, Standard Test Method for Moisture, Ash, Organic Matter of
Peat and Other Organic Soils [35]. The ignition temperature was 440 °C. The total content of
carbon, hydrogen, nitrogen, and sulfur of dry residue and clay was determined by combustion
of samples in O2 atmosphere using the CHNS-O Thermo Finnigan Elementary Analyzer Flash
EA 1112. The higher heating value (HHV) was determined using a Parr 1341 Plain Oxygen
Bomb Calorimeter.
Chemical oxygen demand (COD) were established through potassium dichromate
digestion and, the amount of oxygen required to biodegrade the organic material present in
the waste is evaluated by the biochemical oxygen demand (BOD5) using a BOD sensor Velp
Scientific.
The plasticity of the samples was measured from the Atterberg Limits (Plastic Limit, LP,
and Liquid Limit, LL) and Plasticity Index (IP). The amount of added water in the mixer
depends on the clay plasticity an on the consistency at which the extrusion is performed. The
moisture content for stiff extrusion is 15-20 %, and 21-26 % for soft extrusions [36]. In the
present work, FW, OW or OOW was added to 22 wt %, the results indicated that the plastic
limit or amount of minimal water to mold the mixture decreased when using OW (19.7 %) or
OWW (20,1 %) as mixing water. These results demonstrated that the volume of OW or
OWW amount needed to produce the optimum extrusion performance is smaller than that the
fresh water was used (21,9 %). This would be attributed to an additional lubricating effect
performed by the addition of the wastes. Similar results were previously found in the
extrusion effect when adding olive oil mill wastewater (OMW) [31, 32]. However, the
plasticity index that establishes a range of moisture content in which the clay is moldable,
decreased when FW is replaced by OW or OOW from 9.6 % to 6.1 % and 5.9% respectively.
The Qualitative determination of major crystalline phases was achieved using the Philips
X‘Pert Pro automated diffractometer equipped with a Ge (111) primary monochromator.
Chemical composition was determined by X-ray fluorescence (XRF) using the Philips Magix
Pro (PW-2440). Thermal behaviour was determined by thermogravimetric and differential
thermal analysis (TGA-DTA) with a Mettler Toledo 851e device in oxygen operating at a
ramp of 20 ºC/min from room temperature to 1000 ºC.
2.3. Characterisation of the Bricks
Linear shrinkage was obtained by measuring the length of the samples before and after
the firing stage, using a caliper with a precision of ± 0.01 mm, according to ASTM standard
C326 [37] . Water absorption values were determined from weight differences between the
as-fired and water-saturated samples (immersed in boiling water for 2h), according to ASTM
standard C373 [38]. Open porosity (in vol %) were determined from weight difference
between saturated mass and dry mass with respect to exterior volume and closed porosity (in
vol %) was calculated from weight difference between dry mass and suspended mass in
waster with respect to exterior volume according to ASTM standard C373 [38]. Bulk density
was determined by the Archimedes method [38]. Water suction of a brick is the volume of
water absorbed during a short partial immersion. The test to determine water suction was
implemented according to standard procedure UNE 67-031 [39]. The compressive strength of
bricks is their bulk unit charge against breakage under axial compressive strength. Tests of
compressive strength were performed according to standard UNE-EN 772-1 [40] on a
Suzpecar CME 200 SDC laboratory press. For this trial, six fired samples were studied. The
area of both bearing surfaces was measured and the average taken. All samples were
submitted to a progressively increasing normal strength, applying with the load applied
centred on the upper surface of the sample until breakage. The compressive strength of each
sample was obtained by dividing the maximum load by the average surface of both bearing
surfaces, expressed in MPa with 0.1 MPa accuracy. The thermal conductivity of samples was
determined with a Selecta heating plate using a Crison T-638 digital thermometer and a Pt-
100 probe.
The microstructure of the pieces formed was evaluated by means of a scanning electron
microscope (SEM), using the high-resolution transmission electron microscope JEOL SM
840. Samples were placed on an aluminium grate and coated with gold using the ion
sputtering device JEOL JFC 1100.
3. RESULTS AND DISCUSSION

3.1. Analysis of Raw Materials
Olive mill wastewaters are the main pollutant from two-phase extraction systems. They
are constituted by the water used in different stages of oil extraction. Wastewater from olive
washing (OW) and olive oil washing (OOW) was collected in a local industry operating the
two-phase olive oil extraction method. The OW and OOW have a black-brownish colour with
an unpleasant smell of fermented organic matter, being the colour and smell more intense in
the case of OOW waste. The composition of olive oil wastewater depends on several factors:
as the process used while extracting oil; operation conditions, olive variety and olive
maturation grade [41]. The main physic-chemical features of the used washing wastewater
(OW) and olive oil washing wastewater (OOW) are presented in Table 1. The OW and OOW
are characterized by a high content of organic pollution (COD/BOD ratio between 7 and 11),
pH slightly acid OOW and pH slightly basic near neutral OW. Presence of polyphenols and
organic substances make the treatment of these wastewaters problematic [42].
Concerning the dry residue of the olive washing wastewater (OW) and olive oil washing
wastewater (OOW), 4.4 and 7.2 % respectively, large amounts of carbon and hydrogen were
determined (Table 2). However, the organic matter content of the clay was low. The
incorporation of these wastes with high carbonaceous content might provide a sensible
energetic contribution to the ceramic process. The HHW of dry residue was 1535 kcal/Kg for
OW and 3425 kcal/kg for OOW (Table 2). Brick manufacturing usually includes some
materials with variable organic matter content like olive pomace or coke [33]. Therefore, the
use of waste to replace fresh water could be a saving in water consumption but also in energy
requirements for the firing process in brick manufacturing.
Table 1. Physico-chemical characterization of the used olive oil wastewater
Dry
Residue
Waste PH Density Conductivity Humidity COD BOD5 Organíc Ashes
(Kg/m3) (mScm-1) (%) (mg dm-3) (mg dm-3) Matter (%) (%)
OW 7.7 1006 2.27 95.5 4383 393 78.6 21.4
OOW 4.5 1012 6.54 93.8 37450 5175 88.1 11.9
Table 2. CNHS analysis and Higher Heating Value (HHV) of raw materials: clay and
dry wastes
Sample %C %H %N %S HHV
(kcal/kg)
Clay 2.30 ± 0.004 0.50 ± 0.004 0.05 ± 0.0 0.03 ±0.008 -
OW 15.29±0.03 1.32±0.01 0.49±0.00 1.78±0.02 1535
OWW 35.33±0.21 5.63±0.05 0.78±0.,03 0.56±0.01 3425
In this work three types of clays were used. The chemical composition of the raw yellow,
red and black clay after firing at 950 ºC was determined by XRF (Table 3). Yellow and black
clays are rich in carbonates, still very high in the black clay. The decrease of calcium contents
is correlated to an increase of SiO2 contents, reaching values of 54% SiO2 in yellow clay. Red
clay is characterized by a low carbonate content with higher ferrite and alumina content. Red
clay does not have sufficient plasticity for the shaping of resistant unfired pieces. Regarding
traditional mixture described in Table 3, and used as reference raw material in the present
work, the addition of red clays allows modifying the technological behaviour of yellow and
black clays. Due to the proportions of clays used, the mixture of clays has appropriate
plasticity (plasticity index: 9.6%) for shaping. The mixed clay had high amounts of SiO2
(55.82 %) as the predominant oxide and of Al2O3 (12.13 %) due mainly to the silicate in the
clay. The significant content of CaO (13.52 %) is related to the abundance of carbonates,
which explain the loss of ignition observed. The clay is considered calcareous if CaO>6 [43].
This information is very important for manufacturers as they can adapt the drying and firing
process to avoid cracking. Carbonates acts as a pore-forming agents and generate crystalline
phases during firing that enhance mechanical strength [44]. XRD data show that de mixed
clay materials used to the elaboration of the ceramic pieces is rich in quartz with significant
amounts of clay minerals as phyllosilicates such as muscovite, orthoclase, faujasite, albite and
chlorite. Carbonates as calcite and dolomite (Figure 2) are also presents in small quantities
and traces of hematite can be observed. The comparison of the mineralogical composition of
the used mixture with the overall composition of common raw materials for ceramics
products [45] reveals that this material contains the appropriated phyllosilicates, carbonate
and quartz contents to be considered inside the range of mineralogical compositions
potentially suitable for structural ceramics products.
Figure 2. XRD patterns of clay.
Table 3. Chemical composition of the fired clay
Oxide content (%) Red clay Yellow clay Black clay Mixture of Clays
SiO2, 53.94 53.54 45.50 47.17
Al2O3 15.93 11.78 11.55 12.51
Fe2O3 14.22 7.02 5.82 6.49
CaO 3.84 13.67 21.45 13.52
MgO 1.81 2.20 2.10 2.11
MnO 0.032 0.063 0.10 0.05
Na2O 0.22 1.72 1.40 0.31
K2O 7.95 4.17 3.43 3.61
TiO2 1.54 1.56 1.21 0.78
P2O5 0.21 0.10 0.10 0.14
SO3 - - 2.91 1.58
NiO - - - 0.0086
CuO - - - 0.0017
ZnO - - - 0.0082
Ga2O3 - - - 0.0027
Rb2O - - - 0.017
SrO 0.035 0.096 0.22 0.043
ZrO2 - 0.059 0.141 0.035
Nb2O5 - - - 0.0021
BaO - - - 0.047
LOI - - - 10.6
Figure 3. (Continued).
Figure 3. DTA-TGA analysis of (a) raw clay; (b) wastewater from olive-washing stage (wet OW); (c)
wastewater from oil olive-washing (wet OOW); (d) dry OW and (e) dry OOW.
Thermal behavior of clay and waste was studied by TGA-DTA with maximum
temperature of 1000 ºC. The most significant weight loss processes associated to brick
manufacturing from typical clays were physically-adsorbed or free water, water elimination
by dehydroxilation of clay minerals and carbonate decomposition. Weight loss of free water
mainly occurred at temperatures below 100 ºC, although the process is extended up to 200 ºC
in the clay sample (Figure 3 a). An endothermic DTA effect is observed in the range from
room temperature up to 200 ºC, and the weight loss associated to this process is 1.4 wt %.
There are other thermal events within the range 200-600 ºC, with weight loss of 2.3 wt %,
associated to the combustion of organic matter and dehydroxilation of the silicate layer
identified in this sample. However, a broad exothermic effect is observed by DTA with a
shoulder. A small endothermic DTA peak is detected at ca. 565 ºC, being associated to the
transformation α to β quartz. Calcium carbonate (calcite) decomposition took place in the
range 600-800 ºC with the release of CO2, associated with an endothermic DTA effect,
centered at 750 ºC in the clay sample, with weight loss of 7.8 wt %. Since bricks were fired at
950 ºC, decomposition of carbonates is expected and a development in porosity of the fired
samples.
TGA-DTA curves of wet OW and OWW showed a strong endothermic peak centered at
150 ºC, corresponding to evaporation of the absorbed water in 95.5 and 94.0 wt%,
respectively (Figure 3 b and c), major constituent of the waste. TGA-DTA curves of the dry
wastes are typical for a solid fuel (Figure 3d y f). A total weight loss of about 45.4% and
77.1% was observed at 1000 ºC for OW and OWW, respectively. The first decrease in mass
observed between 25 and 180 ºC is caused by the evaporation of physical water, indicating a
small endothermic reaction. The second mass loss was observed within the 200–700 ºC range,
which may possibly be due to the burning of volatile organic compound (VOCs) and, at the
upper temperature, it may be due to the burning of fixed carbon, as indicated two for OW and
three for OOW exothermic reactions, with maxima at 363 ºC and 474 ºC for OW and 440 ºC,
525ºC and 590 ºC for OOW. Thermal degradability is affected by the composition of any
biomass because different components of lignocellulosic materials have different thermal
behaviour. The three major constituents of lignocellulosic materials (hemicellulose, cellulose
and lignin) are chemically reactive and decompose thermochemically in the temperature
range of 150-500 ºC. Some studies on the thermal decomposition of the individual
components indicated that decomposition of hemicellulose starts first, followed by cellulose
and, finally, the lignin [46, 47]. Organic matter started to leave the material and burns very
rapidly as the strong exothermic peaks centered at 363 ºC and 474 ºC in OW, and 440 ºC and
525 ºC in OOW. Volatile compounds burn first, followed by non-volatile components. The
higher thermal degradation of OWW might be due to larger percentages of volatile matter and
lower ash contents than OW (Table 2). The exothermic peak at 750 ºC could correspond to
the combination of residual carbon with atmospheric oxygen and CO formation. Also the
lightly endothermic reaction between 700-850 ºC represented the thermal decomposition of
carbonates, phosphates and potassium salts [46].
3.2. Characterization of the Bricks
No defects such as cracks and bloating were observed after firing (Figure 4). The colour
of the fired samples probes is similar to that visible in the formulas without wastewaters. Nor
was any black core observed on the fracture section of the residue-containing fired samples.
The presence of organic matter in clays can cause a firing defect known as black core if the
organic carbon is not completely burned during firing. Factors favouring black core
occurrence are a high organic matter and the vitrification of the brick, which can hinder
combustion of organic matter.
Linear shrinkage occurs when the chemically and mechanically bound water vaporates,
so the temperature and time of the drying and firing stages must be controlled [48]. Shrinkage
during the ceramic process is a significant parameter, since structural change and
solidification, implying densification, may create tensions and failures in fired clay bricks
[32]. During this process, open porosity is transformed into closed porosity. According to the
literature, this value must be below 8%. Table 4 shows the differences of linear shrinkage
after firing at 950 ºC for clays that incorporate fresh water and clays that incorporate
wastewater as mixing water by extrusion and compressing techniques. The linear shrinkage of
bricks formed by the addition of FW presented a slightly higher linear shrinkage than OW
and OWW bricks at both conformation processes. The extrusion technique presented higher
values of linear contraction. Replacing FW by OW or OOW produced a slight decrease in the
contraction due to the small content of organic matter in wastewaters.
Figure 4. Bricks made from clay using as mixing water: fresh water, OW or OOW formed by (a)
extrusion and (b) compression.
Mass loss after sintering clay at 950 ºC may be attributed to firing reactions resulting in
phyllosilicates and carbonates decomposition and to the elimination of organic matter by
combustion. The incorporation of OW or OWW produced a slight increase in weight loss
after sintering, due to the higher organic matter content of waste, as indicated in thermal and
elemental analysis (Table 4). On the other hand, can be observed that the compressing
technique allows to obtain weight loss after sintering slightly lower than the extrusion,
however, the differences are not very representative. The cause of this result can be found in
the degree of compaction of the specimens.
Table 4. Technological properties of construction bricks made from using mixing water,
fresh water, olive wastewater and olive oil wastewater formed by extrusion and
compression
Sample Linear Loss on ignition Suction water

Shrinkage (%) (%) (kg/m2 min)
E-C-FW 1.03 ± 0.17 8.96 ± 0.02 2.43 ± 0.19
E-C-OW 0.96 ± 0.10 9.07 ± 0.07 2.24 ± 0.15
E-C-OOW 0.66 ± 0.10 9.23 ± 0.03 1.80 ± 0.08
C-C-FW 0.45± 0.08 8.40± 0.07 2.31± 0.05
C-C-OW 0.30± 0.11 8.24± 0.09 2.17± 0.06
C-C-OOW 0.23± 0.07 8.15± 0.08 2.03 ± 0.16
The importance of bulk density lies in the fact of being directly related to the internal
microstructure of the piece and largely determines the properties of the final product [49]. In
samples obtained by compressing and by extrusion both as shown a slight decrease of the
values of bulk density when using OW or OOW instead of FW as mixing water. This decrease
is more pronounced in specimens obtained by compressing, between a 4.3 and 4.6%,
declining only between a 1 and 1.5% for samples obtained by extrusion, when adding OW
and OOW, respectively. This reduction can be due to a greater porosity of the bodies supplied
by the low content of organic matter of the waste. In Figure 5 can also be seen that the
samples formed by the extrusion technique present a lower bulk density compared with the
technique of compressing. These differences may be due to the lower degree of packing that
is achieved with the process of formed by extrusion compared with the compressing, since the
pressure of compaction in compressing has been higher than in the extrusion.
Water absorption is a key factor affecting the brick durability and a measure to open
porosity. As shown in Figure 6, water absorption slightly decreased when OW or OOW was
added. Therefore, the use of wastewater as mixing water barely alters this property, decreased
slightly the open porosity of ceramic bodies, being this decreased more pronounced when
OOW wastewater was incorporated. The organic matter in OW and OOW wastewaters was
transformed during the thermal process and it lead to a increase in the closed porosity of the
ceramic bodies. The extrusion technique allows to obtain bricks with less open porosity. Thus
the specimens used as water mixing FW presented a water absorption of 18.64% when the
bricks were formed by extrusion, increasing until the 20.32% this property when they were
formed by compression.
Figure 5. Bulk density of the extruded and compressed fired bricks as function of mixing water.
According to data on bulk density of bricks, samples with OW or OOW addition showed
higher total porosity than FW bodies and slightly lower values on water absorption, indicating
that these samples had slightly higher closed porosity values. As can be observed in Figure 7,
the open porosity (in vol. %) decreased slightly when OW or OOW is used as mixing water
according to the water absorption data. The closed porosity (in vol. %) increased slightly with
the addition of waste OW or OOW, according to SEM micrographs. The samples formed by
compression present lower total porosity, since they present higher values of bulk density.
However, these samples have higher proportion of open porosity than samples formed by
extrusion, according to data from water absorption and lower proportion of closed porosity.
Figure 6. Water absorption of the fired bricks as function of mixing water used in the forming process:
extrusion and compression.
Figure 7. Open porosity and closed porosity (vol %) of the extruded and compressed fired bricks as a
function of mixing water used in the forming process.
Water suction affects the quality of the final material and its durability significantly. High
values should be avoided, since they can cause pieces with defects and lower durability. The
substitution of FW by OW or OWW and forming method affected water suction values of
bricks. Water suction decreased when the bricks are formed by pressure. (Table 4). Clay
mixed with FW showed a water suction of 2.43 and 2.31 kg/m2min for extruded and
compressed bricks, respectively. Replacement FW by OW or OOW waste produced a
decrease in the water suction, being the most pronounced decrease when OOW is used as
mixing water. Therefore, the incorporation of OW and OOW as mixing water produced a
decrease in the interconnected surface porosity to a lesser extent by altering its quality and
durability. However, all samples meet the UNE, which states that the value of the suction of
water should be lower than 4.5 kg/m2 min.
Figure 8. SEM micrographs of extruded fired bricks using as mixing water (a) FW; (b) OW waste and
(c) OWW waste; and SEM micrographs of compressed fired bricks using a missing water (d) FW, (e)
OW and (f) OWW.
Figure 9. Compressive strength of the fired bricks fired as a function of mixing water used in the
extrusion and compression process.
The microstructure of fired bricks with FW, OW or OWW formed by extrusion and
compression has been studied by SEM on fracture surface. Figure 8 illustrated the general
features of the microstructures. It can be observed by SEM (Figure 8 a y b) that clay porosity
is due mainly to open porosity, which is formed by fine, interconnected, irregularly-shaped
pores and a lower proportion of small-sized and closed pores. At 950 ºC, the amount of liquid
phase and its viscosity are not sufficient to efficiently close open porosity. According to the
data of water absorption greater open porosity in compression-formed bricks can be observed.
Employment of OW or OWW as mixing water results, in a greater proportion, in the
formation of spherical closed pores, being the amount of closed porosity higher when using
the extrusion technique (Figure 8 c, d, e and f).
Compressive strength is the most critical index for building materials. The compressive
strength of fired products is shown in Figure 9. Compressive strength values indicated that the
mixing water and forming process hardly affect the compressive strength. The compressive
strength of the samples that used as mixing water FW ranged from 32.25 and 26.24 MPa for
extruded and shaped by pressure bricks, respectively. Bricks formed by compression had
lower compressive strength values than extruded bricks, since although these samples had
higher bulk density, and therefore lower total porosity, they have higher proportion of open
porosity, as indicated water absorption data and SEM micrographs. Due to their irregular
shape and microscopic imperfections, open pores can concentrate pressures and decrease
compressive strength in bricks [50]. The used of OW and OWW produced a slight increase in
the compressive strength of bricks, due to the small decrease in open porosity. As a result,
extruded bricks that used OOW as mixing water showed the maximum compressive strength
value of 39.4 MPa due to the type of porosity created (closed porosity) or, more probably, to
the pore shape (spherical) [51]. Nevertheless, in all cases, compressive strength rates are
always significantly exceed the requeriments of UNE-EN 772-1:2002 standard [40], which
establishes that strength of good quality bricks must be above 10 MP.
Figure 10. (a) Thermal conductivity of the fired bricks fired as a function of mixing water used in the
extrusion and compression process. (b) Relationship between thermal conductivity and bulk density of
the extruded and compressed fired bricks.
A key aim of the ceramic industry is to improve thermal insulation of ceramic products
without impairing their mechanical properties. The thermal conductivity of the materials used
plays a key role in avoiding heat loss and conserving energy. The bulk density and porosity
are the major factors governing thermal conductivity [44, 52-55]. Figure 10 shows the
thermal conductivity of samples. Thermal conductivity of extruded bricks is lower than
compressed bricks, according with total porosity and closed porosity data of extruded
samples. The thermal conductivity decreases with increasing total porosity (thus a lower
density values). The use of OW or OWW as mixing water for clay increased total porosity
and decreased bulk density, producing a slight decrease in thermal conductivity. The closed
pores created by the combustion of organic matter of the wastes during brick firing provide
this insulation quality. Figure 10 b showed thermal conductivity versus bulk density of the
fired extruded and compressed clay bricks. In fact, according to data, there seems to be a
good correlation between both factors R2=0.95 and R2=0,99 for extruded and compressed
bricks, respectively.
The relation between the two parameters has been described elsewhere [56]. Also,
mineralogical composition, no modified using OW or OOW as water mixing, and
microstructure of the bricks, are important factors governing thermal conductivity [44, 55, 57,
58]. So, OOW clay extruded bricks had a lower thermal conductivity 0,815 W/m2K, showing
lower bulk density and lower water absorption. Therefore, closed and small pores contribute
to a higher extent to increase thermal insulation properties of bricks.
CONCLUSION
Wastewater from olive-washing stage (OW) and wastewater from olive oil-washing stage
(OOW) represents a highly pollutant effluent obtained in most of olive oil extraction factories
in Mediterranean countries, operating the so-called two-phase extraction procedure. The
valorization and recycling of these wastewaters could be a partial solution to the negative
environmental impact, since these effluents are usually sent to evaporation ponds producing
environmental and economic costs. Disposal of wastewaters obtained from olive oil
extraction, OW and OOW, can be effectively achieved by using these liquid wastes as mixing
water for brick manufacturing.
This work attempted to show that in spite of the solid organic contents of the OW and
OOW wastewaters, its substitution for the fresh water currently used in brick manufacture had
a positive effect on bricks performance. Technological properties were hardly affected by the
use of the wastewater, improving them slightly, being its use beneficial. Test confirms that
the experimental products that incorporated OW or OWW as mixing water decreased the bulk
density and thermal conductivity compared to the brick control used as mixing FW. These
incorporations of waste decreased slightly the values of water absorption and hardly increased
the compressive strength. The improvement in the technological properties may be due to
slight increase in closed porosity when OW or OOW were used as mixing water as indicated
SEM micrographs.
As regards the forming process it is concluded that extrusion is the most appropriate
technique, due to getting specimens with the best technological properties: lower bulk
density, water absorption and thermal conductivity and greater compressive strength than the
bricks formed by compression. The extrusion process is also more versatile and presents a
higher production capacity, to work continuously, important aspect at the industry level.
Therefore, the use of these types of waste is a benefit both on an environmental and
economic basis. The main advantage for the olive oil extraction industry is the elimination of
storage pools, while ceramic industry benefits from reducing fresh water and heating
requirements, along with the production of better materials.
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Chapter 3
FRUIT AND POMACE EXTRACTS: APPLICATIONS

TO IMPROVE THE SAFETY AND QUALITY OF
FISH PRODUCTS
F. Gai* and P. G. Peiretti

Institute of Sciences of Food Production, Italian National Research Council,
Grugliasco, Italy
ABSTRACT
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.
Keywords: Fish, fatty acid, grape, pomegranate, berry, oxidative spoilage, microbial
spoilage.
INTRODUCTION
Fish flesh is considered to have great nutritional value due to its higher levels of long
chain n-3 polyunsaturated fatty acids (PUFA), which have been shown to have various health
*
Corresponding author: Tel.: +39 011 6709232; fax: +39 011 6709297. E-mail address: francesco.gai@ispa.cnr.it.
54 F. Gai and P. G. Peiretti
benefits in human nutrition. Unfortunately, the unsaturated nature of these PUFAs makes
them highly susceptible to oxidation, which is responsible for rancidity and loss of nutritive
value, resulting in off-flavors and structural changes which are unappealing for consumers
(Kulawik et al., 2013).
Moreover, fish products have a short shelf life compared to meat products, due to the
microbiological spoilage that limits the storage, processing and use of this food source
(Speranza et al., 2009). Spoilage of fish muscle results from changes brought about by
biological reactions such as the metabolic activities of the microorganisms present in the
aquatic environment in which fish live. The main bacterial species involved in spoilage are
Pseudomonas spp., Aeromonas spp., Vibrio spp., certain species of psychrotolerant
Enterobacteriaceae, usually found in iced freshwater fish, and Shewanella putrefaciens-like
bacteria, mainly Shewanella putrefaciens, Shewanella algae, and Shewanella baltica, which
are commonly found in iced marine fish (Kulawik et al., 2013).
These microorganisms generate a wide array of compounds that contribute to whole fish
spoilage and produce many off-odor and off-flavor compounds such as biogenic amines
(putrescine, cadaverine, and histamine), hydroxylamine, ketones, aldehydes, alcohols, and
organic acids that are essentially absent or only occur at very low levels in fresh fish (Ghaly
et al., 2010).
In order to contrast the chemical and microbiological deterioration of fish products, the
use of natural or naturally-derived antioxidants has fostered research into identifying new
low-cost antioxidants having commercial potential for the production of foods with a longer
shelf life and a higher degree of safety (Corbo et al., 2008; Maqsood et al., 2013; Widsten et
al., 2014). The fruit-processing industry generates large volumes of solid waste in the form of
peels, kernels, and pulp, which are rich in bioactive phytochemicals, dietary fiber and
unsaturated fatty acids, and hence have the potential to serve as functional food ingredients
(Babbar et al., 2015).
In the following paragraphs, various studies carried out into the application of fruit and
pomace extracts, by-products from the processing of grape, pomegranate and berry fruits, in
limiting the oxidative and microbial spoilage of fish products, are critically reviewed.
GRAPE
In the world, grape (Vitis vinifera L.) is the second largest fruit crop after oranges. During
the production of wines, up to 40% of the grape ends up as by-products (Moreno-Arribas &
Polo, 2005). The principal winery by-product, grape pomace, produced after pressing the
crushed grapes in production of white wine or after fermentation and maceration for red wine,
contains mainly pressed grape skins, pulp, seeds and stems that are rich sources of
inexpensive antioxidative phenolic compounds (Cheynier, 2012; Cheynier et al., 2013). The
grape seeds, which can be recovered from pressing grape pomace and after maceration during
first pressing contain oils rich in linolenic acid (12–20%), protein (11%), non-digestible
carbohydrates (60–70%), and phenolic and non-phenolic antioxidants (Yu & Ahmedna,
2013). Numerous studies have demonstrated that these compounds exhibit health-promoting
effects, with cardioprotective, neuroprotective, and other health benefits (Pozo-Bayón et al.,
2012; Del Rio et al., 2013).
The different studies investigating the properties of grape by-products carried out in fish
muscle models are summarized in Table 1.
Table 1. Recent articles about the effects of grape by-products on fish products
Fish Type of Impact on

Scientific name Reference
product ingredient product
Atlantic Scomber scombrus White grape Inhibition of Pazos et al.,
mackerel pomace ethanol muscle lipid 2005a,b
white extract oxidation during
muscle frozen storage by
flavanol oligomers
Bonito Sarda sarda Grape seed Delayed lipid Yerlikaya &
fillets ethanol extract oxidation Gökoğlu,
during frozen 2010a
storage
Horse Trachurus Grape pomace Inhibition of fillet Pazos et al.,
mackerel trachurus procyanidins lipid oxidation 2006, 2010
fillets Red grape Inhibition of lipid Sánchez-
antioxidant oxidation during Alonso &
dietary fiber frozen storage Borderıás,
2008
Red grape Delayed lipid Sánchez-
antioxidant oxidation in 3 Alonso et al.,
dietary fiber months of frozen 2007a
storage
White grape Augmented Sánchez-
dietary fiber aggregation of Alonso et al.,
concentrate myofibrillar 2007b
proteins, Enhanced
water retention &
improved cooking
yield
White grape Delayed lipid Sánchez-
antioxidant oxidation during Alonso et al.,
dietary fiber frozen storage 2008
Meagre Argyrosomus regius White grape Lowered TBARS Ribeiro et al.,
sausages antioxidant values; 2013
dietary fiber Antimicrobial
effect
Rainbow Oncorhynchus Red grape Delayed lipid Gai et al.,
trout mykiss pomace ethanol oxidation & 2014
minced extract cadaverine
muscle formation after
refrigerated storage
Silver Hypophthalmichthys Grape pomace Delayed lipid Hasani &
carp molitrix acetone extract oxidation Dughikolaei,
fillets during refrigerated 2014
storage
Pazos et al. (2005a) determined the ability of white grape pomace extract fractions,
containing flavanol monomers, oligomers (procyanidins) and glycosylated flavonols, to
inhibit oxidation of fish lipids. The tests were conducted in mackerel (Scomber scombrus)
minced fillets during frozen storage at -10 °C within 6 months. Duplicate samples of frozen
muscle were taken regularly over the course of six months, and oxidation was followed by
measuring peroxide value, conjugated diene and triene hydroperoxides, thiobarbituric acid-
reactive substances (TBARS), fluorescence compounds and degradation of PUFA. The
effectiveness of each antioxidant was evaluated by comparing the inhibition of oxidation with
samples treated with synthetic antioxidant (propyl gallate) used as control. Grape phenolic
fractions showed different effectiveness on decreasing the rate of oxidation and the amount of
oxidation products formed; flavanol oligomers were the most potent inhibitors of oxidation in
frozen fish muscle. The authors concluded that in order to obtain the highest antioxidant
efficacy of grape polyphenols in frozen fish muscle, an optimal combination of procyanidin
degree of polymerization and galloylation percentage should be considered.
In another similar study, Pazos et al. (2005b) investigated the capacity of a phenolic
extract, obtained from pressing destemmed Parellada grapes, and of a purified fraction of
procyanidins to preserve the endogenous antioxidants of minced mackerel (Scomber
scombrus) muscle and horse mackerel (Trauchurus trauchurus) fillets during frozen storage
at -10 °C for a period of 140 days. Grape polyphenols were used in muscle concentrations of
0.01% and compared with propyl gallate, as synthetic antioxidant. The results demonstrated
that grape polyphenols inhibit the depletion of endogenous α-tocopherol, ubiquinone and total
glutathione. Moreover, grape polyphenols showed similar efficiency to the synthetic
antioxidant propyl gallate in the preservation of ubiquinone, in both minced and filleted
muscle, and total glutathione, in minced muscle. Grape polyphenols more efficiently
preserved α-tocopherol, an endogenous antioxidant whose depletion is highly correlated with
the evolution of lipid oxidation. The development of lipid oxidation was repressed, while the
concentration of α-tocopherol was not reduced up to critical levels, so the authors concluded
that grape polyphenols may be an attractive potential food additive because they can stabilize
frozen fatty fish and preserve an important compound such as vitamin E.
Pazos et al. (2006) carried out a study aiming to evaluate the reducing and chelating
capacities, and the affinity for incorporation into frozen horse mackerel fillets stored at -10°C,
of grape procyanidins isolated from grape pomace. The effects of grape procyanidin were
compared against two other phenolic antioxidants, hydroxytyrosol and propyl gallate.
Phenolic antioxidants were supplemented into the fillets in three ways: spraying with an
aqueous phenolic solution, glazing with an aqueous phenolic solution, and pre-washing of
fillets with water plus spraying with an aqueous phenolic solution. Lipid oxidation in the fish
fillets was delayed by all phenolic compounds and the order of antioxidant efficiency in
spraying and glazing was as follows: propyl gallate > hydroxytyrosol > grape procyanidin. As
far as the reducing power of these phenolics is concerned, the order of efficiency was similar
to that observed in the spraying and glazing treatments, but no correlation with their chelating
capacity and their affinity to fish muscle was shown. The antioxidant activity of grape
procyanidin increased synergistically where fillets were washed with water prior to spraying
phenols and relative antioxidant efficiency changed to propyl gallate > grape procyanidin >
hydroxytyrosol. The authors concluded that this synergism could be due to the residual water
that remained on the fillet surface after washing, resulting in a better distribution of grape
procyanidin across the fillet surface.
Pazos et al. (2010) evaluated the influence of polymerization and galloylation of

oligomeric catechins (proanthocyanidins) obtained from grape, pine (Pinus pinaster) bark and
witch hazel (Hamamelis virginiana) on their effectiveness to prevent lipid oxidation in horse
mackerel muscle refrigerated at 4°C. Fractions from pine bark showed non-galloylated
oligomers of catechin with diverse mean polymerization (1.9-3.4 monomeric units) while
grape and witch hazel showed homologous fractions with galloylation ranging from 0.25 to
<1 gallate group per molecule. The formation of lipid oxidation products, lipid peroxides, and
volatiles responsible for rancidity was evaluated by comparing induction periods and
inhibition percentages in order to assess the antioxidant effectiveness of the different
fractions. Lipid oxidation in horse mackerel muscle was better inhibited by the
proanthocyanidins of medium size (2-3 monomeric units) and low galloylation degree (0.15-
0.25 gallate group/molecule). The authors therefore concluded that their study gave
information relevant for achieving optimum use of polyphenols in muscle-based foods such
as pelagic fish muscle.
Neira et al. (2011) investigated the influence of different concentrations (10-100 μg/g) of
polyphenolic fractions extracted from grape pomace to prevent haemoglobin-promoted lipid
oxidation using in vitro experiments carried out with the pelagic fish, horse mackerel. Horse
mackerel light muscles were washed, homogenized and frozen at -80°C for less than one
week and then thawed for 30 min under running cold water. Horse mackerel haemoglobin
was added as lipid oxidation initiator to the light muscles at a concentration of 15 μmol/kg
fish. Peroxide value, TBARS index, and sensory analysis were carried out to monitor lipid
oxidation. All polyphenolic fraction concentrations, except the final concentration of 10 ppm,
were able to prevent haemoglobin-promoted lipid oxidation, proportionally to the amount of
polyphenolic fraction added, with samples supplemented with 100 ppm showing the longest
induction periods (5 days) and inhibition percentages. The same antioxidant efficiency was
showed by the sensory analysis of rancid off-flavors, with the 100 ppm-supplemented group
showing the greatest inhibition of rancidity. In the light of the results, the authors concluded
that new antioxidant ingredients from natural sources could be useful for preventing fish lipid
oxidation in which oxidation is essentially initiated by heme proteins.
Hasani & Dughikolaei (2014) determined the antioxidant effect of red grape pomace
extract supplemented on fillets of silver carp (Hypophthalmichthys molitrix) during a
refrigerated storage challenge. Silver carp samples were treated with different concentrations
of red grape pomace extract (0%, 2%, and 4%) and stored for 15 days in a refrigerator at
+4°C. At 0, 3, 6, 9, 12, and 15 days of storage, the changes in pH, peroxide value, TBARS
and heme iron were measured. Results showed that samples treated with 4% of red grape
pomace extract were better preserved compared with other treatments; indeed, the addition of
this concentration of extract significantly delayed lipid oxidation in silver carp fillet during
refrigerated storage. The authors concluded that red grape pomace extract has the potential to
be used as a natural antioxidant, improving the quality and preventing the deterioration of
stored fish.
Gai et al. (2014) evaluated the effects of the addition of ethanol red grape pomace
extracts on the shelf life of minced rainbow trout (Oncorhynchus mykiss) muscles. Extracts
were added to trout patties to give a final concentration of 0%, 1%, and 3% and after 1 and 6
days of refrigerated storage, the treated muscle samples were analyzed for their physical (pH,
color) and chemical (TBARS, fatty acid and biogenic amine content) properties. Red grape
pomace extracts were effective in delaying lipid oxidation and cadaverine formation in
muscle after six days of refrigerated storage, with the best results obtained in the samples
treated with the 3% extract. The authors concluded that red grape pomace extract in minced
trout muscle can enhance the quality and shelf life of this ready-to-cook fish-based food and
simultaneously provide a functional food with natural antioxidants that are beneficial for
consumers.
Wine by-products are also rich in dietary fiber. Saura-Calixto (1998) proposed to define
antioxidant dietary fiber as a natural product that combines the beneficial effects of dietary
fiber and natural antioxidants, such as polyphenol compounds. This dietary fiber can also be
an effective tool in seafood processing for improving functional properties, such as water
binding, gelling etc.
Sánchez-Alonso et al. (2007a) studied the effect of red grape antioxidant dietary fiber
addition, at concentrations of 0%, 2%, and 4% to horse mackerel (Trachurus trachurus)
minced fillets on flesh lipid stability during a 6-month frozen storage experiment. Red grape
antioxidant dietary fiber was characterized in terms of dietary fiber, total polyphenols and
antioxidant capacity, and multifunctional antioxidant assays were carried out on all the
minced fillets immediately after the preparation of samples and at 30, 60, 90, 120, 150 and
180 days of storage at -20 °C. The formation of conjugated dienes and trienes (products of
lipid oxidation) was monitored during frozen storage and the samples with added dietary fiber
exhibited reduced formation of dienes and trienes until 90 days compared with the control lot.
Moreover, TBARS values were lower in the samples with dietary fiber added for most of the
storage period. The rate of inhibition at 90 days of frozen storage was 57.3% and 54.1% for
the treated sample groups, respectively. At this time of frozen storage, antioxidant action was
not significantly different for the two levels of dietary fiber. The authors concluded that the
addition of red grape fiber considerably inhibits oxidation in minced fillets during the first 3
months of frozen storage, indicating that red grape antioxidant dietary fiber could be used as
an ingredient to prevent oxidation in minced fillets during frozen storage. A possible reason
for this effect could be the chelating action of fiber on some pro-oxidant metals or the action
of grape flavonoids associated with dietary fiber.
In another similar study, Sánchez-Alonso & Borderias (2008) studied the effect of adding
red grape antioxidant dietary fiber to horse mackerel (Trachurus trachurus) minced fillets as
a technological ingredient in a frozen storage trial lasting 6 months. Concentrations of 0, 2
and 4% of red grape antioxidant dietary fiber were added to minced fish muscles. Sensory
analyses, protein solubility, water retention, color, mechanical properties and lipid oxidation
analyses were carried out immediately after preparation of samples and after every 30 days of
storage at -20°C. The presence of fiber significantly increased the water retained after
physical stress while it reduced thawing drip in minced products over the entire storage
period. Texture profile analysis indicated that changes in fiber content significantly affected
the textural characteristics, with lower textural parameters found in the samples with the
highest concentrations of fiber. Regarding the lipid oxidation products formed from PUFAs,
lower levels of conjugated hydroperoxides, dienes and trienes were found in samples with
added red grape antioxidant dietary fiber (2% and 4%) over a period of 6 months at -20 °C.
At 90 days of storage, inhibition of oxidation development was significant in samples with
added red grape antioxidant dietary fiber; development of conjugated dienes was inhibited by
18.2% and by 26.7% in the 2% and 4% extract groups, respectively. As regards sensory
analysis, the panellists detected no significant changes in flavor over the course of frozen
storage. Overall, the sample with 2% fiber addition received the best ratings followed by the
control group and the 4% extract group. In conclusion, in the light of the chemical, physical
and sensory analyses, the authors suggest that red grape antioxidant dietary fiber is a highly
active technological ingredient in frozen dark minced fish.
In another study, carried out again in horse mackerel, Sánchez-Alonso et al. (2007b)
investigated the functional properties of a white grape (var. Airén) dietary fiber concentrate
obtained from peels and seeds. This product was added at 2% and 4% to minced fish muscle
and stored for 6 months at -20 °C. Every month, samples were analyzed for their physical and
mechanical properties, sensory and color attributes, microscopic and electrophoretic profiles.
White grape dietary fiber concentrate showed good functional properties, high water and oil
retention capacity, and considerable swelling properties. Its addition augmented aggregation
of myofibrillar proteins and made samples softer and less springy and cohesive in the course
of frozen storage. Moreover, addition significantly enhanced water retention and improved
cooking yield. In sensory evaluation, according to the hedonic analysis, all samples were
considered acceptable with 4% samples scoring the lowest values. The minced fish muscle
with 2% concentrate scored slightly higher for flavor than the 4% and control groups. Any
odd flavors, throughout frozen storage, were detected by the panellists while some
differences, albeit insignificant, were perceived in the texture. The results indicate that white
grape dietary fiber concentrate could be proposed as a new ingredient in minced fish muscle,
not only for its nutritional and antioxidant properties but also for its technological properties.
In a similar experimental design, Sánchez-Alonso et al. (2008) carried out a study
evaluating the antioxidant properties of white grape dietary fiber concentrate in horse
mackerel minced muscle. The evaluation of this by-product‘s antioxidant capacity when
added to frozen minced muscle over 6 months of storage was similar for all the methods
followed. Addition considerably delayed lipid oxidation in minced fish muscle during frozen
storage. These results indicate that white grape dietary fiber concentrate could be used as a
natural ingredient to prevent oxidation in minced fish during frozen storage and could be used
as a functional ingredient in the design of healthy foods.
Ribeiro et al. (2013) evaluated the effect of antioxidant grape dietary fiber addition in
sausages made with farmed meagre (Argyrosomus regius), during a 98-day storage
experiment at a refrigeration temperature of 2±2°C. Physical, chemical, nutritional,
microbiological and sensory assessment were performed in two experimental groups, one
contained 3.9% of inner pea dietary fiber and the other group contained 0.9% inner pea
dietary fiber plus 3.0% of antioxidant grape dietary fiber. The latter group had an effective
antioxidant capacity, proven not only by the radical scavenging activity and reducing power
measurements, but also by the lower TBARS values over storage time. Moreover, antioxidant
grape dietary fiber seemed to have some antimicrobial effects. Formulation had an effect on
the H2S producer counts over storage time, as control sausages had an average logarithmic
count of 0.59 log CFU/g, while antioxidant grape dietary fiber sausages showed a value of
0.26 log CFU/g. From day 63 onwards, microbial growth in the control sausages was higher
(3 log CFU/g) than in the antioxidant grape dietary fiber sausages (<3 log CFU/g). Sensory
assessment only highlighted more accentuated loss of textural quality in the latter sausages.
Yerlikaya & Gökoğlu (2010a) compared grape seed extract with green tea extract and a
control in an experiment carried out in bonito (Sarda sarda) fillets during a 5-month frozen
storage period. Ethanol extract solutions of 1% were prepared using concentrated extracts and
distilled water and were applied to bonito fillets using two different methods: either dipped
into extract solutions and then frozen or glazed with extract solutions. Lipid oxidation
increased progressively through the storage period, but in samples treated with grape seed
extract and green tea extract, TBARS and para-anisidine (indicators of secondary oxidation)
remained at low levels, with grape seed being more at retarding oxidation. Dipping into
extract solutions before freezing inhibited lipid oxidation of bonito fillets more than using
extracts as glazing solutions.
POMEGRANATE
Pomegranate (Punica granatum L.) belongs to the Punicaceae family and is a deciduous
shrub cultivated throughout the world and in particular in the Mediterranean area.
Historically, the fruits, peels and roots of pomegranate have been commonly used for various
ethnomedical uses by local healers in many countries. On the other hand, modern applications
of pomegranate-derived products (seed oil, juice, fermented juice and peel or seed extract)
include uses for cardiovascular protection, oral hygiene and as an anti-obesity agent, as
recently reviewed by Viuda-Martos et al. (2010). The antimicrobial activity (in vitro and in
situ) of tannin-rich peels has been demonstrated in a study carried out by Al-Zoreky (2009),
revealing the relationship between microbial inhibition and total phenolic content of various
peel extracts including phenolics and flavonoids against some food-borne pathogens (Listeria
monocytogenes, Staphylococcus aureus, Escherichia coli and Yersinia enterocolitica).
Table 2. Recent articles about the effects of pomegranate by-products on fish products
Fish Type of Impact on

Scientific name Reference
product ingredient product
Bonito Sarda sarda Pomegranate Increased adhesion Yerlikaya &
fillets peel extract values of fillets Gökoğlu, 2010 b
Chub Scomber japonicus Pomegranate Inhibition of lipid Özalp Özen et al.,
mackerel seed extract hydroperoxides & 2011
minced thiobarbituric acid-
muscle reactive substances
Greenland Reinhardtius Pomegranate Antioxidant Ünalan et al., 2011
halibut hippoglossoides ethanol extract effect/No
fillets beneficial effect on
microbial spoilage
Marinated Engraulis Pomegranate Better oxidative Gökoğlu et al.,
anchovy encrasicholus sauce stability, higher 2009
taste & flavor &
lower appearance
scores
Pomegranate Prolonged lipid Topuz et al., 2014
juice sauce oxidation &
improved sensory
properties
Rainbow Oncorhynchus Pomegranate Firmer texture & Demirok et al.,
trout mykiss juice sweeter taste 2014
fillets
The various studies, investigating the properties of the pomegranate by-products, carried
out in fish muscle models, are summarized in Table 2.
Demirok et al. (2014) investigated the suitability of pomegranate juice, produced by
pressing fresh pomegranate arils, on the proteolytic and sensory changes of rainbow trout
flesh marinated in three different marinating solutions for 12 days at a storage temperature of
4°C. 100% acetic acid, 50% acetic acid/50% pomegranate juice, and 100% pomegranate juice
marinating solutions were tested. At 1, 3, 6, 9, and 12 days during the marinating process, on
raw fish fillets and marinated rainbow trout flesh, proximate composition, chemical and SDS-
PAGE analysis as well as color measurement and sensory evaluation were performed. Results
indicated that fish flesh marinated in 100% pomegranate juice had lower protein content and
the highest significant ash content per marination day. Color measurement showed that fish
flesh traditionally marinated in 100% acetic acid was lighter than fish marinated in 50%
acetic acid/50% pomegranate juice and 100% pomegranate juice, while groups containing
pomegranate juice had higher redness and lower yellowness values due to migration of color
components present in juice to the fish tissues. In light of the results, the authors concluded
that the fish flesh thus produced would combine the positive effects of pomegranate juice
with the organoleptic and textural properties of traditionally-marinated fish flesh.
In order to prolong fish marinade shelf life, Gökoğlu et al. (2009) investigated the effects
of the addition of pomegranate sauce on the quality of marinated anchovy during storage at
+4°C. Anchovies (Engraulis encrasicolus) were marinated with 30 g/L acetic acid and 150
g/L salt, put into glass jars, filled with either sunflower oil or pomegranate sauce, prepared
with a commercial pomegranate juice concentrate, and stored at 4°C. At monthly intervals,
fish samples from each jar were randomly taken, drained, homogenized and analyzed to
determine chemical and sensorial quality changes. pH, total volatile basic nitrogen,
trimethylamine, free fatty acid, conjugated diens and para-anisidine values were recorded at
0, 1, 2 and 3 months of storage. Samples with sunflower oil showed higher values for free
fatty acid, conjugated diens and para-anisidine than those with pomegranate sauce. Moreover,
samples in pomegranate sauce showed better oxidative stability, higher taste and flavor and
lower appearance scores than those in sunflower oil. The authors concluded that even though
pomegranate sauce produced desirable taste and flavor, pomegranate sauce was at least as
effective as traditional sunflower oil at maintaining marinated anchovy quality.
Topuz et al. (2014), in order to produce a new polyphenol-rich marinade sauce by
emulsifying pomegranate juice in different proportions (25%, 35% and 50%) with olive oil,
evaluated the effects of these sauces as an antioxidant, preservative and flavoring agent in
anchovy marinades. In order to evaluate these effects, chemical, oxidative and sensory
analyses were carried out at 0, 20, 40, 60, 80 and 100 days of storage at 4°C. The results
enabled the authors to conclude that these sauces have the ability to delay chemical changes,
inhibit lipid oxidation, maintain sensory attributes and extend shelf life in addition to their
desirable taste and flavor.
Özalp Özen et al. (2011) studied the effect of adding pomegranate seed extract and grape
seed extract on lipid oxidation during frozen storage of chub mackerel (Scomber japonicus)
minced muscles. These samples were supplemented with a concentration of 2% for each
extract and then stored at -18°C for a 3-month storage period. Pomegranate seed extract and
grape seed extract addition significantly inhibited the formation of lipid hydroperoxides and
TBARS as analyzed each month, when compared with the control group. According to the
results of TBARS, both extracts significantly retarded fish lipid oxidation even though a
significant reduction in color values was detected during frozen storage. Samples with
pomegranate seed extract showed the lowest redness and the highest yellowness values. The
authors concluded that to prevent oxidation in minced muscles during frozen storage, grape
seed and pomegranate seed could be used as an antioxidant source.
Ünalan et al. (2011) evaluated the effect of using an ethanol pomegranate extract (1%)
and rosemary extract (1%) on the shelf-life extension of chilled Greenland halibut
(Reinhardtius hippoglossoides) fillets stored in modified atmosphere packaging at 2 °C.
Microbiological (aerobic plate counts, lactic acid bacteria, Lactobacillus spp., and
Photobacterium phosphoreum), biochemical (pH, TBARS, trimethylamine and total-volatile-
nitrogen) parameters and sensory (color, flavor and texture) attributes were monitored at 0, 3,
7, 10, 14, 18 and 23 days of storage. The spoilage microflora of fillets in modified atmosphere
packaging was dominated by lactic acid bacteria, but the concentration of Lactobacillus was
very low while aerobic plate counts reached levels of >107 CFU/g during storage, irrespective
of treatments. Among the chemical indices examined, TBARS values of control samples
exceeded the limit of 2 mg malondialdehyde/kg on days 18 and 23 of storage, while samples
treated with extracts showed values below the limit throughout the storage period.
Trimethylamine and total-volatile-nitrogen final values did not exceed the upper acceptability
limit set by the European Union for all treatments. Sensory analysis correlated well with
trimethylamine and total-volatile-nitrogen values, indicating a shelf life of longer than 23
days for all samples. The authors concluded that despite no significant antimicrobial activity,
pomegranate extracts provide their antioxidant effect on chilled halibut fillets in modified
atmosphere packaging.
Yerlikaya & Gökoğlu (2010b) carried out a study on the sensory and physical properties
of bonito (Sarda sarda) fillets, comparing the use of pomegranate peel extract with grape
seed extract and green tea extract and a control group at maintaining fish quality
characteristics during frozen storage. Fillets were dipped into plant ethanol extract solutions
before freezing, then glazed and stored at -18°C for 5 months. Sensory analysis showed that
the preference of panellists focused on steam-cooked fillets treated with green tea extract
followed by the samples with grape seed extract, in terms of odor and texture at the end of the
storage period. Regarding the color results, the redness and yellowness parameters of the
fillets were affected by the natural color of the pomegranate peel extract and grape seed
extract, respectively. Stiffer and less adhesive product was obtained within the control groups,
followed by fillets to which green tea extract had been applied. Deformation of muscle
structure was considerably decreased by dipping fillets into plant extracts before glazing.
OTHER FRUITS AND BERRIES

The high antioxidant capacity of berry extracts is particularly due to their content of
various phenols, anthocyanins, and ascorbic acid (Pantelidis et al., 2007). Besides the health
benefits related to their natural antioxidants, the color attributes of berry extracts are also of
interest in food processing, as color plays a vital role in the acceptability of foods.
The different studies, investigating the properties of berries and other fruits, carried out in
fish muscle models are summarized in Table 3.
Table 3. Recent articles about the effects of berries and other fruits on fish products
Fish Scientific Impact on

Type of ingredient Reference
product name product
Fish oil Not Ethanolic extracts of Higher inhibition Sekhon-Loodu et
indicated apple of lipid oxidation al., 2013
peel compared to
synthetic
antioxidants
Marinating Clupea Concentrated Enhanced quality Sampels et al.,
herring harengus solutions of either & shelf life of the 2010
fillets elderberry, cranberry fillets
or blackcurrant
Menhaden Not Black raspberry seed Suppressed lipid Luther et
Fish oil indicated flours oxidation & al.,2007
rancidity
development in
fish oil
Preserved content
of total n-3 fatty
acids & PUFA
Sekhon-Loodu et al. (2013) studied the potential use of polyphenols isolated from frozen
and dried apple peel extract as natural antioxidants to stabilize a fish oil enriched with n-3
PUFA. The ethanolic apple peel extracts were fractionated by reversed-phase
chromatography and the total phenolic content and antioxidant capacity of each fraction were
evaluated by Folin–Ciocalteu, ferric reducing antioxidant power and 1,1-diphenyl-2-
picrylhydrazyl (DPPH) radical scavenging assays. Inhibition of fish oil oxidation, compared
with butylated hydroxytoluene (BHT) and α-tocopherol as reference antioxidants, was studied
using the TBARS assay. Significantly higher scavenging values were found using
polyphenols fractionated from frozen apple peel extract than those of dried apple peel extract.
Fish oil oxidation was inhibited by 40–62% with the flavonol-rich fractions at a total phenolic
concentration of 200 µg/ml. The fractionated polyphenols from both dried and frozen apple
peels extract showed higher inhibition of lipid oxidation compared to BHT, α-tocopherol, and
raw apple peel extract.
Sampels et al. (2010) evaluated inhibition of the oxidation of lipids and proteins and also
the degradation of tocopherol in marinating herring (Clupea harengus) fillets treated with
powder of elderberry (Sambucus nigra), cranberry (Vaccinium spp.), or blackcurrant (Ribes
nigrum). Solutions made from concentrates of either elderberry, cranberry and blackcurrant
were used to marinate the fillets; additionally, two groups of fillets were injected with a salt
solution before they were marinated in blackcurrant solution. After treatment, all of the fillets
were vacuum-packed, stored on ice for 7 days, and then frozen at -20 °C for 6 months.
Chemical composition and oxidation markers as volatile lipid compounds (propanal, hexanal,
2-penten-1-ol, and 1-penten-3-ol) and biogenic amines (agmatin and tyramine) were
determined in the treated herring fillets. Cranberry and blackcurrant appeared to be more
efficient than elderberry at inhibiting the degradation of tocopherol and the formation of
ammonium. Significantly increased levels of carbonyls, ammonium, and biogenic amines
were found in fillets injected with a 5% salt solution, whereas formation of volatile lipid
compounds was lowest in fillets marinated in blackcurrant following injection of the salt
solution. The authors concluded that the quality and shelf life of herring fillets marinated in
solutions containing berry powder can be enhanced and enriched with natural antioxidants
that are beneficial for consumers.
Luther et al. (2007) evaluated the capacity of ethanolic seed flour extracts from defatted
black raspberry (Rubus occidentalis) and Chardonnay grape to suppress lipid oxidation and to
preserve important fatty acids in menhaden fish oil. A low 10% and a high 20% dose of
extract were tested for each treatment. Mixed tocopherols and fish oil containing no
antioxidant were used as the positive and negative control, respectively. The results of the
oxidative stability index, that measures the secondary products of lipid peroxidation by total
volatile carbonyl compounds, were expressed as the % extension time, defined as the hours
required for an oil sample to develop measurable rancidity. Moreover, relative concentrations
of docosahexaenoic acid, eicosapentaenoic acid, and total n-3 PUFA in fish oil samples
containing the antioxidant extracts collected at 4.5 h of oxidation reaction time at 80°C were
analyzed and compared with the original fish oil for their fatty acid composition. Results
showed that black raspberry and Chardonnay grape seed flour extracts were able to suppress
lipid oxidation and rancidity development in fish oil under the experimental conditions, with
Chardonnay grape seed showing the best results. Moreover, the black raspberry and
Chardonnay grape seed flour extracts were able to reduce the loss of eicosapentaenoic acid
and total PUFA in the fish oil under the accelerated oxidation conditions, with the black
raspberry being more effective than the Chardonnay grape seed extract at preserving total n-3
fatty acids and PUFA maintaining higher docosahexaenoic acid content. The authors
concluded that natural food preservatives able to stabilize food products and maintain
essential fatty acids may be developed from edible fruit seed flours.
ACKNOWLEDGMENTS
The author would like to thank Dr. Anthony Green for the English revision of the paper.
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Topuz, O. K., Yerlikaya, P., Ucak, I., Gumus, B. & Büyükbenli, H. A. (2014). Effects of
olive oil and olive oil–pomegranate juice sauces on chemical, oxidative and sensorial
quality of marinated anchovy. Food Chemistry, 154, 63-70.
Ünalan, U., Dalgaard, P. & Korel, F. (2011). Effect of pomegranate (Punica granutum) and
rosemary (Rosmarinus officinalis L.) extracts on shelf-life for chilled Greenland halibut
(Reinhardtius hippoglossoides) fillets in modified atmosphere packaging at 2°C.
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extracts of fruit byproducts: antibacterial activity against foodborne bacteria and
antioxidant capacity. Journal of Agricultural and Food Chemistry, 62, 11146-11156.
Yerlikaya, P. & Gökoğlu, N. (2010a). Inhibition effects of green tea and grape seed extracts
on lipid oxidation in bonito fillets during frozen storage. International Journal of Food
Science and Technology, 45, 252-257.
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and physical properties of frozen bonito (Sarda sarda) fillets. Turkish Journal of
Fisheries and Aquatic Sciences, 10, 341-349.
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biological properties and potential applications. International Journal of Food Science
and Technology, 48, 221-237.
Chapter 4
SUPERCRITICAL FLUID EXTRACTION

OF PHARMACEUTIC COMPOUNDS FROM WASTE
MATERIALS DERIVED
FROM VINIFICATION PROCESSES
Cleofe Palocci and Laura Chronopoulou

Department of Chemistry, Sapienza University of Rome,
Rome, Italy
ABSTRACT
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,
trans-resveratrol 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.

cleofe.palocci@uniroma1.it
70 Cleofe Palocci and Laura Chronopoulou
ECONOMIC IMPORTANCE AND POTENTIAL OF WASTE

MATERIALS OF THE WINE INDUSTRY
According to the OIV, International Organisation of Vine and Wine, 7519 mha of the
world were dedicated to grape cultivation in 2013 [1]. While globally the vineyards extention
is reducing, grape production is increasing and has reached 751 Mq in 2013. Approximately
71% of world grape production is used for wine producion, 27% is consumed as fresh fruit,
and 2% as dried fruit.
The top ten grape producing countries are currently China (13% of world grape
production), the United States (9%), Italy (8%), France (7%), Spain (7%), Turkey (6%), Chile
(4%), Argentina (4%), Iran (3%) and South Africa (2%). However, these figures change
significantly when we consider wine production. Currently, Europe is the world‘s first source
of wine, with France, Italy and Spain being the top three wine producers in the world,
followed by american countries such as the United States and Argentina. Australia, China,
South Africa, Chile and Germany complete the list of the top ten wine producing countries in
the world.
The residue of wine making is named grape pomace or grape marc and is produced in
large quantities, accounting for approximately 20% of grapes used in winemaking (w/w). Its
disposal is therefore a major economic and environmental issue. Grape pomace is composed
of seeds, 38 to 52% on a dry matter basis, but also of stems, pulps and skins. Traditionally,
grape pomace has been used to produce pomace brandy. However, in 2008, the European
Community has revoked the compulsory distillation of the by-products of winemaking (EC
Regulation 479/2008 and 555/2008) and this has created a great problem on winery waste
handling and disposal. Grape marc has a heavy environmental impact for its high content of
phenols that considerably increase chemical and biochemical oxygen demands. Currently, the
industrial recovery of grape pomace is performed by its partial use for tartaric acid extraction
or ethanol production [2] and the final solid residue is sometimes cast off as fertilizer,
although the high levels of phenolics constitute a problem because they inhibit seed
germination [3]. Grape pomace has also been used as an additive in animal feeding, but the
presence of polymeric polyphenols (lignin) reduces digestibility because they inhibit
cellulolytic and proteolytic enzymes as well as the growth of rumen bacteria [4].
Considering the increasing awareness of consumers towards the use of additives in foods
and the ongoing demand for functional foods in recent years, the identification of alternative
natural and safer sources of food antioxidants is an important goal.
Parallely, modern industries are focused on diminishing the environmental impact of
industrial byproducts. Therefore, more and more attention is being paid to the recovery of
bioactive phenolics from grape byproducts from the winemaking industry [5]. When grape
berries are processed for red winemaking, the skins and seeds are usually in contact with the
fermenting broth for several days. Thus, grapes are subjected to a slight but prolonged
extraction with a hydroethanol mixture that provides the red wine with a variable content of
polyphenols. However, the residue remaining after fermentation, which mainly consists of
skins and seeds, still contains high levels of polyphenols. Such polyphenols have known
health-promoting effects and other properties in different biological and food systems.
Supercritical Fluid Extraction of Pharmaceutic Compounds … 71
These features are related to the antioxidant characteristics as reducing agents (i.e., by
donating hydrogen-quenching free radicals such as singlet oxygen), that play a role in
inhibiting and delaying lipid oxidation in diverse food systems [6-8].
As a consequence, GP is considered a valuable source of phytochemicals that may be
recovered as functional compounds for the pharmaceutical, cosmetic, and food industries as
well as used as biopesticides. In this way, the recovery of phenolics from grape byproducts
from the winemaking industry has attracted increasing attention in the past years, since
industries may find a high value and sustainable alternative to the current management of
industrial residues.
GRAPE POMACE COMPOSITION

Grape pomace consists mainly of peels (skins), seeds and stems and accounts for about
20–25% of the weight of the grape crushed for wine production [8]. It contains significant
amounts of substances that are of interest for the pharmaceutic, food and cosmetic industries
because of their antioxidant, antimicrobial, antidiabetic, anticarcinogenic and
antiinflammatory activities.
Grape pomace is rich in extractable phenolic antioxidants (10–11% of dry weight). Grape
seed contains abundant phenolic acids, flavonoids, procyanidins and resveratrol, while grape
skins are rich in anthocyanins.
Phenolics are the secondary metabolites of plants. Chemically, they are substances
containing a benzene ring bearing one or more hydroxyl groups. Polyphenols are compounds
that have more than one phenolic hydroxyl group attached to one or more aromatic rings.
Four major classes of polyphenols found in foods are phenolic acids, flavonoids, lignans and
stilbenes [9]. Major stilbenoids found in foods of plant origin are resveratrol and it
glycosides. Resveratrol is a phytoalexin produced by plants in response to pathogen attack,
i.e. a naturally occurring fungicide.
Phenolic acids are related compounds (aromatic carboxylic acids), divided into
hydroxycinnamic and hydroxybenzoic acids. Hydroxycinnamic acids are more common and
they mainly include gallic acid, p-coumaric, caffeic, chlorogenic acid, ferulic and sinapic
acids.
Flavonoids are the largest and best studied class of naturally occurring polyphenols,
comprising different subclasses such as anthocyanidins, proanthocyanidins and isoflavones.
Grape pomace polyphenol composition is affected by many different factors. It is variety
dependant [10-11] and, within the same variety, different parts have significantly different
compositions. For example, the grape skins, have been found to be rich in anthocyanins,
hydroxycinnamic acids, flavanols and flavonol glycosides, whereas the seed portion is a good
source of gallic acid and flavanols [3, 11]. Polyphenol composition is also influenced by the
growing location, climate, maturity and fermentation time [12].
Similarly to other plant materials, grape pomace also contains a fraction of non-
extractable polyphenols.
The biggest portion of grape pomace polyphenols has been reported to be highly
polymerised condensed tannin, and some polyphenols form complexes with fibers and are
non-extractable unless strong acidic treatments are applied [13]. Thermal processing may
increase the extractability and bioavailability of some polyphenols but it would as well
destroy heat sensitive compounds.
Grape seeds found in grape pomace also contain high amounts of grape seed oil (11.6–
19.6%) [14]. The main fatty acids contained in grape seed oil are linoleic acid (66.76–
73.61%), oleic acid (17.8–26.5%), palmitic acid (6.35–7.93%) and stearic acid (3.64–5.26%)
[15-16].
Grape seeds also contain non-phenolic antioxidants such as tocopherols and β-carotene,
that are mainly concentrated in grape seed oil [17].
Grape pomace also contains significant amounts of proteins, nondigestible fibres and
minerals. Grape seeds contain about 60–70% of non-digestible carbohydrates and 11%
protein, however no evidence on the digestibility of purified grape seed protein has been
reported [18].
GENERAL ASPECTS OF SUPERCRITICAL FLUID EXTRACTION

A supercritical fluid is any substance above its critical point, that is defined by
characteristic temperature and pressure values. At the critical point and above no distinct
liquid and vapor phases exist and a different phase of the matter arises, the supercritical one.
The properties of supercritical fluids can be seen as intermediate between the ones of liquids
and gases. Supercritical fluids are highly compressible, similarly to gases, but they have high
densities, comparable with those of liquids. The combination of some of the properties of
liquids with those of gases provides supercritical fluids of some very interesting features. For
example, supercritical fluids can effuse through solid materials like a gas but they can also act
like a liquid and dissolve substances. Supercritical fluid extraction (SFE) is based upon this
characteristic. In SFE, the organic phase used in typical solid-liquid extractions (SLE) is
substituted by a supercritical fluid. The manipulation of both temperature and pressure of the
fluid can solubilize the substance of interest in a complex matrix and selectively extract it.
SFE may be an environmentally sustainable alternative to the conventional organic solvent
extraction because it avoids the use of large amounts of toxic solvents, being also rapid,
automatable, and selective. Moreover, the absence of light and air during the extraction
reduces the degradation processes that may occur during the traditional SLE.
The most common supercritical fluid used for SFE is carbon dioxide (CO2). Its critical
temperature and pressure are modest, respectively 304.25 K and 7.39 MPa, it is non toxic and
available at a low cost. Moreover, it is not flammable, odorless and has a low environmental
impact.
Thanks to its low critical temperature, supercritical CO2 (SCCO2) is a valuable solvent
for the extraction of thermolabile compounds, such as many natural compounds that undergo
degradation at high temperatures. Also, SCCO2 is particularly useful in the extraction of
compounds meant for cosmetic, pharmaceutic or food applications, where the presence of
organic solvents in the extracts must be avoided. In fact, being CO2 a gas at atmospheric
pressure, it can be readily removed from the extraction products by depressurization.
SCCO2 solvent properties can be modulated by varying its density, that can be easily
achieved by modifying the temperature and pressure in a SFE process. At higher pressures,
intramolecular distances are reduced thus favoring solute-solvent interactions and enhancing
solubility. Viceversa, as temperature increases, density decreases, lowering solubility. Other

key parameters in SFE are the processing time and the pretreatment of the materials (i.e.
particles dimensions, humidity content). Also, it is possible to use small amounts (5-10%) of
polar co-solvents (i.e. ethanol, water) in order to enhance the otherwise low polarity of CO2.
The main elements of a SF extractor are a pump for the CO2, i.e. a syringe pump, a
pressure cell that contains the sample and a collecting vessel (see Figure 1).
Figure 1. Schematic diagram of the main elements of a SF extractor.
The CO2 used is liquid. It is first pressurized to the target pressure and then heated to
reach supercritical conditions. SCCO2 is then directed into the extraction vessel, where it
diffuses into the sample, selectively dissolving substances according to their hydrophobicity.
A static extraction phase is then followed by a dynamic one, opening the extraction chamber
and letting SCCO2 and the materials dissolved in it flow into a separator at lower pressure.
The employed CO2 can be cooled, re-compressed and recycled, or discharged into the
atmosphere.
Processing of natural products with supercritical fluids-based technologies has been an
extensive area of research during the past two decades. In fact, since many valuable products
occurring in natural compounds, such as vitamins, aromas, natural pigments or essential oils,
are soluble in supercritical fluids, their extraction from natural materials is one of the most
widely studied applications of supercritical fluids. To date, SFE technology is used at
industrial levels in economically relevant processes such as decaffeination of both coffee and
tea and extraction of hop constituents and spices [19].
BIOLOGICAL ACTIVITY OF SUPERCRITICAL FLUID EXTRACTS FORM

WASTE MATERIALS OF THE WINE INDUSTRY
As described above, grape pomace is considered a valuable source of polyphenols. The
biological and functional properties of these compounds are intensively studied. Over the
years, researchers of different disciplines have investigated the mechanisms of
chemoprevention, anticardiovascular disease and other disease prevention activities of grape
polyphenols, revealing that these compounds indeed possess appealing properties [20-23].
Therefore, grape pomace has a great potential to serve as a source of pharmaceutical
compounds as well as of functional food ingredients.
The composition as well as the quality of grape pomace extracts are strongly dependant
on the extraction technique, the solvent used, the origin of the raw material, its storage
conditions and the pre-treatment applied [24-26]. The quality of an extract can be represented
by its properties, i.e. its biological activities. Despite the generally recognized antioxidant
ability of substances present in grapes, specific studies on the biological activities of grape
pomace extracts obtained by SFE are still needed.
Comparing the properties of extracts obtained with different extraction methods is very
useful for assessing the optimal extraction conditions, able to extract valuable compounds
while maintaining their biological activity. Oliveira and coworkers studied the global
extraction yield, antimicrobial activity and composition profile of grape pomace extracts
(Merlot and Syrah varieties) obtained with different extraction methods and conditions: SFE
with pure SCCO2 and with the addition of ethanol, at pressures up to 300 bar and
temperatures of 50 and 60°C, and Soxhlet extraction [27]. The main components of the
extracts, analyzed by HPLC, were gallic acid, p-OH-benzoic acid, vanillic acid and
epicatechin. All extracts were tested to assess their antimicrobial and anifungal activity on
four bacterial strains (Staphylococcus aureus, Bacillus cereus, Escherichia coli, Pseudomonas
aeruginosa) and on three fungi strains (Candida albicans, Candida parapsilosis, Candida
krusei). All extracts obtained by SFE were effective against S. aureus (inhibition zone >9
mm). Among the microorganisms studied, P. aeruginosa was the most resistant and only one
SF extract (obtained at 250 bar and 60°C) gave a positive result (halo size >9 mm). Such
extract was the only one effective against all bacteria tested (both Gram-positive and Gram-
negative). Besides that, the extract obtained by SCCO2 at 150 bar and 60°C was also very
effective against the bacteria tested, except P. aeruginosa (7 mm halo size), while also being
effective against the fungus C. albicans with a halo size of 21 mm. The extracts obtained by
Soxhlet extraction, on the other hand, were effective only against ona bacterial strain, B.
cereus, with an inhibition zone of 14 mm. Moreover, SFE is performed in the absence of air
and light, which protects the bioactivity of the extracts [28]. The best performance of SFE in
comparison with other extraction methods was also reported by Kitzberger et al. [29] for
shiitake extracts and by Palma et al. for grape seed extracts [30].
The extracts were also submitted to the test of microdilution in culture broth to determine
the minimum concentration able to inhibit the growth of certain microorganism, i.e., the
minimum inhibition concentration (MIC). The supercritical extracts obtained from Merlot
pomace by SFE at 300 bar and 50°C and at 150 bar and 50°C showed the lowest MIC values
against S. aureus, 625±375 and 750 ± 250 g/mL, respectively. The extracts were more
effective (lowest MIC values) against Gram-positive bacteria, mainly S. aureus, comparing to
Gram-negative ones (E. coli and P. aeruginosa). Only the supercritical extract obtained from
Merlot by SCCO2 (50 ◦C, 300 bar) showed moderate activity against E. coli and P.
aeruginosa, inhibiting their growth with a MIC value of 1000 g/mL, while all other extracts
behaved as weak inhibitors against the Gram-negative bacteria (MIC above 1600 g/mL).
Thus, in general, supercritical extracts from grape pomace could be classified as moderate
inhibitors against Gram-positive bacteria (S. aureus and B. cereus).
The best antifungal activity was presented by the SCCO2/200 bar/50°C extract,
presenting a moderate inhibition against all three fungi tested. Two grape pomace extracts
were considered relevant in terms of quality aspects: the Merlot extract by SCCO2 at 200 bar
and 50°C, which presented medium inhibition power against all three fungi and the two
Gram-positive bacteria tested; and the Merlot extract by SCCO2 at 300 bar and 50°C, a
moderate inhibitor against all bacteria tested. The antimicrobial activity could be related to
the plant cultivation conditions since the active components are usually synthesized as a
response to stress such as microorganisms attack or strong UV radiation [31-32]. Thus, the
better antimicrobial results provided by the selected Merlot SF extracts (50°C at 200 bar and
300 bar) may be attributed to the phenolic substances identified such as gallic, p-OH-benzoic
and vanillic acids, whose antimicrobial abilities are well documented [33-34]. Besides the
above mentioned components, other bioactive substances may have participated as
antimicrobial agents. In fact, natural extracts may be more beneficial than isolated
constituents, because of the synergic positive interactions among compounds [35].
The antioxidant properties of natural extracts are currently considered the most appealing
thanks to possible large scale applications in the food and cosmetic industries. In a recent
work, the extraction of proanthocyanidins from grape pomace was conducted using SCCO2
[36]. The effect of pressure, flow rate and quantity of co-solvent were analyzed. The
performance of the extractions was evaluated in terms of phenolic yield, proanthocyanidins
content and antioxidant activity. SFE was compared with conventional SLE with methanol as
the solvent. The highest total antioxidant activity for grape pomace extracts was obtained with
SFE, employing a CO2 flow rate of 6 kg/h and 10% cosolvent (a ethanol-water mixture at
57% v/v ethanol). In such conditions, the total antioxidant activity, evaluated by the total free
radical scavenger ability, was 8703 mgα tocopherol /100 g dried matter, increasing by 20%
compared to the conditions employing a flow rate of 4 kg/h and 7.5% of cosolvent (7187 mgα
tocopherol /100 g dried matter), and it was about 13-folds that of the methanol extract (678
mgα tocopherol /100 g dried matter). This finding suggests that different CO2 flow rate and
percentage of cosolvent, as well as the extraction methods, affect the extraction of phenols
responsible for the antioxidant activity of the extracts. Both SF extracts presented a high level
of total proanthocyanidins (703.7 and 630.2 mgcatechin /100 gdried matter respectively),
compared to the methanol extract (159.0 mgcatechin /100 gdried matter). Moreover, it is
interesting to note that flow rate and cosolvent amount have an influence on the
proanthocyanidins fraction that is extracted (monomeric, oligomeric and polymeric fractions).
Polymeric fractions of proanthocyanidins always showed the highest antioxidant activity.
This can be attributed to the structure of polymeric flavan-3-ols characterized by the presence
of several hydroxyl functions exhibiting a higher ability to donate a hydrogen atom and to
support the unpaired electron as compared to the low molecular weight phenols [37].
Comparing SFE to SLE, proanthocyanidins could account for about 97% of the total
antioxidant activity of SL extracts but only for about 60% in SF extracts. This indicates that
SFE could extract not only selectively the proanthocyanidins, but a great amount of other
antioxidant compounds, not extractable with conventional methods.
Such findings are in agreement with other works that compare different extraction
methods [38-39]. Overall, SFE affords richer extracts, with higher number of separated
compounds; the use of EtOH as co-solvent increases the antioxidant potential of the extract, if
compared with pure CO2 extraction, due to the increase in solvent polarity; the supercritical
fluid is able to extract important compounds not detected in conventional extracts, such as
oleic acid and phytol, used in cosmetic products.
In a recent work, grape pomace SF extracts were added to samples of fish oil, and
experiments of accelerated oxidation were monitored by formation of dienes and trienes [40].
Fish oils are often using for enriching soup, milks, and other products because of their high
content in ω-3 polyunsaturated fatty acids. For this reason, oils are readily oxidized and the
addition of natural antioxidants as rosemary extracts, catechins from green tea or polyphenols
is common [41-42]. SF extracts were able to prevent significantly trienes formation (and
dienes formation to a minor extent), being promising for substituting the synthetic
preservatives in food processing and manufacture. Results indicated that the most polar
molecules were more effective for preventing fish oil oxidation, due to the fact that they are
disposed in the active air/oil interphase.
CONCLUSION
The wine industry is rapidly changing, with new regulations being applied, increasing
worldwide demand but also new challenges due to the emergence of new producers on the
global market. Residues of the wine industry have so far been considered mostly as a waste
management issue, while in the future they will more and more be seen as valuable natural
sources for biologically active compounds such as antioxidants, whose demand not only in
the pharmaceutical sector, but also in the food and cosmetic industry, is steadily increasing.
SFE is a promising methodology for the production of high-quality extracts. It is a green and
safe technique that, although requiring initially high investments, could provide valuable
alternatives to conventional extraction methods. Some parameters have already been
identified as crucial for the quality of SF extracts (i.e. pressure, amount and nature of
cosolvent), that, though the works studying their properties are still limited, are generally
richer and of higher quality if compared to those obtained by conventional techniques.
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Chapter 5
PASSION FRUIT POMACE POWDER: POTENTIAL

APPLICATIONS OF EMERGING TECHNOLOGIES FOR
EXTRACTION OF PECTIN
Cibele Freitas de Oliveira1* and Poliana Deyse Gurak2†

1
Departamento de Engenharia Química, Universidade Federal do Rio Grande do Sul,
Porto Alegre, Porto Alegre, RS, Brazil
2
Departamento de Nutrição, Universidade Federal de Ciências da Saúde de Porto Alegre,
Porto Alegre, RS, Brazil
ABSTRACT
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 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,
*
Email: ci-oliveira@hotmail.com.
†
Email: poligurak@hotmail.com (corresponding author).
82 Cibele Freitas de Oliveira and Poliana Deyse Gurak
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 (15-20% 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.
1. INTRODUCTION
The use of fruit waste as raw material for the production of functional ingredients that
can be used by the food and pharmaceutical industries is an area of research that has been
increasing in the last three decades. An example of these ingredients are pectins that have
been isolated from orange peels, lemon, passion fruit and apple pomace.
Brazil is the largest producer of passion fruit (Passifloraedulisvar. Flavicarpa),
producing around 776.000 tons per year (IBGE, 2012). 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 (Bruckner &
Picanço, 2001). The most important variety cultivated in Brazil for commercial purposes is
the yellow passion fruit, Passifloraedulis Sims f. flavicarpaDegener (Teixeira et al., 1994),
which is used for pulp and juice processing. The P. edulis var. flavicarpa fruits are round,
with a diameter between 8 and 10 cm and a green peel at maturity. The edible part of the
passion fruit (40 %) consists of pulp with seeds, and 60 % of the peel consists of mesocarp
and epicarp. They contain many seeds (as do the other Passifloraceae species) surrounded by
a gelatinous yellow pulp that has an intense aroma and a sweet-acid taste (López-Vargas et
al., 2013).
The valorization of pomace has grown in the last years, since the conversion of plant
residues into food ingredients with added value is important for industry and the environment.
Within this context, pectin is present in significant amounts in passion fruit peel; and it is
widely used as a gelling agent and stabilizer in a variety of food, pharmaceutical, and
cosmetic products (Espitia et al., 2014; Guo et al., 2014; Thakur et al., 1997).This soluble
fiber is poorly digested in the small intestine but it is fermented by bacteria in the colon,
leading to the production of short chain fatty acids. This characteristic has led to interest in
pectin as a prebiotic (Manderson et al., 2005). Due to its simple and compatibility gelling
mechanism, pectin has been recently exploited for different biomedical applications,
including drug delivery, wound healing and tissue engineering (Munarin et al., 2012). Min et
al. (2010) used pectin-enriched materials from apple pomace as a fat replacer in a model food
system, and Tripathi et al. (2010) studied pectin as a material for food-packaging applications.
Passion Fruit Pomace Powder 83
The composition and physicochemical properties of pectin depend on source material,

technology and purification process used during the extraction.
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 (Guo et al., 2014; Jiang et
al., 2012; Koubala et al., 2008; Levigne et al., 2002; Xu et al., 2014; Yapo et al., 2007; Yapo,
2009a). The most commonly used methods for the extraction of pectin include direct boiling
and microwave heating (Fishman et al., 1999; Guo et al., 2014; Joye & Luzio, 2000;
Kratchanova et al., 2004; Guo et al., 2012). In industrial scale, pectin is normally extracted
using hot acid water, low pH, high temperature and a lot of time. Due to a long period of
direct heating, the extracted pectin undergoes degradation in their structure that can be
resulted in undesirable changes in the physicochemical and technological properties of this
fiber (Einhorn-Stoll et al., 2014; El-Nawawi & Shehata, 1987; Xie et al., 2011). There is an
industrial importance to explore new methods for extraction of pectin with good yield and
quality attributes.
Several kinds of new technologies, such as microwave heating at ambient and moderate
pressures, moderate electric field, high pressure, ultrasonication, and super high frequency
electromagnetic field are currently studied, either sequentially or independently, in the
extraction of pectin. This review will discuss about the passion fruit, the chemical
composition of the yellow passion fruit peel and its nutritional and functional implications in
human health, and finally the potential of passion fruit pomace powder for extraction of
pectin using two emerging technologies (moderate electric field and high pressure).
2. DEFINITIONS AND CHARACTERISTICS OF PASSION FRUIT

2.1. Passion Fruit
Passion fruit (Passiflora) remains to the botanic family of Passifloraceae and is found in
tropical climate. The plant has its origin in North America, but it is also spontaneous in
Brazil, Peru, Mexico, the Mediterranean region of Europe and North Africa (Rain Tree
Nutrition, 2011). In Brazil, 150 varieties are known (Embrapa, 2011) and the productivity in
Brazil is high with around 20–40 tons per hectare (IAC, 2011).
The cultivation of yellow passion fruit (Passifloraedulis var. flavicarpa Deg.,
Passifloraceae) has been preferred for industrial production of juice, jelly and dessert due the
nutritional quality of this variety; there are a number of characteristics which consider the
yellow passion fruit superior to the others varieties, for example, higher fruit size, weight,
total acidity, pest resistance and increased productivity per hectare (Carvalho-Okano et al.,
2001; Zibadi & Watson, 2004). Figure 1, shows the parts of yellow passion fruit.
Furthermore, this fruit is rich in vitamin C, vitamins B1 and B2 and the pro-vitamin A, β-
carotene, as well as minerals such as K, P, Ca, Fe and fibers. In the Food Composition and
Nutrition Tables (Souci et al., 2008) the following values are presented for 100 g of passion
fruit: 75.8% water, 63 kcal, 9.5 g carbohydrates, 0.4 g lipids, 2.4 g proteins, 1.5 g dietary
fibers, 3.9 organic acids and 0.9 g minerals. Vitamin C values have been reported as 40 mg in
100 g of natural passion fruit juice (Suntornsuk et al., 2001). Other studies showed the
presence of polyphenols (López-Vargas et al, 2013) and polinsaturated fatty acids. Due the
presence of these compounds in fruit, the ingestion of products derived of passion fruit can
exhibit beneficial effects for health.
Figure 1. Constituent parts of the yellow passion fruit. Source: Personal archive.
Research has been conducted showing the potential of passion fruit (pulp, skin and seeds)
for various purposes, but the most studied purpose is the biological activity regarding the
antioxidant action. The antioxidant activity in juices is attributed to polyphenols, particularly
the flavonoids (Heim et al., 2002). Kuskoski et al., (2006) determined the antioxidant activity
in pulps of tropical fruits, including the passion fruit, using the method of radical 2,2-
diphenyl-1-picrylhydrazyl (DPPH), and found a value of 1.02 ± 0 4 µmol g-1, value expressed
in equivalent antioxidant activity equivalent to Trolox after 60 min of reaction. Also, the total
polyphenols were determined by the Folin-Ciocalteu method (20.0 ± 2.6 mg 100 g-1). Araujo
et al. (2004) studied the biological activity of total proteins present in the pulp of tropical
fruit, and the pulp of passion fruit showed the greatest amount of protein (0.8 mg of protein
per gof pulp) among the samples tested and showed no inhibitory activity of mammalian
digestive enzymes.
Studies have showed the high content of dietary fiber (DF) in passion fruit. Dietary fiber
decreases the risk for type 2 diabetes mellitus, cardiovascular disease, and colon cancer
(Sierra et al., 2002; Eshak et al., 2010) by reducing the digestion and absorption of
macronutrients and decreasing the contact time of carcinogens within the intestinal lumen
(Sierra et al., 2002; Lattimer et al., 2010; Behall. 2006; Raninen et al., 2011). In addition, the
United States Food and Drug Administration has approved health claims supporting the role
of DF in the prevention of cancer and coronary heart disease (CHD) (Code of Federal
Regulations, 2010). More recently, and perhaps more interestingly, epidemiological studies
have found the benefits of DF to extend beyond type 2 diabetes mellitus, CHD, and colon
cancer (Park et al., 2011).
Based on scientific evidence of the health benefits provided by fruits and vegetable, the
food industry is very active, introducing new products as alternatives for health-conscious
consumers. This is quite pertinent in Latin America, where the population follows diets high
in carbohydrates and saturated fats, with low content of fruits and vegetables. Some studies
were conducted in this way in order to introduce fruits and vegetables in diet. Solar et al.
(2014), used passion fruit pulp (Passifloraedulis Sims.) to develop a texturized product
employing a natural hydrocolloid, with added probiotics and prebiotics as functional food
ingredients. The final product was accepted by the sensory panel, it was physically,
chemically and microbiologically stable during the evaluated period.
2.2. Passion Fruit Peel
The wide-scale use of passion fruit, inevitably leads to the generation of vast quantities of
the fruit peels, which constitutes about half of the fruit mass. These fruit peels are discarded
as a major waste, causing a substantial burden on the environment. It is therefore imperative
to find adequate disposal of these peels or means to convert the peels into useful products
(Liu et al., 2006; Pinheiro et al., 2008). Recent studies have shown that the pericarp (epicarp
and mesocarp) of passion fruit, even though processed and stored, could be used as raw
material for obtaining co-products in food industry, such as dietary fiber and other bioactive
compounds (Yapo & Koffi, 2006).
The passion fruit pericarp is rich in soluble and insoluble fibers (Cazarin et al., 2011).
Dietary fibers increase fecal bulking and viscosity, reducing the contact time between
potential pathogens and mucosal cells, and acting in glycemic control, they are able to
regulate energy intake, thus, increasing weight loss or maintaining healthy bodyweight
(Lattimer & Haub, 2010). The ideal consumption of dietary fiber is considered to be
approximately 25 - 35 g day-1, of which 6 g should be soluble fiber (Lattimer & Haub, 2010).
Pectin is a soluble fiber that is present in large amounts in passion fruit peel; this fiber is a
complex carbohydrate from plants with technological and physiological functions (Yapo &
Koffi, 2008). The passion fruit peel has been tested with success in feed and food, and also as
an ingredient in the preparation of food products. Table 1 shows some studies performed with
passion fruit peel.
Table 1. Studies with passion fruit peel
Autors Objective Results

Otagaki & Matsumoto To use passion fruit peel in feed Higher milk production and less
(1958) supplementation of dairy cows intestinal problems probably by the
and other animals. presence of significant amounts of
fiber in the peel of passion fruit.
Guertzentein (1999) To check the benefits of passion The yellow passion fruit was efficient
fruit peel to reduce blood glucose. in the treatment of diabetes mellitus
type II, and the probable mechanism
of this action was the high content of
pectin, which helps to decrease the
level of glucose and cholesterol in the
blood.
López-Vargas et al. To determine the physicochemical Passion fruit peel has significant
(2013) and technological characteristics, amounts of dietary fiber, phenolic
phenolic compounds, antioxidant compounds and interesting
and antibacterial properties of technological, antioxidant and
passion fruit peel. antibacterial properties; can be used
as an ingredient in developing
functional foods.
Table 1. (Continued)
Autors Objective Results

Contreras-Calderón et To determine the antioxidant The results for passion fruit peel
al. (2011) capacity and total phenolic showed that the content of phenolic
compounds of the pulp, peel and compounds were 288 ± 8.41 mg of
seeds of 24 species of fruits. gallic acid / 100 g of sample; the
antioxidant capacity was 36.7 ± 0.07
μmolTrolox / g of sample. The results
observed for passion fruit pulp were
highest in comparison with the peel,
but compared with banana and tomato
peel the values were higher.
Agra et al. (2007) To study the medicinal uses of It was showed that the dried and
native, naturalized and cultivated pulverized mesocarp of P. edulis
plant species, which are utilized (unspecified array) can be used in the
for therapeutic purposes in all control of diabetes.
States of Northeast Brazil
extending from Maranhão to
Bahia.
Ramos et al. (2007) To make a clinical study using the It was observed a decrease in the
passion fruit powder (P. edulisfo. cholesterol levels in women between
flavicarpa) 30 and 60 years who had
hypercholesterolemia (cholesterol
levels ≥200mg/dL)
Watson et al. (2008) To study patients with asthma for The effects of the extracts were
four weeks it was given extracts evaluated by means of clinical
of purple passion fruit peel or symptoms of asthma. It was found
placebo pills, for later that the purple passion fruit peel
comparison. extract, administered orally in
humans, improved clinical symptoms
of asthma, such as reduced wheezing
and cough and caused an
improvement over the lack of air, the
patients showed no collateral effects.
The passion fruit pomace processing can be converted in different commercial products,
either as raw materials or just as an ingredient for the development of new products (Sánchez-
Zapata et al., 2008); Lira Filho (1995) used the peel of passion fruit for the elaboration of a
common jelly and the result was a product of good texture, acceptable taste and color.
Other relevant research in the area showed that the albedo of passion fruit have higher
amounts of soluble and insoluble dietary fibers when compared with the pulp, as well as
significant amounts of ash, protein and lipids. The oil and water holding capacity was also
higher when compared to the pulp and seeds (Lopez-Vargas et al., 2013). Table 2 shows the
results of the chemical composition of the yellow passion fruit peel obtained by different
authors. The variations of their constituents depend mainly on the fruit maturation stage,
given that the maturation leads to loss of moisture, which causes variations in the
concentration of other constituents. Furthermore, there are also other factors such as the place
of planting and genetic conditions of the plants.
Table 2. Nutrient content in dried peel of yellow passion fruit

(P. edulisfo. Flavicarpa)
Components (%) Passionfruit Passionfruit peel2 Passionfruit

peel1 peel3
Moisture 9.93 4.60 9.48
Ash 7.52 3.36 6.88
Lipids < 0.01 0.57 0.31
Proteins 4.05 5.15 3.94
Total fiber 57.36 64.80 65.22
Solublefiber 19.20 15.81 17.11
Insolublefiber 38.00 34.37 48.12
Carbohydrates* 21.28 26.17 14.17
*Carboidrtatoscalculatedbydifference
1
Pinheiro (2007)
2
Kliemann (2006)
3
Cazarin et al., (2011)
3. PECTIN
3.1. General Aspects and Characteristics
Carbohydrates are widely available in nature since they constitute more than 90% of the
dry matter of plants; they are food components and can occur naturally or added as an
ingredient in food products. The molecular structures, sizes and configurations of
carbohydrate have large variation, therefore different physical and chemical properties can be
observed, which results in changes in its application in the food industry and physiological
effects when ingested. For this reason, carbohydrates are amenable to chemical modifications
for improving their properties and expanding its applications (Fenemma, 2010). Usually, they
are subdivided according to the size of the chemical structure, into monosaccharides,
oligosaccharides and polysaccharides (Ribeiro & Seravalli, 2007).
Pectin, a family of complex heteropolysaccharides consisting predominantly of partially
methoxylatedgalacturonic acid residues (Figure 2), is extensively distributed in almost all of
the fruits and vegetables as the structural unit of fresh cells and the junction between the cells
(Ridley et al., 2001; Thakur et al., 1997). Its structure is based on 1,4-linked α-D-galacturonic
acid, interrupted by L-rhamnose residues with side-chains of neutral sugars (mainly D-
galactose and L-arabinose) (Mohnen, 2008; Ridley et al., 2001). Industrial pectins have
particular specifications that include no less than 65 % galacturonic acid as stated by the Food
and Agricultural Organization.
Pectin is naturally found in the primary cell walls of plant cells and in the intercellular
layers (middle lamella), contributing for adhesion between cells, firmness and strength of the
tissue, whose schematic is shown in Figure 3 (Carpita et al., 2000). Beyond its importance for
the cells growth, pectins are involved in interactions with pathogens, and their quantity and
source are important for texture of fruit during growth, maturation, storage and processing
(Mesbahi et al., 2005). Pectin is linked to cellulose, hemicellulose and lignin, this complex is
called protopectin and its function is to give rigidity to the plant structure; this soluble fiber
can be extracted with plenty of apple pomace (15-20% dry matter), citrus albedo (30-35% dry
matter) and beet pulp (15-20% dry matter) (Thibault & Petit, 1979).
The passion fruit pomace has been studied due its high amount of pectin, researchers
found the content of this soluble fiber around 6.3 % when the extraction conditions were 80
°C, 60 minutes, pH 2 and solid:liquid ratio 1:30 (Kulkarni & Vijayanand, 2010). It is
important to emphasize, in accordance with López-Vargas et al. (2013), that the fiber content
depends on degree of ripeness, harvesting season, storage time and extraction method.
Figure 2.Structure of the unity of galacturonic acid.
Figure 3. Structure of the cell wall of plants. Source: IPPA (2008).
The discovery of pectin, as a chemical compound, was made by Vauquelin in 1790. In

1824, Bracon not was the first to characterize the pectin as a composed of fruit responsible for
the gel formation; and the same author suggested the name pectin from greek, which means
thick. Until 1930, pectin was considered as a small cyclic structure; Smolenski in 1923, was
the first to suggest that pectin was a complex polymer, similar the starch structure. Later, X-
ray analysis indicated it to be more consistent with cellulose (Owens et al., 1946). According
to Meyer and Mark (1930), the pectin contains in its structure methyl ester groups attached in
the position 1-4 (Baker, 1948) and the basic formula was established by Schneider and Bock,
1937 (Cybercolloids, 2008). In the beginning of the studies with pectin, the difficulty in
establishing comparative studies about the composition of pectin due the different method of
extraction and the analyses used had already been discussed (Elwell & Dehnt, 1939). Until
now the difficulty of extraction even exists, for this reason the studies of new technologies
and the improvement of traditional technologies for extraction of pectin have received more
attention by researchers in recent years.
3.2. Molecular Structure
The structure of pectin is based on 1,4-linked α-D-galacturonic acid (Figure 4) and has L-
rhamnose residues with side-chains of neutral sugars (mainly D-galactose and L-arabinose)
(Mohnen, 2008). The carboxyl acid groups of the monomers of galacturonic acids (GalA)
may or may not be esterified with methyl groups where the percentage of esterified groups is
expressed as degree of methoxylation (Yapo et al., 2006).
Figure 4. Structure of units of galacturonic acid linked in position α- (1-4).
The fine structures of pectins can be extremely heterogeneous between plants, between
tissues, and even within a single cell wall. The content and kind of sugar, size of chain and
esterification degree may interfere on extraction yield (Yapo et al., 2006).
Pectins are composed of three major fractions: homogalacturonana (HG),
ramnogalacturonana I (RGI) and ramnogalacturonana II (RGII). Homogalacturonan is a linear
polymer consisting of 1,4-linked α -D-GalA, whilst rhamnogalacturonan I consists of the
repeating disaccharide to which a variety of different glycan chains (principally arabinan and
galactan) are attached to the ramnose residues. The confusingly named rhamnogalacturonan II
has a backbone of HG rather than RG, with complex side chains attached to the GalA
residues (Ridley et al., 2001; Willats et al., 2001a). Until recently it was accepted that
rhamnogalacturonan and homogalacturonan domains constitute the ‗backbone‘ of pectic
polymers as shown in Figure 5(A). However, an alternative structure has recently been
proposed in which HG is a long side chain of RGI (Figure 5(B)) (Vincken et al., 2003).
Pectins are an extremely complex and structurally diverse group of polymers; the chain
lengths of the various domains can vary considerably, and the sugar composition of RGI can
also be highly heterogeneous. In contrast, RGII is thought to have a highly conserved
structure. Galacturonic acid in HG may be esterified, the degree of methyl- esterification
(DE) has a profound impact on functional properties and pectins are traditionally categorized
as high-ester or low-ester with DEs of >50% an <50% respectively (Rinaldo, 1996; Voragen
et al., 1995).
Figure 5.The basic structure of pectin. Schematic representations of the conventional (A) and recently
proposed alternative (B) structures of pectin. Source:Willats et al. (2006).
The esterification degree performs an important role in the ability of gelling of pectin,
being a parameter to indicate the physical and functional properties of pectins; other factors
also determine gelling properties including temperature, pectin type, pH, sugar, calcium
(Willats et al., 2001b). A pectin gel is formed when portions of HG are crossed-linked to form
a three dimensional crystalline network in which water and solutes are trapped. The pectins
with high esterification degree and junction zones are formed by the cross-linking of HG by
hydrogen bridges and hydrophobic forces between methoxyl groups, both promoted by high
sugar concentration and low pH. Pectins with high esterification degree represents a high-
value functional food ingredient widely used as gelling agent and stabilizer, particularly in
jams and jellies (Willats et al., 2006). On the other hand, the pectins with low esterification
degree and junction zones are formed by calcium cross-linking between free carboxyl groups;
this kind of pectin is used as a gelling agent in products with low content of sugar (Iglesias &
Lozano, 2004).
The source of extraction can affect the gelling and functional properties of pectin
extracted and consequently the applications of the fiber. The next step of this review will be
to highlight this subject.
3.3. Sources and Applications
The most important sources for extraction of pectin are apple pulp and pomace of citric
fruits (co-products of juice industry), which give rise to pectins of high methoxyl (Thakur et
al., 1997; Thibault & Petit, 1979). Some sources of vegetables, such as sunflower, has a large
amount of pectin of low methoxyl, which is extracted by using chelating agents (Wicsenborn
et al., 1999; Yokoi et al., 2002) and pulp of beet has high content of pectin with low gelling
capacity (Turquois et al., 1999). Commercial pectin is usually produced from pomace of plant
material remaining after the extraction of juice, such as (15 – 20 % d.m), beet pomace (15 –
20 % d.m) (Thakur et al., 1997; Thibault & Petit, 1979) and passion fruit pomace (3- 15 %)
(Yapo 2009a, 2009b; Fishman et al., 2006).
Pectin is a soluble fiber, and it can be used as a gelling agent and stabilizer in a variety of
foods (including meat products, breakfast cereals, bakery products and dairy products),
pharmaceutical, and cosmetic products (Espitia et al., 2014; Guo et al., 2014; Thakur et al.,
1997).
This fiber is poorly digested in the small intestine but it is fermented by bacteria in the
colon, leading to the production of short chain fatty acids. This characteristic has led to
interest in pectin as a prebiotic (Manderson et al., 2005). Due to its simple and
cytocompatible gelling mechanism, pectin has been recently exploited for different
biomedical applications, including drug delivery, wound healing and tissue engineering
(Munarin et al., 2012). Min et al. (2010) used pectin-enriched materials from apple pomace as
a fat replacer in a model food system, and Tripathi et al., (2010) studied pectin as a material
for food-packaging applications.
The health effects of pectin are receiving more interest. It is generally accepted that a
high fiber diet is beneficial to one‘s health. There is clear evidence that pectin can lower
cholesterol levels, serum glucose levels and may also have anti-cancer activities (Yamada,
1996; Behall & Reiser, 1986).
3.4. Extraction of Pectin
Extraction is a method used for obtaining components from a solid mixture or solution.
Generally the extraction consists of two steps, the material to be extracted is first soaked in a
solvent to swell and hydrate it, and then the soluble components move into the extraction
solvent by the mass transfer actions of diffusion and permeation (Mustafa & Turner, 2011).
2009a).
Pectin extraction is a multiple stage physicochemical process which involves hydrolysis
and extraction of pectin macromolecules from plant tissue, purification of the liquid extract
and isolation of the extracted pectin from the liquid. These processes are influenced by
various factors, mainly temperature, pH, and time (Pagán et al., 2001). The most commonly
used methods for the extraction of pectin include direct boiling (Guo et al., 2014; Guo et al.,
2012) at 80 – 100 °C, pH 1 – 3 (phosphoric acid, acetic acid, sulfuric acid, nitric acid or
hydrochloric acid) and 20 – 240 minutes. (Pagán et al., 2001; Levigne et al., 2002, Koubala et
al., 2008). Studies recommend the use of hydrochloric acid (Iglesias & Lozano, 2004;
Mesbahi et al., 2005) and nitric acid (Pagán et al., 2001; Levigne et al., 2002) as an extractor
agent.
According to Mesbahi et al. (2005), long periods of direct boiling (T>90°C) during the
extraction of pectin can result in the hydrolysis and degradation of pectin. Canteri-Schemin et
al. (2005) extracted and characterized the pectin from apple pomace and obtained 17.82 %
(d.m) of yield when the extraction conditions were 100 °C, 153 minutes and nitric acid
concentration 6.2 % (w/v); the esterification degree of pectin extracted was 68.84 %. Marcon
(2004) got a similar result, the yield of extraction was 16.80 % (d.m) when the extraction
conditions were 100 °C, 80 minutes and the nitric acid concentration 5 % (w/v).
Klieman et al. (2009) found values of yield for extraction of pectin from passion fruit
pomace between 1.1 – 27.7 %, using different conditions. Yapo & Koffi (2006) extracted
pecin from the same source described above and found a yield of 14.2 % (d.m), while Yapo
(2009a) found 6.5 %. The difference between the values obtained for different authors can be
result of the maturity of fruit, climatic conditions during the plant growth, treatments before
extraction (drying and grinding), time and temperature of extraction, solvent extractor used
during the extraction. Another important factor for food industry is the fast dehydration of
pomace to avoid enzymatic action (Thakur et al., 1997).
D‘Addosio et al. (2005) extracted and characterized the pectin from passion fruit pomace
in different maturation stages and using different acid, such as, hydrochloric acid, phosphoric
acid and a mixture of these two acids. The pectin with highest esterification degree and
galacturonic acid content was obtained when hydrochloric acid was used during the
extraction.
Seixas et al., 2014 studied the extraction of pectin from passion fruit pomace by
microwave-induced heating. Three types of acids (tartaric, acetic and nitric acid) were
employed as extracting agents. The effect of extraction time and microwave-power on yield
of pectin was studied using the response surface methodology. The results indicate that
exposure time and microwave-power significantly affects the yield of pectin extraction with
both nitric and tartaric acids. However, the extractions using acetic acid were significantly
affected only by the exposure time. For all scenarios, the highest yields were obtained when
the highest levels of power and time were used (628 W and 9 min). Under these conditions,
the yield of pectin obtained with nitric and acetic acids were 13 and 12.9% respectively.
Tartaric acid emerged as the best extracting agent in terms of yield (18.2%), however, the
obtained pectin exhibited low purity and low degree of esterification. Pectin extracted from
passion fruit by employing acetic and nitric acid presented better properties: high molar mass
(4.63x105 for acetic acid and 4.96x105 for nitric acid), degree of esterification (64.56% for
acetic acid and 64.15% for nitric acid) and content of uronic acids (62.5% for acetic acid and
82.3% for nitric acid).
Liew et al., 2014 studied the influence of pH and extraction time on pectin yield and
composition was studied in a citric acid extraction process. The pectin yield and degree of
esterification (DE) of the extracted pectin ranged from 2.25 to 14.60% and 41.67 to 67.31%
respectively. It was found that pH extraction was the most important parameter influencing
yield. DE was significantly affected by extraction time. Morphological analysis performed
using scanning electron microscopy suggested that the dried passion fruit pectin has a smooth
surface with little mound-shaped pellets on it.
4. EMERGING TECHNOLOGIES FOR EXTRACTION OF PECTIN

2009b). Several kinds of new technologies have been receiving more attention. In this chapter
the moderate electric field and high pressurewill be highlighted.
4.1. Moderate Electric Field
Moderate electric field (MEF) treatment involves the application of an electric field,
usually at frequencies on the order of 50 Hz and at field strengths below 1 kV/cm. MEF fields
are lower than those for pulse electric field (PEF), which are usually current direct pulses
measured in field strengths of kV/cm. MEF treatment times are also typically measured in
seconds, whereas PEF treatments involve exposure times at the microsecond order of
magnitude. Some advantages of MEF are summarized below:
• Volumetric heating of food by internal heat generation, and without the limitations
and non-uniformities of the conventional heat.
• Reduction in the burning of the product, resulting in minimal mechanical damage
and better retention of vitamins and nutrients.
• High energy efficiency, 90% of the electrical energy is converted into heat (Ghnimi
et al., 2008).
• Easy control of the process, it can be turned on and off instantly.
• System less aggressive to the environment (Ruan et al., 2001).
MEF treatment has been shown to non-thermally increase the permeability of cell tissue
and diffusion of water and soluble matter through cell membranes, thus opening the door for
potential applications, such as improvements in extraction, dehydration, and fermentation
operations. Under most conditions, ohmic heating (ohmic heating is based on the passage of
an alternating current through a sample which responds by generating heat internally due to
its inherent resistance) occurs during MEF treatment, but here we are concerned with the non-
thermal electrical effects. (Fryer et al., 1993; Palaniappan & Sastry, 1991)
During MEF treatment, the electric field may cause changes in the permeability of plant
cell membranes at temperatures below those at which thermal permeabilization occurs
(Personius & Sharp, 1938). Diffusion is enhanced, electrical conductivity changes are more
linear during heating, and moisture migrates more easily out of the tissue. (Lima et al., 2001;
Schreier et al., 1993). Electropermeabilization is a mechanism that can account for these
effects. According to this hypothesis, pores formed in the cell membranes upon electric field
exposure cause a drop in resistance as ions are allowed to pass through the membrane (Coster,
1965). If electropermeabilization occurs in a vegetable tissue, the permittivity and
conductance should be affected due to the changes in membrane permeability. Thus, low-
frequency measurements may be used for examining the effects of MEF treatment and other
processes on cell membranes (Kulshrestha et al., 2010).
A lot of studies have been performed using this emerging technology. Kulshrestha &
Sastry (2010) investigated the changes in permeability of vegetable tissue treated by moderate
electric field (MEF). The results showed that electrical treatments permeabilize vegetable
tissue, permitting enhanced diffusion; a microscopic examination of beet tissue given a
moderate electric field treatment shows that some cells lose their membrane selectivity while
others remain intact. Kusnadi & Sastry (2012), researched about the effect of moderate
electric fields on salt diffusion into vegetable tissue and the results showed that the salt
diffusion coefficient was significantly increased by moderate electric field for all materials
(celery, mushroom, and water chestnut). The enhanced diffusion under electric field appears
to have been caused by electroporation under low temperature and a combination of
electroporation and thermal denaturation effects under high temperature.
Figure 6. Moderate electric field equipament. Source: personal archive.
Sensoy & Sastry (2004) studied the solid extraction of black tea leaf and mint using
moderate electric field and extraction with hot water. In this study it was observed that there
was an improvement in the leaching of solute of the fresh plant material as compared to only
heating. However, no difference was observed when the sample was previously dried; it was
also observed that a decrease in the frequency implies an increase in the permeability of the
membrane.
3.2.1. Operation of Moderate Electric Field Equipment

The typical food processing unit for electrical field is a simple system comprising an
energy supply system, a data acquisition system and a treatment chamber (Wan, 2009). An
example of moderate electric field equipment is presented in Figure 6.The energy supply
system includes a stabilizer, a manual voltage regulator ranging and a circuit breaker; data
acquisition system monitors and registers data related to electric current, voltage and
temperature as a function of time. Data is recorded at constant time intervals by a data logger
linked to a computer. The temperature is monitored by two platinum resistance thermometers
covered with stainless steel. The voltage can be measured using a voltage transducer. The
electrical current can be monitored using a current transducer. The treatment chamber (glass
cell) has two electrodes, among which the sample to be treated is placed, and it has a set of
monitoring and control devices.
4.2. High Pressure
High pressure (HP) has been recognized as an environmentally friendly technology by

the U.S. Food and Drug Administration (U.S. FDA 2007), and it has been in use in the
chemical, ceramic, and plastic industries for many years, but the food industry recognized its
potential application only in the late 1980 (Otero & Sanz, 2003; Zhang et al., 2004). HP as a
relatively novel technique is used for extraction of active compounds from plant materials.
High pressures ranging from 100 to 1000 MPa are considered an alternative extraction
method, which has been proven to be fast and effective. The advantages of HP are
summarized below:
• HP acts immediately and equally through a food mass, independentof its size, shape,
or composition (Cheftel, 1995).
• HP requires minimal heat and can prevent thermal degradation; due to this does not
produce deterioration of thermolabile nutrients, such as vitamins and does not alter
low molecular weight compounds (Tellez-Luis et al., 2001).
• HP influences the secondary and tertiary structures of proteins and polysaccharides,
and can alter the functional properties of these compounds (Télléz-Luis et al., 2001 ).
• HP can shorten extraction times and increase the extraction yields (Lebovka et al.,
2011).
• HP does not induce the Maillard reaction or browning, therefore, it does not alter the
natural taste and the food coloring (Gross & Jaenicke, 1994);
The first record of the use of high pressure to food processing was done in 1899 by Hite,
who investigated the application of HP as a means of preserving milk and the results obtained
were satisfactory (Hite, 1899). Later, in 1914, studies were conducted to the conservation of
fruits and vegetables that also appeared to be effective (Hite et al., 1914). Unfortunately, the
lack of technological development became a disadvantage in the development of this
technique. For this reason, eighty years later, Japan rediscovered the industry interest by high
pressure and since then, this technology has been adapted to the specific needs of the food
industry (Zimmerman & Bergman, 1993).
Currently, this technology is applied to the conservation and food processing, including
the destruction of microorganisms, inactivation of the enzyme activity, change in the
conformation of biopolymers and extraction of bioactive compounds (Bang & Chung, 2010).
Weak bonds such as the hydrogen bond, the electrostatic bond, the van der Waals bond, and
the hydrophobic bond can be broken by high pressure, but small molecules cannot be changed
under high pressure conditions (Butz & Tauscher, 2002; Zhang et al., 2004; Corrales et al.,
2008).
During the extraction high pressure can enhance the efficiency of mass transfer and
improve solvent permeability in cells as well as diffusion of the secondary metabolites, but it
has no significantly negative effect on the structure and activity of bioactive compounds
(Dornenburg & Knorr 1993; Ahmed & Ramaswamy 2006; Lopes et al., 2010). Based on the
phase behavior theory, the dissolution is faster at higher pressure. The differential pressure
between the inner part and the exterior of the plant cells is very large under HP conditions.
Under this large differential pressure, the solvent can permeate very fast through the broken
membranes into plant cells, and the mass transfer rate of solute or the rate of dissolution is
very large, which leads to a very short extraction time using HP, compared to conventional
extraction processes (Zhang et al., 2004). Furthermore, HP provides the possibility of
inactivating degrading enzymes, which may explain the higher extraction yield and
antioxidant activity compared to other extraction methods (Ahmed & Ramaswamy 2006).
High pressure has many advantages for extraction of bioactive products. Various
researchers have successfully used the HP technique for the extraction of corilagin from
longan fruit pericarp (Prasad et al., 2009), catechins from green tea (Jun et al., 2010),
anthocyanins from grapes (Corrales et al., 2008) and flavonoids from litchi fruit pericarp
(LFP) (Prasad et al., 2009).
Another research performed by Briones-Labarca et al. (2015) studied the use of papaya
seeds as a source of bioactive compounds by using different types of assisted extractions such
as high hydrostatic pressure, ultrasound and conventional extractions. The results showed that
high pressure was more effective than ultrasound and conventional extractions in extracting
bioactive compounds and it had a shorter extraction time.
Strati et al. (2014) studied the extraction of carotenoids (lycopene) from tomato pomace
assisted by enzyme and high pressure. Total carotenoid and lycopene extraction yields were
increased by the use of pectinase and cellulase enzymes, when compared to the non enzyme
treated solvent extraction process.
HP assisted extraction led to higher extraction yields (from 2 to 64% increase depending
on the solvent used) compared to conventional solvent extraction process performed at
ambient pressure for 30min.
HP assisted solvent extraction was successfully performed at 700 MPa by using
significantly (P < 0.05) lower ratios of solvent:solid (6:1 and 4:1) and reduced processing
time (10min), compared to solvent extraction performed at ambient pressure, solvent:solid
ratio 10:1 and 30min extraction time.
High pressure processed products are commercially available in the United States,
European, and Japanese retail markets. Examples of high pressure processed products
commercially available in the United States include fruit smoothies, guacamole, ready meals
with meat and vegetables, oysters, ham, chicken strips, fruit juices, and salsa. Low acid, shelf-
stable products such as soups are not commercially available yet because of the limitations in
killing spores with HP. High pressure results in foods with fresher taste, and better
appearance, texture and nutrition; this technology is especially beneficial for heat-sensitive
products (http://ohioline.osu.edu/fse-fact/0001.html).
4.2.1. Operation of High Pressure Equipment

The main aspect of high pressure technology is the fluid medium; water is relatively
incompressible under high pressure and because of its biological compatibility, it is a
preferred medium for high pressure application (Lopes et al., 2010). During extraction using
high pressure equipment (Figure 7(A)), a sample with the extraction solvent is packed into a
flexible plastic bag (Figure 7(C)), and then loaded into a high pressure vessel (Figure 7(B)),
surrounded by a pressure transmitting medium. Pressure is applied by automatic system
driven piston, while the pressure increases the temperature also increases 3°C each 100 MPa
applied.
Even with an increase in the temperature, this technology is considered non-thermal by
the fact that the temperature during the process is relatively low compared with the thermal
process.
Pressurization cycles, pressures, and time can be programmed by program, which can
also control the automatic locking of the safety box and the alarms. After depressurization the
temperature decreases rapidly and the plastic bag with the sample can be collected.
Figure 7. (A) High pressure equipment (B) high pressure vessel (C) sample with extraction solvent
packed in flexible plastic bag. Source: personal archive.
CONCLUSION
The constant increasing in production of passion fruit juice and others derived products
have been producing a large amount of pomace that can be reuse in the food industry for
elaboration of new products; and consequently decrease the environmental problems. Besides,
the strong investments in research, technology of production (such as modetare eletric field
and high pressure equipament), and the trend for consumption of foods that present functional
properties and sensory quality have also influenced positively the enhancement of production,
processing and ingestion of passion fruit derived products. This chapter also meant to show
technologies emerging for extraction of bioactive compounds using less time and energy than
conventional technology.
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Chapter 6
HESPERETIN: SIMPLE NATURAL COMPOUND

WITH MULTIPLE BIOLOGICAL ACTIVITY
José Valdo Madeira Junior1, Vânia Mayumi Nakajima2,

Fabiano Jares Contesini1, Camilo Barroso Teixeira1,
Juliana Alves Macedo2 and Gabriela Alves Macedo1,2
1
Food Science Department
2
Food and Nutrition Department, Faculty of Food Engineering,
University of Campinas, Campinas – SP, Brazil
ABSTRACT
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.

Author e-mail: madeira_jr@hotmail.com.
108 J. V. Madeira Junior, V. M. Nakajima, F. J. Contesini et al.
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 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.
Hesperetin 109
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
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, O-
glycosylated 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
Hesperetin 111
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 micro-
agitation 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 environmental-
friendliness [41, 42]. The mechanism is based on heating dipolar compounds by microwave-
irradiation 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
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 pre-

treat 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.
Hesperetin 113
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 β-
glucosidase 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].
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 1,2-
dimethylhydrazine (DMH)-treated rats decreased the tumor incidence and the number of
aberrant crypt foci with simultaneous enhancement of tissue lipid peroxidation, glutathione S-
transferase (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- β-
D-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 protein-
1) 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
Hesperetin 115
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,
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, anti-
atherogenic, antimicrobial and anti-inflammatory properties.
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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,
2
Department of Biology, University of Guyana, South America
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
*
raymondjagessar@yahoo.com.
122 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.
A Review of the Antimicrobial Activity of Various Solvent Type Extracts ... 123
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
isolated [17, 18]. The structure of two of these compounds
N C Sare shown in Figure 1.
(1)
N C S
(1)
O O
N O O N
O O
S N O O N S
(2)
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 3-
cyclohexyliden-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-D-
glucopyranoside-(4'->O->4''')-galangin-3''-O-beta-D-glucopyranoside on the basis of
physicochemical and spectroscopic techniques, including 2D NMR spectral techniques [20].
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 alpha-
glucosidase 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-
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, anti-
inflammatory, cytotoxic, negative inotropic effect, anti-cancer, antitubercular and
antiplasmodial activities. An ethanol extract of the leaves showed potent ACE (Angiotensin-
converting 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 ACE-
inhibitory 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 ACE-
inhibitory 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-benzopyran-
5-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].
OH OH
H3CO O
H3CO
O
OH
O
OH O
OH O
(2)
(1)
Artocarpin
Cycloartocarpin
OH
OH
H3CO O
O
OH O
OH O (4)
(3) OH
Morusin
Chaplashin
OH HO OH
HO (6)
O O
O O O
OH O O
OH O
(5)
Cycloartobiloxanthone
Cudraflavone HO OH
(7)
O O
OH O O
Artonin
Figure 2. Isolates from Artocarpus altilis.
Flavonoids, 10-oxoartogomezianone, 8-geranyl-3-(hydroxyprenyl)isoetin, hydroxylarto-

flavone, isocycloartobiloxanthone, and furanocyclocommunin, together with 12 known
compounds, were isolated from heartwood and cortex of Artocarpus altilis, and were
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 n-
C6H14. 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
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].
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 Weight of Type of State of Weight of % yield

ground plant extract Extract Extract of Extract
material (g)
(g)
Solanum melongena 387 g n-C6H14 Black viscous 3.6 0.9
extract
Solanum melongena 387g CH3CH2OH Green 4.1 1.1
Viscous
Extract
Moringa oleifera 350g n-C6H14 Green semi 3.7 1.1
viscous
Extract
Moringa oleifera 361g CH3CH2OH Green 4.5 1.2
Viscous
Extract
Brassica rapa 375g n-C6H14 Green viscous 2.5 0.7
Chinensis solid
Brassica rapa 375g CH2Cl2 Light green 0.7 0.2
Chinensis
Brassica rapa 375g CH3CH2OH Dark green 21.9 5.9
Chinensis
Altocarpus altilis 361g n-C6H14 Off-White 2.5 0.69
yellow
361g CH2Cl2 Green 1.4 0.39
361g CH3CH2OH Viscous 22.5 6.23
light brown
Table 2. TLC profile for Solanum Melongena, Moringa oleifera, Brassica rapa chinensis
and Artocarpus altilis
Solvent Solanum Moringa oleifera Brassica rapa Altocarpus altilis

Extract melongena Rf Chinensis Rf
Rf Rf
CH3CH2OH 0.21, 0.35, 0.56, 0.35, 0.41, 0.61, 0.8, 1.88 0.8, 0.6
0.61 0.75
C6H12 0.35, 0.5, 0.75, 0.25, 0.41, 0.49, 0.32, 0.30, 0.51, 0.63
0.81 0.61 0.44,0.88,0.96
Rf: Retention factor.
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)
Solanum melogena E.coli 5 4.43 4.43 ±3.85 15.04
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
Solanum E.coli 5 7.2 7.2 ± 0.71 40.69
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
Moringa oleifera E.coli 5 4.4 4.4 ±3.81 15.19
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
Sample 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
Moringa oleifera E.coli 5 6.73 6.73 ±0.25 33.55
Ethanol
10 4.76 4.76 ± 4.12 17.78
20 7.73 7.73 ± 0.11 46.9
S.aureus 5 5 5 ± 4.35 38.46
10 8.1 8.1 ±0.79 51.5
20 8.1 8.1 ±0 51.5
Klebsiella 5 6.93 6.93 ±0.05 37.69

pneumoniae
10 7.33 7.33 ±0.05 42.17
20 7.93 7.93 ±0.11 49.36
Positive Control
Table 4. Area of Zone of Inhibition, AZOI for the positive control, tetracycline against
human pathogens
Microorganism Area of zone of inhibition

(mm2)
Escherichia.coli 36cm2
Staphylococus. aureus 37cm2
Klebsiella. pneumoniae 35cm2
Table 5.
Plant Extracts Tested Diameter of DZOI Mean Diameter Area of

Microorganism of ZOI ZOI
Hexane extract of E. coli 10mm, 9mm,14mm 11± 2.65 94.9
Brassica rapa
chinensis at low S. aureus 13mm, 14mm, 8mm 11.67± 3.22
concentration 106.9
(0.01 g/ml)
Bacillus subtilis 8mm, 6mm, 6mm 6.67 ± 1.16 34.9
C. albicans 18mm, 8mm, 11mm 12.33 ± 5.13 119.3

Hexane extract of E. coli No Inhibition 0
Brassica rapa
chinensis at high S. aureus 8mm, 6mm, 0 7 ± 4.16 38.5
concentration
0.1g/ml Bacillus subtilis No Inhibition 0
C. albicans 11mm, 10mm, 8mm 9.67 ± 1.53 73.4

Table 6. Antimicrobial activity of n-C6H14 extract of A. altilis at low and high

concentration
Plant Extracts Tested Diameter of ZOI Mean Diameter of AZOI

Microorganism ZOI
Hexane extract of A. E. coli No Inhibition 0 0
altilis at low S. aureus No Inhibition 0 0
concentration Bacillus subtilis No Inhibition 0 0
(0.01g/ml) C. albicans No Inhibition 0 0
Hexane extract of A. E. coli No Inhibition 0 0
altilis at high S. aureus No Inhibition 0 0
concentration Bacillus subtilis 12mm, 10mm, 10.67 ± 1.16 89.2
(0.1 g/ml) 10mm
C. albicans No Inhibition 0 0
Table 7. Antimicrobial activity of CH2Cl2 extract of A. altilis at low, high and very high
concentration
Plant Extracts Tested Diameter of ZOI Mean Diameter Area of ZOI

Microorganism
Dichloromethane E. coli No Inhibition 0 0
Extract of A. altilis S. aureus No Inhibition 0 0
At low concentration Bacillus subtilis 9mm, 8mm, 9mmm 8.67 ± 0.58 59.0
(0.01g/ml) C. albicans No Inhibition 0 0
Extract of A. altilis S. aureus 7mm, 9mm, 0 5.33 ± 4.73 22.3
At high concentration Bacillus subtilis 8mm, 7mm, 8mm 7.67 ± 0.58 46.2
(0.05 g/ml) C. albicans No Inhibition 0 0
Extract of A. altilis S. aureus 8mm, 10mm, 7mm 8.3 ± 1.5 54.1
At a very high Bacillus subtilis No Inhibition 0 0
concentration C. albicans No Inhibition 0 0
0.1g/ml
Table 8. Antimicrobial activity of CH3CH2OH extract of Brassica rapa chinensis at low

and high concentratio

Microorganism of ZOI (mm2)
Ethanol extract of E. coli 17mm, 17mm, 16.33 ± 2.7 209.3
Brassica rapa 15mm
chinensis at low S. aureus 9mm, 17mm, 13.67 ± 3.73 146.7
concentration 15mm
0.01g/ml Bacillus subtilis 7mm, 5mm, 0 4 ± 3.61 12.6
C. albicans 16mm, 15mm, 14 ± 2.6 153.9
11mm
Ethanol extract of E. coli 9mm, 8mm, 7mm 8 ± 1 50.2
Brassica rapa

Microorganism of ZOI (mm2)
chinensis at high S. aureus 15mm, 13mm, 12.33 ± 3.05 119.3
concentration, 9mm
0.1g/ml Bacillus subtilis 5mm, 9mm, 9.67± 5.03 73.4
15mm
C. albicans 15mm, 14mm 12.67± 3.27 126.0
9mm
Table 9. Antimicrobial activity of CH3CH2OH extract of A. altilis at low and high

concentration
Plant Extracts Tested Diameter of ZOI Mean Diamter Area of ZOI

Microorganism ZOI
Ethanol extract of E. coli 9mm, 8mm, 9mm 8.67 ± 0.6 59.00
A. altilis at low S. aureus 13mm, 11mm, 9mm 11 ± 2 94.9
concentration Bacillus subtilis 9mm, 8mm, 6mm 7.66 ± 1.5 46.7
0.01g/ml C. albicans No Inhibition 0 0
Ethanol extract of E. coli No Inhibition 0 0
A. altilis at high S. aureus No Inhibition 0 0
concentration Bacillus subtilis No Inhibition
Table 10. Combined fruit extracts of Brassica rapa chinensis and Artocarpus altilis
Plant Extracts Tested Diameter of ZOI Mean Diameter of Area of ZOI

Microorganism ZOI
Ethanol extracts of E. coli 11mm, 11mm, 10.67 ± 0.58 89.4
A. altilis + Brassica 10mm
rapa chinensis at
high concentration S. aureus 19mm, 12mm, 14 153.9
0.1g/ml 11mm
Bacillus subtilis 10mm, 10mm, 9.3 ± 1.16 67.8

8mm
C. albicans 18mm, 13mm, 14.67 ± 5.78 168.9

13mm
Hexane extracts of E. coli No Inhibition 0 0

A. altilis + Brassica S. aureus No Inhibition 0 0
rapa chinensis at Bacillus subtilis 13mm, 8mm, 9.67 ± 2 73.4
high concentration, 8mm
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
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 n-
C6H14 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.
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.
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 n-
C6H14 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.
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..
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
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.
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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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: mlimbad@aut.ac.nz.
146 Mansi Jayantikumar Limbad, Noemi Gutierrez-Maddox and Nazimah Hamid
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 Young coconut water

Energy value 19 kcal
Water 94.99 g/100g
Dry 5.01 g/100g
Ash 0.39 g/100g
Protein 0.72 g/100g
Total lipid, fats 0.2 g/100g
Total dietary fibre 1.1 g/100g
Carbohydrates 3.71 g/100g
Total sugar 2.61 mg/ml
Sucrose 9.18 mg/ml
Fructose 5.25 mg/ml
Glucose 7.25 mg/ml
Mannitol 0.8 mg/ml
Sorbitol 15 mg/ml
Myo-inositol 0.01 mg/ml
Scyllo-inositol 0.05 mg/ml
Calcium 24 mg/100g
Magnesium 30 mg/100g
Phosphorus 37 mg/100g
Iron 0.29 mg/100g
Sodium 105 mg/100g
Potassium 312 mg/100g
Manganese 0.142 mg/100g
Zinc 0.1 mg/100g
Copper 0.04 mg/100g
Chloride 183 mg/100g
Selenium 0.001 mg/100g
Sulfur 24 mg/100g
Vitamin C, total ascorbic acid 2.4 mg/100g
Thiamin (B1)sx 0.03 mg/100g
Folate, total 0.03 mg/100g
Coconut Water 147

Pyridoxine (B6) 0.032 mg/100g
Folate, food 0.003 mg/100g
Niacin (B3) 0.08 mg/100g
Nicotinic acid (Niacin) 0.64 mg/100g
Riboflavin (B2) 0.057 mg/100g
Pantothenic acid (B5) 0.52 mg/100g
Folate, Dietary Folate Equivalent (DFE) 3 (μg_DFE)
Biotin 0.02 mg/100g
Folic acid 0.003 mg/100g
Alanine 312 µg/ml
γ-Aminobutyric acid 820 µg/ml
β-Alanine 12 µg/ml
Aspartic acid 65 µg/ml
Cystine 0.97-1.17 µg/ml
Arginine 133 µg/ml
Asparagine and glutamine ca. 60 µg/ml
Glutamic acid 240 µg/ml
Homoserine 5.2 µg/ml
Glycine 13.9 µg/ml
Lysine 150 µg/ml
Leucine 22 µg/ml
Isoleucine 18 µg/ml
Histidine 0.017 g/100g
Methionine 8 µg/ml
Ornithine 22 µg/ml
Phenylalanine 12 µg/ml
Proline 97 µg/ml
Serine 111 µg/ml
Tyrosine 16 µg/ml
Tryptophan 39 µg/ml
Threonine 44 µg/ml
Valine 27 µg/ml
Ethanolamine 0.01 µmol/ml
Pyridoline 0.39 mg/ml
Malic acid 34.31 meq/ml
Citric acid 0.37 meq/ml
Shikimic and quinic acids 0.57 meq/ml
Total lipids 0.2 g/100g
Total saturated fatty acids 0.176 g/100g
Total monounsaturated fatty acids 0.008 g/100g
Total polyunsaturated fatty acids 0.002 g/100g
Auxin 0.07 mg/ml
Acid phosphatase Present (units not given)
Catalase Present (units not given)
Dehydrogenase Present (units not given)
(Continued)

Diastase Present (units not given)
Peroxidase Present (units not given)
RNA polymerase 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 anti-
ageing, 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
Coconut Water 149
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).
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 Percentage range (%)

Total solids 10.6-14.9
Crude protein 2.9-6.4
Carbohydrate 3.8-4.7
Ash 0.7-1.1
Moisture 86.3
Fats 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 auto-
aggregation, 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 Strains/type of isolates References

kefir‘s microbial profile
Biochemical techniques Saccharomyces sp., Kluyveromyces sp., Candida (Kolakowski &
sp., Mycotorula sp., Torulaspora sp., Cryptococcus Ozimkiewicz, 2012)
sp., Pichia sp. etc.
PCR-based DGGE and L. acidophilus, B. bifidum, S. thermophiles, S. (Theunissen, Britz,
species specific PCR thermophiles, L. bulgaricus, Streptococcus spp., S. Torriani, & Witthuhn,
thermophiles, B. adolescentis, B. longum, B. lactis. 2005)
Coconut Water 151
Technique used to study Strains/type of isolates References

kefir‘s microbial profile
Lb. kefiranofaciens subsp. kefirgranum, Lb.

kefiranofaciens subsp. kefiranofaciens, Lb. kefiri,
Lb. parakefiri, Lactobacillus parabuchneri, (Leite et al., 2012)
Lactobacillus amilovorus, Lactobacillus crispatus
and Lactobacillus buchneri.
Leuconostoc mesenteroides, Lactobacillus mali,

Lactobacillus hordei, Leuconostoc mesenteroides,
Enterococcus faecalis, Lactococcus lactis,
Bifidobacterium psychraerophilum,
Zygosaccharomyces fermentati, Saccharomyces (Hsieh, Wang, Chen,
cerevisiae, Dekkera bruxellensis, Pichia Huang, & Chen,
fermentans 2012)
DNA sequencing of Lact. acidophilus complex, Lact. amylovorous, (Kullen, Sanozky-
16srRNA region Lact. crispatus, Lact. galinarium, Lact. gasseri, Dawes, Crowell, &
and Lact. jonsonii Klaenhammer, 2000)
Pulse Field gel Strains of Lact. acidophilus complex, including (Singh, Goswami,
electrophoresis (PFGE) Lact. Delbrueckii subspecies (Lact. delbrueckii Singh, & Heller,
subsp. bulgaricus, Lact. Delbrueckii subsp. 2009)
delbrueckii, Lact. delbrueckii subsp. lactis), Lact.
plantarum, Lact. fermentum, Lact. rhamnosus, and
Lact. sakei
PCR DGGE, 16s rRNA Kluyveromyces maxianus, Torulaspora delbrueckii, (Leite et al., 2012;
sequencing for yeast Saccharomyces cerevisiae, Candida kefir, Magalhães et al.,
Saccharomyces unisporus, Pichia fermentans, 2010; Simova et al.,
Kazachastania aerobia, Lachanceae meyersii, 2002; Wang, Chen,
Yarrowia lipolytica, and Kazachstania unispora 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.
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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Atatürk Üniversitesi Veteriner Bilimleri Dergisi, 1(1).
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, anti-
inflammatory, 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: muntean@chem.ubbcluj.ro.
158 Luminiţa David and Bianca Moldovan
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 anti-
inflammatory 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
Extraction, Characterization and Potential Health Benefits … 159
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.
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: hydroxy-
acids, 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
chromatography (HPLC) and nuclear magnetic resonance (NMR) are necessary for the
unambiguous characterization of these compounds.
3'
2' 4'
8 1
7 O 2 5' O O
6'
6 3
5 4 OH
O O O
Flavones Flavonols Isoflavones
O O O
OH OH
O O
Flavanones Flavanonols Flavan-3-ols
OH
Cl-
HO O
OH
OH O
Anthocyanidins Chalcones
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
commonly present in higher plants: cyanidin, delphinidin, malvidin, peonidin, pelargonidin

and petunidin (Figure 2) [Andersen and Jordheim, 2006].
R3'
3'
OH
2'
1
B 4'
8
HO 7 9 O 2
1' 5' R5'
A C 3 6'
6
10
4
OH
5
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
R3'
HO O+ HO OH OH
R5' OR2
OR2 R5'
OR1 I + H 2O OR1 O III
- H+ - H2O
R3' R3'
+ H+
OH OH
OH
O O HO O
R5' R5'
OR2 OR2
OR1 OR1
IV R1 = H or glycosyl II
R2 = glycosyl
R3', R5'= H, OH or OCH3
Figure 3. Chemical structures of anthocyanins at different pH values.

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 co-
extraction 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 3-
galactosides 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
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-3-
galactoside, cyanidin-3-rhamnosylgalactoside, delphinidin-3-galactoside, pelargonidin-3-
galactoside 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-β-
galactoside, 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 UV-
VIS detection three anthocyanins in Cornus mas berries: cyanidin-3-O-glucoside, cyanidin-3-
O-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-O-
galactosyl-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-O-
galactoside [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-O-
glucoside 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.
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 Cornus spp. References

Cyanidin-3-galactoside C. mas Du and Francis, 1973a
OH Seeram et al., 2002
Cl-
OH Jayaprakasam et al., 2005
HO O Jayaprakasam et al., 2006
Pawlowska et al., 2010
O OH
O C. suecica Slimestad and Andersen, 1998
OH HO
HO C. alba Bjoroy et al., 2007
OH
C. officinalis Seeram et al., 2002
C. controversa Seeram et al., 2002
C. florida Vareed et al., 2006
C. kousa Vareed et al., 2006
Cyanidin-3-glucoside C. mas Tural and Koca, 2008
OH Capanoglu et al., 2011
OH C. suecica Slimestad and Andersen, 1998
Cl-
C. controversa Vareed et al., 2006
HO O
C. alternifolia Vareed et al., 2006
OH C. florida Vareed et al., 2006
O O
OH HO OH C. kousa Du et al., 1974a
HO Vareed et al., 2006
Cyanidin-3-rutinoside C. mas Tural and Koca, 2008
OH Capanoglu et al., 2011
Cl- OH
HO O
OH
HO OH
O O
HO
O
Me O
HO
HO
OH
Cyanidin-3-galactosyl-3‘-glucoside C. alba Bjoroy et al., 2007

HO
HO OH
O O
OH
OH
Cl-
HO O
O OH
O
OH HO
HO
OH

Cyanidin-3-(2-glucosylgalactoside) C. suecica Slimestad and Andersen, 1998
OH
OH
Cl-
HO O
O OH
O
OH O
HO
O OH
HO OH
OH
OH
Cyanidin-3-(2-glucosylglucoside) C. suecica Slimestad and Andersen, 1998

OH
OH
Cl-
HO O
OH
O O
O OH
OH
HO
O
HO OH
OH
OH
Cyanidin-3-robinobioside C. mas Du and Francis, 1973a

OH
C. canadensis Du et al., 1974b
OH
Cl-
HO O OH
OH
HO
O O
OH O
Me O
HO
HO
OH
Delphinidin-3-galactoside C. mas Du and Francis, 1973a

OH Seeram et al. 2002
Cl-
OH Jayaprakasam et al., 2005
OH
C. alba Bjoroy et al., 2007
O OH
O C. officinalis Seeram et al., 2002
OH HO
HO C. controversa Seeram et al., 2002
OH
Delphinidin-3-glucoside C. controversa Vareed et al., 2006

OH
OH
Cl - C. kousa Du et al., 1974a
HO O
OH
O OH
O
HO OH
OH
HO

Delphinidin-3- rutinoside C. controversa Vareed et al., 2006
OH
Cl- OH
HO O
OH OH
HO OH
O O
HO
O
Me O
HO
HO
OH
Delphinidin-3- galactosyl-3‘- C. alba Bjoroy et al., 2007

glucoside
HO
HO OH
O O
OH
OH
-
Cl
HO O
OH
O OH
O
OH HO
HO
OH

Delphinidin-3- galactosyl- C. alba Bjoroy et al., 2007
3‘,5‘-diglucoside
HO
HO OH
O O
OH
OH
Cl-
HO O O OH
O
HO OH
O OH HO
OH OH
OH
HO
Pelargonidin-3-galactoside C. mas Du and Francis, 1973b

-
OH Seeram et al., 2002
Cl
O OH C. officinalis Seeram et al., 2002

O
OH HO C. controversa Seeram et al., 2002
HO
OH
Pelargonidin-3- robinobioside C. mas Du and Francis, 1973b

OH
Cl- C. canadensis Du et al., 1974b
HO O OH
OH
HO
O O
OH O
Me O
HO
HO
OH

Pelargonidin-3-glucoside C. mas Tural and Koca, 2008
OH Pawlowska et al., 2010
Cl-
HO O C. kousa Du et al., 1974a
O OH
O
HO OH
OH
HO
Pelargonidin-3-rutinoside C. mas Pawlowska et al., 2010

Cl- OH
HO O
OH
HO OH
O O
HO
O
Me O
HO
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-O-
glucoside, 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 delphinidin-
3-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-O-
galactoside 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].
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-O-
glucoside), 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-O-
glucoside) 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 OH
HO O HO O
OH
OH OH
OH O OH O
Quercetin Myricetin
OH OH
HO O HO O
OH OH
OH O OH O
Kaempferol Aromadendrin
Figure 4. Chemical structures of flavonoids (aglycones) from Cornus fruits.

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 OH
Gallic acid Caffeic acid Chlorogenic acid
HO COOCH3 OH
O OH HO
OH OH
3,5-Dihydroxy-2-(2-methoxy-2oxoethyl)- Resveratrol
phenyl 4-hydroxy-benzoate
OH
H3CO
OCH3
HO
OH HO
OH O
HO O OH
O O OH
HO HO
OH OH
Tachioside Guaiacylglycerol-3-O-glucopyranoside
COOH
O OH OH
OH HO
O OH
HO OH OH O
HO
OH Tyrosol OH
p-Coumaric acid 7-O-Galloyl-D-sedoheptulose
Figure 5. Chemical structures of phenolic compounds isolated from Cornus fruits.

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 high-
speed 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 4-
hydroxy-benzoate, tachioside and guaiacylglycerol-3-O-glucopyranoside [Ma et al., 2014].
Zhang isolated for the first time a low molecular weight polyphenol, 7-O-galloyl-
sedoheptulose 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-D-
sedoheptulose 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].
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
OCH3
HO
O
OH
O
H3OC O
OCH3
OCH3 Balanophonin
O
OH
Pinoresinol
HO OH OH
OCH3 OH
OH
O HO
H3OC O
O O
OCH3 OCH3
OH
Cornuskoside A
OH
Lariciresinol
OH
OCH3 HO
O OCH3
HO
OH O
O OH
OCH3 HO
OCH3
Dihydrodehydroconiferyl alcohol Guaiacylglycerolconiferyl aldehyde ether
Figure 6. Lignans isolated from Cornus kousa L. fruits.

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 OH
O
O
OH
O OH O
O O
O O
O
O O
C C OH
O
HO OH OH
OH OH OH
HO
HO
Figure 7. Chemical structure of cornusiin B.

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
indicated the presence of morroniside and loganin as major constituents as well as of 7-O-
butyl 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
HO HO HO
O O O
O O
OH H3C OH
CH3 O OH O OH
HO HO
Morroniside Loganin
O O O O
HO HO HO
O O
O O
OH OH
O OH O OH
HO HO
Sweroside Swertiamarin
O COOCH3 COOCH3
HO O
O O O
O
CH3 OH
O OH CH3
HO Dehydromorroniaglycone
Cornin
COOH
OH
HO
HO HO
COOCH3 O
O
HO COO H3C OH
O OH
HO HO
O Loganic acid
OH
O OH
Cornuside 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.
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.
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-α-
hydroasiatic 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-3-
one (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].
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 COOH H COOH H COOH
H H H H H
HO HO HO
H H
Ursolic acid Oleanoic acid Betulinic acid
HO
H COOH H COOH H COOH

HO HO HO
H H H
HO HO HO
H H
HO HO
Tormentic acid Arjunolic acid Asiatic acid
H H
H
H H H
H H H H
HO HO HO
H H
Lupeol Taraxasterol Sitosterol
H
H
HOOC COO H H
O O
HO 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
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.
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. Biological activity References

C. alternifolia Inhibition of lipid peroxidation Vareed et al., 2006
C. controversa Inhibition of lipid peroxidation as well as astringent Vareed et al., 2006
and tonic effect
C. kousa Inhibitory activity on tumor cell proliferation, lipid Vareed et al., 2006
peroxidation and cyclooxygenase enzymes (COX) Vareed et al., 2007
Lee et al., 2007b
Anticarcinogenic activity Yan et al., 2002
Lee et al., 2007a
Lee et al., 2008a
Lee et al., 2008b
Lee et al., 2010b
Inhibitory activity against low density lipoprotein Lee et al., 2007c
(LDL) oxidation Lee et al., 2010
Anti-inflammatory, hepatoprotective and anti-HIV Puppala et al., 2007
activity Lee et al., 2007b
Inhibitory activity against human acyl-CoA: Lee et al., 2009
cholesterol acyltransferaze
Inbititory of metastatic properties of tumor cells Lee et al., 2010a
C. mas Treatment of diarrhea and gastrointestinal disorders Celic et al., 2006
Amelioration of obesity and insulin resistance, Jayaprakasam et al., 2005

reduction of the body weight and decrease of the level Jayaprakasam et al., 2006
of plasma cholesterol and liver lipids
Antigenotoxic activity Deng et al., 2013
Hepatoprotective activity Alavian et al., 2014
Diuretic effect Bijelic et al., 2011
Anti-inflammatory properties Yilmaz et al., 2009
Cardioprotective effects Eshaghi et al., 2012
C. officinalis Tonic, analgesic and diuretic activity Kean and Hwan, 1998
Cornus spp. Biological activity References

Antitumoral activity Nawa et al., 2007
Telang et al., 2012
Antiarrithmic, anti-shock, antineoplastic, anti- Kang et al., 2007
inflammatory, hepatoprotective and antidiabetic Cao et al., 2009
effects Zhao et al., 2010
Antibacterial activity Wu et al., 2008
Hepato- and renoprotective activity on diabetes Yamabe et al., 2009
Park et al., 2010
Antiviral and antitumor activity; α-glucosidase Kakiuchi et al., 1985
inhibitory effect Myamoto et al., 1987
Omar et al., 2012
Prevention and therapy of diabetic nephropathy Xu and Hao, 2004
Yokozawa, 2008
Ma et al., 2014
Antibacterial, antifungal and antispasmodic activity Kang et al., 2007
Dinda et al., 2007b
Wei et al., 2013
Jang et al., 2014
Amelioration of diabetic cardiomyopathy 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.
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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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: tbahorun@uom.ac.mu.
190 Theeshan Bahorun, Darshini Narrain, Piteesha Ramlagan et al.
CHAPTER HIGHLIGHTS
 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, anti-
tumor, 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
Kumquat (Fortunella Spp.) 191
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-
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: Plantae
Phylum: Tracheophyta
Class: Magnoliopsida
Order: Sapindales
Family: Rutaceae
Genus: Fortunella
Botanical Species Common Names
F. hindsii Hong Kong or Golden Bean
kumquat
F. japonica Marumi or round kumquat
F. X crassifolia Meiwa or large round kumquat
F. margarita Nordmann Nordmann Seedless kumquat
F. margaritaNagami Nagami, or oval kumquat
F. hindsii Hong Kong or Golden Bean
kumquat
F. japonica Marumi or round kumquat
Common Hybrids
Calamondin = Tangerine x Kumquat
Citrangequat = Citrange x Kumquat
Limequat = Key Lime x Kumquat
Mandarinquat = Mandarin x Kumquat
Orangequat = Satsuma
x Kumquat
Mandarin
Procimequat = Limequat x Kumquat
Sunquat = Lemon x Kumquat
Yuzuquat = Yuzu x Kumquat
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 β-
cryptoxanthin while pulps include additionally auroxanthin and luteoxanthin (Agόcs et al.,
2007). β-carotene and β-cryptoxanthin have also been identified in Fortunella hindsii
(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 α-
bergamotene (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> β-
elemene.
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-C-
glucoside, vicenin-2, lucenin-2 4′-methyl ether, narirutin 4′-O-glucosideand apigenin 8-C-
neohesperidoside were the main ones. Ogawa et al. (2001) found 3‘,5‘-di-C-beta-
glucopyranosylphloretin 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, anti-
inflammatory, neuro-protective and anti-H.pylori agents. Chlorogenic acid seems to be one of
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, anti-
hypertensive activities and modulation neurodegenerative diseases (Figure 5). Some of the
outcomes of these investigations are discussed in the subsections that follow.
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.
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‘-di-
C-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
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 glia-
mediated 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-amyloid-
induced 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
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.

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).
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 monophosphate-
activated 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
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 membrane-
bound 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.
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).
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 Rodriguez-
Casado, 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
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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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, 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.
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: f.mahomoodally@uom.ac.mu; mmfawzi@gmail.com.
212 Taukoorah Urmeela and Mahomoodally Mohamad Fawzi
(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, anti-
oxidant, 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 freeze-
dried 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
Aloe Vera Extracts: From Traditional Uses to Modern Medicine 213
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).
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
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 Functions and mode of action (where applicable)

Acemannans 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.
Aloetic acid The specific properties are not fully known but it seems to act as a natural
antibiotic.
Cinnamic acid It procures antiseptic, germicidal, anaesthetic and analgesic effects. Moreover,
it has a strong detergent action due to molecular similarity with saponins.
Chrysophanic acid It is a good purifying agent with strong laxative and diuretic effects. It also
stimulates bile secretion and has fungicidal action.
Salicylic acid 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.
Alo-emodin This compound is present in the yellow exudates found in lining of Aloe vera
leaf and has bactericidal and laxative properties.
Aloin or Barbaloin It has purging, detoxifying and markedly antibiotic properties.
Isobarbaloin Acts as a natural antibiotic.
Alprogen 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.
Vitamins It is rich in all vitamins except vitamin D. Vitamins A, C, E and B12 act as
antioxidants.
Enzymes 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.
Minerals 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:
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 double-
blind, 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
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
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
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 24-
methylene-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
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).
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 freeze-
dried 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
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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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.
228 Mihaela Mirela Bratu and Ticuta Negreanu-Pirjol
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-3-
sambubioside (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].
Elderberries Extracts 229
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.
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.
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 flavonoid-
concentrated 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
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 p-
coumaric 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-5-
glucoside, 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
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
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 anti-
influenza flavonoids were identified: 5,7-dihydroxy-4-oxo-2-(3,4,5-
trihydroxyphenyl)chroman-3-yl-3,4,5 trihydroxycyclohexanecarboxylate and 5,7,30,40-tetra-
O-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 mode-
of-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,
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.
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 anti-
inflammatory 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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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 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 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: ddaaggeerr@gmail.com.
242 Dragana Ĉetojević-Simin
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 H-
6), flavonoids (flavan-3-ols and their oligomers, quercetin) and anthocyanins (cyanidin-3-
sophoroside, cyanidin-3-(2-glucosylrutinoside), cyanidin-3-glucoside, pelargonidin-3-
sophoroside, cyanidin-3-rutinoside, pelargonidin-3-(2-glucosylrutinoside), pelargonidin-3-
glucoside, 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, anti-
proliferative (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.
Tumor Cell Growth Activity of Fruit and Pomace Extracts 243
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 by-
products, 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
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
(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.
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 non-
favorable 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
(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=11.74-
38.91µg/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)
(IC50<31.25 µg/ml). The most favorable non-tumor/tumor cell growth ratios were obtained by
ascorbic acid (NT/T=14.93 µg/ml) and synthetic antioxidant trolox (6-hydroxy-2,5,7,8-
tetramethylchroman-2-carboxylic acid) (NT/T=14.74 µg/ml) in cervix carcinoma cell line
(HeLa) (Table 2).
Table 1. Tumor cell growth activity of fruit and pomace extracts
Apoptotic
IC50 values (mg/ml)
Fruit and Extraction procedure/ Genotype/ Increase (AI)
References
Pomace solvent Extract NT/T NT/T NT/T MRC
HeLa MCF7 HT-29 HeLa MCF7
HeLa MCF7 HT-29 -5
Raspberry 80% Methanol with Willamete 0.06 2.16 0.04 3.25 0.16 0.81 0.13 2.8 1.0- Ĉetojević-Simin et al.
pomace 0.05% Acetic Acid Meeker 0.07 2.14 0.06 2.50 0.19 0.79 0.15 3.1 3.1 2015
Raspberry
80% Methanol with
fruit freeze Meeker 0.60 1.60 0.40 2.50 0.72 1.39 1 - - Vulić et al. 2014
0.05% Acetic Acid
dried
Fr 1 1 - 1 - 1 - - - -
Rosehip fruit 80% Acetone/Solid
Fr 2 0.08 - 0.25 - 0.36 - - - - Tumbas et al. 2012
dried Phase Extraction (SPE)
Fr 3 0.19 - 0.35 - 0.47 - - - -
Fr 1 1 - 1 - 1 - - - -
Bilberry fruit 80% Acetone/Solid Tumbas Šaponjac et al.
Fr 2 0.17 - 0.17 - 0.20 - - - -
dried Phase Extraction (SPE) 2015
Fr 3 0.13 - 0.17 - 0.30 - - - -
Baĉka 0.51 0.92 0.60 0.78 - - 0.47 - -
Knjaz 0.57 0.75 0.68 0.63 - - 0.43 - -
Tomato
Hexane Novosadski Stajĉić et al. 2015
pomace 3.08 0.24 1 0.75 - - 0.75 - -
niski
Rutgers 1.20 0.39 0.88 0.53 - - 0.47 - -
Saint Pierre 0.81 0.65 0.85 0.62 - - 0.53 - -
Baĉka 20.70 0.65 20.50 0.65 - - 13.40 - -
Knjaz 18.10 0.66 20.90 0.57 - - 11.90 - -
Novosadski
18.80 0.80 20.60 0.73 - - 15.00 - -
80% Ethanol niski Ćetković et al., 2012
O2 20.90 0.57 23.10 0.52 - - 12.00 - -
Rutgers 20.70 0.90 23.70 0.79 - - 18.70 - -
Saint Pierre 13.70 0.96 20.30 0.65 - - 13.20 - -
Hexane (Soxhlet) SX 28.07 - 14.28 - 25.90 - - - -
Pepper fruit SF 20 28.75 - 30.01 - 33.10 - - - - Tumbas Šaponjac et al.
Supercritical Extraction
dried SF 30 25.64 - 29.29 - 45.00 - - - - 2014
at 20, 30 and 40 MPa
SF 40 23.54 - 30.03 - 27.47 - - - -
NT/T- non-tumor/tumor IC50 ratio.
Table 2. Tumor cell growth activity of standards
IC50 values (µg/ml)

Standard References
HeLa NT/THeLa MCF7 NT/TMCF7 HT-29 NT/THT-29 MRC-5
Flavonoid
Apigenin 7-O-glucoside 500 1 500 1 500 1 500 -
Catechin 189.95±32.35 2.07 348.20±16.70 1.13 500 0.79 393.16±8.97 -
Epicatechin 265.66±20.69 1.17 456.46±30.73 0.68 500 0.62 311.22±62.76 -
Kaempferol 2.84±0.62 1.13 11.10±2.96 0.29 14.083.15 0.23 3.20±0.11 Simin et al. 2013
Morin 54.06±7.89 1.18 159.71±41.66 0.40 216.1654.48 0.30 64.01±2.68 -
Myricetin 17.57±1.66 2.87 33.93±2.49 1.48 92.573.62 0.54 50.39±3.94 -
Naringenin 9.93±1.75 8.40 68.70±10.83 1.21 102.4318.75 0.81 83.40±13.59 -
Phloridzin 500 1 500 1 500 1 500 -
Quercetin 12.77±1.23 0.92 12.21±0.60 0.96 38.91±5.89 0.30 11.74±2.98 Simin et al. 2013
Quercitrin 500 1 500 1 500 1 500 -
Rutin >500 1 >500 1 >500 1 >500 Simin et al. 2013
Phenolic acid
Caffeic acid 19.73±4.93 1.73 75.02±10.42 0.46 274.937.91 0.12 34.16±1.77 Simin et al. 2013
Chlorogenic acid  250 1 203.99±29.11 1.22 221.97±6.93 1.13  250 -
Cinnamic acid >500 0.21 164.75±21.92 0.65 8.74±19.22 12.22 106.80±16.86 -
Ellagic acid 2.47±0.40 1.17 11.10±0.73 0.26  25 0.12 2.89±0.91 Ĉetojević-Simin et al. 2015
Ferulic acid >500 0.41 114.35±16.46 1.77 271.79±24.60 0.74 202.69±15.43 Simin et al. 2013
Gallic acid 2.33±0.30 8.22 6.59±0.27 2.91 169.7413.03 0.11 19.16±6.12 Ĉetojević-Simin et al. 2015
Gentisic acid 31.25 1 500 0.06 152.5127.10 0.20 31.25 -
p-Coumaric acid 144.71±21.70 1.91 284.91±42.43 0.97 242.470.78 1.14 276.19±49.19 -
Protocatechuic acid 194.35±31.95 1.45 313.96±51.26 0.90 500 0.56 281.43±29.35 -
Rosmarinic acid  250 1 185.80±9.10 1.34 165.42±35.66 1.51  250 -
Sinapic acid 103.44±13.98 3.44 299.12±86.99 1.19 376.3787.89 0.95 355.93±82.73 -
Syringic acid 500 1 500 1 482.1417.18 1.04 500 -
Vanillic acid 94.69±38.48 5.28 298.84±35.26 1.67 338.0563.47 1.48 500 -
Synthetic Antioxidant
BHA 7.87±0.056 1.39 8.23±0.40 1.33 7.620.66 1.43 10.94±2.26 Stajĉić et al. 2015
BHT 1.11±0.14 2.71 1.09±0.28 2.76 6.102.33 0.49 3.01±0.49 -
Trolox 241.13±62.18 14.74 248.61±11.12 1.58 275.5122.17 1.43 393.56±45.78 -
Provitamin and Vitamin
Ascorbic acid 26.69±5.07 14.93 32.93±1.84 12.10 98.3635.64 4.05 398.60±24.96 Ĉetojević-Simin et al. 2015
-Carotene 13.83±10.87 1.25 10.65±1.71 1.63 21.085.23 0.82 17.33±1.31 Tumbas Šaponjac et al. 2014
NT/T- non-tumor/tumor IC50 ratio.
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)
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-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.
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.
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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
1
Laboratory of Biochemistry, UR ‗‗Human Nutrition and Metabolic Disorder‖ Faculty of
Medicine, Monastir, Tunisia
2
Institute of Olivier of Sousse, Sousse, Tunisia
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: snebrifati@yahoo.fr, mohamed.hammami@fmm.rnu.tn.
256 Faten Brahmi, Chehab Hechmi, Imed Chraief et al.
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.
Influence of Two Maturation Stages and Three Irrigation Regimes … 257
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
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
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.
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 ha-
1. 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].
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.
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
AIDS, xii, 211

A alcohols, 54, 174, 192
aldehydes, 15, 20, 21, 54, 192
A. altilis, x, 121, 125, 127, 132, 133, 134, 137, 138,
aldosterone, 204
139, 140
algae, 54
Abraham, 149, 153
alkaloids, ix, 107, 124
accessions, 239
allergy, 216
accounting, ix, 70, 107, 109, 116, 242, 259
aloe, 213, 220, 221, 223, 225, 226
acetaldehyde, 149
alters, 42
acetic acid, 61, 92, 149, 150, 152, 243, 247
aluminium, 35
acetone, 23, 55, 163, 173, 230, 236, 244
amine(s), 9, 11, 20, 21, 24, 25, 54, 57, 63
acetylcholine, x, 108, 114
amino, 11, 21, 146, 213, 244
acidic, 71, 149, 163, 244
amino acid(s), 146, 213, 244
acidity, 34, 83
ammonium, 63
acne, 212, 213, 215, 217, 222, 223
amplitude, 111
acne vulgaris, 213, 217, 222, 223
amylase, 206, 214
active compound, 95, 239
anaerobic digestion, 49
activity level, 213, 220
analgesic, xi, 157, 158, 180, 200, 214, 215
adaptability, 256
androgen, 217
additives, vii, viii, 1, 2, 6, 21, 24, 53, 65, 70
angiogenesis, 221
adenine, 114
angiotensin II, 204
adenocarcinoma, 245, 247, 250, 251
anomalities, 31
adenosine, 215, 221
ANOVA, 258
adhesion, x, 60, 87, 104, 108, 114, 150, 184, 234
anthocyanin(s), xi, xii, 17, 22, 62, 65, 71, 96, 99,
adipocyte, 116
108, 110, 157, 158, 159, 160, 161, 162, 163, 164,
adiponectin, 202
165, 168, 169, 180, 181, 182, 183, 184, 185, 186,
adolescents, 217, 224
188, 209, 227, 228, 229, 230, 231, 232, 234, 236,
adrenal gland, 204
237, 238, 239, 240, 241, 242, 250, 251
adults, 104, 202
antibiotic(s), ix, 107, 122, 128, 141, 196, 215
advancements, xi, 145
antibody, 215, 220, 225
adverse effects, 9
anti-cancer, 91, 125, 148, 152, 200, 212
adverse soil conditions, 256
anticancer activity, 123, 125, 184, 253
aerobic bacteria, 12
anticarcinogenic, viii, 69, 71, 200, 233
aetiology, 216
antidiabetic, viii, 69, 71, 158, 181, 213, 218
aflatoxin, 114
antigen, 215, 220, 225
Africa, 70, 113, 212, 223
anti-inflammatory, viii, ix, xi, xii, 69, 77, 107, 113,
agar, 127, 128, 187
114, 115, 116, 123, 125, 142, 157, 158, 168, 170,
age, 213, 219, 222, 242
176, 181, 190, 194, 195, 198, 200, 213, 214, 215,
aggregation, 55, 59, 150, 208
216, 218, 225, 227, 228, 236, 237, 240, 242, 253
agonist, 115
266 Index
antimicrobial, vii, viii, x, xi, 1, 4, 10, 14, 18, 26, 53, bacteria, xi, 6, 7, 8, 14, 54, 62, 67, 70, 74, 75, 82, 91,
59, 60, 62, 65, 71, 74, 75, 78, 99, 108, 113, 116, 122, 128, 134, 145, 148, 149, 150, 152, 154, 155,
121, 122, 127, 128, 129, 134, 135, 136, 137, 139, 181, 196, 218, 234
141, 142, 148, 151, 154, 158, 196, 197, 205, 206, bacterial colonies, 128
235, 242, 253 bacterial pathogens, 239
antioxidative activity, 27 bacterial strains, 74, 128
antioxidative potential, 124 bactericides, 196
antipyretic, 123 bacteriocins, 151
antitumor, 177, 181, 185, 187, 247, 250, 251 bacterium, 217, 235
antiviral, ix, 108, 113, 220, 233, 234, 235 Bangladesh, 222
apoptosis, x, xiii, 77, 108, 114, 115, 181, 197, 198, base, 162
200, 203, 204, 207, 208, 209, 210, 241, 243, 246, basement membrane, x, 108, 115
247, 250 BBB, 198
appetite, 197 beef, vii, 1, 5, 6, 9, 10, 11, 13, 14, 15, 16, 17, 20, 21,
apple pomace, ix, 2, 3, 25, 26, 82, 88, 91, 92, 102, 23, 24, 25, 26, 27, 28, 197
252 beet pulp, ix, 82, 88, 104, 105
apples, 26 beneficial effect, viii, xii, 6, 58, 60, 69, 84, 181, 198,
aptitude, 263 211, 222
aquifers, 31 benefits, ix, xi, 2, 17, 26, 48, 54, 62, 84, 85, 107,
Argentina, 51, 70 145, 152, 157, 159, 186, 189, 200, 242, 251, 259,
arginine, 179 263
aromatic rings, 71, 108 benzene, 71, 125
ARS, 142, 205 benzo(a)pyrene, 200
arterial hypertension, 219 beta-carotene, 214
arteries, 219 beverages, 108, 116, 153, 154, 155, 190
Artocarpus altilis, x, xi, 121, 122, 124, 125, 126, bilberry, xii, 230, 236, 240, 241, 242, 243, 244, 247,
127, 128, 129, 130, 133, 137, 140, 141, 143 250, 253
aryl hydrocarbon receptor, 186 bile, 215
ascorbic acid, xi, 11, 62, 65, 146, 157, 179, 180, 181, bioactive compounds, ix, xi, 1, 17, 64, 81, 85, 96, 97,
185, 233, 241, 242, 243, 245, 247, 250, 251 107, 111, 112, 157, 159, 161, 163, 177, 181, 190,
Asia, xi, 21, 113, 157, 158, 159, 160, 207, 228 191, 210, 242, 252
Asian countries, 190 bioassay, 125
aspartic acid, 179 bioavailability, 65, 72, 109
assessment, 59, 66, 78, 117, 186 biochemistry, 65, 182, 189
asthma, 86, 104, 124 bioconversion, 119
astringent, 180, 222 bioflavonoids, 5
atherosclerosis, 115, 218, 253, 259 biogas, viii, 69
atmosphere, 8, 12, 26, 34, 50, 62, 66, 67, 73, 245 biological activity(s), vii, xi, 74, 84, 112, 157, 158,
atmospheric pressure, 72 168, 174, 180, 181, 188, 243
atomic emission spectrometry, 179 biologically active compounds, 76, 116
Austria, 231, 237 biomarkers, 182
authentication, 127 biomass, 40, 49, 154
authenticity, 222 biomaterials, 111
autoimmune disease, 213, 218 biomedical applications, 82, 91, 102
autooxidation, 6, 17 biomolecules, 173
auxins, 148 biopolymers, 96
avian, 174 biosynthesis, 64, 102, 103, 156
awareness, 70 biotechnology, 112, 252
biotic, 197
biotin, 246
B birds, 158, 159
black tea, 94
Bacillus subtilis, 18, 131, 132, 133, 137, 139
bladder incontinence, xi, 157
Index 267
bleaching, 180, 233 carboxyl, 89, 90

bleeding, xi, 157, 216 carboxylic acid(s), 71, 247
blood, x, 85, 108, 114, 125, 146, 148, 151, 155, 176, carcinogen, 198, 200
179, 197, 198, 202, 203, 204, 205, 210, 218, 219, carcinogenesis, 114, 200, 201
222, 225, 231, 235, 236, 240 carcinoma, 125, 158, 172, 178, 208, 245, 247, 250
blood pressure, x, 108, 114, 125, 146, 151, 155, 176, cardiac diseases, ix, 108, 114
204, 219, 225, 231 cardiac dysfunction, x, 108
blood-brain barrier, 198, 210 cardiac output, 204
body fat, 213, 219 cardiomyopathy, 179, 181
body mass index (BMI), 202 cardiovascular disease(s), 84, 99, 113, 148, 190, 197,
body weight, 114, 115, 168, 180, 198, 203, 213, 219 199, 204, 206, 209, 220, 222, 242
bonds, 96 cardiovascular disorders, xii, 181, 211, 242
bone, 115, 179 carotene, 72, 83, 123, 180, 193, 198, 199, 200, 206,
bone marrow, 115 209, 232, 241, 242, 247, 250
Bosnia, 183 carotenoids, vii, viii, xii, 1, 4, 53, 96, 103, 113, 123,
bowel, 221 174, 190, 193, 199, 200, 208, 231, 241, 242, 253
bowel obstruction, 221 cascades, 198
brain, 176, 198, 205 cascara, 221
Brassica rapa chinensis, x, xi, 121, 122, 124, 127, catalysis, 111
128, 129, 130, 131, 132, 133, 134, 137, 138, 139, catechin, viii, 14, 57, 69, 125, 148, 207
140 cation, 161, 162
Brazil, viii, 14, 77, 81, 82, 83, 86, 98, 107, 190 C-C, 41
breakdown, 202 cell culture, 114, 115, 186, 235, 245, 246
breast cancer, 125, 186, 200, 208 cell death, 116, 203, 243, 245, 246, 247
breast carcinoma, 172, 178, 250 cell differentiation, 116
bronchitis, 124, 235, 239 cell division, xii, 227
bulk density, vii, 29, 41, 42, 45, 46, 47, 51 cell line(s), 125, 142, 170, 172, 176, 178, 184, 200,
burn, 40, 217, 225, 226 208, 243, 245, 246, 247, 250
by-products, vii, viii, xii, 1, 2, 4, 5, 6, 8, 17, 18, 25, cell membranes, 93
29, 49, 50, 53, 54, 55, 58, 60, 61, 66, 70, 78, 81, cell signaling, 196
99, 109, 117, 241, 243 cell surface, 150
cellulose, 40, 50, 87, 88, 110, 113, 218
Central Europe, 187, 253
C central nervous system, 176
ceramic, vii, viii, 29, 30, 31, 32, 35, 36, 40, 42, 46,
C. albicans, x, 74, 121, 122, 128, 131, 132, 133, 139,
48, 49, 50, 51, 95
197
ceramic materials, vii, 29, 32, 51
Ca2+, 204
cervix, xii, 172, 178, 241, 245, 247, 250
Cairo, 222
challenges, 76
calcium, 36, 90, 123, 179, 193, 220, 242, 257
cheese, 149, 154
calcium carbonate, 257
chemical characteristics, 238
calorie, 197
chemical properties, 87, 187
cancer, xii, 84, 98, 114, 124, 142, 152, 170, 177,
chemical structures, 116, 125, 161, 170, 171, 172,
178, 180, 181, 184, 190, 194, 200, 205, 207, 208,
173, 176, 177, 197
211, 212, 213, 220, 221, 222, 231, 242, 253
chemoprevention, 73, 208, 220
cancer cells, 114, 253
chemopreventive agent(s), ix, 107, 114, 177
cancerous cells, 220
chemotherapy, 220
candidates, 202, 233
chicken, vii, 1, 3, 4, 5, 6, 8, 9, 10, 13, 14, 15, 18, 19,
capillary, 109, 186, 257
21, 24, 26, 27, 28, 96
carbohydrate(s), 2, 19, 54, 72, 83, 84, 85, 87, 110,
childhood, 216
124, 160, 173, 202, 206, 219
children, 15, 22, 202, 216, 224
carbon, 34, 35, 40, 72, 77, 154, 183, 223, 245
Chile, 70
carbon dioxide, 72, 77, 223
carbon tetrachloride, 154, 183
268 Index
China, xii, 70, 142, 158, 159, 187, 190, 211, 212, communication, 200, 201
239 compaction, 41, 42
Chinese medicine, xi, 157, 197, 208 compatibility, 82, 97
chitosan, 104 compilation, 117
chloroform, 23, 257 complement, 212, 221
cholera, 124 complications, x, 108, 115, 202, 209, 218, 224
cholesterol, x, 21, 85, 86, 91, 108, 115, 124, 155, composites, 110
168, 177, 179, 180, 185, 203, 204, 219 compression, vii, 29, 32, 41, 42, 43, 45, 46, 47, 111
choline, 214 computer, 95
chromatographic technique, 163 condensation, 173
chromatography, 63, 161, 171, 173, 174, 177, 185, conductance, 94
186, 188 conductivity, vii, 29, 34, 35, 46, 47, 50, 51
chromium, 214 conflict, 251
chronic diseases, ix, 65, 107, 202, 242 conflict of interest, 251
cigarette smoke, 186 congress, 67, 76, 263
citizens, 218 conjugated dienes, 58, 124
citrus albedo, ix, 82, 88 connective tissue, 115
Civil War, 159 conservation, 30, 95, 96
classes, 22, 26, 71, 108, 160, 161, 171, 173, 190 conserving, 46
clay minerals, 36, 39 constipation, xii, 211, 221, 228
cleavage, 113 constituents, ix, xi, xii, 4, 40, 73, 75, 86, 107, 145,
climate(s), 30, 71, 77, 83, 208, 212, 256 152, 158, 169, 175, 176, 179, 182, 187, 190, 197,
climate change, 30 211, 212, 213, 215, 218, 223, 228, 229, 262, 263
clinical symptoms, 86, 235 construction, 30, 31, 41, 48, 49
clinical trials, 204, 218 consumers, 2, 5, 11, 14, 30, 54, 58, 64, 70, 84, 180
closure, 213, 218 consumption, xii, 12, 35, 85, 97, 115, 141, 155, 189,
CO2, viii, 40, 69, 72, 73, 75, 78, 230, 234, 245 231, 242
cocoa, 149, 155 contact time, 84, 85
coconut water, xi, 145, 146, 148, 149, 150, 151, 152, contamination, 152
153, 154, 155 control group, 59, 61, 62, 114, 220
coconut water kefir, xi, 145, 149, 151, 152 cooking, 3, 4, 5, 6, 13, 16, 17, 20, 22, 24, 25, 55, 59,
Code of Federal Regulations, 84, 98, 99 236
coffee, 12, 73 copper, 179, 214
cognition, 198 Cornaceae plant, xi, 157
cognitive function, 198, 206 Cornus, vi, xi, 157, 158, 159, 160, 161, 163, 164,
cognitive impairment, 176 165, 166, 167, 168, 169, 170, 171, 172, 173, 174,
coke, 35 175, 176, 177, 178, 179, 180, 181, 182, 183, 184,
colitis, 183 185, 186, 187, 188
collagen, 115, 218, 221, 236 coronary artery disease, 219
collateral, 86 coronary heart disease, 84, 99, 146
Colombia, 99 correlation(s), 47, 51, 56, 232
colon, ix, xii, 82, 84, 91, 107, 114, 172, 176, 178, cortex, 126
200, 208, 221, 231, 241, 242, 245, 247, 250 cosmetic(s), ix, xii, 71, 72, 75, 76, 82, 91, 211, 212,
colon cancer, 84, 200, 208, 231 221, 222, 223, 243
colon carcinogenesis, ix, 107, 114 cost, xii, 31, 54, 72, 112, 122, 217, 234, 241
color, xii, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17, cough, 86, 104, 122, 191
19, 20, 21, 22, 23, 24, 25, 26, 57, 58, 59, 61, 62, coughing, 197, 234
86, 158, 160, 161, 162, 192, 227, 231, 246 cracks, 32, 40
combined effect, 8 critical value, viii, 69
combustion, vii, 30, 34, 39, 40, 41, 47 crop(s), viii, 54, 69, 104, 122, 158, 181, 190, 241,
commercial, viii, 3, 4, 12, 13, 14, 54, 61, 81, 82, 86, 242, 256, 257, 262
105, 109, 117, 122, 150, 153, 158, 182, 186, 230, crown, 124
245 crystalline, 34, 36, 90
Index 269
crystals, 123 diabetes, ix, xi, 84, 85, 86, 108, 113, 114, 115, 124,
cultivars, 105, 158, 160, 169, 171, 191, 230, 238, 125, 157, 158, 168, 171, 181, 187, 190, 197, 199,
243, 251, 252, 256, 263 201, 202, 204, 205, 207, 209, 213, 218, 220, 222,
cultivation, viii, 69, 70, 75, 83, 148, 156, 231, 256 224, 225, 226, 259
cultivation conditions, 75, 148 diabetic nephropathy, 158, 176, 181, 185
culture, 74, 128, 148, 149, 150, 153, 154, 235, 246 diabetic patients, 103, 204, 218, 223
culture media, 148, 235 diarrhea, 180, 202, 228
cure, 148, 197, 216, 228, 234 diastole, 219
curing process, 7 diastolic blood pressure, 219
CV, 143, 233 dienes, 58, 76
cycles, 97 diet, 3, 77, 84, 91, 102, 178, 184, 196, 203, 205, 209,
cyclooxygenase, 168, 170, 176, 180, 215, 218 221, 224, 233
cytochrome, 203 dietary fiber, vii, viii, ix, 1, 3, 4, 5, 6, 8, 19, 25, 26,
cytokines, 197, 202, 217, 234, 235, 236, 239 28, 53, 54, 55, 58, 59, 66, 81, 83, 84, 85, 86, 101,
cytoplasm, 148 102, 242
cytotoxicity, 114, 125, 170, 172, 176, 187, 197, 252, diffusion, 91, 93, 94, 96, 101, 103, 110, 111, 128,
253 134, 137, 140, 142
Czech Republic, 169, 186 digestibility, 49, 70, 72, 77
digestion, xi, 34, 84, 145, 151, 152, 182, 197, 215
digestive enzymes, 84
D discrimination, 155, 260
discs, x, 121, 128
data set, 261
diseases, ix, xi, xii, 107, 113, 114, 116, 122, 148,
deaths, 219
152, 157, 158, 181, 190, 197, 198, 204, 211, 219,
decay, 65
220, 231, 242, 259
decomposition, 39, 40, 41, 112, 236
disorder, 216, 259
defecation, 221
distillation, 70, 110
defects, 32, 40, 43
distilled water, 17, 59, 112, 127, 128, 244
defense mechanisms, 235, 236
distribution, xii, 56, 119, 141, 142, 154, 156, 183,
deficiency, 242
189, 205, 256
deficit, 3
diuretic, 158, 179, 180, 191, 215, 228
degradation, 56, 63, 72, 83, 92, 99, 110, 120
diversity, 141, 142, 154, 200, 232, 251
degradation process, 72
DNA, 148, 151, 154, 176, 190, 200, 209, 220, 246
dehydration, 92, 93
DNA damage, 148, 176, 200
Dekkera bruxellensis, 150
DNA sequencing, 151
denaturation, 94
docosahexaenoic acid, 64
density values, 47
Dogwoods, xi, 157, 158, 183
deoxyribose, 180
DOI, 65, 182, 185, 186, 253
Department of Agriculture, 152
donors, 235
depolymerization, 173
dopamine, 77
deposition, 176
dosage, 13, 222
depth, 111, 128, 149, 152
down-regulation, 200
derivatives, 110, 160, 161, 164, 169, 171, 175, 176,
drought, 256
183, 194, 197, 198, 202, 205, 256
drug delivery, 82, 91
dermatitis, 216
drug interaction, 236
dermatology, 225
drug metabolism, 236
destruction, 96, 111
drug resistance, 198
detectable, 14
drugs, 122, 152, 181, 187, 202, 221, 231, 237, 250
detection, 116, 117, 118, 164, 246
dry matter, ix, 19, 21, 70, 82, 87, 88
detergents, 122
drying, 6, 7, 36, 40, 92, 127, 163, 243, 247
detoxification, 213, 220, 231
DTA curve, 40
developed nations, 216
durability, 42, 43
deviation, 130
dyes, 161
270 Index
dysuria, 124, 148 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,
E 200, 202, 203, 204, 205, 206, 207, 208, 209, 212,
213, 214, 215, 219, 220, 223, 231, 236, 246, 263
E.coli, x, xi, 121, 122, 128, 130, 131, 134, 135, 137,
enzyme immunoassay, 246
139, 140
enzyme inhibitors, 231
Eastern Europe, 159
epicarp, viii, 81, 82, 85, 191
ECM, 176
epicatechin, viii, 14, 69, 74, 76, 148
ecology, 30, 153
epidemiologic, 253
economic growth, 30
epidemiologic studies, 253
eczema, 216, 223
epidermis, 216
edema, 115, 216
equilibrium, 162
effluent(s), 47, 182, 187, 244
equipment, 95, 97, 105
effluents, viii, 30, 32, 47
Eschericia coli, xi, 122, 142
efflux transporters, 198
ESI, 164, 169, 174, 175
Egypt, xii, 211, 212
essential fatty acids, 64
eicosapentaenoic acid, 64
ester, xii, 88, 89, 102, 110, 123, 209, 227, 228
elaboration, 36, 86, 97
estrogen, 186
elastin, 221
ethanol, 22, 23, 55, 57, 60, 62, 70, 73, 74, 75, 78, 79,
elbows, 216
111, 115, 125, 134, 137, 140, 141, 163, 174, 181,
elderberries, xii, 227, 228, 229, 230, 231, 232, 233,
230, 234, 235, 244, 247
234, 235, 236
ethers, 171
electric current, 95
ethyl acetate, 23, 125, 163, 169, 171, 173, 174, 177
electric field, ix, 82, 83, 93, 94, 95, 99, 101, 103, 104
Europe, xi, 70, 83, 157, 158, 160, 228, 231
electrical conductivity, 34, 93
European Community, 70
electrodes, 95
European Union, 30, 62
electrolyte, 148
evaporation, 31, 40, 47
electromagnetic, 83
evapotranspiration, xiii, 255, 257, 263
electron, 27, 35, 75
evidence, 65, 72, 84, 91, 114, 125, 190, 205, 216,
electrophoresis, 151
225, 258
electroporation, 94
evil, 228
elucidation, 163, 174, 223
evolution, 7, 12, 50, 56, 262
e-mail, 29, 107, 241
exothermic peaks, 40
emission, 30
expectorant, 228
emotion, 198
experimental condition, 64
employment, 31
experimental design, 59, 101
endocrine, 115
exploitation, 220
endosperm, xi, 145
exposure, 11, 92, 93, 94, 216, 222, 226
endosperm fluid, xi, 145
expulsion, 215
endothelial cells, x, 108, 114, 181, 184, 185, 221
extracellular matrix, 176, 218
endothelial dysfunction, 114
extrusion, vii, 29, 32, 34, 41, 42, 43, 45, 46, 47, 50
endothelium, x, 108, 114, 204
endothermic, 39, 40
energy, viii, ix, 31, 35, 46, 48, 50, 69, 82, 85, 93, 95, F
97, 110, 111, 112, 198
energy consumption, 31 fabrication, 33
energy efficiency, 93 factories, 47
energy supply, 95 families, 212
enlargement, 202, 217 fasting, 203, 218
environment, 30, 31, 48, 54, 82, 85, 93, 174, 256 fat, 1, 3, 4, 5, 6, 8, 9, 11, 14, 15, 16, 17, 19, 20, 22,
environmental factors, 160, 216 24, 28, 82, 91, 102, 146, 168, 184, 203, 209, 220,
environmental impact, viii, 30, 31, 47, 70, 72 224
environmental issues, ix, 30, 81
Index 271
fatty acids, xiii, 54, 63, 64, 72, 82, 84, 91, 98, 179, food spoilage, 2, 5, 14
181, 255, 257, 258, 259, 260, 261, 262, 263 force, 16
FDA, 95, 104, 221 formation, 5, 6, 7, 8, 9, 10, 11, 12, 17, 20, 21, 22, 24,
fecal impaction, 221 25, 40, 45, 55, 57, 58, 61, 63, 76, 115, 152, 197,
fermentation, 7, 54, 70, 71, 77, 93, 110, 112, 149, 198, 217, 218, 219, 220, 221
150, 153 formula, 88
ferrite, 36, 149 fragments, 246
fever, 158, 181, 228 France, 70, 159
fiber(s), ix, 2, 3, 5, 7, 8, 15, 19, 24, 25, 28, 58, 59, free radicals, 71, 190, 199, 202, 220, 232, 243, 253
71, 81, 82, 83, 84, 85, 87, 88, 90, 91, 99, 102, freezing, 14, 60, 62
103, 105, 221 freshwater, viii, 53, 54
fiber content, 3, 5, 58, 88 fructose, 213, 228
fibroblasts, 155 fruit-processing, vii, viii, 53, 54, 243
fibrosis, x, 108, 114 functional analysis, 104
fillers, 5 functional food, xi, 25, 54, 58, 70, 74, 79, 85, 90,
film thickness, 258 145, 190, 202, 204, 208, 222, 243
films, 99 fungi, 5, 74, 119, 128, 218
filters, 244, 245 fungus, 74, 122, 134, 197
filtration, 112, 231 furan, 184
fingerprints, 191
Finland, 118, 246
fish, viii, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, G
64, 65, 66, 76, 77
gastritis, 148
fish oil, 63, 64, 65, 66, 76
gastrointestinal disorders, xi, 157, 158, 180, 181
flame, 257
gastrointestinal tract, 215, 228
flammability, 23
gel, 3, 26, 88, 90, 103, 104, 151, 212, 213, 216, 217,
flatulence, 202
218, 219, 220, 221, 222, 223, 224, 225, 226
flavanones, ix, 107, 108, 110, 116, 118, 125, 160,
gel formation, 3, 88
194, 198, 199, 201, 204
gelling agent, ix, 81, 82, 90, 91
flavonol, xii, 22, 63, 71, 169, 192, 194, 227, 228, 232
gene expression, 203, 218, 220
flavor, 3, 4, 5, 8, 9, 11, 13, 14, 15, 16, 20, 22, 54, 58,
genes, 203, 224
59, 60, 61, 62, 158, 159, 160, 194
genetic alteration, 243
flavour, 152, 263
genetic diversity, 187
flexibility, 230
genetics, 64
flight, 117
genital herpes, 225
flocculation, 123
genomics, 252
flora, 122, 141
genotype, ix, 108, 204, 244, 252
flour, 64, 104, 119
genus, viii, xi, 81, 82, 109, 123, 157, 158, 189, 190,
flowers, xii, 123, 158, 227, 228
191, 194, 208, 212, 228, 230
fluid, xi, 72, 75, 78, 79, 97, 112, 118, 145, 148, 177,
geographical origin, 206
221, 230, 245
Germany, 70, 156, 243, 244, 245
fluid extract, 72, 78, 79, 112, 177, 230
germination, 70
fluorescence, 34, 56
glia, 198, 199, 210
folic acid, 214
glucose, 85, 91, 98, 109, 113, 115, 149, 160, 161,
follicles, 217
169, 173, 184, 201, 203, 207, 210, 213, 218, 220,
food additive, 56
222, 228, 234, 245
Food and Drug Administration, 84, 95, 221
glucose tolerance, 203
food industry, vii, viii, xi, 4, 6, 8, 53, 84, 85, 87, 92,
glucoside, xii, 109, 113, 117, 164, 165, 166, 167,
95, 97, 118, 145, 152, 163, 189
168, 169, 170, 183, 194, 195, 196, 197, 227, 228,
food intake, x, 108, 115, 168
229, 232, 242, 249, 250, 251
food production, 186
glucosinolates, 123, 124, 141, 142
food products, ix, 2, 6, 64, 85, 87, 107, 149
glutamate, 176, 184
food safety, 104
glutamic acid, 179
272 Index
glutamine, 147 hemoglobin, 65

glutathione, 56, 114, 115, 200, 217, 219, 220, 241 hepatocellular carcinoma, 172
glycerol, 116 hepatocytes, 220
glycine, 179 hepatotoxicity, 182
glycosaminoglycans, 218 herbal medicine, 207
glycoside, 109, 110, 112, 123, 142, 172, 176, 185, hesperetin, ix, 107, 108, 109, 112, 113, 114, 115,
187, 188, 192, 200, 201, 230 116, 120
glycosylation, 109, 117, 160 hexane, 134, 137, 139, 140, 169, 174, 177, 244, 245,
granules, xi, 145, 151 247
grape, viii, 1, 9, 10, 11, 12, 13, 14, 24, 25, 26, 27, 28, high blood pressure, 204
53, 54, 55, 56, 57, 58, 59, 61, 62, 64, 65, 66, 67, high fat, 179
69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 99 histamine, xi, 54, 157, 215, 220
grape pomace, viii, 9, 11, 14, 24, 25, 28, 54, 55, 56, histidine, 179
57, 65, 66, 67, 69, 70, 71, 72, 73, 74, 75, 76, 77, histone(s), 246
78, 79 history, 100, 212
grape seeds, viii, 54, 69, 77, 78 HIV, 170, 180
graph, 135, 136 HM, 77
GRAS, 196 homeostasis, 219
Greece, 109, 117, 212 Hong Kong, 192
growth, ix, xii, 5, 7, 9, 18, 19, 23, 59, 70, 74, 87, hormone(s), x, 108, 113, 114, 115, 214
108, 115, 124, 128, 134, 142, 146, 154, 155, 177, host, 234, 235
185, 197, 220, 225, 235, 236, 241, 243, 245, 246, House, 153
247, 248, 249, 250, 253, 256, 259, 263 human body, 221, 231
growth dynamics, 236 human health, viii, ix, 30, 65, 67, 69, 83, 107, 152,
growth factor, 115, 146 190, 206, 242
guidelines, 31, 224 human neutrophils, 220, 223
Guyana, 121, 122, 127, 141, 142 human subjects, 204, 236
humidity, 10, 24, 73, 245
hybrid, 171
H hydration, xi, 145, 212, 221
hydrocarbons, 20, 21, 192
hair, 192
hydrocortisone, 216, 236
hair cells, 192
hydrogen, x, 34, 35, 71, 75, 90, 96, 108, 114, 187,
hardness, 3, 4, 6, 21
190, 232
harvesting, 88, 256
hydrogen peroxide, x, 108, 114, 187, 190
healing, 125, 212, 213, 216, 217, 226
hydrolysis, 49, 91, 92, 112, 113, 120, 206, 232
health, vii, xi, xii, 2, 17, 53, 54, 62, 66, 70, 84, 91,
hydroperoxides, 56, 58, 60, 61, 124
101, 102, 105, 145, 148, 149, 152, 153, 157, 159,
hydrophobicity, 73
160, 163, 181, 186, 189, 190, 201, 204, 207, 212,
hydroxyl, 71, 75, 112, 161, 163, 173, 181, 185, 190,
225, 231, 241, 242, 243, 250, 251, 253, 259, 263
217, 250
health benefits, xi, 2, 17, 54, 62, 84, 145, 152, 157,
hydroxyl groups, 71, 112, 161, 163, 173
159, 186, 189, 242, 251, 263
hygiene, 60
health care, 105
hypercholesterolemia, 86, 191, 220
health condition, 201
hyperemia, 216
health effects, vii, xi, 91, 157, 160, 231
hyperglycemia, 218, 220, 224
health promotion, 153
hyperinsulinemia, 218
heart disease, 180, 219, 231, 259
hyperlipidemia, 181, 220
heat loss, 46
hypersensitivity, 217
heat transfer, 102
hypertension, ix, 108, 114, 125, 204, 207, 209, 219,
height, 191, 212, 233
222
Helicobacter pylori, 231
hypertrophy, x, 108, 114
heme, 57
hypotensive, 114, 123
hemicellulose, 40, 50, 87
hypothalamus, 198
hemisphere, 158
Index 273
hypothesis, 94 inhibitor, 22, 75, 186, 221

initiation, ix, 107, 114
injury(s), 77, 154, 184, 213, 216
I inoculum, 128
inositol, 146
ICAM, 114
insulation, 46, 47
ID, 182
insulin, 98, 115, 116, 168, 179, 180, 198, 201, 202,
ideal, 85
203, 204, 209, 213, 218, 224
identification, 70, 78, 124, 154, 155, 160, 177, 182,
insulin dependent diabetes, 218
202, 258
insulin resistance, 115, 168, 180, 202, 209, 213, 218
IL-8, 235
integrity, 222
illumination, 197
interphase, 76
immersion, 35
intervention, 198, 223
immune function, 218
intestine, 201
immune modulation, 235
intracellular calcium, x, 108, 115
immune response, 235
investment(s), 31, 76, 97
immune system, 220, 234
ion channels, 207
immunity, 78, 207, 213, 220, 226
ionization, 257
immunomodulator, 224
ions, 94, 148, 149
immunomodulatory, 235, 240
Iowa, 209
improvements, 93
Iran, 70
in vitro, xii, 49, 57, 60, 76, 77, 110, 113, 115, 116,
Ireland, 257, 258
119, 120, 122, 127, 128, 134, 143, 201, 203, 210,
iron, 18, 57, 179, 193
211, 213, 218, 219, 220, 222, 223, 227, 231, 234,
irradiation, 5, 6, 10, 13, 27, 111, 197
235, 236, 237, 239, 240, 245, 246, 252
irrigation, xiii, 255, 256, 257, 258, 259, 260, 261,
in vivo, ix, xii, 77, 107, 113, 114, 116, 119, 120, 211,
262, 263
213, 219, 221, 222, 227, 231, 235, 236, 240
ischemia, 187, 207
incidence, 114, 231, 235
Islam, 222
India, xii, 153, 190, 211, 212
isoflavonoids, 108
individuals, 213, 219, 235
isolation, 49, 91, 122, 124, 125, 143, 160, 163, 164,
induction, x, 57, 108, 114, 210, 220
171, 175, 177, 185, 223
induction period, 57
isomers, 176, 177
industrial processing, xii, 241
isoprene, 174, 176
industrial wastes, 31, 109
Israel, 262
industry(s), vii, viii, xi, 2, 7, 8, 14, 21, 28, 29, 30, 31,
issues, 30
32, 35, 46, 47, 49, 50, 54, 70, 71, 75, 76, 78, 82,
Italy, 1, 50, 53, 69, 70, 262, 263
91, 95, 104, 109, 113, 118, 122, 145, 181, 211,
212, 215, 222, 230, 243, 252
infants, 216 J
infection, 139, 234, 235, 239
inflammasome, 218, 222 Japan, 95, 158, 159, 190, 209, 212
inflammation, 197, 198, 199, 207, 210, 212, 213, joints, 216
214, 215, 216, 217, 231, 235
inflammatory disease, 179, 181
inflammatory responses, 197, 242 K
influenza, 231, 233, 234, 235, 239
K+, 221
influenza virus, 234, 239
kaempferol, 169, 170, 186, 194, 197, 200, 206, 232,
ingestion, 84, 97
247, 250, 251
ingredients, viii, 2, 3, 5, 14, 15, 16, 27, 54, 57, 74,
keratin, 217
81, 82, 85, 103, 148, 186, 204, 208, 228, 243
keratinocyte, 217
inhibition, ix, x, xi, xii, 18, 22, 27, 56, 57, 58, 60, 63,
ketones, 54, 192, 236, 240
74, 108, 115, 121, 122, 125, 128, 131, 134, 135,
kidney, xi, 157, 181, 187
136, 139, 140, 168, 179, 198, 200, 202, 203, 206,
kinetics, 149, 155, 186
210, 215, 218, 220, 233, 235, 236, 241
274 Index
Klebsiella pneumoniae, xi, 122, 130, 131, 134, 136, Luo, 142, 197, 198, 207, 208
139 lutein, 193, 242
knees, 216 lycopene, 96, 242, 253
Korea, 158, 159 lymphatic system, 113
kumquats, xii, 189, 190, 193, 194, 196, 197, 198, lymphocytes, 215
202, 203, 209 lysis, 246
L M
lactic acid, xi, 6, 7, 8, 12, 62, 145, 149, 150, 152, 155 machinery, 196
Lactobacillus, 62, 150, 154, 155, 156, 219, 224 macromolecules, 91
lactose, 150 macronutrients, 84
Latin America, 84 macrophages, ix, 107, 114, 115, 183, 206, 213, 215,
laxatives, 221 218, 220, 222
LC-MS, 164, 168 magnesium, 123, 146, 179, 193, 214, 236, 242
LDL, 124, 125, 142, 172, 180, 184, 185, 204 magnetic properties, 149
leaching, 94 magnitude, 93, 235
lead, 7, 31, 42, 113, 122, 152, 197, 216, 217, 221, Maillard reaction, 95
236 majority, 15, 30, 111, 194, 247, 250
leakage, x, 108, 115 malaria, 158, 185, 235
learning, 198 malignant cells, 201
leishmaniasis, 235 man, 117, 228
lesions, 115, 176, 236 management, x, xii, 71, 108, 115, 202, 204, 205,
life quality, 30 207, 211, 213, 224, 225, 258, 262
ligand, 203 Mandarin, 118, 192
light, 9, 57, 59, 61, 72, 74, 129, 179, 196, 204, 230 manganese, 179, 214
lignans, 71, 160, 171, 172, 188, 190 manipulation, 4, 72
lignin, 40, 50, 70, 87, 185, 213 manufacturing, 35, 39, 47, 102
linoleic acid, xiii, 72, 232, 242, 255, 258, 259, 260, marine fish, 54
261 Mars, 141, 142
linolenic acid, xiii, 54, 255, 259, 260, 261 Maryland, 104, 221, 225
lipid metabolism, 155, 207 mass, 34, 40, 85, 91, 92, 95, 96, 109, 116, 117, 156,
lipid oxidation, 2, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 163, 176, 177, 213, 219, 224
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, mass loss, 40
55, 56, 57, 58, 59, 60, 61, 63, 64, 65, 66, 67, 71 mass spectrometry, 117, 163
lipid peroxidation, 64, 114, 168, 170, 176, 180, 202, mast cells, 220, 225
208, 233, 243 materials, viii, 30, 31, 32, 35, 36, 40, 45, 46, 48, 71,
lipid peroxides, 57 72, 73, 81, 82, 91, 94, 95, 99, 102, 110, 112, 113,
lipids, 21, 23, 56, 63, 66, 83, 86, 147, 168, 179, 180, 127, 160
219, 256, 257, 263 matrix, 49, 72, 104, 110, 112, 149, 150, 171, 174,
lipolysis, 116 240
lipophilic compounds, viii, 69 matrixes, 187
liquid chromatography, 116, 117, 161, 163, 175, 183, matter, ix, 35, 40, 41, 72, 75, 82, 88, 93
252 Mauritius, 189, 190, 211, 212, 215, 224
liquid phase, 45 MCP, 114
liquids, 72, 100 MCP-1, 114
Listeria monocytogenes, 9, 18, 26, 60, 197 measurement(s), 12, 20, 59, 61, 94, 102, 225
liver, 10, 12, 27, 124, 154, 168, 178, 179, 180, 181, meat, vii, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
191, 201, 203, 209, 220, 242 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
low-density lipoprotein, 206 28, 54, 91, 96
LTD, 237 mechanical properties, 46, 58
lumen, 84, 221 media, 30, 153, 187
lung cancer, 77 medical, xi, 189, 191, 219, 221, 225
Index 275
medication, 234, 236 model system, 11, 26

medicine, 17, 122, 125, 148, 158, 171, 181, 187, modelling, 49
191, 211, 212, 215, 219, 228, 237 models, 55, 61, 62, 198, 213, 226, 235
Mediterranean, vii, 20, 25, 29, 30, 31, 47, 48, 60, 83, moderate activity, 74, 247
256 modifications, 87, 108
Mediterranean countries, 30, 47, 256 moisture, 3, 4, 7, 8, 10, 14, 16, 17, 32, 34, 50, 86, 93,
melanin, 143 221
melanoma, 172, 176, 178 moisture content, 3, 10, 16, 34
mellitus, 85, 213, 218, 224 mold, 6, 7, 34
membrane permeability, 94 molecular structure, 87, 99
membranes, 23, 94, 96, 99, 231, 235 molecular weight, 75, 95, 171, 173, 212
memory, 188, 198 molecules, vii, viii, 53, 76, 96, 102, 111, 163, 184,
mesocarp, viii, 81, 82, 85, 86, 179, 191 197, 198, 200, 202, 231
meta-analysis, 253 monocyte chemoattractant protein, x, 108, 114
Metabolic, 223, 224, 255 monomers, 56, 89
metabolic disorder(s), 209 monoterpenoids, 174
metabolic disturbances, 202 monounsaturated fatty acids, xiii, 147, 255, 259, 260,
metabolic pathways, 214, 215 261
metabolism, ix, 2, 98, 100, 107, 115, 124, 146, 203, Moon, 184
214, 220, 223 Moringa oleifera, x, xi, 121, 122, 123, 127, 128, 129,
metabolites, ix, xii, 71, 96, 99, 107, 111, 112, 114, 130, 131, 134, 135, 136, 139, 140, 142
118, 174, 183, 198, 227, 230, 231, 251, 256 morphology, 222
metabolized, 112 mortality, 99, 102, 224
metabolizing, 205, 219 moulding, 32
metal ion(s), 149, 202 MR, 186, 253
metals, 58 mRNA, x, 108, 114, 116, 197, 201, 203, 207
meter, 34 mucosa, 221
methanol, 23, 75, 110, 111, 112, 125, 160, 163, 168, mucus, 234
171, 172, 174, 177, 178, 230, 243, 244, 247, 257 multivariate analysis, 260
methodology, 33, 76, 92, 101, 102 muscles, 15, 16, 57, 58, 61
methyl group(s), 89, 174 mutagenesis, 200
Mexico, 83, 212 mutation, 190
Miami, 208 mycotoxins, ix, 107
mice, x, 108, 114, 168, 179, 183, 184, 186, 203, 205, myocardial infarction, 152
207, 209, 219, 220, 222, 224, 235 myocardial ischemia, 184
microbial communities, 154 myocardium, 179
micronutrients, 196, 242 myoglobin, 8
microorganism(s), 14, 54, 74, 75, 96, 100, 112, 122,
128, 134, 141, 150, 151, 155, 156
microsatellites, 187 N
microscope, 35
Na+, 201, 204, 207, 221
microstructure(s), vii, 29, 32, 35, 41, 45, 47
Na2SO4, 127
microwave heating, 83, 99, 101
NaCl, 6, 9, 10, 245
microwaves, 111, 112
nanoparticles, 236, 240
middle lamella, 87
National Academy of Sciences, 208
migration, 61
national product, 30
Ministry of Education, 182, 251
National Research Council, 1, 53
mitochondria, 207
natural compound, 12, 64, 72, 73
mitogen, 179
natural food, vii, viii, 1, 2, 27, 53, 64, 65, 101, 197,
mixing, vii, 29, 32, 34, 41, 42, 43, 44, 45, 46, 47, 50
202
MMP, 218, 226
natural resources, 30
MMP-3, 218
necrosis, xiii, 220, 235, 241, 243, 246, 247
MMP-9, 226
negative effects, 7, 31
276 Index
neolignans, 171, 172 olive oil, vii, viii, 29, 30, 31, 32, 34, 35, 36, 41, 47,
nephropathy, 176, 218 48, 49, 50, 61, 66, 171, 256, 257, 259, 260, 262,
neuralgia, 124 263
neuroblastoma, 176, 187, 199 olive oil wastewater, vii, 29, 32, 35, 36, 41
neurodegeneration, 197, 210 olive wastewater, vii, 29, 32, 41
neurodegenerative diseases, 190, 195, 197, 198, 199, operations, 93
207, 209 opportunities, 4, 118
neuroinflammation, x, 108, 115, 198, 199, 208 organ(s), 179, 198
neurons, 77, 209 organic compounds, 108, 160, 174, 176
neuropathy, 218 organic matter, vii, 30, 35, 39, 40, 41, 42, 47, 50, 257
neuroprotection, 181, 198, 199, 210 organic soils, 50
neurotoxicity, 77, 176, 209 organic solvents, viii, 69, 72, 78
neutral, ix, 35, 81, 87, 89, 244 organism, 128, 236
New Zealand, 145 ornithine, 200
niacin, 193 osmotic pressure, 148
nickel, 149 overproduction, 213, 218
nicotinamide, 114 overweight, 202, 206
Nigeria, 222 ox, 4
nitrates, 5 oxalate, 123
nitric oxide, 114, 125, 197, 204, 206 oxidation, 5, 6, 7, 9, 10, 12, 13, 14, 16, 17, 20, 21,
nitric oxide synthase, 197, 204 22, 23, 25, 54, 56, 57, 58, 59, 61, 63, 64, 66, 76,
nitrite, 5, 6, 7, 8, 21, 28 124, 142, 172, 180, 184, 185, 190, 203, 232
nitrogen, 8, 34, 61, 62, 218, 257 oxidation products, 12, 20, 56, 57, 58
nitrosamines, 5 oxidative damage, 197, 198, 231, 243
NMR, 123, 161, 163, 164, 170, 171, 173, 174, 177 oxidative reaction, xii, 241
nodules, 217 oxidative spoilage, vii, 1, 53
non-communicable diseases, ix, 108, 114 oxidative stress, x, 108, 115, 176, 185, 186, 187,
non-insulin dependent diabetes, xii, 211 197, 198, 199, 200, 202, 208, 209, 210, 231, 236,
non-polar, 163, 244 240, 242
normal development, 179 oxygen, 8, 13, 27, 34, 40, 70, 71, 108, 174, 190, 206,
North Africa, xii, 83, 211 207, 232, 243
North America, xi, 83, 153, 157, 158, 159, 160, 228 oysters, 96
Norway, 234
nuclear magnetic resonance, 161, 163
nutraceutical, xii, 22, 64, 116, 118, 227, 233, 241 P
nutrients, xi, 93, 95, 145, 190, 193, 205, 259
Pacific, 113, 153, 207
nutrition, 54, 77, 97, 145, 189, 204, 209
pain, 148, 181, 213, 215, 217
nutritional status, 141
Pakistan, 24, 152, 155, 190, 225
Panama, 233, 239
O pancreas, 215, 219
parallel, 21
obesity, x, 60, 108, 113, 115, 168, 178, 180, 181, parasite(s), ix, 107, 113, 185, 235
184, 190, 195, 197, 202, 205, 206, 218, 219, 224 partition, 11, 23, 163, 169, 173, 174, 177
oedema, x, 108 Passiflora edulis Sims f. flavicarpa Degener, viii, 81
OH, 74, 75 Passiflora L., viii, 81, 82
oil, vii, 4, 7, 8, 11, 28, 29, 30, 31, 32, 35, 39, 47, 48, Passifloraceae, viii, 81, 82, 83
49, 50, 59, 60, 61, 63, 64, 66, 72, 76, 77, 86, 104, passion fruit, viii, 81, 82, 83, 84, 85, 86, 87, 88, 91,
113, 119, 194, 196, 228, 237, 256, 258, 262, 263 92, 93, 97, 98, 101, 102, 103, 104, 105
oil production, vii, 29, 256, 262 passion fruit pomace, viii, 81, 83, 86, 88, 91, 92
oil samples, 64 pathogenesis, 190, 198, 202, 216
oleic acid, xiii, 72, 75, 255, 258, 260, 261 pathogens, x, xii, 5, 9, 10, 14, 60, 85, 87, 121, 124,
oligomers, 55, 56, 57, 198, 242 128, 131, 137, 139, 140, 196, 227
pathology, 201
Index 277
pathways, 179, 200 plants, ix, x, xi, xii, 21, 48, 64, 71, 81, 85, 86, 87, 88,
PCA, 258, 259, 260 89, 98, 102, 107, 109, 112, 113, 116, 119, 120,
PCR, 150, 151, 154, 155 121, 124, 129, 141, 142, 148, 157, 158, 160, 161,
peat, 50 162, 171, 173, 174, 176, 181, 183, 211, 212, 225,
pectin, ix, 2, 4, 81, 82, 83, 85, 88, 89, 90, 91, 92, 93, 227, 231, 235, 237, 239, 257
98, 99, 100, 101, 102, 103, 104, 105, 110, 163, plaque, 216, 223
228 plasma membrane, 207, 235
penicillin, 122, 245 plasma proteins, 3, 25
peptide(s), 148, 154, 198, 209 plasticity, vii, 29, 32, 34, 36
periodontal, 152 platinum, 95
peripheral blood, 226 PM, 262
peripheral blood mononuclear cell, 226 pneumonia, xi, 122, 128
permeability, 93, 94, 96, 101, 210, 221 Poland, 156, 243
permeation, 91 polar, 19, 73, 76, 111, 163, 198, 244
permittivity, 94 polarity, x, 73, 75, 110, 111, 112, 121, 127, 134, 163
peroxidation, 187 pollination, 158
peroxide, 12, 56, 57, 181, 231 pollutants, 118, 231
peroxynitrite, 198, 207 pollution, 30, 35, 48
Peru, 83 polycyclic aromatic hydrocarbon, 118
petroleum, 77 polymer(s), 71, 88, 89, 98, 104, 163, 226
pH, 3, 4, 6, 7, 8, 10, 11, 14, 17, 22, 34, 35, 57, 61, polymerase, 148
62, 83, 88, 90, 91, 92, 100, 148, 149, 150, 162, polymerization, 56, 57
163, 231, 238, 244, 257 polyphenols, 7, 11, 19, 24, 35, 56, 57, 58, 63, 65, 66,
pharmaceutical, ix, xii, 71, 74, 76, 82, 91, 109, 113, 70, 71, 73, 76, 77, 78, 84, 118, 160, 163, 169,
158, 174, 181, 187, 211, 215, 219, 222, 227, 230, 181, 198, 199, 206, 229, 230, 233, 235, 237, 238,
243 240, 242
pharmacological research, 174 polysaccharide(s), ix, 81, 87, 95, 104, 120, 149, 150,
pharmacology, 77 151, 179, 213, 218, 220, 223, 226
pharmacotherapy, 225 Polysaccharides, 217
phenol, 65, 76, 185, 188 polyunsaturated fat, 53, 76, 147, 258, 260, 261, 262
phenolic compounds, vii, viii, ix, xi, xii, 1, 7, 14, 19, polyunsaturated fatty acids, 53, 76, 147, 258, 260,
22, 23, 53, 54, 56, 65, 76, 85, 86, 107, 109, 110, 261, 262
112, 116, 119, 124, 125, 157, 158, 163, 170, 173, pomace extracts, vii, viii, xii, 1, 2, 11, 14, 53, 54, 57,
180, 210, 211, 214, 231, 232, 242, 244, 252 74, 75, 78, 241, 246, 247, 248, 250, 252
phenotype, 206 pomegranate, viii, 1, 17, 18, 19, 24, 25, 26, 27, 28,
phosphate(s), 15, 16, 26, 40, 114 53, 54, 60, 61, 62, 64, 65, 67
phosphorous, 146 ponds, 31, 47
phosphorus, 179, 193 pools, 48
physical and mechanical properties, 59 population, 84, 156, 182, 216
physical phenomena, 111 porosity, vii, 30, 34, 40, 42, 43, 44, 45, 46, 47
physical properties, 50, 51, 62, 67 Portugal, 237
physicochemical characteristics, ix, 82, 83, 91, 93 positive correlation, 234
physicochemical properties, 24, 27, 83, 155, 160 positive interactions, 75
Physiological, 209 potassium, 4, 8, 34, 40, 146, 148, 179, 193, 204, 214,
physiology, 104 242
phytochemicals, xii, 54, 71, 123, 143, 189, 190, 193, potato, 3, 25, 102, 104, 209
195, 197, 198, 199, 200, 202, 204, 206, 211, 231, poultry, 12, 14, 15, 22, 26, 27
239, 241, 242, 253 pregnancy, 148
phytosterols, 219, 220, 224 preparation, 58, 85, 149, 209, 229, 234, 240
placebo, 86, 206, 216, 219, 225, 233, 234 preservation, 23, 56, 65, 98, 100, 105
placenta, 148 preservative, vii, 1, 5, 6, 9, 10, 11, 12, 18, 61, 197
plant growth, ix, 92, 107 prevention, ix, 66, 73, 84, 107, 112, 115, 116, 153,
plant phenolics, ix, 107, 110, 116 176, 187, 202, 242, 250, 253
278 Index
principal component analysis, 258, 261 radicals, 108, 180, 181, 185, 217, 236, 250
principles, xii, 31, 118, 223, 227, 233, 256 rainfall, 257
probe, 35 ramp, 34, 258
probiotic(s), 85, 103, 149, 151, 152, 153, 155, 156 rancid, 9, 11, 57
process innovation, 119 rash, 216
procyanidin B1, viii, 69 raspberry, xii, 63, 64, 65, 234, 236, 238, 240, 241,
producers, 30, 70, 76, 229, 243 243, 247, 250, 251, 252, 253
profitability, 2 raw materials, viii, 30, 32, 36, 50, 51, 69, 86
pro-inflammatory, 184, 197, 202, 213, 218 RCD, 51
project, 182, 251 RE, 184, 186, 187
proliferation, xi, xii, 31, 114, 157, 168, 176, 180, reaction time, 64
187, 200, 201, 204, 207, 218, 241 reactions, 40, 41, 54, 215, 225, 232, 234
promoter, ix, 107, 114, 200 reactive oxygen, 148, 201, 220, 236
propagation, 111, 216, 235 reactivity, 7, 204
prophylactic, xii, 189, 190, 191, 194, 195, 200, 204, reading, 219
205, 208 receptors, 202, 203, 234
prophylaxis, 189 recommendations, 204
prosperity, 30 recovery, 49, 70, 71, 76, 99, 109, 110, 113, 115, 118
prostate cancer, 177, 184, 242 recycling, 47
protection, xii, 10, 12, 14, 17, 19, 60, 66, 77, 176, red wine, 54, 70, 233, 242, 252
180, 196, 198, 199, 200, 204, 210, 212, 227, 228, regression, 220
231, 235, 243 regulations, 76
protein oxidation, 18, 19, 20, 22, 26, 206 rehydration, 148
proteinase, 3 relaxation, 219
proteins, 3, 55, 57, 59, 63, 72, 83, 84, 95, 98, 100, relevance, 256, 259
110, 115, 124, 146, 148, 163, 198, 244 relief, 214, 217
proteolytic enzyme, 70 repair, 213, 218
Pseudomonas aeruginosa, 74 replication, 235, 239
psoriasis, 213, 216, 223, 224, 236 requirements, 31, 35, 48
psoriatic arthritis, 216, 224 researchers, viii, 2, 30, 31, 73, 81, 88, 89, 96, 114,
pulp, viii, 3, 4, 17, 19, 28, 54, 81, 82, 84, 86, 88, 91, 174, 196
99, 104, 105, 149, 155, 177, 190, 191, 193, 196, residues, viii, 2, 14, 28, 51, 64, 71, 81, 82, 87, 89,
200, 205, 213, 219, 221 109, 110, 112, 119
purification, 2, 83, 91, 109, 163, 169, 174, 177, 185, resins, 163
263 resistance, 83, 93, 94, 95, 122, 141, 152, 204, 219
purity, 11, 92, 105 resolution, 35
PVC, 11 resources, 30, 31, 186, 243
pyridoxine, 193 response, x, 71, 75, 92, 101, 102, 108, 114, 117, 235,
243
resveratrol, viii, 69, 71, 76, 78, 171, 210
Q retail, 96
retention rate, 204
quality of life, 213, 218
reticulum, 235
quantification, 164
retinopathy, 218
quartz, 36, 39
reverse transcriptase, 174
quercetin, 21, 22, 23, 76, 123, 169, 194, 197, 200,
RH, 143, 183
203, 204, 205, 206, 210, 230, 232, 242, 247, 250,
riboflavin, 193
251
ribosome, 237
rice husk, 50
R rings, 108, 160
risk, viii, 5, 81, 84, 99, 146, 148, 168, 197, 202, 206,
race, 234 208, 219, 242
radiation, xii, 212, 216, 220, 222, 227 RNA, 146, 148, 184
Index 279
Romania, 157, 182, 227 shortness of breath, 104

room temperature, 32, 34, 39, 163, 230, 243, 244 showing, xii, 30, 47, 57, 64, 84, 112, 168, 178, 181,
root(s), 60, 123, 124, 125, 142, 228 227, 260
routes, 215 shrubs, 158
Royal Society, 141 Siberia, 159
rubbers, 174 side chain, 89, 104, 171
side effects, xi, 122, 145, 202, 220
signal transduction, 200
S signaling pathway, 200, 202, 203, 205
signalling, 207, 242
S.aureus, x, 121, 122, 130, 131, 134, 135, 139
signals, 215, 236
safety, vii, viii, 1, 2, 7, 20, 53, 54, 65, 66, 97, 105,
signs, 213
234
silver, 57, 65, 217, 225, 236, 240
Salmonella, 9, 18
sintering, 41
salts, 8, 40, 236
SiO2, 36, 37
Sartorius, 245
skeletal muscle, 201
saturated fat, 84, 147, 178, 258, 259, 260, 261
skeleton, 160, 171, 174
saturated fatty acids, 147, 258, 259, 260, 261
skin, xii, 10, 12, 22, 84, 125, 127, 200, 211, 212,
savings, 112
213, 214, 216, 217, 221, 222, 223, 226, 228, 236,
scabies, 212, 224
242
scanning electron microscopy, 93
skin diseases, 228
scavengers, 231
Slovakia, 182
science, 119
sludge, 49
scientific publications, ix, 82, 83, 91, 93
small intestine, 82, 91, 115, 203
SDS-PAGE, 61
SNP, 114
sea level, 257
society, 211
seafood, 58, 65, 66
sodium, 8, 10, 14, 15, 21, 28, 114, 148, 179, 193,
seborrheic dermatitis, 216, 225
214, 257
sebum, 217
Solanum melongena, x, xi, 121, 122, 124, 127, 130,
secondary metabolism, 160
134, 142
secretion, x, 108, 115, 184, 198, 202, 204, 215, 219,
solid phase, 163, 244
221, 235
solid state, 110
sedentary lifestyle, 218
solid waste, 31, 48, 49, 50, 54, 252
sedimentation, 123
solidification, 40
seed, 8, 9, 10, 11, 12, 13, 14, 17, 18, 24, 25, 26, 27,
solubility, 10, 58, 73, 78, 110, 112
55, 59, 60, 61, 62, 63, 64, 65, 67, 70, 71, 72, 74,
solution, 19, 28, 31, 32, 47, 48, 56, 63, 91, 103, 127,
77, 99, 104, 117, 123, 160, 179, 183, 200, 242
149, 171, 220, 222, 234, 236, 243, 244, 245, 257
seeding, 245
solvents, x, 23, 72, 73, 110, 111, 121, 127, 163, 169,
selectivity, viii, 69, 94, 110, 122, 139, 141
174, 230, 244, 245
selenium, 193, 214
South Africa, 70, 155, 190
SEM micrographs, 42, 44, 45, 47
South America, 113, 121
sensation, 258
South Asia, 190
sensitivity, 18, 114, 256
South Korea, 190
sepsis, 213, 218
soybeans, 112
sequencing, 151
soymilk, 154
Serbia, 182, 241, 243, 244, 245, 251, 252
SP, 107, 188
serine, 179
Spain, 29, 30, 31, 32, 70
serum, 91, 182, 203, 204, 219, 245
specifications, 87
services, 30
spectral techniques, 123
shape, viii, 45, 81, 95, 159, 191
spectroscopic techniques, 123
shear, 16, 24, 111
spectroscopy, 173
shelf life, vii, viii, 2, 5, 6, 8, 9, 10, 13, 18, 19, 24, 26,
sponge, 236
28, 53, 54, 57, 61, 62, 63, 64, 65
shock, 176, 181
280 Index
stability, 3, 4, 6, 7, 8, 9, 10, 12, 13, 17, 19, 20, 21, synaptic plasticity, 198
24, 26, 28, 58, 60, 61, 64, 66, 149, 185, 231, 243, synergistic effect, 196
262 synthesis, ix, 107, 115, 116, 194, 240
stabilization, 2, 3 systolic blood pressure, 204, 206, 219
Staphyloccocus aureus, xi, 122
starch, 3, 25, 88, 98
state(s), x, 32, 4, 44, 101, 108, 110, 112, 127, 158, T
190
Taiwan, 206, 208, 210
steel, 95, 109
tannins, 4, 17, 19, 110, 124, 160, 173, 181, 183, 185,
sterile, 128
190, 241
steroids, 124, 177, 214
target, 73, 112, 202, 203, 218, 224
sterols, 185
techniques, 32, 41, 65, 76, 110, 112, 141, 150, 163,
stimulant, xii, 124, 215, 227
169, 170, 177, 223, 230, 232, 233, 256
stimulation, 116, 212, 215, 220, 235
technology(s), viii, ix, 2, 31, 50, 69, 73, 82, 83, 89,
stock, 245
93, 94, 95, 96, 97, 103, 104, 105, 110, 111, 112,
stomach, 176, 228, 242
116, 118
stomata, 212
temperature, viii, ix, 3, 34, 39, 40, 50, 51, 59, 61, 69,
storage, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19,
72, 82, 83, 90, 91, 92, 94, 95, 97, 101, 104, 110,
20, 21, 22, 24, 25, 26, 28, 31, 48, 54, 55, 56, 57,
185, 198, 230, 231, 232, 243, 244, 257, 258
58, 59, 61, 62, 65, 66, 67, 74, 77, 87, 88, 99, 148,
tension(s), 40, 204
150, 256
terpenes, 174
Streptozotocin, x, 108, 115
textbook(s), 211, 225
stress, x, 58, 75, 108, 197, 198, 202, 221, 256
textural character, 58
stroke, 155, 199, 208
texture, 3, 4, 6, 7, 13, 15, 17, 20, 21, 25, 59, 60, 62,
structural changes, 54
86, 87, 97
structure, ix, 26, 62, 75, 81, 83, 87, 88, 89, 90, 96,
TGA, 34, 39, 40
100, 102, 110, 123, 125, 150, 154, 160, 161, 163,
Thailand, 226, 235
168, 173, 179, 194, 195, 199, 204, 221, 223, 229,
therapeutic agents, xii, 209, 227
237
therapeutic effects, xii, 181, 212, 227
style, 5, 7, 152
therapeutic use, xii, 181, 211
styrene, 109, 194
therapy, 113, 176, 181, 201, 216, 250
subgroups, 108, 160
thermal analysis, 34, 50, 99
substitutes, 196
thermal conductivity, vii, 29, 35, 46, 47, 51
substitution, 43, 47
thermal decomposition, 40
substrate, 112, 246
thermal degradation, ix, 40, 82, 95
sucrose, 4, 148
thermal properties, 30
sugar beet, 101, 104, 105
thermal resistance, 149
sulfur, 34
thermal stability, 22
sulfuric acid, 92
thiamin, 193
sulphur, 218
thickening agents, 2
Sun, 117, 119, 141, 185, 187, 224
threonine, 179
supercritical fluids, viii, 69, 72, 73, 77, 78
thymus, 215
supplementation, ix, 85, 107, 114, 198, 206
TIMP, 218
suppression, 198, 199, 210, 217
tissue, 20, 21, 24, 82, 87, 91, 93, 94, 101, 102, 110,
surface properties, 105
114, 218, 250
survival, 150, 188, 198, 204, 256
tissue engineering, 82, 91
susceptibility, xi, 23, 111, 122, 128, 139
TNF, 115, 116
suspensions, 105
TNF-α, 115, 116
sustainability, 30
tobacco, 105, 237
sustainable development, 31
tocopherols, 1, 64, 72, 242
swelling, 59, 215
tonic, xi, 157, 158, 180, 191
Switzerland, 207, 245
total cholesterol, 21, 203
symptoms, 86, 216, 234, 239
toxic effect, 114
Index 281
toxicity, viii, 23, 69, 153, 172, 184, 187, 198 vapor, 72
traditions, 228 variables, 117, 219, 260
traits, 10 variations, 86, 154, 180, 252, 256, 259
transcription, 115, 116, 197, 200, 201, 203, 220 varieties, 14, 21, 28, 74, 76, 77, 83, 148, 196, 259
transcription factors, 116, 200, 203 vascular system, 114
transducer, 95, 197 vasodilation, x, 108, 114
transformation, 39, 186 vegetables, 24, 84, 87, 91, 95, 96, 98, 99, 100, 108,
transforming growth factor, 115, 187 116, 117, 122, 124, 141, 143, 190, 231, 238
transition metal, 108 vein, 114, 184
transmission, 35 Venezuela, 212
transport, 146 vessels, x, 108, 115
trial, 35, 58, 206, 216, 218, 223, 225, 226 viral infection, 228
trifluoroacetic acid, 163 virus infection, 234
triggers, 242 virus replication, ix, 108
trypsin, 245 viruses, 181, 218, 220, 225, 234, 235, 239
tumor(s), xi, xii, 78, 113, 114, 151, 157, 168, 176, viscosity, 45, 85
177, 178, 180, 187, 190, 195, 200, 201, 220, 241, vitamin A, 83, 123, 193, 214
243, 246, 247, 248, 249, 250, 251 vitamin B6, 123
tumor cells, xii, 177, 178, 180, 201, 241 vitamin C, 1, 4, 19, 83, 99, 103, 123, 146, 159, 179,
tumor necrosis factor, 220 193, 202, 208, 242, 244
tumorigenesis, 213, 220 Vitamin C, 83, 146, 193
tumours, 220 vitamin D, 215
turbulence, 111 vitamin E, 12, 56, 181
Turkey, 14, 15, 67, 70, 153, 156, 159, 186, 239 vitamins, vii, viii, 1, 53, 73, 83, 93, 95, 148, 190,
type 2 diabetes, 84, 209 193, 212, 213, 214, 215, 228, 231, 233, 241, 242,
tyramine, 63, 124 251
Tyrosine, 147
W
U
Washington, 98, 100, 263
Ukraine, 159 waste, vii, viii, xii, 2, 29, 30, 31, 32, 34, 35, 39, 40,
ulcer, 212, 213, 226 41, 42, 43, 44, 47, 48, 49, 50, 69, 70, 76, 82, 85,
ultrasound, 96, 98, 105, 109, 110, 111, 112, 118, 103, 109, 241, 244, 252, 253, 262
119, 174, 177 waste management, 30, 76
United, 70, 84, 96, 118, 152, 159, 187, 208, 212, 239 waste water, 48, 49, 109, 262
United States, 70, 84, 96, 118, 152, 159, 187, 208, wastewater, vii, 29, 31, 32, 34, 35, 36, 39, 41, 42, 47,
212, 239 48, 49, 50, 262
urban, 49 water absorption, vii, 29, 42, 45, 47, 221
urea, 218 water purification, 123
urokinase, 221 water supplies, 256
USA, 24, 159, 185, 205, 209, 243, 245, 246, 257 water vapor, 40
USDA, 146, 152, 193, 205, 209 weight loss, 39, 40, 41, 85
uterine bleeding, xi, 157 wellness, xii, 211, 222
UV, 75, 160, 163, 164, 174, 177, 197, 213, 216, 225 wells, 245
UV radiation, 75 wheeze, 104
UVB irradiation, 236 wheezing, 86
wildlife, 159
wine making process, viii, 69
V wine production, viii, 69, 70, 71
workers, 168, 169, 171, 173, 177
vacuum, 8, 12, 13, 14, 15, 16, 27, 28, 63, 234, 244,
World Health Organization (WHO), 202, 210
245
World War I, 159
valorization, viii, 31, 47, 48, 81, 82
worldwide, vii, viii, 1, 53, 69, 76, 109, 201, 218
282 Index
worms, ix, 108, 113 yield, ix, 3, 4, 10, 12, 16, 17, 24, 32, 55, 59, 74, 75,
wound healing, 82, 91, 214, 218, 222 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
yeast, xi, 6, 7, 14, 145, 149, 150, 151

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FOOD AND BEVERAGE CONSUMPTION AND HEALTH

Additional books in this series can be found on Nova‘s website

Additional e-books in this series can be found on Nova‘s website

FRUIT AND POMACE EXTRACTS

NOTICE TO THE READER

Library of Congress Cataloging-in-Publication Data

Published by Nova Science Publishers, Inc. † New York

Chapter 9 Extraction, Characterization and Potential Health Benefits

The use of natural or naturally-derived antioxidants, instead of synthetic antioxidants, to

FRUIT AND POMACE EXTRACTS:

P. G. Peiretti and F. Gai

Meat product Type of ingredient Impact on product Reference

Meat product Type of ingredient Impact on product Reference

Meat product Type of ingredient Impact on product Reference

Meat product Type of ingredient Impact on product Reference

Meat product Type of ingredient Impact on product Reference

Meat product Type of ingredient Impact on product Reference

OTHER FRUITS AND BERRIES

Meat product Type of ingredient Impact on product Reference

Because of the susceptibility of muscle membrane lipids to oxidation, the ability of

Alesón-Carbonell, L., Fernández-López, J., Pérez-Alvarez, J.A. and Kuri, V. (2005).

González-Aguilar, G., Robles-Sánchez, R.M., Martínez-Téllez, M.A., Olivas, G.I., Alvarez-

VALORIZATION OF LIQUID EFFLUENTS FROM OLIVE

D. Eliche-Quesada*1,2 and F. A. Corpas-Iglesias2

process; x=E: extrusion or x=C: compression. In Figure 1 can be observed a scheme a

2.2. Characterisation of Brick Raw Materials

2.3. Characterisation of the Bricks

3. RESULTS AND DISCUSSION

Table 1. Physico-chemical characterization of the used olive oil wastewater

Figure 2. XRD patterns of clay.

Table 3. Chemical composition of the fired clay

3.2. Characterization of the Bricks

Sample Linear Loss on ignition Suction water

FRUIT AND POMACE EXTRACTS: APPLICATIONS

F. Gai* and P. G. Peiretti

Fish Type of Impact on

Pazos et al. (2010) evaluated the influence of polymerization and galloylation of

Fish Type of Impact on

OTHER FRUITS AND BERRIES

Fish Scientific Impact on

quality decay of packed fish hamburger. International Journal of Food Microbiology,

SUPERCRITICAL FLUID EXTRACTION

Cleofe Palocci and Laura Chronopoulou

ECONOMIC IMPORTANCE AND POTENTIAL OF WASTE

GRAPE POMACE COMPOSITION

GENERAL ASPECTS OF SUPERCRITICAL FLUID EXTRACTION

solubility. Viceversa, as temperature increases, density decreases, lowering solubility. Other

Figure 1. Schematic diagram of the main elements of a SF extractor.

BIOLOGICAL ACTIVITY OF SUPERCRITICAL FLUID EXTRACTS FORM

[7] Sánchez-Alonso, I; Jim nez-Escrig, A; Saura-Calixto, F; Borderías, AJ. Antioxidant

[23] Alvarez, E; Rodino-Janeiro, BK; Jerez, M; Ucieda-Somoza, R; Nunez, MJ; Gonzalez-

[39] Da Porto, C; Decorti, D; Natolino, A. Water and ethanol as co-solvent in supercritical

PASSION FRUIT POMACE POWDER: POTENTIAL

Cibele Freitas de Oliveira1* and Poliana Deyse Gurak2†

The composition and physicochemical properties of pectin depend on source material,

2. DEFINITIONS AND CHARACTERISTICS OF PASSION FRUIT

2.2. Passion Fruit Peel

Table 1. Studies with passion fruit peel

Autors Objective Results

Autors Objective Results

Table 2. Nutrient content in dried peel of yellow passion fruit

Components (%) Passionfruit Passionfruit peel2 Passionfruit