Polyphenols: Properties, Occurrence, Content in Food and Potential Effects HUSSEIN I. ABDEL-SHAFY * AND MONA S.M. MANSOUR

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232 Environ. Sci. & Engg. Vol. 6: Toxicology

11

Polyphenols: Properties, Occurrence, Content
in Food and Potential Effects

HUSSEIN I. ABDEL-SHAFY1* AND MONA S.M. MANSOUR2

ABSTRACT
Recently, polyphenol compounds occupy a unique place in environmental
science as important class of bioactive natural products worldwide. The
most abundant polyphenols are the condensed tannins. Polyphenols found
almost in all families of plants and are concentrated in leaf tissue, the
epidermis, bark layers, flowers and fruits. The biological properties of
polyphenols include anticancer, antioxidant and anti-inflammatory effects.
Polyphenols posses anti-microbial and anti-cariogenic properties and is an
important source as anti-infective agents against antibiotic-resistant human
pathogens. As antioxidants, polyphenols are the most abundant in Man diet.
Dietary intake requirement is 1 g/d that can be achieved by consuming a
wide array of plant foods. Cereals, vegetables, dry legumes and chocolate
contribute to the intake of polyphenols. Olive mill wastewater (OMW) contains
phenolic compounds. Their recovery is an advantage for several industrial
sectors, including medicine, cosmetic, and food preservation. Therefore, OMW
could represent an important alternative source of biologically active
polyphenols.
Key words: Polyphenols, Potential health effects, Antioxidants, Bioactivity

of polyphenols, Olive mill wastewater, Recovery of polyphenols

1Water Research & Pollution Control Department, National Research Centre, Dokki,
Cairo, Egypt.

2Analysis & Evaluation Department, Egyptian Petroleum Research Institute, No. 1 Ahmed
El-Zomor Street, Nasr City, Cairo, Egypt.

* Corresponding author: E-mail: [email protected]

Polyphenols: Properties, Occurrence, Content in Food and Potential Effects 233

1. INTRODUCTION

Polyphenols is known as Polyhydroxyphenols. They are not only a structural
class of mainly natural, but also synthetic or semi synthetic organic chemicals
that are characterized by the presence of large multiples of phenol structural
units. The characteristics of these phenol structures underlie the unique
physical, chemical, and biological properties of particular members of this
class (i.e., metabolic, toxic, therapeutic, etc.). Examples of these phenol
structures include tannic acid and ellagitannin. Therefore, the chemical
class of tannins is an important subset of the polyphenols (Quideau et al.,
2011).

The name “poly” derives from the ancient Greek word ðïëýò (polus,
meaning “many, much”). The word phenol refers to a chemical structure
formed by attaching a hydroxyl (–OH) group; that found in alcohols (hence
the “–ol” suffix); to an aromatic benzenoid-(phenyl) ring. The term polyphenol
has been in use since 1894 (Bate-Smith, 1954).

2. DEFINITION OF POLYPHENOL COMPOUNDS

The scientist of organic chemistry Edwin Haslam and co-workers (Haslam
and Cai, 1994) offered and justified the earliest widely accepted definition of
polyphenols, as natural product. Based on this earlier natural products
research of Edgar Charles Bate-Smith (Bate-Smith, 1954), Haslam (1998)
studied the characterization of specific structural common to plant phenolics
used in tanning (i.e., the tannins). The polyphenol class has been described
by White–Bate-Smith–Swain–Haslam (WBSSH) as:

• The class of polyphenols are generally moderately water-soluble
compounds,

• Their molecular weight of 500–4000 Da,
• With >12 phenolic hydroxyl groups, and
• Polyphenol class range from 5–7 aromatic rings per 1000 Da,

The limits to these ranges were found to be somewhat flexible (Quideau
et al., 2011; Haslam and Cai, 1994). The definition is further states that
polyphenol compounds is also display unique physical/chemical behaviors.
Such behaviors are attributed to their high molecular weights as well as
profusion of phenolic substructures—precipitation of proteins and particular
amine-containing organics (e.g., in particular the alkaloid of natural
products), and the formation of certain metal complexes (e.g., intense blue-
black trivalent iron (III) complexes).

The need to clarify the definition of ‘polyphenols’ in the light of the
extensive research into this large substance class and due to the increasingly
ambiguous use of the “polyphenol” term, Stéphane Quideau, (Bordeaux 1

234 Environ. Sci. & Engg. Vol. 6: Toxicology

University, France), offered a definition that was not given formal status
by International Union of Pure and Applied Chemistry (IUPAC) (Quideau,
2014) . The “polyphenol” term must be used to define compounds exclusively
derived from the shikimate (from Shikimic acid)/phenylpropanoid and/or
the polyketide pathway. This features that is more than one phenolic unit
is deprived of nitrogen-based functions (Quideau, 2014).

The core molecule is Gallic acid dimer, ellagic acid (M.W. 302, right), in
the naturally occurring phenolic compounds of varying sizes. It is not defined
as polyphenol by itself according to Haslam and Cai (1994). It is defined as
polyphenol according to the Quideau (2014).

On the other hand, the raspberry ellagitannin (M.W.~2450), that is
characterized by its 14 gallic acid moieties (most in ellagic acid-type compone-
nts), and more than 40 phenolic hydroxyl groups, meets the criteria of both
definitions of a polyphenol. Examples of other compounds that fall under
the definitions of both WBSSH and Quideau include the black tea antioxidant
theaflavin-3-gallate, and the hydrolysable tannin, tannic acid (Fig. 1).

Fig. 1: Chemical structure of Raspberry ellagitannin

Furthermore, raspberry ellagitannin is a tannin that is composed of 14
gallic acid units around a core of three units of glucose, with two gallic acids
as simple esters, and the remaining 12 appearing in 6 ellagic acid-type
units. Ester, ether, and biaryl linkages are present in this compound.

The reactions of an individual polyphenols are related to their core
phenolic structures, their linkages and the types of glycosides they form.
Standard phenolic reactions include ionization, oxidations to ortho- and
para-quinones and underlying aromatic transformations related to the
presence of the phenolic hydroxyl (Drynan et al., 2010). The reactions
phenolic hydroxyl is related to their linkages include nucleophilic additions,

Polyphenols: Properties, Occurrence, Content in Food and Potential Effects 235

and oxidative and hydrolytic bond cleavages (Drynan et al., 2010). The
ionization contributes to solubility and complexation. The oxidation to ortho-
and para-quinones contributes to antioxidant characteristics (Drynan et al.,
2010). In addition, as noted above, a traditional feature of polyphenols was
their ability to form particular, characteristic metal complexes (Haslam
and Cai, 1994).

3. CHEMICAL STRUCTURE AND SYNTHESIS OF POLYPHENOLS
3.1. Features of Chemical Structure
Polyphenols are large molecules (i.e., macro-molecules). For small
molecules, the upper limit weight is approximately 800 Daltons. This weight
allows the possibility of such molecule to diffuse rapidly across cell
membranes where they can reach intracellular sites of action. Otherwise,
they may remain as pigments once the cell senesces. For this reason, many
larger polyphenol molecules are biosynthesized in-situ originated from
smaller polyphenols to non-hydrolyzable tannins. Thus they remain
undiscovered in the plant matrix.

Most polyphenols contain repeating phenolic moieties of pyrocatechol,
resorcinol, pyrogallol and phloroglucinol connected by esters (hydrolyzable
tannins) or more stable C-C bonds (non-hydrolyzable condensed tannins).
The proanthocyanidins are mostly polymeric units of catechin and
epicatechin. On the other hand, catechol- and resorcinol-(benzenediol-) types
of polyphenols have two phenolic hydroxyl groups. Furthermore, pyrogallol-
and phloroglucinol-(benzenetriol-) types have three phenolic hydroxyl groups.
Though mixing of these types within polyphenols is also possible. The
substructures of phenolic compounds arise from various biosynthetic
pathways (i.e., WBSSH definition), particularly phenylpropanoid and
polyketide molecules aimed at plant and related to the secondary
metabolites.

Fig. 2: Chemical structure of phenolic moieties (a) Pyrocatechol (b) Resorcinol (c)
Pyrogallol (d) Phloroglucinol

The chemical structure of polyphenols has always heteroatom substituent
other than hydroxyl groups. Ester and ether linkages are common, as well
as various carboxylic acid derivatives. Meanwhile, ester linkages are
common in the hydrolyzable tannins compounds. Regardless of the simple
heteroatom links, the carbon frameworks can become complex. In this

236 Environ. Sci. & Engg. Vol. 6: Toxicology

respect, various carbon-carbon bond linkages join hydrolytically labile ethers
and esters as common in non-hydrolyzable condensed tannins.

For example, the chemical structure of theaflavin-3-gallate is given in
Fig. 3. It is a plant derived polyphenol that formed by esterification of two
equivalents gallic acid in the core of a theaflavin. Note that, two of the
phenolic hydroxyl groups are required to meet the phenol-count criterion
of the WBSSH definition points and they are engaged in ether linkages. In
this respect, diverse biosynthetic steps: the 7-atom ring (7-membered ring)
appearing in theaflavin chemical structure (Fig. 3) is an example of a
“carbocycle” that belongs to a non-benzenoid aromatic tropolone type.

Fig. 3: Chemical structure of Theaflavin-3-gallate

Furthermore, there are the following periodic occurrences:
• C-glucoside derivatives is illustrated in Fig. 4.
• Different biaryls and triaryls (as biphenyls), shown in Fig. 5
• Spiro-type structures as given in Fig. 6.

Fig. 4: Structure of C-glucoside

The C-glucoside (Fig. 4) is substructure of polyphenol compounds. As
exemplified by the phenol-saccharide conjugate puerarin. It is a mid-
molecular weight plant natural product. Here the attachment of the phenol

Polyphenols: Properties, Occurrence, Content in Food and Potential Effects 237

Fig. 5: Illustration of Ullmann reaction

Fig. 6: R (rectus) and S (sinister) of spiro- type in polyphenols

molecule to the saccharide is by a carbon-carbon bond. On the other hand,
isoflavone and its 10-atom benzopyran “fused ring” system is common in
polyphenols.

Biphenyl/biaryl (Fig. 5) is substructure of polyphenols. The given reaction
is illustrating the preparation by synthetic chemists using the copper-
mediated “Ullmann reaction”. The carbon-carbon bond in biaryls in nature
is synthesized also through a metal-mediated coupling reaction, which is
involving iron in most cases.

On the other hand, Fig. 6 is an example of substructure Spiro-type that
found in polyphenols in which the two rings are joined at a single shared
point. They are two stereoisomers. Based on the Cahn–Ingold–Prelog priority
(CIP) system. They are labeled as R (rectus) and S (sinister) to be described
according to stereochemistry.

Further example is tannic acid as shown in Fig. 7. It is a plant-derived
polyphenol. It can be formed by esterification of ten equivalents of the
phenylpropanoid-derived gallic acid (Fig. 7) to a monosaccharide (as glucose)
core from primary metabolism. Natural chiral (stereo) centers are highly
available, due to the preponderance of saccharide-derived core structures
(e.g., tannic acid), as well as spiro- and other structure types.

3.2. Chemical Synthesis of Polyphenols
Typical polyphenols from the tannin and other WBSSH types are
biosynthesized in the natural sources from which they derive. Using
standard “laboratory bench scale” organic chemical methods, their chemical
syntheses were somewhat limited until the first decade of the new
millennium. These syntheses involve challenging regioselectivity as well
as stereoselectivity issues (Krohn et al., 2010). In the late 70’s, early work
focused on the achiral synthesis of phenolic-related components of
polyphenols. Nelson and Meyers synthesized the permethyled derivative of
the ubiquitous diphenic acid core of ellagitannins in 1994 (Nelson and Meyers,

238 Environ. Sci. & Engg. Vol. 6: Toxicology

Fig. 7: Chemical structure of Tannic acid

1994). In the same year, Lipshutz and coworkers followed by stereo selective
synthesized the more complex permethylated structures such as a (+)-
tellimagrandin II derivative (Lipshutz et al., 1994). Furthermore, Itoh and
coworker’s synthesized a permethylated pedunculagin with particular
attention to axial symmetry issues in 1996 (Itoh et al., 1996). It was reported
in 1994 by Feldman, Ensel and Minard (Feldman and Ensel, 1994) that the
“total synthesis” of a fully unmasked polyphenol compounds, that of the
ellagitannin tellimagrandin I, was the diastereo selective sequence.

Feldman group conducted further study in the “total syntheses” of
“deprotected” polyphenols (Feldman et al., 2000). Thus, Feldman and
Lawlor’s synthesized the ellagitannin, coriariin A and other tannin relatives
(Feldman et al., 2000). Furthermore, Khanbabaee and Grosser (2003)
accomplished an efficient “total synthesis” of pedunculagin by using a twofold
intra-molecular double esterification strategy (Feldman, 2004).

Figure 8 is an example of a synthetically achieved small ellagitannin,
tellimagrandin II, derived biosynthetically and sometimes synthetically by
oxidative dimerization of two of the galloyl moieties of 1,2,3,4,6-pentagalloyl-
glucose.
4. CHEMICAL PROPERTIES AND USES OF POLYPHENOLS
4.1. Chemical Properties of Polyphenols
Polyphenols are molecules owing their UV/V absorptive character to the
aromatic structures with large conjugated systems of the pi-electron

Polyphenols: Properties, Occurrence, Content in Food and Potential Effects 239

Fig. 8: Tellimagrandin II chemical structure

configurations. In addition, they have auto-fluorescence properties,
particularly lignin and the phenolic part of suberin (Force, 2013). Meanwhile,
polyphenols are reactive species toward oxidation reaction.

On the other hand, 2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid)
(ABTS) as described by Osman et al. (2006) can be used to characterise the
products of polyphenol oxidation. In addition, polyphenols possess a significant
binding affinity for proteins compounds. This characteristic can lead to the
formation of soluble as well as insoluble protein polyphenol complexes
(Papadopoulou and Frazier, 2004).

4.2. Chemical Uses of Polyphenols

Some polyphenols compounds are traditionally used as dyes. For example,
in India, the pomegranate peel, or its juice, that are high in tannins and
other polyphenols are employed in dyeing of non-synthetic fabrics (Jindal
and Sharma, 2004).

Traditionally, polyphenols, particularly tannins, were used for tanning
leather. Today, polyphenols are still used as precursors in green chemistry
(Olshettiwar and Varma, 2008). The objectives are to make use of plant
residues from olive (called pomaces), grape or pecan shells left after
processing.

For instance, Cashew nut shell liquid (CNSL) is an important source of
phenolic raw material that is rich in cardol, cardanol and anacardic acid.
Strictly speaking, it is used mainly in polymer-based industries for friction
paints, linings, laminating resins, varnishes, rubber compounding resins,
surfactants, polyurethane based polymers, epoxy resins and wood
preservatives (Edoga et al., 2006).

240 Environ. Sci. & Engg. Vol. 6: Toxicology

5. THE BIOLOGY OF POLYPHENOL COMPOUNDS

5.1. Occurrence of Polyphenols in Nature
The most abundant polyphenols are the condensed tannins. Polyphenols
found almost in all families of plants and they are often concentrated largely
in leaf tissue, the epidermis, bark layers, flowers and fruits. In addition,
polyphenols play important roles in the decomposition of forest litter, and
nutrient cycles in the environment. Concentrations of phenols in plant tissues
differ widely depending on the source, assay and type of polyphenols. In
plant they are in the range of 1–25% as total natural phenols and polyphenols,
calculated according to the mass of dry green leaf (Hättenschwiler and
Vitousek, 2000).

The natural protection and preservation of woods against rot can be
explained by the high levels of polyphenols in some woods. The submerged
aquatic plant, namely Flax and Myriophyllum spicatum are the secrete
polyphenols that are involved in allelopathic interactions (Popa et al., 2008).

Meanwhile, polyphenols are found in animals. They play a role in
epicuticle hardening (sclerotization) of the arthropods such as insects and
crustaceans. The hardening of the cuticle is attributed to the presence of a
polyphenol oxidase (Schulbach et al., 2013).

5.2. Biological Role Polyphenols in Plants
It is worth mentioning that both natural phenols and polyphenols play
important roles in the ecology of most plants. The effect of phenols and
polyphenols in plant tissues can be divided into the following (Lattanzio et
al., 2006):

• Suppression and release of growth hormones such as auxin.
• To provide coloration (or plant pigments) and UV screens to protect

against ionizing
• “Sensory properties”: Deterrence of herbivores.
• To be used as “Phytoalexins”: Prevention of microbial infections (Huber

et al., 2003).
• “Signaling molecules”: In ripening and other growth processes.

6. BIOSYNTHESIS AND METABOLISM OF POLYPHENOLS

Polyphenol compounds incorporate smaller parts and building blocks
originated from simpler natural phenols. They are originated from the phenyl
propanoid pathway for the shikimic acid (from shikimate) or the phenolic
acids pathway for gallo-tannins and analogs. Flavonoids and caffeic acid

Polyphenols: Properties, Occurrence, Content in Food and Potential Effects 241

derivatives are biosynthesized from phenyl-alanine and malonyl-CoA. The
gallo-tannins complex develop through the in-vitro oxidation of 1, 2, 3, 4, 6-
pentagalloyl-glucose or dimerization processes, this resulting in hydrolyzable
tannins. For anthocyanidins, precursors of the condensed tannin
biosynthesis, dihydroflavonol reductase and leucoanthocyanidin reductase
(LAR) (as crucial enzymes that catalyze the chemical reaction) with
subsequent addition of catechin and epicatechin moieties for larger, non-
hydrolyzable tannins (Tanne et al., 2003).

The form of glycosylated develops from glucosyltransferase activity and
it increases the solubility of polyphenols (Krasnow and Murphy, 2004).

Important enzyme is Polyphenol oxidase (PPO). This enzyme catalyzes
the oxidation of o-diphenols to produce o-quinones. It is worth mentioning
that such rapid polymerisation of o-quinones is to produce black, brown or
red polyphenolic pigments. That is the cause of fruit browning. In insects,
on the other hand, PPO serves for the cuticle hardening (Krasnow and
Murphy, 2004).

A major enzyme is Laccase which initiates the cleavage of hydrocarbon
rings, that catalyzes the addition of a hydroxyl group to the phenolic
compounds. This major enzyme can be found in fungi including Panellus
stipticus. These organisms are able to break down lignin. The later is a
complex aromatic polymer in wood that is highly resistant to degradation
by conventional enzyme systems (Hertweck, 2009).

6.1. Content of Polyphenols in Food
Foods, generally, contain complex mixtures of polyphenols (D’Archivio et
al., 2010). According to Mennen et al. (2005) the most important sources of
polyphenols in food commodities are widely consumed in large quantities
including fruits, vegetables, black tea, green tea, coffee, chocolate, red wine,
olives, and extra virgin olive oil are all rich with polyphenols. Nuts, spices,
herbs and algae are also potentially significant for supplying certain
polyphenols. Some polyphenols are specific to particular food (phloridzin in
apples, flavanones in citrus fruit, isoflavones in soya). Others, including
quercetin, are found in all plant products such as fruit, vegetables, tea,
wine, cereals and leguminous plants (D’Archivio et al., 2010). Some
polyphenols are considered as anti-nutrients in food. Such compounds
interfere with the absorption of essential nutrients, including iron and other
metal ions. These compounds are binding to digestive enzymes and other
proteins, particularly in ruminants (Mennen et al., 2005).

Miglio et al. (2008) studied the effects of different cooking methods on
nutritional and physicochemical characteristics of selected vegetables, they
reported that phenolic and carotenoid compounds with antioxidant properties
in vegetables are to be retained significantly better through cooking by

242 Environ. Sci. & Engg. Vol. 6: Toxicology

steaming than frying (Miglio et al., 2008). It was further reported that
Polyphenols in beer, wine, various nonalcoholic juice beverages can be totally
removed using finings, substances that are usually added at or near the
completion of the processing of brewing.

7. POLYPHENOLS AND THE POTENTIAL HEALTH EFFECTS
Different polyphenolic extracts, for example from olive pulp, grape seeds,
grape skin and maritime pine bark are sold as ingredients as dietary
supplements, functional foods, and cosmetics without any legal health
claims. It is worth mentioning that there is no recommended “Dietary
Reference Intake” levels established for polyphenols (Watson, 2014).

On the other hand, the diverse structures of phenolic compounds prohibit
broad statements regarding their specific health effects. Furthermore, some
claims concerning potential health effect for specific polyphenol-enriched
foods remain unproven (Halliwell, 2007). Some of the phyto-estrogens
compounds are dietary polyphenols with significant affinities to the estrogen
receptors. Such phyto-estrogens compounds have positive or negative health
effects on humans and livestock (Woclawek-Potocka et al., 2013).

Information on the ongoing research regarding the effects of polyphenols
in vitro, the effects in vivo are limited and vague. The main reasons for this
are: (1) the absence of validated in vivo biomarkers, particularly in terms of
carcinogenesis or inflammation; (2) long-term studies did not prove or
demonstrate effects with an action mechanism, efficacy or specificity; and
(3) invalid applications of un-physiological test concentrations in the in vitro
studies. Such applications are subsequently irrelevant for the design of in
vivo experiments (Williamson and Manach, 2005). In rats, experiments
showed that polyphenols absorbed in the small intestine (Carbonaro et al.,
2001). It was suggested that polyphenols may be bound in protein-polyphenol
complexes modified by intestinal microflora enzymes that is allowing
derivative compounds formed by ring-fission to be better absorbed (Del Rio
et al., 2010).

8. ANTIOXIDANT PROPERTIES OF NATURAL PHENOLS AND
POLYPHENOLS

Polyphenols antioxidants consist of the natural phenol substructure of over
4,000 distinct species. Many of these compounds have antioxidant activity
in vitro. They are unlikely to have antioxidant roles in vivo (Williams et al.,
2004). However, they may affect cell-to-cell signalling, receptor sensitivity,
inflammatory enzyme activity or gene regulation (Virgili and Marino, 2008).

Historically, about 500 million years ago, freshwater and terrestrial plants
slowly optimized the production of “new” endogenous antioxidants, including

Polyphenols: Properties, Occurrence, Content in Food and Potential Effects 243

ascorbic acid (vitamin C), polyphenols, natural phenols (including flavonoids)
and tocopherols, etc. More recently, a few of these appeared in fruits and
flowers of angiosperm plants during the last 50–200 million years. As a
matter of fact, the angiosperms (the flowering plants), the dominant type of
plant today (and most of their antioxidant pigments) evolved during the
late Jurassic period.

Polyphenols are found in a wide array of all phytochemical-bearing foods.
Therefore, the main source of polyphenols is dietary. This dietary includes
honey, most legumes and fruits are all rich in polyphenols. Meanwhile,
fruits such as apples, pomegranate, strawberries, blackberries, aronia
berries, cranberries, blueberries, raspberries, cherries, cantaloupe, grapes,
plums, and pears are all rich in polyphenols. In addition, vegetables such as
parsley, broccoli, celery, cabbage, and onion are rich in polyphenols. Green
tea, white tea, black tea, chocolate, as well as olive oil, bee pollen, argan oil
and many grains and red wine are sources of polyphenols (Breton, 2008).
Intake of polyphenols can be achieved by consuming a wide array of plant
foods.

According to Bors et al. (1990)the regulation theory considers a polyphenol
antioxidant’s ability is to scavenge the free radicals and to up-regulate certain
metal chelation reactions. Singlet oxygen, peroxynitrite and hydrogen
peroxide, as various reactive oxygen species, must be continually removed
from cells to maintain healthy metabolic function. Several benefits possibly
associated with ion transport systems by diminishing the concentrations of
reactive oxygen species. This may affect redox signaling.

With regard to food systems that are deteriorated by peroxyl radicals
(R•), the “deactivation” of oxidant species by polyphenolic antioxidants (POH)
is based on the donation of hydrogen, which interrupts chain reactions (Bors
et al., 1990):

R• + POH  R-H + PO•
According to this reaction, the generated phenoxyl radicals (PO•) may
be stabilized through resonance and/or intra molecular hydrogen bonding,
as proposed for quercetin, or combine to yield dimerisation products. Thus
terminating the chain reaction (Bors et al., 1990):

PO• + PO•  PO-OP
The advantages of consuming dietary polyphenols may be associated
with benefit effects in higher animal species as follow:
• Possible reduction in inflammation: such as the effect in coronary

artery disease including specific research on endothelial cells via down
regulation of oxidative Low-density lipoprotein (LDL) (Serafini et al.,
2000).

244 Environ. Sci. & Engg. Vol. 6: Toxicology

• Among other possible advantages is anti-aging effects in skin (Owen
et al., 2000). However, this point is not yet proved scientifically in
humans. Thus, it is not allowed as health statements by regulatory
authorities like the U.S. Food and Drug Administration (FDA).

Therefore, further research may discern if polyphenol antioxidants have
biological roles in vivo (Ferrazzano et al., 2009).

9. ANTI-MICROBIAL PROPERTIES OF POLYPHENOLS
According to Ferrazzano et al. (2009), there is evidence to suggest that
plant polyphenols have anticariogenic properties. More recently, there has
been much discussion about the possibility that plant-derived phytochemical
compounds may have a role as potential antimicrobial substances (Ferrazzano
et al., 2011). Phytochemicals originally produced as defensive molecules in
order to discourage animals from eating plants such as flavonoids, including
resveratrol, tea catechins. Phenolic acids and other botanical molecular
byproducts, have recently attracted much attention by researchers and
clinicians worldwide. It has been proved that such phytochemicals are
excellent factors for promoting human health (Kay, 2010). It is believed
that some of the health benefits that are gained from eating vegetables,
fruits and other plant derived products are due to their polyphenol content.
It is important to elucidate the mechanisms behind metabolism of
polyphenols to understand their health effects in vivo. Depending on their
relationship with intestinal microbes, gut absorption and metabolism of
these compounds takes place. The main source of polyphenols is the food;
this has also raised a debate about the role of plant-derived phytochemicals
on the gut microflora. It was suggested by Selma et al. (2009) that such
phytochemicals may modify the gut microbial composition and/or biological
activity. It was proposed by investigators that the plant-derived
phytochemicals may be converted by the colonic microbiota to bioactive
compounds that can influence host health (Selma et al., 2009).

Such health issue underlines one aspect of a wider, more important and
complex relationship between microbes, plant products and animal species.
This might hamper the interpretation of these products as considering them
real antibacterial chemicals in human’s life. It is also important in medical
microbiology to address the role of plant polyphenols as potential drugs
against microbes (Chirumbolo, 2010). With respect to this complex biological
issue, several studies show the role of plant derived nutrients in preventing
infectious diseases and inflammatory ailments. For instance, tea catechins,
especially epigallocatechin-3-gallate, proved effective in treating important
nosocomial bacterial infections (Gordon and Wareham, 2010). Flavonoids
and some phenolic acids including caffeic acid, quinic acid, chlorogenic acid
and gallic acid exert a potent antimicrobial action against typical microbial
strains that are affecting the human respiratory system or urinary tract

Polyphenols: Properties, Occurrence, Content in Food and Potential Effects 245

system, including Candida species (Chirumbolo, 2010). The flavonoid
galangin is able to inhibit Klebsiella pneumoniae (Gram-negative bacterium)
replication by suppressing bacterial DNA-B helicase (enzyme in bacteria)
(Gordon and Wareham, 2010).

The main problem of using the phenolic derivatives as high antimicrobial
efficacy in vitro and/or in animal models (Paolillo et al., 2011) is their
metabolic route in the organism that includes their interaction with gut
microflora. The main role of plant polyphenols lies in the action that they
play in the immune system and inflammation. Their action is by affecting
signaling pathways strategic in immune system and inflammatory responses,
rather than having a direct antibacterial or antimicrobial impact.

Nowadays, there is a great interest in the use of natural herbal remedies,
as the Man tends to return to his ancestral relationship with the surrounding/
natural environment. As a result most people agree with taking natural
products rather than drugs. Many further studies are needed to clarify
whether phytochemicals really represent key molecules that are involved
in the antimicrobial potential of plant-derived foods. In the near future,
further molecular and pharmacological investigation could possibly reveal
and prove that they deserve an important place among others well known
as antibacterial and/or antiviral chemicals.

10. ANTI-CARIOGENIC PROPERTIES OF POLYPHENOL
COMPOUNDS

Extensive survey was carried out on varieties of compounds that are capable
of controlling dental caries. However, only limited numbers of compounds
from natural products were found to be available because of effectiveness,
odour, taste, economic feasibility and stability (Llorach et al., 2010). The
effects of polyphenols have been surveyed through both in vitro and in vivo
studies. Further investigation was carried out on the effect of polyphenols
against mutans streptococci and in vivo studies in animals and humans
(Luczaj et al., 2005; Milgrom et al., 2000). Due to their antibacterial action,
Polyphenol compounds occurring in tea, coffee and cocoa can have a role in
the prevention of cariogenic processes. Studies by Milgrom et al. (2000) on
cocoa polyphenol pentamers found that it significantly reduce biofilm
formation and acid production by Streptococcus mutans and S. sanguinis.
Similarly, trigonelline, caffeine and chlorogenic acid occurring in roasted
coffee and green interfere with S. mutans adsorption to saliva-coated
hydroxyapatite beads. Investigations carried out on black tea, green and
oolong indicate that tea polyphenols exert an anti-caries effect via an
antimicrobial mode-of-action. Furthermore, it was found that galloyl esters
of ()-epicatechin, ()-epigallocatechin and ()-gallocatechin exhibit increasing
antibacterial activities. Nevertheless, the anti-cariogenic effects against -
haemolytic streptococci exhibited by polyphenols from tea, coffee, and cocoa

246 Environ. Sci. & Engg. Vol. 6: Toxicology

suggest the importance of further studies to a possible application of such
beverages in the prevention of pathogenesis of dental caries (Ferrazzano
et al., 2009).

11. EXTRACTION OF POLYPHENOLS FROM OLIVE OIL
Lozano-Sánchez et al. (2014) extracted the polyphenols as a by-products
that is generated during extra-virgin olive oil (EVOO) filtration. The study
was evaluated using pressurized liquid extraction (PLE) and considering
mixtures of two solvents (ethanol and water) and the studied temperatures
ranged from 40 to 175°C. Characterization of the extracts was carried out
by high-performance liquid chromatography (HPLC) coupled with diode array
detection (DAD) and electrospray time-of-flight mass spectrometry (HPLC-
DAD-ESI-TOF/MS). The phenolic-composition of the filter cake was then
determined. It was found that the best isolation procedure is using ethanol
and water (50:50, v/v) at 120°C. The main identified phenolic compounds
were following: phenolic alcohols or derivatives (hydroxytyrosol and its
oxidation product), and elenolic acid derivatives and flavones (luteolin and
apigenin) secoiridoids (decarboxymethylated and hydroxylated forms of
oleuropein and ligstroside aglycones). Meanwhile, PLE extraction process
can also be applied to obtain enriched extracts with applications as bioactive
food ingredients.

12. POLYPHENOLS IN OLIVE MILL WASTEWATER
12.1. Introduction
Crude olive oil is produced traditionally in Mediterranean area by different
systems. The most common one is pressing through three-phase process
that produces a press cake and a great amount of olive mill wastewater.
The other one is mainly used in Spain which is two-phase system. This
produces either a paste-like waste called “alperujo” or “pomace”. The later
has higher water content that is very difficult to treat. The water content is
about 30% of the pomace, press cake, husk, and composed of crude olive
cake, if it is produced by traditional pressing technology. The water content
is about 45–50% if it is produced by decanter centrifuges. However, some
oil has to be recovered from the press cake by separate installation. The
exhausted olive cake is normally used as a soil conditioner in olive groves
or incinerated.

Meanwhile, pomace is used as fuel in a cogeneration system. It is also
used as fertilizer after a composting operation. Composting the pomace is
achieved by transforming the organic waste into stable humus via controlled
biologic process. It is worth mentioning that adding such composted olive
mill pomace as fertilizer to the olive groves allows the soil to get back the
lost nutrients after each olive crop.

Polyphenols: Properties, Occurrence, Content in Food and Potential Effects 247

12.2. Olive Oil Mill Wastewater

Olive oil mill wastewater (OMW) generated during olive oil production in
Mediterranean countries causes a serious environmental problem. This
wastewater is characterized by turbid dark color and a foul smelling. It is
called “Alpecin” in Spain (Madejon et al., 2009) and “Karasu” in Turkey
(Yesilada et al., 1999). OMW contains a large number of organic compounds
and a complex composition. Meanwhile, OMW is characterized by high
concentrations of biological oxygen demand (BanOdDp5)h, eCnholeimc iccoaml pOoxuyngdesn.
demand (COD), total suspended solid (TSS)
Generally, these characteristics are within the following range: pH (4.0
tgo/l6),)0, .B3O23Dg5/l(3o5f to 100 g/l), COD (40 to 195 g/l), total organic matter (40–165
lipid, and mineral matter (5 to 14 g/l). The seasonal discharge
of OMW is during November to February each year with large volume of
wastewater effluent of about 3 × 107 m3/y. This amount is equivalent of
pollution produced by more than 22 million people per year. The dark colour
and foul smelling of this OMW comes from lignin components polymerized
and the phenolic compounds (Jamoussi et al., 2005).

OMW is characterized by elevated toxic effect to the aquatic and terrestrial
organisms due to its high organic load, low biodegradability and high
concentrations of phenol components. In addition, the high levels of electrical
conductivity (EC), salinity, and (TSS) makes OMW not suited to be discharged
in the waterways. Nevertheless, large fraction of organic compounds is lost
in mill wastewater during olive oil processing. Still, large volumes of such
wastewater are poured into the Mediterranean environment (Niaounakis
and Halvadakis, 2006).

Niaounakis and Halvadakis (2006) reported that there is archaeological
evidence that OMW effluent that discharged for thousands of years around
Mediterranean has been damaging delicate shoreline environments. The
roman author Varro (c.116–27 B.C.); according to Niaounakis and Halvadakis
(2006); had observed that where amurca (the watery residue obtained when
the oil is drained from olive fruits) resulted from the olive presses and
flowed onto the fields where the groundwater became barren. Theophrastus
(c. 372–c. 287 B.C.) the Greek philosopher mentioned that pouring olive oil
over roots could kill trees and that young trees are more affected to such
treatment than mature ones (Niaounakis and Halvadakis, 2006).

Also the use of OMW is known since antiquity. Several ancient authors
have described Amurca as a universal remedy against insects, weeds, and
plant diseases. For the purpose of greater yields would be forthcoming,
Virgil (70 B.C.–19 B.C.) recommended that seeds be soaked in a mixture of
amurca and native soda before planting. It was used as a means of protecting
clothes from moths as well as preserver for dried fruits. Meanwhile, Amurca
has also been recommended and used for soothing out plaster floors, oiling
leather, etc. Nowadays, many of these uses are not applicable (Niaounakis

248 Environ. Sci. & Engg. Vol. 6: Toxicology

and Halvadakis, 2006). Traditionally, solid wastes of olive mill have been
used as fuel, and as animal feed. They are also used as fertilizer and as
construction materials.

12.3. Chemical Structure and Matrix of OMW

12.3.1. Organic compounds
The organic constitutes of OMW are mainly hydrocarbons, various phenolic
compounds and long chain fatty acids. The chemical compositions are organic
acids, polyphenols, polyalcohols, nitrogen compounds, lipids, tanins, and
pectines. The final chemical composition is variable and depends mainly on
the oil extraction procedure, olive fruit maturation, storage time and so
forth (Lanciotti et al., 2005).

Toxicity of OMW can be attributed to more than one compound. However,
phenols are the most important molecules accountable for toxicity. Phenolic
compounds are responsible for the phyto-toxicity of OMW as well as other
several biological effects including antibiosis. Such toxicity of the phenols
makes the biological treatment of OMW problematic. However, decreasing
the concentration of total phenol decreases toxic potential of OMW (Isidori
et al., 2005).

Isidori et al. (2005) studied the toxicity of OMW fractioned via
ultrafiltration (UF) and reverse osmosis techniques (RO) on aquatic
organisms. They found that most toxic fractions were that from RO
containing different compounds of low molecular weight (<350 Da).

Furthermore, the study provided strong evidence that the high toxicity
was due to both catechol and hydroxytyrosol. These compounds are the
most abundant compounds of reverse osmosis and constantly present in
OMW. In addition, the variable sensitivity was exhibited by the aquatic test
organisms, namely, (algae, rotifers, and crustaceans). Furthermore, it was
also demonstrated that a multispecies approach was required. For the
purpose of completing scenario on the effects of the most toxic OMW fraction,
it is recommended to extend the investigation to further tests that include
terrestrial producers and aquatic reducers.

Chemical structure of 15 low molecular phenolic compounds is given
in Fig. 9.

12.3.2. Instrumental methods for the determination of phenolic
compounds

The lack of an appropriate methodology is responsible for the major
shortcoming of the qualitative/quantitative evaluation of phenol compounds

Polyphenols: Properties, Occurrence, Content in Food and Potential Effects 249

Fig. 9: Chemical structure of the low molecular phenolic compounds: (1) Catechol, (2)
4 hydroxybenzoic acid, (3) 3,4- dihydroxybenzoic acid (protocatechuic acid), (4)
4-hydroxy-3-methoxybenzoic acid (vanillic acid), (5) 4- hydroxy-3,5-
dimethoxybenzoic acid (syringic acid), (6) 4 hydroxyphenylacetic acid, (7) 3,4-
dihydroxyphenylacetic acid, (8) 4-hydroxy-3methoxyphenylacetic acid
(homovanillic acid), (9) 4-hydroxyphenylethanol (tyrosol), (10) 3,4-
dihydroxyphenyl ethanol (hydroxytyrosol), (11) 3,4-dihydroxyphenylethylene
glycol, (12) 4-hydroxycinnamic acid (p-coumaric acid), (13) 3,4
dihydroxycinnamic acid (caffeic acid), (14) 4-hydroxy-3methoxycinnamic acid
(ferulic acid), and (15) 4-hydroxy-3,5-dimethoxycinnamic acid (sinapic acid).

in OMW. Recently, the most important analytical methods for evaluating
the phenolic content in OMW are the high performance liquid
chromatography (HPLC) and GC-MS as well as colorimetric method via the
reaction between phenol compounds and 4-aminoantipyrine dyes (Jamoussi
et al., 2005). The later is simple and economical to perform. However, the
method is limited by the low specificity of the reagent toward phenolics. In
addition, it does not provide qualitative information of single phenolic
compounds.

250 Environ. Sci. & Engg. Vol. 6: Toxicology

On the other hand, HPLC is very sensitive and specific. Nevertheless,
one run of this method lasts for 1 hour, thus, it is time-consuming. In
addition, it does not provide information on phenolic compound molecules.
Nevertheless, gas chromatography (GC) method has been widely employed
to detect the phenols. Finally, phenolic compounds are amenable to GC
without derivatization. However, at lower phenols concentrations peak
tailing might occur. It is important to mention that GC is efficient and
superior method of separation. If GC is coupled with a mass selective detector
(GC-MS), it allows much lower detection limits in addition to chemical
structural information (Jamoussi et al., 2005).

12.4. Environmental Studies and Aspects
Discharge of OMW on the land represents several drawbacks on the
environment. Uncontrolled dispersing of different substances that cause
foul smelling and possible pathogenic effect on land. The higher OMW
production and disposal, the more hazardous changes to the environmental,
microorganisms in soil and water ways. However, adequate treatment of
OMW should be optimized for the purpose of safe reuse as well as protecting
the environment.

Oleuropein and its derivatives are compounds that are readily degraded
into simpler products. These derivatives are considered non-polluting and
non-toxic to the environment. During the extraction process of olive oil,
almost 80% of all Oleuropein compounds are degraded.

12.5. Recovery and Extraction of Polyphenols from Olive Mill
Wastewater

12.5.1. Introduction
OMW contain significant quantities of phenolic compounds that possess
ideal structural chemistry for free the radical-scavenging including metal-
chelating properties. These phenolic compounds have been proved to be
more effective antioxidants in vitro than both vitamins E and C on a molar
basis (Rice-Evans et al., 1997). Recently, the interest and demand in natural
antioxidants have been increased due to the evidence that indicates the
involvement of oxygen-derived free radicals in different pathological
processes (Tapiero et al., 2002). Epidemiological research has suggested a
strong connection between consumption of polyphenol-rich food and
prevention of diseases associated with oxidative stress, including
cardiovascular diseases, cancers, inflammations and others. It is confirmed
that OMW is representing alternative source of biologically active
polyphenolic compounds. Environmental issues raised the need for
detoxification of OMW. Meanwhile, recovery and exploitation of polyphenols

Polyphenols: Properties, Occurrence, Content in Food and Potential Effects 251

as by-products at all stages of olive oil industry. It is well known that
Mediterranean countries are the main producer of OMW. Many of these
countries are encouraged to develop several wastewater treatment
technologies for the purpose of reducing the impact of pollution as well as
the possible recovery and exploitation of polyphenols (Owen et al., 2003).

On the other hand, hydroxytyrosol is one of the most abundant
polyphenols present in OMWW. This compound has been extensively studied
where it demonstrated its antioxidant and health-beneficial properties. The
studies showed that hydroxytyrosol has the following advantages: inhibits
human LDL oxidation (Fernandez-Bolanos et al., 2008), scavenges free
radicals during the oxidation process, the production of leukotriene for human
neutrophils (Petroni et al., 1995), inhibits platelet aggregation (De La Puerta
et al., 1999) and shows in-vitro antimicrobial activity (Bisignano et al., 1999).
It is worth mentioning that hydroxytyrosol as food additive is not
commercially available in large amounts and is highly expensive for scientific/
experimental purposes (Obied et al., 2005). Different studies have been
proposed for the production of hydroxytyrosol chemically (Tuck et al., 2000)
or enzymatic synthesis (Espin et al., 2001). Nevertheless, the achievements
are slow and expensive to obtain a commercially available pure
hydroxytyrosol. The objective is a by-products of biological origin from olive
mills wastewater such as hydroxytyrosol including other antioxidant
polyphenols.

On the other hand, the biocide effect of polyphenol extracts at variable
concentration for the treatment of E. olivina and A. citricola showed that it
was effective after 24 h and that the mortality increased gradually by
increasing the concentration. These results solve environmental problems
and indicate that the polyphenol extract is a biopesticide for the protection
against diseases of citrus and olive trees. Also it avoids using the synthetic
insecticides. This can enable a sustainable agro-economical and
environmental development (Larif et al., 2012).

12.5.2. Techniques for the recovery of polyphenols
Evaluation of several studies for recovering olive phenols from OMW or
solid wastes were carried out by several researchers, focusing on the
feasibility and the economy of the processes (Fki et al., 2005). The main
proposed systems to recover the phenols from OMW are the following: resin
chromatography; extraction with solvents; solid–liquid or liquid–liquid
extraction, supercritical fluid extraction; selective concentration by ultra-
filtration and reverse osmosis (Teresa et al., 2006).

12.5.2.1. Polyphenols recovery using liquid–liquid extraction process
Liquid–liquid solvent extraction represents a convenient alternative and
simple technique for the recovery of polyphenols. In pilot-scale it is widely

252 Environ. Sci. & Engg. Vol. 6: Toxicology

used for the production and commercial recovery (Tura and Robards, 2002)
resulted in a polyphenol rich extract and residue. This exhausted fraction
residue is a complex mixture of water, organic acids, sugars, nitrogenous
substances, residual polyphenols and tannins, pectins, lipids, mucilages,
and inorganic substances. Treatment of this exhausted fraction via physico-
chemical or biological techniques is necessary before discharging into water
ways.

Liquid–liquid extraction was employed by Khoufi et al. (2008) to recover
phenolics from centrifuged OMW while reducing their toxicity for further
treatment by aerobic or anaerobic digestion. They could recover over 90%
of the phenolics by using ethyl acetate extraction. This pre-treatment
resulted in the removal of the major low molecular mass (LMM) phenolic
compounds and a small part of high molecular mass (HMM) polyphenols.

In further investigation by Kalogerakis et al. (2013), various polyphenolic
compounds such as hydroxytyrosol and tyrosol with known antioxidant
properties were yield.

More recently, Oral et al. (2014) used medium pressure liquid
chromatography (MPLC) for purifying some phenolic compounds such as
hydroxytyrosol, punicalagin and chlorogenic acid from OMW. They also
used European cranberrybush (Viburnum opulus L.) juice, and pomegranate
peel to extract some phenolic compounds. Extraction of polyphenols from
OMW and European cranberry bush juice was achieved by ethyl acetate
(EtOAc). Meanwhile, methanol was employed for pomegranate peel
extraction. The solvents were removed using rotary evaporator. The residue
was passed into the water phase followed by centrifugation and
microfiltration. Afterwards chlorogenic acid, and punicalagin were
concentrated using a rotary evaporator and were transformed into particle
form via freeze drying process. Hydroxytyrosol was extracted as concentrated
liquid using a rotary evaporator. The purities of the extracted compounds
were determined as 97.1% for punicalagin, 92.5% for chlorogenic acid, and
90.2% for hydroxytyrosol.

12.5.2.2. Polyphenols recovery using solid phase extraction process
Solid phase extraction process is an effective process for extracting
polyphenols through solid phase extraction (SPE). Solid phase recovery of
polyphenols from apple wastes (Lozano-Sanchez et al., 2014), OMWs
(Kammerer et al., 2010) and grape pomace (Maier et al., 2009) was achieved
using acrylic ester-based Amberlite XAD7 and the styrene- divinylbenzene-
based XAD16 resins.

Agalias et al. (2007) treat the OMW in a pilot scale study aiming to recover
polyphenols and to reduce the environmental problems. The treatment
technology includes three successive sections: The 1st one consists of
sequence filtration stages. The purpose is to reduce the suspended solids of

Polyphenols: Properties, Occurrence, Content in Food and Potential Effects 253

the OMW down to a limit of 25 µm. The 2nd consists of passing the filtered
OMW through a series of adsorbent resins (namely, XAD16 and XAD7HP)
for the purpose of achieving the de-odoring and de-colorization of the OMW
as well as removing and recovering polyphenols compounds and lactone.
The 3rd procedure consists evaporating and recovery of the organic solvents
mixture that has been used in the resin regeneration. Finally using fast
centrifuge partition chromatography for the separation of the polyphenols
and other organic substance. The final obtained extract was rich in
polyphenols and lactones as high antioxidant compounds with high added
value as well as pure hydroxytyrosol.

Deeb et al. (2012) studied the separation of polyphenols from Jordanian
OMW with the possibility of pharmaceutical applications in industry. Silica
column chromatography was used to isolate the phenolic compounds by using
deferent solvents after extracting the acidified solution with normal-hexane
and ethyl-acetate. The concentrations of the isolated phenolic compounds in
the Jordanian OMW were hydroxytyrosol (315.9 mg/L), tyrosol (210.6 mg/L),
caffeic acid (140.4 mg/L), vanillic acid (128.7 mg/L), p-coumaric acid (117.0
mg/L), trans-cinnamic acid (105.3 mg/L), and ferulic acid (93.6 mg/L).

Bertin et al. (2011) employed the solid phase extraction using commercial
resins; ENV+ gave rise for the recovery of natural polyphenols from real
industrial OMW. The extracted phenols from OMW were achieved by
employing acidified ethanol. While the extraction of lower molecular weight
phenols (such as HT) was better achieved by ethanol. However, the study
revealed that efficiency of extraction procedure remarkably influenced by
the physical-chemical characteristics of OMW. Thus, they confirmed that
recovery procedure is successful but they recommended that the procedure
has to be adjusted from time to time.

Sannino et al. (2013) proposed and demonstrated liquid-liquid extraction
using ethyl acetate followed by solid phase chromatographic fractionation.
The concept of this study aimed to produce, at small-scale, high grade purified
hydroxytyrosol.

Shadabi et al. (2013) proposed bulk liquid membrane - High performance
liquid chromatography/ultraviolet (BLM-HPLC/UV) method for the
separation of essential phenolic compounds from real OMWW samples, using
liquid-liquid Extraction (LLE) and Solid Phase Extraction (SPE) methods.
For the first time, the BLM system was successfully employed for selective
transport and separation of essential phenolic compounds of caffeic acid
(CA), hydroxytyrosol (HTY), and tyrosol (TY) from OMW samples. The
advantages of this method are to provide separation of analysts from complex
matrices, as well as the possibility of using a variety of organic liquids. The
employed liquids were inert, insoluble, and harmless. They used selective,
single step and simple process. The efficiency of the studied procedure
depends on the pH of the OMWW, organic membrane, stirring rate, transport
time and the receiving phases.

254 Environ. Sci. & Engg. Vol. 6: Toxicology

12.5.2.3. Polyphenols recovery using membrane filtration technology
Recently, the integrated membrane filtration became an important tool to
recover polyphenols. Paraskeva et al. (2007b) employed Ultra filtration (UF),
Nano filtration (NF) and/or Reverse Osmosis (RO) for the treatment of OMW.
They used NF for the separation of phenols. Meanwhile, a membrane
filtration for the selective fractionation and the recovery of polyphenol
compounds, water and organic substances from OMW was also studied by
Russo (2007) and Abdel-Shafy et al. (2014).

Mazzuca et al. (2006) reported a new combined method to localize the
sites of enzyme immobilization. They also determine the enzyme catalytic
activity on a polymeric capillary membrane reactor. -Glucosidase enzyme
from olive fruit was selected due to its suitable relevance in the foods
industrial processing, in pharmaceuticals and in biotechnology. -Glucosidase
enzyme was selected due to its activity against the synthetic substrate 5-
brome-4-chloro-3-indolyl--d-glucopyranoside that develops an insoluble dyed
product.

Garcia-Castello et al. (2010) reported the use of integrated membrane
separation including micro-filtration (MF) and nano-filtration (NF), osmotic
distillation (OD) and vacuum membrane distillation (VMD) for the
purification, recovery, and concentration of polyphenol compounds from
OMW. The study was successful to obtain an enriched concentrated solution
of polyphenols via treating the NF permeate by OD. They produced free
low molecular weight of polyphenols, with hydroxytyrosol that representing
56% of the total weight. The purified solution is of great interest for preparing
formulations that can be used in food, pharmaceutical industry and cosmetic.

Mazzei et al. (2010) studied the combination of biocatalytic membrane
reactor and membrane emulsification system. Moreover, the hydrophilic
components (including glucose and non-reacted oleuropein) were kept in
water phase. The lipophilic molecule; namely, oleuropein aglycone; was
extracted by the organic phase. The process aimed to extract; in a pure
solvent; the non-commercially oleuropein aglycones by separation from
unreacted oleuropein, glucose and enzyme. This concept opens doors to
control intermediate reaction products in multi-step reaction procedures.

Mazzei et al. (2012) employed a biocatalytic membrane system that was
operated in water phase, while extracting the water reaction product in
organic phase using water-in-oil droplet formation via membrane
emulsification. The system was, then, optimized and tested. The efficiency
of the system was evaluated according to operation parameters including of
pH, temperature, residence time. Furthermore, the amount of immobilized
enzyme has been investigated in order to identify the optimal values of
performance. In addition, other aspects were also taken into account
including energy and chemical consumptions. On a lab scale study, the
stability of the catalytic process was evaluated showed no activity decay

Polyphenols: Properties, Occurrence, Content in Food and Potential Effects 255

within three days of continuous operation. From OMW, the conversion of
oleuropein as simple phenolic compounds was achieved at constant
production of oleuropein aglycone of about 9 g/m2 h.q.

Petrotos et al. (2012) subjected OMW to clarification first using membrane
technology. By employing selective macro-porous resins as adsorption-
desorption technique, the polyphenols were successfully isolated. Finally
the polyphenolic compounds were recovered in dry form after been processed
by RO technique followed by freeze drying. Furthermore, in order to mask
the color and bitterness effect, the polyphenols were encapsulated in modified
starch by freeze drying. The phenolic products were used successfully as
additives in yogurt. Such produced products were highly acceptable by the
consumers.

Further study by Petrotos et al. (2014) in which they determined the
effect of microfiltration (MF) for the separation of polyphenols from OMW.
The treatment system consisted of a preliminary centrifugation, followed
by MF to separate both fats and polyphenols. The results of this study
indicated that a 3-month storage of OMW prior to treatment exhibited 20%
decrease in permeate flux. This indicated that direct processing of the OMW
is important. Nevertheless, the obtained microfiltrate was an excellent
antioxidant. It consists of useful polyphenols, including tyrosol,
hydroxytyrosol, catechin, caffeic acid, and p-coumaric acid.

Abdel-Shafy et al. (2014) studied integrated membrane systems to recover
and concentrate valuable polyphenol compounds from OMWW. The study
was extended to conduct the effect of enzymatic hydrolysis using -
glucosidase in order to release higher concentration of pure hydroxytyrosol
(HT). The OMWW was filter through microfiltration (MF). The polyphenolic
compounds in raw OMWW were 2.5 g/l. The polyphenols was recovered and
concentrated in the Retentate of MF, UF and NF at the concentration of
2.3, 2.0 and 2.4 g/l, respectively. The study revealed that almost all
polyphenolic compounds were recovered and concentrated in the NF
Retentate. When the enzymatic hydrolysis was employed using -glucosidase
on NF Retentate followed by acid hydrolysis, an amount of 1.3 g/l of pure
HT could be produced. Based on these findings OMWW can be used as
alternative source of valuable polyphenolic compounds that are necessary
for industrial and pharmaceutical applications. It was then recommended
that the permeate of NF can be further treated using RO for recovering
extra amount of the polyphenol compounds.

13. CONCLUSIONS

1. Polyphenols are naturally occurring chemical compounds found largely
in the fruits, vegetables, cereals and beverages. Fruits like berries,
grapes, pear, apple, cherries and strawberry contains up to 200–300

256 Environ. Sci. & Engg. Vol. 6: Toxicology

mg polyphenols per 100 grams fresh weight. The natural products
manufactured from these fruits, also contain significant amounts of
polyphenols.
2. In food, polyphenols may contribute to the color, odor, bitterness,
flavor, astringency, and oxidative stability.
3. Polyphenols are a group of chemical compounds that may protect
against some common health problems of Man and possibly certain
effects of aging (at least in theory).
4. Polyphenols compounds act as antioxidants to Man. They protect
Human cells and body chemicals against damage caused by free
radicals. Possible reduction in inflammation as well as the effect in
coronary artery disease including specific research on endothelial cells
via down regulation of oxidative LDL
5. Polyphenolic compounds posses the ability to block enzymes action
that cancers need for growth. They also can inhibit and deactivate
substances that promote the growth of cancers. The polyphenolic
compound that is most strongly associated with cancer prevention is
epigallocatechin-3-gallate (or EGCG).
6. All different types of tea contain polyphenols. Laboratory studies
indicate that polyphenols isolated from tea act as scavengers of oxygen
and nitrogen-free radicals, thus it protects the fatty membranes of
cells, proteins and DNA.
7. The dietary intake requirement is as high as 1 g/d. This amount is
approximately 10 times higher than the intake of vitamin C and about
100 times higher than the intakes of both carotenoids and vitamin E.
8. Typically one cup of coffee or tea contains about 100 mg polyphenols.
9. Generally, polyphenols are involved in defense against ultraviolet
radiation and against aggression by pathogens.
10. In food, polyphenols may contribute to the color, odor, bitterness,
flavor, astringency, and oxidative stability.
11. Olive mill wastewater (OMWW) contains high concentrations of
polyphenols, up to 10 g L–1. Such chemicals are able to exert acute
toxic effects towards microorganisms that hinder the use of biological
wastewater treatment system.
12. Recovery of polyphenolic compounds from OMWWs can achieve two
major advantages: (a) reduction of the intrinsic wastewater
environmental toxicity and (b) recovery of high added value molecules,
mainly, hydroxytyrosol (HT) and tyrosol. These two compounds are
among the most economically relevant components of OMWW
phenolic fractions.
13. Polyphenols are natural antioxidants of special relevance for several
industrial sectors, including medicine, cosmetic, and food preservation.

Polyphenols: Properties, Occurrence, Content in Food and Potential Effects 257

13.1. Recommendation

• Further research should be conducted to clarify if polyphenol
antioxidants have biological roles in vivo.

• It is important to elucidate the mechanisms behind metabolism of
polyphenols to understand their health effects in vivo.

• Research on the role of flavonoids and other polyphenols in
inflammation in humans is increasing and encouraging but still
insufficient.

• Clinical pharmacology trials, as in vivo investigation, should not focus
only on flavonoid-rich foods but also on purified molecules.

REFERENCES

Abdel-Shafy, H.I., Schories, G., Mohamed-Mansour, M.S. and Bordei, V., Integrated
membranes for the recovery and concentration of antioxidant from olive mill
wastewater. Desalination and Water Treatment, www.deswater.com, doi: 10.1080/
19443994.2014.935807.

Agalias, A., Magiatis, P., Skaltaounis, A.L., Mikros, E., Tsarbopoulos, A., Gikas, E., Spanos,
I. and Manios, T. (2007). A new process for the management of olive waste water and
recovery of natural antioxidants. J. Agric. Food Chem., 55: 2671–2676.

Bate-Smith, E.C. (1954). Polyphenol on www.merriam-webster.com online dictionary.
Leuco-anthocyanins. 1. Detection and identification of anthocyanidins formed leuco-
anthocyanins in plant tissues. The Biochemical Journal, 58(1): 122–5. PMC 1269852.
PMID 13198862

Bertin, L., Ferri, F., Scoma, A., Marchetti, L. and Fava, F. (2011). Recovery of high added
value natural polyphenols from actual mill wastewater through solid phase extraction
olive, Chemical Engineering Journal, 171: 1287–1293.

Bisignano Tomaino, A., Lo Cascio, R., Crisafi, G., Uccella, N. and Saija, A. (1999). On the
in-vitro antimicrobial activity of oleuropein and hydroxytyrosol. Journal of Pharmacy
and Pharmacology, 51(8): 971–974.

Bors, W., Heller, W., Michel, C. and Saran, M. (1990). Flavonoids as antioxidants:
Determination of radical Scavenging efficiencies. Methods in Enzymology, 186:
343–355.

Breton, F. (2008). Polyphenol antioxidants in red wine.
Carbonaro, M., Grant, G. and Pusztai, A. (2001). Evaluation of polyphenol bioavailability

in rat small intestine. European Journal of Nutrition, 40(2): 84–90.
Chirumbolo, S. (2010). The role of quercetin, flavonols and flavones in modulating

inflammatory cell function. Inflamm Allergy Drug Targets, 9: 263–285.
D’Archivio, M. et al. (2010). Bioavailability of the polyphenols: Status and controversies.

Int. J. Mol. Sci., 11(4): 1321–1342.
De La Puerta, R., Gutierrez, V.R. and Hoult, J.R.S. (1999). Inhibition of leukocyte 5-

lipoxygenase by phenolics from virgin olive oil. Biochemical Pharmacology, 57(4):
445–449.
Deeb, A.A., Fayyad, M.K. and Alawi, M.A. (2012). Separation of polyphenols from Jordanian
olive oil mill wastewater. Chromatography Research International, 2012: Article ID
812127, 8 pages. doi:10.1155/2012/812127.
Del Rio, D., Costa, L.G., Lean, M.E.J. and Crozier, A. (2010). Polyphenols and health:
What compounds are involved? Nutrition, Metabolism and Cardiovascular Diseases,
20: 1.
Drynan, J.W., Clifford, M.N., Obuchowicz, J. and Kuhnert, N. (2010). The chemistry of
low molecular weight black tea polyphenols. Nat. Prod. Rep., 27: 417–462.

258 Environ. Sci. & Engg. Vol. 6: Toxicology

Edoga, M.O., Fadipe, L. and Edoga, R N. (2006). Extraction of polyphenols from cashew
nut shell. Leonardo electronic. Journal of Practices and Technologies, 9: 107–112.

Espin, J.C., Soler-Rivas, C., Cantos, E., Tomas-Barberan, F.A. and Wichers, H.J. (2001).
Synthesis of the antioxidant hydroxytyrosol using tyrosinase as biocatalyst. Journal
of Agricultural and Food Chemistry, 49(3): 1187–1193.

Feldman, K.S. and Ensel, S.M. (1994). Ellagitannin chemistry. Preparative and mechanistic
studies of the biomimetic oxidative coupling of galloyl esters. J. Amer. Chem. Soc.,
116(8): 3357–3366.

Feldman, K.S., Lawlor, M.D. and Sahasrabudhe, K. (2000). Ellagitannin chemistry.
Evolution of a three-component coupling strategy for the synthesis of the dimeric
ellagitannin coriariin A and a dimeric gallotannin analogue. J. Org. Chem., 65(23):
8011–8019.

Feldman, K.S. (2004). Recent progress in ellagitannin chemistry, Proceeding of 4th Tannin
Conference held at the Fall Meeting of the American-Chemical-Society, Philadelphia,
PA,. Phytochemistry, 66(17): 1984–2000.

Fernandez-Bolanos, J.G., Lopez, O., Juan, F., Fernandez–Bolanos, J. and Rodriguez-
Gutierrez, G. (2008). Hydroxytyrosol and derivatives: Isolation, synthesis, and biological
properties. Current Organic Chemistry, 12(22): 442–463.

Ferrazzano, G.F., Amato, I., Ingenito, A., Zarrelli, A., Pinto, G. and Pollio, A. (2011). Plant
polyphenols and their anti-cariogenic properties: A review. Molecules, 16: 1486–1507.

Ferrazzano, G.F., Amato, I., Ingenito, A., De Natale, A. and Pollio, A. (2009). Anticariogenic
effects of polyphenols from plant stimulant beverages (cocoa, coffee, tea). Fitoterapia,
80: 255–262.

Fki, I., Allouche, N. and Sayadi, S. (2005). The use of polyphenolic extract, purified
hydroxytyrosol and 3,4-dihydroxyphenyl acetic acid from olive mill wastewater for
the stabilization of refined oils: A potential alternative to synthetic antioxidants. Food
Chem., 93(2): 197–204.

Force, A. (2013). See to Act. Fluorescence and Polyphenols. Retrieved.
Garcia-Castello, E., Cassano, A., Criscuoli, A., Conidi, C. and Drioli, E. (2010). Recovery

and concentration of polyphenols from olive mill wastewaters by integrated membrane
system. Water Research, 44: 3883–3892.
Gordon, N.C. and Wareham, D.W. (2010). Antimicrobial activity of the green tea
polyphenols (–)-epigallocatechin-3-gallate (EGCG) against clinical isolates of
Stenotrophomonas maltophilia. Int. J. Antimicrob. Agents, 36: 129–131.
Halliwell, B. (2007). Dietary polyphenols: Good, bad, or indifferent for your health.
Cardiovasc. Res., 73(2): 341–347.
Haslam, E. and Cai, Y. (1994). Plant polyphenols (vegetable tannins): Gallic acid metabolism.
Natural Product Reports, 11(1): 41–66.
Haslam, E. (1998). Practical polyphenolics. ISBN 05-521-46513-3.
Hättenschwiler, S. and Vitousek, P.M. (2000). The role of polyphenols in terrestrial
ecosystem nutrient cycling. Trends in Ecology and Evolution, 15(6): 238–243.
Hertweck, C. (2009). The biosynthetic logic of polyketide diversity. Angewandte Chemie
International Edition, 48(26): 4688.
Huber, B., Eberl, L., Feucht, W., Polster, J. and Naturforsch, C. (2003). 58 (11-12): 879–84.
Isidori, M., Lavorgna, M., Nardelli, A. and Parrella, A. (2005). Model study on the effect of
15 phenolic olive mill wastewater constituents on seed germination and Vibrio fischeri
metabolism. J. Agric. Food Chem., 53: 8414–8417.
Itoh, T., Chika, J., Shirakami, S.S. et al. (1996). Synthesis of trideca-O-methyl-alpha-
pedunculagin. Diastereo-favoritism studies on intramolecular ester-cyclization
of axially chiral diphenic acids with carbohydrate core. J. Org. Chem., 61(11): 3700–
3705.
Jamoussi, B., Bedoui, A., Ben Hassine, B. and Abderraba, A. (2005). Analyses of phenolic
compounds occurring in olive oil mill wastewater by GC-MS. Toxicological and
Environmental Chemistry, 87(1): 45–53.

Polyphenols: Properties, Occurrence, Content in Food and Potential Effects 259

Jindal, K.K. and Sharma, R.C. (2004). Recent trends in horticulture in the Himalayas:
Integrated Development Under the Mission Mode. Indus Publishing, New Deldi, India,
p. 333.

Kalogerakis, N., Politi, M., Foteinis, S., Chatzisymeon, E. and Mantzavinos, D. (2013).
Recovery of antioxidants from olive mill wastewaters: A viable solution that promotes
their overall sustainable management. Journal of Environmental Management, 128:
749–758.

Kammerer, J., Kammerer, D.R., Jensen, U. and Carle, R. (2010). Interaction of apple
polyphenols in a multi-compound system upon adsorption onto a food-grade resin. J.
Food Eng., 96: 544–554.

Kay, C.D. (2010). The future of flavonoid research. Br J. Nutr., 104 (3): S91–S95.
Khanbabaee, K. and Grosser, M. (2003). An efficient total synthesis of pedunculagin by

using a twofold intramolecular double esterification strategy. Eur. J. Org. Chem., 11):
2128–2131.
Khoufi, S., Aloui, F. and Sayadi, S. (2008). Extraction of antioxidants from olive mill
wastewater and electro-coagulation of exhausted fraction to reduce its toxicity on
anaerobic digestion. Journal of Hazardous Materials, 151: 531–539.
Krasnow, M.N. and Murphy, T.M. (2004). Polyphenol glucosylating activity in cell
suspensions of grape (Vitis vinifera). Journal of Agricultural and Food Chemistry,
52(11): 3467–3472.
Krohn, K., Ahmed, I., John, M., Letzel, M.C. and Kuck, D. (2010). Stereoselective synthesis
of benzylated prodelphinidins and their diastereomers with use of the mitsunobu
reaction in the preparation of their gallocatechin precursors. Eur. J. Org. Chem.,
2010: 2544–2554.
Lanciotti, R., Giannoti, A. and Baldi, D. (2005). Use of Yarrowia lipolytica strains for the
treatment of olive mill wastewater. Bioresource Technology, 96: 317–322.
Larif, M., Zarrouk, A., Soulayman, A. and Elmidaoui, A. New innovation in order to
recover the polyphenols of olive mill wastewater extracts for use as a biopesticide
against the Euphyllura olivina and Aphis citricola. Res. Chem. Intermed., DOI 10.1007/
s11164-012-0947-5.
Lattanzio, V. et al. (2006). Role of phenolics in the resistance mechanisms of plants
against fungal pathogens and insects. Phytochemistry. Advances in Research, pp.
23–67.
Lipshutz, B.H., Liu, Z.P. and Kayser, F. (1994). Cyanocuprate-mediated intramolecular
biaryl couplings applied to an ellagitannin - synthesis of (+)-O-Permethyltellimagrandin
II. Tet. Lett., 35(31): 5567–5570.
Llorach, R., Urpi-Sarda, M., Rotches-Ribalta, M., Rabassa, M. and Andres-Lacueva, C.
(2010). Resveratrol: From dietary intake to promising therapeutic molecule. Agro
Food Ind. Hi-Tech, 21: 42–44.
Lozano-Sanchez, J., Castro-Puyana, M., Mendiola, J.A., Segura-Carretero, A., Cifuentes,
A. and Ibanz, E. (2014). Recovering bioactive compounds from olive oil filter cake by
advanced extraction techniques. Int. J. Mol. Sci., 15: 1627–16283.
Luczaj, W. and Skrzydlewska, E. (2005). Antioxidative properties of black tea. Prev. Med.,
40: 910–918.
Madejon, E., Romero, A.S., Lopez, R. and Cabrea, F. (2009). Characterization of the
wastewater from the two-phase centrifugation system for olive oil extraction.
Sustainable Organic Waste Management for Environmental Protection and Food
Safety Seminar, IRNAS-CSIC.
Maier, T., Fromm, M., Schieber, A., Kammerer, D.R. and Carle, R. (2009). Process and
storage stability of anthocyanins and non-anthocyanin phenolics in pectin and gelatin
gels enriched with grape pomace extracts. Eur. Food Res. Technol., 299: 949–960.
Mazzei, R., Drioli, E. and Giorno, L. (2010). Biocatalytic membrane reactor and membrane
emulsification concepts combined in a single unit to assist production and separation

of water unstable reaction products. Journal of Membrane Science, 352: 166–172.

260 Environ. Sci. & Engg. Vol. 6: Toxicology

Mazzei, R., Drioli, E. and Giorno, L. (2012). Enzyme membrane reactor with heterogenized
-glucosidase to obtain phytotherapic compound: Optimization study. Journal of
Membrane Science, 390–391: 121–129.

Mazzuca, S., Giorno, L., Spadafora, A., Mazzei, R. and Drioli, E. (2006). Immunolocalization
of -glucosidase immobilized within polysulphone capillary membrane and evaluation
of its activity in situ. Journal of Membrane Science, 285: 152–158.

Mennen, L et al. (2005). Risks and safety of polyphenol consumption. Am. J. Clin. Nutr.,
81(1): 3265–3295.

Miglio, C., Chiavaro, E., Visconti, A., Fogliano, V. and Pellegrini, N. (2008). Effects of
different cooking methods on nutritional and physicochemical characteristics of
selected vegetables. J. Agric. Food Chem., 56(1): 139–47.

Milgrom, P., Riedy, C.A., Weinstein, P., Tanner, A.C., Manibusan, L. and Bruss, J. (2000).
Dental caries and its relationship to bacterial infection, hypoplasia, diet, and
oral hygiene in 6- to 36-month-old children. Community Dent. Oral Epidemiol., 28:
295–306.

Nelson, T.D. and Meyers, A.I. (1994). A rapid total synthesis of an Ellagitannin [sic].
J. Org. Chem., 59(9): 2577–2580.

Niaounakis, M. and Halvadakis, C.P. (2006). Olive processing waste management, litrature
review and patent survey. Waste Management Series 5, Second edition, Elsevier,
2006: 340.

Obied, H.K., Allen, M.S., Bedgood, D.R., Prenzler, P.D., Robards, K. and Stockmann, R.
(2005). Bioactivity and analysis of biophenols recovered from olive mill waste. Journal
of Agricultural and Food Chemistry, 53(4): 823–837.

Olshettiwar, V. and Varma, R.S. (2008). Greener and expeditious synthesis of bioactive
heterocycles using microwave irradiation. Pure and Applied Chemistry, 80(4):
777–90.

Oral, R.A., Dogan, M. and Sarioglu, K. (2014). Recovery of bioactive phenolic compounds
from olive mill waste water, pomegranate peel, and european cranberrybush
(Viburnum opulus L.) juice by preparative MPLC. Journal of Liquid Chromatography
and Related Technologies, 37: 1827–1836.

Osman, A.M., Wong, K.K.Y. and Fernyhough, A. (2006). ABTS radical-driven oxidation of
polyphenols: Isolation and structural elucidation of covalent adducts. Biochemical
and Biophysical Research Communications, 346(1): 321–329.

Owen, R.W., Giacosa, A., Hull, W.E., Haubner, R., Spiegelhalder, B. and Bartsch, H.
(2000). The antioxidant/anticancer potential of phenolic compounds isolated from
olive oil. Eur. J. Cancer, 36(10): 1235–47.

Owen, R.W., Haubner, R., Mier, W., Giacosa, A., Hull, W.E., Spiegelhalder, B. and Bartsch,
H. (2003). Isolation and structure elucidation of the major individual polyphenols in
carob fibre. Food Chem. Toxicol., 41: 1727–1738.

Paolillo, R., Carratelli, C.R. and Rizzo, A. (2011). Effect of resveratrol and quercetin in
experimental infection by Salmonella enterica serovar Typhimurium. Int. Immuno
pharmacol., 11: 149–156.

Papadopoulou, A. and Frazier, R.A. (2004). Characterization of protein-polyphenol
interactions. Trends in Food Science and Technology, 15(3–4): 186–190.

Paraskeva, C.A., Papadakis, V.G., Tsarouchi, E., Kanellopoulou, D.G. and Koutsoukos,
P.G. (2007b). Membrane processing for olive mill wastewater fractionation.
Desalination, 213: 218–229.

Petroni, A., Blasevich, M., Salami, M., Papini, N., Monte-doro, G.F. and Galli. C. (1995).
Inhibition of platelet aggregation and eicosanoid production by phenolic components
of olive oil. Thrombosis Research, 78(2): 151–160.

Petrotos, K.B., Karkanta, F.K., Gkoutsidis, P.E., Giavasis, I., Papatheodorou, K.N. and
Ntontos, A.C. (2014). Production of novel bioactive yogurt enriched with olive fruit
polyphenols, World Academy of Science. Engineering and Technology, p. 64.

Polyphenols: Properties, Occurrence, Content in Food and Potential Effects 261

Petrotos, K.B., Lellis, T., Kokkora, M.I. and Gkoutsidis, P.E. (2014). Purification of olive
mill wastewater using microfiltration membrane technology. Journal of Membrane
and Separation Technology, 3: 50–55.

Popa, V., Dumitru, M., Volf, I. and Anghel, N. (2008). Lignin and polyphenols as
allelochemicals. Industrial Crops and Products, 27(2): 144–9.

Quideau, S.P., Deffieux, D., Douat-Casassus, C.L. and Pouységu, L. (2011). Plant
polyphenols: Chemical properties, biological activities, and synthesis. Angewandte
Chemie International Edition, 50(3): 586.

Quideau, S. (2011). Why bother with polyphenols?. Group Polyphenols. 2011. Retrieved
2014 March 26.

Rice-Evans, C., Miller, N.J. and Paganga, G. (1997). Antioxidant properties of phenolic
compounds, Trends Plant Sci., 2: 152–159.

Russo, C. (2007). A new membrane process for the selective fractionation and total
recovery of polyphenols, water and organic substances from vegetation waters (VW).
Journal of Membrane Science, 288: 239–246.

Sannino, F., De Martino, A., Capasso, R. and El Hadrami, I. (2013). Valorisation of organic
matter in olive mill wastewaters: Recovery of highly pure hydroxytyrosol. Journal of
Geochemical Exploration, 129: 34–39.

Schulbach, K.F., Johnson, J.V., Simonne, A.H., Kim, J.M., Jeong, Y., Yagiz, Y. and Marshall,
M.R. (2013). Polyphenol oxidase inhibitor from blue mussel (Mytilus edulis) extract.
Journal of Food Science, 78(3): C425–C431.

Selma, M.V., Espin, J.C. and Tomas-Barberan, F.A. (2009). Interaction between phenolics
and gut microbiota: Role in human health. J. Agric. Food Chem., 57: 6485–6501.

Serafini, M., Laranjinha, J.A., Almeida, L.M. and Maiani, G. (2000). Inhibition of human
LDL lipid peroxidation by phenol-rich beverages and their impact on plasma total
antioxidant capacity in humans. J. Nutr. Biochem., 11(11–12): 585–590.

Shadabi, S., Ghiasvand, A.R. and Hashem, P. (2013). Selective separation of essential
phenolic compounds from olive oil mill wastewater using a bulk liquid membrane.
Chemical Papers, 67(7): 730–736.

Tanner, G. et al. (2003). Proanthocyanidin biosynthesis in plants. The Journal of Biological
Chemistry, 278: 31647–31656.

Tapiero, H., Tew, K.D., Nguyen, G.B. and Mathe, G. (2002). Polyphenols: Do they play a
role in the prevention of human pathologies. Biomed. Pharmacother., 56: 200–207.

Teresa, M., Reis, A., Ondina, M.F., Freitas, D., Ferreira, L.M. and Jorge Carvalho, M.R.
(2006). Extraction of 2-(4-hydroxyphenyl) ethanol from aqueous solution by emulsion
liquid membrane. J. Membr. Sci., 269(1/2): 161–170.

Tuck, K.L., Tan, H.W. and Hayball, P.J. (2002). Synthesis of tritium labeled hydroxytyrosol,
a phenolic compound found in olive oil. Journal of Agricultural and Food Chemistry,
48(9): 4087– 4090.

Tura, D. and Robards, K. (2002). Sample handling strategies for the determination of
biophenols in food and plants. J. Chromatogr. A, 975: 71–93.

Virgili, F. and Marino, M. (2008). Regulation of cellular signals from nutritional molecules:
A specific role for phytochemicals, beyond antioxidant activity. Free Radical Biology
and Medicine, 45(9): 1205–16.

Watson, E. (2012). Who has self-affirmed GRAS. FOOD navigator-usa.com.
Williams, R.J., Spencer, J.P. and Rice-Evans, C. (2004). Flavonoids: Antioxidants or

signalling molecules? Free Radical Biology and Medicine, 36(7): 838–49.
Williamson, G. and Manach, C. (2005). Bioavailability and bioefficacy of polyphenols in

humans. II. Review of 93 intervention studies. Am J. Clin. Nutr., 81(1): 243S–5S.
Woclawek-Potocka, I., Mannelli, C., Boruszewska, D., Kowalczyk-Zieba, I., Wasniewski,

T. and Skarzynski, D.J. (2013.). Diverse effects of phytoestrogens on the reproductive
performance: Cow as a model. International Journal of Endocrinology, Article ID
650984, 15 pages.
Yesilada, O., Sik, S. and Sam, M. (1999). Treatment of olive oil mill with fungi. Turkish
Journal of Biology, 23: 231–240.

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