polifenoles lectura rch

Chapter II ____________________________________________________________________

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II.1. Oxidative stress

The importance of oxidation in the body and in foodstuffs has been widely recognized. Oxidative metabolism is essential for the survival of cells. A side effect of this dependence is the production of free radicals and other Reactive Oxygen Species (ROS) that cause oxidative changes (Antolovich et al., 2002).

Oxygen is the primary oxidant in metabolic reactions designed to

obtain energy from the oxidation of a variety of organic molecules. Oxidative stress results from the metabolic reactions that use oxygen, and it has been defined as a disturbance in the equilibrium status of pro-oxidant/anti-oxidant systems in intact cells (Sies, 1991; Thomas, 1999). This definition of oxidative stress implies that cells have intact pro-oxidant/anti-oxidant systems that continuously generate and detoxify oxidants during normal aerobic metabolism (Thomas, 1999). All forms of life maintain a reducing environment within their cells. The cellular redox environment is preserved by enzymes that maintain the reduced state through a constant input of metabolic energy. When disturbances in this normal redox state are produced, toxic effects through the excessive production of free radicals or ROS may occur. Consequently, the pro-oxidant systems outbalance the anti-oxidants, producing oxidative damage to lipids, proteins, carbohydrates and nucleic acids. This may ultimately lead to cell death in case of severe oxidative stress (Halliwell, 1994; Wood et al., 2006).

ROS generation is essential for many important biological processes.

Pincemail et al. (2002) and Havsteen (2002) indicated that ROS, have an important physiological role, even at very low concentrations, acting as secondary messengers capable of (1) regulating apoptosis; (2) activating transcription factors (e.g. Nuclear factor kappa-beta) able to activate genes implicated in immune and inflammatory responses; (3) modulating gene expressions in order to activate antioxidant enzymes. However, when there is an over-production of these ROS that exceeds the capacity of defense,

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damage to valuable biomolecules may occur. The following important cellular damages can be produced: (1) ruptures and mutations at the DNA level; (2) denaturation of proteins; (3) oxidation of sugars; (4) induction of lipid peroxidation processes (Rice-Evans et al., 1996).

A number of chemical and physical events can initiate oxidation,

which proceeds then continuously in the presence of a suitable substrate until a blocking defense mechanism occurs. In vivo, many biochemical systems can lead to high ROS production (inflammation processes, hemoglobin oxidation, among others) (Pincemail et al., 2002). Besides, the environment where we live and our life style are also sources oxidative stress increase in our organism. Exercise can increase the levels of free radicals as can environmental stimuli such as ionizing radiation (from industry, sun exposure, cosmic rays, and medical X-rays), environmental toxins, altered environmental conditions (e.g. hypoxia and hyperoxia; ozone and nitrous oxide primarily from automobile exhaust), infectious agents, inadequate eating. Lifestyle stressors such as cigarette smoking and excessive alcohol consumption are also known to affect oxidative levels in our organism (Aruoma, 1998; Pincemail et al, 2002).

Oxidation can also affect foods. It is in fact one of the major causes of chemical spoilage (Colbert and Decaer, 1991), resulting in rancidity and/or deterioration of the nutritional quality, colour, flavour, texture and safety of foods (Shahidi et al., 1992). Also, photo-oxidation (Hamilton et al., 1997; Robards et al., 1999) and the temperature increase (Halliwell, 1994; Aruoma et al., 1997) are processes inducing radical formation that causes oxidative deterioration in foods.

As an example, it is known that lipid oxidation is considered one of

the main causes of food deterioration. Lipids (e.g. triacylglycerols, phospholipids) are naturally found in most biological materials consumed as foods and added as functional ingredients in many processed foods contributing to their texture, structure, mouth-feel and flavor. However,

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19

lipids are also one of the most chemically unstable food components and will readily undergo free-radical chain reactions (German, 1999). Polyunsaturated fatty acids (PUFA) oxidation can cause unpleasant odors, flavors and colors in foods, thereby reducing food shelf-life (Richards et al., 2005). Thus, because of the abundance of unsaturated lipids in muscle foods, such as meat and fish products and vegetable and fish oils, they are especially susceptible to the undesirable effects of lipid oxidation (Frankel, 1996; Richards et al., 2005). Moreover, a special attention must be given to the growing number of food products enriched in PUFA (omega-6 and omega-3). In addition, the lipids are dispersed in an aqueous phase in most of these foods. This dispersed state of lipids increases susceptibility to oxidation by favouring their contact with oxygen and pro-oxidant species (e.g. metal ions) dissolved in the aqueous phase. This increases the risk of development of oxidation during technological processes or storage (Villire et al., 2005; Villire et al., 2006).

Montero et al. (2005) mentioned that protein oxidation may occur

more rapidly than lipid oxidation in systems such as muscle, because protein is within the aqueous phase where many free radicals are formed. Furthermore, protein radicals can react with susceptible lipids to enhance the rate of lipid oxidation.

Taking into account the previous considerations, it appears of utmost

importance to produce and characterize components that may protect the food matrices from oxidative damage. On the other hand, mechanistic studies on these protective properties may be helpful for the design of optimized antioxidant agents.

II.1.1. Reactive oxygen or nitrogen species

A free radical can be defined as any chemical species having one or more unpaired electrons (Hamilton et al., 1997). Free radicals are thus


20

highly unstable, reactive and energized molecules that have electrons available to react with various organic substrates (Rice-Evans and Gopinathan, 1995; Schmidl and Labuza, 2000; Lee et al., 2004). One of characteristics of free radical reactions is that frequently they are not selective, but random.

There are numerous types of free radicals that can be formed within

the body. Most free radicals in biological systems are derivatives from oxygen, but there are also derivatives of nitrogen. One of the most reactive and damaging free radical is the hydroxyl radical (OH). Hydroxyl radical is considered to be a principal actor in the toxicity of partially reduced oxygen species since it is very reactive with all kinds of biological macromolecules. ROS is a collective term describing radicals and other non-radical reactive oxygen derivatives. These are responsible for the majority of radical degradations (Aruoma et al., 1997). Important ROS in living organisms are presented in Table 1.

Reactive Nitrogen Species (RNS) are nitrogen-based molecules that can act to facilitate nitrosylation reactions. RNS include among others: nitric oxide (NO), peroxynitrite (ONOO), peroxynitrous acid (ONOOH), nitroxyl anion (NO), nitrogen dioxide (NO2), nitrous acid (HNO2) (Schmidl and Labuza, 2000).

Both ROS and RNS may participate in reactions giving rise to free

radicals or damage to organic substrates.

Most free radicals are produced by mitochondria where 0.4 to 5% of the oxygen to generate energy results in the formation of ROS, such as superoxide and hydrogen peroxide. It is thus no wonder that most of the free radical damage is to mitochondrial membranes and mitochondrial DNA (Davies 1995; Lee et al., 2004).

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Table 1. Some important radical and non-radical reactive oxygen species in living organisms

Radicals Non-Radicals

Hydroxyl (OH) Hypochloric acid (HOCl)

Superoxide (O2) Hydrogen

Peroxide (H2O2)

Peroxyl (ROO) Singlet Oxygen (1O2)

Lipid peroxyl (LOO) Ozone (O3)

Lipid peroxide (LOOH)

Source: Adapted from ArUoma et al. (1997) and Schmidl and Labuza (2000).

Almost all cell types are capable of producing ROS during the

mitochondrial oxidation. In addition, several cell types do specifically generate ROS or RNS for dedicated purposes. For example, ROS such as superoxide and hydrogen peroxide are produced by phagocytes (macrophages and neutrophils) in order to kill some types of bacterias. The NO is produced within cells by the actions of a group of enzymes called nitric oxide synthases. NO has the ability to dilate blood vessels and relax smooth muscle tissue (Drew and Leeuwenburgh, 2002).

Free radicals react with target organic substrates such as lipids,

proteins, and DNA. Lipids, particularly the PUFA located in the membrane lipophilic section are subject to free radicals attack, which may lead to the production of lipid peroxides. This process is initiated by the action of an oxidizing radical, such as hydroxyl radical or superoxide anion (R) (Aruoma, 1998):


22

LH (substrate) + R L (lipidic radical) + RH L + O2 LOO (lipid peroxyl radical) LOO + LH LOOH (lipid peroxide) + L

II.1.2. Antioxidant defenses and antioxidants The aerobic organisms are protected from ROS and RNS by means

of defensive systems. These include various antioxidants, which have different functions. An antioxidant may be defined as any substance that when present at low concentration, compared with those of the oxidisable substrate, significantly delays or inhibits oxidation of that substrate (Halliwell, 1990; Gutteridge, 1994). The antioxidants comprise enzymes (e.g. superoxide dismutase, glutathione peroxidase, and catalase), proteins (albumin, ferritin) and small molecules (ascorbic acid, glutathione, uric acid, tocopherol, carotenoids, polyphenols) (Prior et al., 2005). Foods are an essential source of small-size antioxidants, as well as of trace elements, which play an important role as enzyme or protein cofactors (Shi and Noguchi, 2001). Table 2 shows some antioxidants, which constitute the defense system in vivo.

As shown in Table 2, there are several lines of defense. The first line

of defense is to inhibit the formation of ROS. This defense system is composed by preventive antioxidants that retard the rate of oxidation. This may be achieved in a number of ways including removal of substances, chelating transition metals or singlet oxygen quenching (Frankel and Meyer, 2000).

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Table 2. Defense systems in vivo against oxidative damage

1. Preventive antioxidants: suppress the formation of free radicals.

(a) Non-radical decomposition of hydroperoxides and hydrogen peroxide:

Catalase Glutathione peroxidase

(cellular) Glutathione peroxidase

(plasma) Glutathione-S-transferase Peroxidase

Decomposition of hydrogen peroxide 2 H2O2 2 H2O + O2 Decomposition of hydrogen peroxide and free fatty acid hydroperoxides H2O2 + 2 GSH 2 H2O + GSSG LOOH + 2GSH LOH + H2O + GSSG Decomposition of hydrogen peroxide and phospholipid hydroperoxides PLOOH + 2 GSH PLOH + H2O + GSSG Decomposition of lipid hydroperoxides Decomposition of hydrogen peroxide and lipid hydroperoxides LOOH + AH2 LOH + 2 H2O + A H2O2+ AH2 2 H2O + A

(b) Sequestration of metal by chelation: Transferrin, lactoferrin Haemopexin Haptoglobin Ceruloplasmin, albumin

Sequestration of iron Stabilisation of haem Sequestration of haemoglobin Sequestration of copper

(c) Quenching of superoxide and singlet oxygen: Superoxide dismutase (SOD) Carotenoids, vitamin E

Disproportionation of superoxide 2O2 + 2H+ H2O2 + O2 Quenching of singlet oxygen

2. Radical-scavenging antioxidants: scavenge radicals to inhibit chain initiation and break chain propagation.

Hydrophilic: vitamin C, uric acid, bilirubin, albumin Lipophilic: vitamin E, ubiquinol, carotenoids, flavonoids

3. Repair and de novo enzymes: repair the damage and reconstitute membranes.

Source: Adapted from Shi and Noguchi (2001)


24

The radical-scavenging antioxidants are the second line of defense. When these antioxidants are present in the system, they either delay or inhibit the initiation step by reacting with the free radical (L) or inhibit the propagation step by reacting with peroxyl (LOO) or alkoxyl (LO) radicals:

The antioxidant free radical (A) may further interfere with chain-

propagation reactions by combining with other radicals (termination reactions):

The third-line of defense is the repair. Various enzymes such as

lipases, proteases and DNA repair enzymes are responsibles for such defence (Schmild and Labuza, 2000).

Gutteridge (1994) indicated that other mechanisms of action for antioxidants include: enhancing endogenous antioxidant defenses by up-regulating the expression of the genes encoding the antioxidant enzymes, increasing elimination of damaged molecules and not repairing excessively damaged molecules in order to minimize introduction of mutations. Inhibition of oxidative enzymes (e.g. cyclooxygenase) is another mechanism of action of antioxidants (Huang et al., 2005).

The most suitable antioxidants are those that perform one or more of

the above functions, without generating any toxic or reactive end-products. As long as adequate amounts of antioxidants are present to provide sufficient

A + LOO LOOA

A + LO LOA

L + AH LH + A

LOO + AH LOOH + A

LO + AH LOH + A

Chapter II ____________________________________________________________________

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protection, the antioxidant-oxidant balance is maintained (Gutteridge, 1994; Halliwell, 1996).

When the classical antioxidant defenses or biological antioxidants

are not sufficient to avoid the oxidative damage in our organism, the consumption of external antioxidants must be increased through the diet or through food supplements (e.g. vitamins C and E, carotenoids and polyphenols) (Niki et al., 1995; Vansant et al., 2004). Compelling epidemiological evidence is available on the benefits of consuming diets rich in plant-based foods. This has led to a current advice consisting in the daily ingestion of several portions of fruits and vegetables for optimal health (Hooper and Cassidy, 2006). The consumption of fruits and vegetables containing high amounts of natural antioxidant molecules has indeed been associated with an improvement of the balance between free radicals and antioxidants, which helps to minimize the oxidative stress in the body and to reduce the risks of degenerative diseases (Prior, 2003; Lee et al., 2004). Natural antioxidant compounds derived from plants, such as ascorbate, tocopherol, carotenoids, bioactive plant phenols, are of the considerable interest from the viewpoint of dietary antioxidants supplementation, as well as of food preservation (Halliwell et al., 1995). As a result result, a lot of research has been conducted to identify and evaluate antioxidants from agricultural by-products, ethnic and traditional products, herbal teas, cold pressed seed oils, exudate resins, hydrolysis products, not evaluated fruits and edible leaves and other raw materials rich in antioxidants that have nutritional importance and/or the potential for applications in the promotion of health and prevention against damages caused by radicals (Dimitrios, 2006).

II.1.3. Methods for testing antioxidant capacity

There are a great variety of chemical and biological methods to evaluate the efficacy of antioxidants against oxidation. In oxidative processes, multiple reactions and mechanisms as well as different steps are


26

usually involved. Therefore, no single assay will accurately reflect all the radical sources or all the effects of antioxidants in a mixed or complex system (Frankel and Meyer, 2000; Awika et al., 2003; Prior et al., 2005). The features involved in oxidation are a substrate, an oxidant and an initiator, intermediates and final products. The measurement of any one of these can be used to assess antioxidant capacity (Clarkson, 1995). Thus, the antioxidant capacity of a sample can vary as a function of the choice for a particular substrate, oxidant, initiator or product formed (that indicates the end-point of reaction), as well as with the particular combination of them in the reaction medium. The medium conditions (e.g. pH, hydrophilicity, temperature, polarity) where oxidation occurs are another factor that may affect the antioxidant capacity of molecules (Moure et al., 2001). Taking all these constraints into account, it is recommended to evaluate the efficiency of antioxidant molecules on the basis of the results obtained by several methods, taking into account the different oxidative pathways, the analytical methods used to determine the extent and end-point of oxidation and different physico-chemical properties of the oxidisable substrates (Frankel, 1993a; Arnao et al., 1999, Frankel and Meyer, 2000; Sanchez-Moreno, 2002).

It is very important that methods to assess the antioxidant status reflect the oxidative processes associated with both the food and physiological systems (Silva, 2006). In foods, it is necessary to determine the efficacy of natural antioxidants for food protection against oxidative damage, to avoid deleterious changes and loss of commercial and nutritional value (Halliwell et al., 1995; Halliwell, 1997). On the other hand, Wood et al. (2006) mentioned that is important to evaluate the intake of dietary antioxidants and their real contribution to the antioxidant status of the human organism.

Prior et al. (2005) and Roginsky and Lissy (2005) listed the major

requirements neccesary for a good method of antioxidant capacity evaluation: (1) its mechanism of action must be based on a well-developed

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27

theory and be described by a definitive kinetic scheme; (2) it must make use of a biologically relevant radical source; (3) it has to be simple to allow the determination to be carried out in any laboratory; (4) the instrumentation must be readily available; (5) the repeatability of determinations should be sufficient within each scientific work and the reproducibility of the assay must be acceptable among all different laboratories; (6) it should be adaptable for both hydrophilic and lipophilic antioxidants and for different radical sources; (7) the mode of quantification must be clearly defined; (8) it should be adaptable to high-throughput analysis for routine quality control analyses.

In a review over the methodologies for the determination of

biological antioxidant capacity in vitro, MacDonalds-Wicks et al. (2006) mentioned that ideally, the antioxidant capacity should be tested using both in vitro and in vivo techniques but due to the high cost of conducting animal and human feeding trials, many products undergo only in vitro testing. Different in vitro assays have been described by the same authors such as: methods that measure the inhibition of induced lipid autoxidation (possible susbtrates could be: low density lipoproteins (LDL) or unsaturated fatty acids) and others as oxygen radical absorbance capacity (ORAC) assay, total radical-trapping antioxidant parameter (TRAP) assay, trolox equivalent antioxidant capacity (TEAC) assay (or ABTS assay), ferric iron reducing antioxidant power (FRAP) assay, 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) scavenging capacity assay. These same assays have been proposed by Sanchez-Moreno (2002) to estimate the oxidation in foods and biological systems. On the other hand, Antolovich et al. (2002) examined various methods of measuring antioxidant capacity related to lipid oxidation such as: accelerated stability tests, peroxide value, diene conjugation, thiobarbituric acid reactive substances (TBARS) and measurement of hexanal.

An excellent compilation of methods currently used to determine the

in vitro antioxidant capacity has been performed by Prior et al. (2005) and Souza (2007) and is presented in Table 3. The following sections describe


28

the in vitro methodologies that have been used in this thesis to determine the antioxidant capacity. II.1.3.1. ORAC assay

The oxygen radical absorbance capacity (ORAC) assay has been

widely used in measuring the net resultant antioxidant capacity of botanical and other biological samples (MacDonald-Wicks et al., 2006). Initially, this method was developed by Cao et al., (1993) and is based on the measurement of the free radical damage to a fluoresence probe through the change in its fluorescence intensity. The change in fluorescent intensity is an index of the degree of free radical damage. The inhibition of free radical damage by an antioxidant, visualized throug the protection against the change of probe fluorescence, is a mesure of its antioxidant capacity against the free radical (Cao et al., 1993; Ou et al., 2001; Huang et al., 2002a).

In the ORAC assay, different generators can be used to produce

different radicals such as the peroxyl radical (ROO), the hydroxyl radical (OH), or the transition metal Cu+2. However, the method has adopted the ROO as standard radical, since it is the most common in biological systems. Initially, the target protein was -phycoerythrin (-PE), whose loss of fluorescence was an indication of the extent of damage from its reaction with the peroxyl radical (Cao et al., 1993). Ou et al. (2001) adopted a new fluorescent substance (fluorescein, FL) to replace -PE as a probe, because -PE showed large inter-batch differences and interacted with polyphenols, through non-specific protein binding, resulting thus in a loss of fluorescence even without the added radical generator (Ou et al., 2001).

In general, either the sample, or a control or a Trolox standard (a water soluble analogue of vitamin E, at four or five different concentrations, used to construct a standard curve), are mixed with the FL solution and

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Table 3. Comparison of methods for assessing in vitro antioxidant capacity based upon mechanism, ROS generator, medium of reaction, endpoint, quantitation method and the adaptability of assays to measure lipophilic and hydrophilic antioxidants.

Antioxidant assay

Mechanism ROS generator

Medium of

reaction

Endpoint Quantitation Lipophilic and

hydrophilic AOC

ORAC HAT AAPH pH 7.4 Fixed time AUC LP/HP TRAP HAT AAPH pH 7.4 Lag phase IC50, lag time HP FRAP SET TPTZ/Fe+3 pH 3.6 Times,

varies DO fixed time HP

TEAC SET/HAT ABTS pH 7.0/ EtOH

Time DO fixed time LP/HP

FC SET FC reactive pH > 10 Fixed time DO fixed time HP DPPH HAT DPPH MeOH IC50 DO fixed time HP

CUPRAC SET Cu+2 H2O Time DO fixed time HP TOSC HAT KMBA-

ABAP pH 7.4 IC50 AUC HP

TBARS-LDL

HAT AAPH pH 7.4 Lag phase Lag time HP

Dines-LDL

SET Cu+2 pH 7.4 Lag phase Lag time HP

Hmolyse HAT AAPH pH 7.4 Lag phase DO fixed time HP AAPH: 2,2-azobis(2-amidinopropane) dihydrochloride ; ABAP: 2,2-Azobis(2-methylpropionamidine) dichloride ; ABTS: 2,2-azinobis(3-ethylbenzothiazoline)-6-sulfonate; AOC: Antioxidant capacity; AUC: Area under the courve; CUPRAC: Cupric-reducing antioxidant capacity; DO: absorbance; DPPH: 2,2-diphenyl-1-picrylhydrazyl; EtOH :Ethanol; FC: Folin-Ciocalteu; FRAP: Ferric reducing ability of plasma; KMBA: -keto--methiolbutyric acid; LDL: Low density lipoproteins; LP: Lipophilic; HP: Hydrophilic; ORAC: Oxygen radical absorbance capacity; HAT: Hidrogen atom transfer; TBARS: Thiobarbituric acid reactive substances; SET: Single electron transfer; TEAC: Trolox equivalent antioxidant capacity; TOSC: Total oxidant scavenging capacity; TPTZ: 2,4,6-Tripyridyl-1,3,5-Triazine; TRAP: Total radical-trapping antioxidant parameter (Source: Prior et al., 2005; Souza, 2007). incubated at 37C, before 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH) initiates the reaction. The reaction is measured at excitation: 485 nm and emission: 525 nm for changes in fluorescence every minute for 35 min. As the reaction progresses, FL is consumed and the fluorescence diminishes. In the presence of an antioxidant, the decay of FL is retarded. The data from the


30

assay are obtained by calculating the area under curve (AUC) and net AUC (AUCsample AUCblank (without sample)) (Figure 1). A standard curve is built up by plotting AUC against Trolox concentrations. The ORAC value of a sample is expressed in Trolox equivalents thanks to the use of the standard curve (Ou et al., 2001; Huang et al., 2002a).

Figure 1. Calculation for ORAC assay: Antioxidant capacity of tested sample expressed as the net area under the curve (AUC) (Source: Prior et al., 2005).

ORAC can be automated and can test hydrophilic and lipophilic

antioxidant capacity (Huang et al., 2002b). The ORAC method is reported to mimic the antioxidant capacity of phenols in biological systems better than other methods since it uses biologically relevant free radicals and integrates both time and degree of activity of antioxidants (Cao et al., 1993; Ou et al., 2001).

II.1.3.2. ABTS assay The ABTS assay was developed by Miller et al. (1993) and Re et al.

(1999) and has been used widely for testing antioxidant capacity in food samples. The ABTS assay measures the relative ability of an antioxidant to scavenge the ABTS+ radical (compound intensely colored). The ABTS+ radical is generated by the reaction of a strong oxidizing agent (e.g., potassium permanganate or potassium persulfate) with the ABTS salt (Re et

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al., 1999; Cano et al., 2002). The principle of the method is to monitor the decay of the radical-cation ABTS+ (disseapareance of blue-green color) caused by the addition of an antioxidant (Miller and Rice-Evans, 1997) (Figure 2).

Figure 2. Reaction between ABTS+ and antioxidant (Source: Miller and Rice-Evans, 1997; Huang et al., 2005).

ABTS+ has a strong absorption at the wavelength of 734 nm and can be easily determined spectrophotometrically. In the absence of antioxidant, ABTS+ is rather stable, but it reacts energetically in the presence of a scavenger antioxidant, such as a phenolic compound. The extent of color loss of ABTS+ is determined as a function of the concentration of an antioxidant-containing sample and time and calculated relative to the Trolox


32

standard determined under the same conditions (Arnao et al., 2001; Awika et al., 2003).

The ABTS assay is rapid and can be used in both aqueous and

organic solvent systems (Arnao et al., 1999; Lemanska et al., 2001). It has also good repeatability and is simple to perform; hence, it is widely reported (Arnao et al., 2001). Several limitations can be listed: 1) ABTS+ is not found in biological systems and is not similar to radicals found in those systems. 2) The reaction with ABTS+ occurs rather slowly with many phenolics and samples of natural products (Campos and Lissi, 1997; Lissi et al., 1999). Thus, the result of the determination of the ABTS assay is expected to be dependent on the time of incubation as well as on the ratio of sample quantity to ABTS+ concentration. 3) ABTS+ reacts with any hydroxylated aromatic compound independently of its real antioxidative potential (Arts et al., 2003).

II.1.3.3. Methods based on lipid peroxidation Several assays have been designed to evaluate lipid peroxidation. In

these assays a lipid (e.g. unsaturated fatty acid) or lipoprotein (LDL) substrate is used under standard conditions and the degree of inhibition of oxidation given by an antioxidant is measured (Snchez-Moreno and Larrauri, 1998).

Conjugated dienes assay. In this method, the autoxidation of unsaturated fatty acids (e.g linoleic acid) or LDL is induced by Cu+2/Fe+3 or an azo initiator as AAPH. Conjugated dienes are intermediate compounds of lipid oxidation (Figure 3). These compounds present a conjugated double-bond and their resonance can be measured at 234 nm (Puhl et al., 1994). Thus, the progress of oxidation in this reaction is monitored by UV absorbance at 234 nm (Pryor et al., 1993).

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Tipically, the assay solution contains a free radical initiator, a substrate and an antioxidant. A characteristic kinetics of conjugated diene formation is represented by three phases: a latence phase (lag time), a propagation phase (conjugated diene oxides accumulate rapidly) and a decline phase (degradation phase). Thus, when the antioxidant inhibits the oxidation process, longer lag times are obtained with respect to the reaction without antioxidant. The duration of the lag time is dependent on the concentration and capacity of the antioxidant molecule to inhibit oxidation (Puhl et al., 1994; Huang et al., 2005). The quantification of the conjugated dienes may be achieved by calculating the increase in absorbence per mass of sample at a fixed time (Frankel et al., 1996), lag phase measurements or as inhibition percentage (Antolovich et al., 2002).

Figure 3. Mechanism of lipid peroxidation (Source: Horton, 1987) The measurement of the formation of conjugated dienes has the

advantage that it measures an early stage in the oxidation process.


34

Conjugated diene measurements often cannot be performed directly on tissues and body fluids because many other interfering substances are present, such as haem proteins, purines, pyrimidines that strongly absorb in the same UV region (Maldhavi et al., 1996).

Thiobarbituric acid reactive substances (TBARS) assay. This method is widely used to detect lipid oxidation (Puhl et al., 1994). This procedure measures the malondialdehyde (MDA) formed as the split product of an endoperoxide of unsaturated fatty acids resulting from oxidation of a lipid substrate (Figure 3). MDA is a final compound of lipid oxidation (degradation step). To evaluate the antioxidative efficacy of the compounds against lipid oxidation, a substrate (e.g. linoleic or other fatty acids or LDL), an initiator (Cu+2/Fe+3, or AAPH), an antioxidant and thiobarbituric acid (TBA) are required. The MDA reacts with TBA to form a pink pigment (Figure 4) that is measured spectrophotometrically at its maximum absorption at 532 535 nm (Fernndez et al., 1997). Measurements can be made also by fluorescence at a excitation of 515 nm and emission of 555 nm (Janssens et al., 2002; Walter et al., 2004; Warnier, 2006; Souza et al., 2008). The procedure involves two steps: the substrate is oxidized with addition of the initiator and then the extent of oxidation is determined by the addition of TBA and a spectrophometric measurement of the product (TBARS) (Esterbauer and Cheeseman, 1990; Puhl et al., 1994). Oxidation is inhibited by addition of an antioxidant and therefore a reduction in the absorbance is seen.

Results are typically quantified against a calibration curve for

malondialdehyde bis-(dimethylacetal) or malondialdehyde bis-(diethylacetal), which acts as a source of MDA. Results may also be described in terms of inhibition percentage of the oxidation. The reaction in the TBARS assay is not very specific because TBA forms complexes with other compounds (e.g. aldehydes, oxidized lipids, etc) and also the reaction conditions have a significant effect on colour development (Esterbauer and Cheeseman, 1990).

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Figure 4. Chromophore formed by condensation of MDA with TBA (Source: Antolovich et al., 2002)

Erythrocyte oxidation assay. This assay was proposed by Yamamoto et al. (1985) as a mechanism to evaluate oxidation of biological membranes. Erythrocytes are vulnerable to lipid peroxidation due to their high content of polyunsaturated lipids, their rich oxygen supply, and the presence of transition metals (Miki et al., 1987; Niki, 1990; Tedesco et al., 2000). ROS generated in the aqueous or lipid phases can attack erythrocyte membranes and can induce the oxidation of lipids and proteins, triggering disruptions of the membrane and hemolysis (Miki et al., 1987; Niki et al., 1988, Tedesco et al., 2000). Numerous investigations have used erythrocytes as model systems for studying biomembrane oxidative damage (Miki et al., 1987; Niki et al., 1988; Janssens et al., 2002; Dai et al., 2006). In many of these studies, free radical initiators such as AAPH have been used to generate free radicals in the aqueous phase in order to attack the erythrocyte


36

membrane and propagate lipid peroxidation, leading to hemolysis (Miki et al., 1987; Niki et al., 1988).

The assay consists in measuring the resistance of red blood cells in

the presence of an initiator (e.g. AAPH) and antioxidant compounds (e.g. polyphenols, vitamin E or C) to the lipid peroxidation of their membranes. The quantity of disrupted red cells (hemolysis) can be measured at 540 nm (absorbance of oxyhemoglobin) (Mc Donald, 1994). This measurement describes the erythrocyte degradation in function of time (Zhu et al., 2002; Janssen et al., 2002; Dai et al., 2006). Protection of erythrocytes by an antioxidant is deduced from the time required for half-hemolysis, as compared to a control (without antioxidant). The inhibition of hemolysis of erythrocytes is calculated as % inhibition (Miki et al., 1987; Zhu et al., 2002).

Niki et al. (1988) mentioned that in the erythrocyte oxidation assay,

the water-soluble, chain-breaking antioxidants such as ascorbic acid was able to scavenge the oxygen radicals present in the aqueous phase, while lipid-soluble, chain-breaking antioxidants such as -tocopherol scavenged predominantly the radicals within the lipid region of the membranes. In addition, Liao and Ying (2000) indicated that the interaction of flavonoids with bio-membranes was an important factor in determining their structure-activity relationship. The interaction of an agent with membranes, or the uptake of an agent into the membranes is strongly related to its partition coefficient. Flavonols such as myricetin, quercetin and rutin which possess an ortho-dihydroxyl functional group have shown much more effective anti-hemolysis activity than other flavonols (morin and kaempherol) bearing no such functional group (Dai et al., 2006).

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II.2. Phenolic compounds

In the last years, researchers and food manufacturers have become increasingly interested in phenolic compounds. The main reason for this interest is the recognition of the antioxidant properties of phenolics (Yang et al, 2001), their great abundance in our diet, and their probable role in the prevention of various diseases associated with oxidative stress, such as cancer and cardiovascular and neurodegenerative diseases (Manach et al., 2004).

Many properties of plant and plant products are associated with the presence, type and content of their phenolic compounds (Robards et al., 1999; Cheynier, 2005). In plants, phenolics play among others the role of attractants for polynators, contributors to plant pigmentation, antioxidants, protective agents against UV light (Robards and Antolovich, 1997; Harborne and Williams, 2000), signal substances for establishment of symbiosis with rhizobia, insulating materials to make cell walls impermeable to gas and water and structural materials to give plant stability (Shahidi and Nackz, 2004). Besides, polyphenols are responsible for the major organoleptic characteristics of plant-derived food and beverages, particularly in terms of bitterness, astringency, colour, flavour and odour of products. They are also reported to contribute to the health benefits associated with the consumption of diets high in fruits and vegetables or plant-derived beverages (such as tea and wine) (Cheynier, 2005).

II.2.1. Classification and chemical structures Phenolic compounds form one of the main classes of secondary

metabolites in plants with a large range of structures and functions. They are found in fruits, vegetables, grains, barks, roots, stems, flowers, as well as in their respective derived products.


38

Phenolics are not uniformly distributed in plants at the tissue, cellular and subcellular levels. Insoluble phenolics are the components of cell walls while soluble phenolics are compartmentalized within the plant cell vacuoles (Yamaki, 1984; Beckman, 2000). At the tissue level, the outer layers of plants contain higher levels of phenolics than those located in their inner parts (Fernndez de Simn et al., 1992; Bengochea et al., 1997). Cell wall phenolics, such as lignins (the polymer of monolignol units) and hydroxycinnamic acids are linked to various cell components. These compounds contribute to the mechanical strength of cell walls and play a regulatory role in plant growth and morphogenesis and in the cell response to stress and pathogens (Lewis and Yamamoto, 1990; Nackz and Shahidi, 2004). The definition of phenolic compounds in terms of its metabolic origin is: substances derived from the shikimate pathway and phenylpropanoid metabolism (Robards et al., 1999; Shahidi and Nackz, 2004) (Figure 5).

Plant phenolics comprise a great diversity of compounds. These compounds may be classified into different groups as a function of the number of phenol rings that they contain and of the structural element that binds these rings to one another. Distinctions are thus made between the phenolic acids, stilbenes, lignins (nonflavonoids) and flavonoids (Figure 6) (Robards et al., 1999; Manach et al., 2004). In addition to this diversity, phenolic compounds may be associated with various carbohydrates and organic acids and with one another (Cheynier, 2005). Next, a brief description of some phenolic compounds found in the vegetal kingdom is presented.

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39

Figure 5. Production of phenylpropanoids, stilbenes, lignans, lignins, suberins, cutins, flavonoids and tannins from phenylalanine. PAL denotes phenylalanine ammonia lyase (Source: Shahidi and Nackz, 2004).


40

Figure 6. Classification of the major phenolic compounds (Source: Robards et al., 1999).

II.2.1.1. Phenolic acids

Phenolic acids have recently received considerable attention for their protective antioxidant behavior and potential health benefits (Robbins, 2003; Biroova et al., 2007). It has been reported that some phenolic acids posses antitumor activity against colon carcinogenesis (Olthof et al., 2001), besides inhibiting the AP-1 transcriptional activity implicated in the control of inflammation, cell differentiation, and proliferation (Maggi-Capeyron et al., 2001) and are potentially considered as protective compounds against cancer and heart diseases (Wen et al., 2005).

Phenolic compounds

Simple phenols(C6)

Phenolic acids Flavonoids(C6-C3-C6)

Hydroxybenzoicacids

(C1-C6)

Hydroxycinnamicacids

(C3-C6)

Stilbenes(C6-C2-C6)

Lignins(C6-C3)n

Chalcones Flavones Flavanones Flavonols Flavanonols Anthocyanins FlavanolsIsoflavones

Proanthocyanidins (Polymerization)

Phenolic compounds

Simple phenols(C6)

Phenolic acids Flavonoids(C6-C3-C6)

Hydroxybenzoicacids

(C1-C6)

Hydroxycinnamicacids

(C3-C6)

Stilbenes(C6-C2-C6)

Lignins(C6-C3)n

Chalcones Flavones Flavanones Flavonols Flavanonols Anthocyanins FlavanolsIsoflavones

Proanthocyanidins (Polymerization)

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41

The name phenolic acids, in general, describes phenols that possess one carboxylic acid functionality. Phenolic acids are subdivided into derivatives of hydroxycinnamic acids and derivatives of hydroxybenzoic acids (Figure 7), on the bases of C3-C6 and C1-C6 skeletons, respectively (Cheynier, 2005).

As seen in Figure 7(a), cinnamic acids present hydroxylations and

methylations in their structures. These compounds possess a phenyl ring (C6) and a C3 side chain and are thus collectively termed phenylpropanoids. They serve as precursors for the synthesis of lignins and many other compounds (Shahidi and Nackz, 2004).

The most common hydroxycinnamic acid derivatives are p-

coumaric, caffeic, sinapic and ferulic acids. These acids are rarely found in the free form, except in processed food that has undergone freezing, sterilization, or fermentation. The bound forms are glycosylated (glucose) derivatives or esters of quinic acid, shikimic acid and tartaric acid. Likely, the most familiar of these is chlorogenic acid (caffeic acid and quinic acid esterified) (Herrmann, 1989; Manach et al., 2004). The hydroxybenzoic acid content of edible plants is generally very low, with the exception of certain red fruits, black radish and onions, which can have concentrations of several tens of milligrams per kilogram of fresh weight (Shahidi and Nackz, 1995). Similar to cinnamic acids, hydroxylation and methylation of hydroxybenzoic acid leads to the formation of dihydroxybenzoic acid (protocatechuic acid), vanillic acid, syringic acid, p-hydroxybenzoic acid and gallic acid. Hydroxybenzoic acids are commonly present in the bound form in foods and are often the component of a complex structure like lignins and hydrolyzable tannins (e.g. gallotannins, ellagitannins) (Khanbabaee and van Ree, 2001).


42

(a)

(b)

Figure 7. Phenolic acids: members of the cinnamic (a) and benzoic acid (b) families found in food and nutraceuticals (Source: Nackz and Shahidi, 2004).

II.2.1.2. Flavonoids The flavonoids are typical phenolic compounds that act as potent

metal chelators and free radical scavengers (Harborne and Williams, 2000; Heim et al., 2002) and they are powerful chain-breaking antioxidants. Besides, some flavonoids have long been recognized to possess anti-inflammatory, antiallergic, hepatoprotective, antithrombotic, antiviral and anticarcinogenic activities (Middleton et al., 2000).

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43

Flavonoids are built upon a C6-C3-C6 flavone skeleton in which the three-carbon bridge between the phenyl groups is commonly cycled with oxygen (Figure 8). Several classes of flavonoids are differentiated on the basis of the degree of unsaturation and degree of oxidation of the three-carbon segment. Within the various classes, further differentiation is possible on the basis of the number and nature of substituent groups attached to the rings (Robards et al., 1999). Thus there are numerous substitution patterns in which primary substituents (e.g. hydroxyl, methoxyl, aromatic or glycosyl groups) can themselves be substituted (e.g. additionally glycosylated or acylated), sometimes yielding highly complex structures (Cheynier, 2005).

Figure 8. Nuclear structure of flavonoids (Source: Heim et al., 2002).

Flavonoids are subdivided in several groups. Flavanols, flavonols

and anthocyanins are the quantitatively dominant groups in plants (Robards and Antolovich, 1997). Flavanols. Flavanols are also known as flavan-3-ols, flavans or catechins. These compounds exist in the monomeric form as well as in the form of oligomers and polymers, which are both referred to as condensed tannins or proanthocyanidins. The main flavanols are catechins and they are found in many types of fruits (e.g. apricots, apples), as well as in green tea, red wine and chocolate (Yang et al., 2001). Catechin and epicatechin are the main flavanols in fruits, while gallocatechin, epigallocatechin and epigallocatechin gallate are found in certain seeds of leguminous plants, grapes and more importantly in tea (Arts et al, 2000a, Arts et al., 2000b). The flavan-3-ols (flavanol monomer) such as: catechin, epicatechin,


44

gallocatechin and epigallocatechin are found in fruits, generally in the free form rather than in the glycosylated forms (Robards et al., 1999; Manach et al., 2004). The chemical structure of different flavan-3-ol units is shown in Figure 9.

Figure 9. Structure of flavan-3-ol units (Source: Gu et al., 2003).

Structurally, the proanthocyanidins are composed of mixtures of oligomers and polymers containing flavan-3-ol units, linked mainly through C4C8 and/or C4C6 bounds (both are called B-type, Figure 10). The flavan-3-ol units can also be doubly linked by an additional ether bond between C2O7 (A-type) (Porter et al., 1991). In addition the proanthocyanidins family also comprises forms with ester bonds to gallic acid (Hammerstone et al., 2000; Nuez et al., 2006). The proanthocyanidins consisting exclusively of (epi)catechin are designated as procyanidins, whereas the proanthocyanidins containing (epi)gallocatechin as subunits are named prodelphinidins (Lazarus et al., 1999; Xie and Dixon, 2005; Gu et al., 2006), this last form being less common in nature.

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45

Figure 10. Representative structures of procyanidin dimers (B-type procyanidins). (a) Procyanidin B2 (b) Procyanidin B5. Where: R1 = OH, R2 = H (Source: Hammerstone et al., 2000).

The molecular weight of proanthocyanidins expressed as degree of polymerization (DP) is one of the most important characteristics of these compounds. Gu et al. (2002) defined proanthocyanidins with DP = 2-10 and DP > 10 as oligomers and polymers, respectively.

Flavonols. These are the most ubiquitous flavonoids in foods and the main representatives are quercetin and kaempherol. The richest sources are onions, leek, brocoli, tea and blueberries (Manach et al., 2004). These compounds posses a double bound between C-2 and C-3 and also possess a hydroxyl group in the 3-position (Figure 11). Flavonols may vary in the number and distribution of hydroxyl groups as well as in their degree of alkylation or glycosylation. The formation of flavonol glycosides depends on the action of light; therefore, they are found mainly in leaves and fruit skins with only trace amounts in parts of plants below the soil surface (Hermann, 1976).

(a) (b)


46

Figure 11. Structures of the main flavonol compounds (Source: Shahidi and Nackz, 2004).

Flavonols are present mainly as mono-, di- and triglycosides. The monoglycosides occur mainly as 3-O-glycosides. The 3-O-diglycosides and 3,7-di-O-glycosides are also found frequently. Rutin, which present a rutinose molecule (6-O--rhamnosyl-D-glucopyranose) in its structure, is an example of a diglycoside quercetin (Shahidi and Nackz, 2004). The sugar moieties of glycosides are usually composed of D-glucose, D-galactose, L-rhamnose, L-arabinose and their combinations. A number of flavonol glycosides acylated with phenolic acids such as p-coumaric, ferulic, caffeic, p-hydroxybenzoic and gallic acids have been found in fruits (Harborne and Williams, 2000).

Anthocyanins. After the flavanols and flavonols, the anthocyanins

are the next most abundant and widely distributed group of flavonoids. They are water-soluble pigments responsable for the bright red, blue and violet colors of fruits and other plant organs (Mazza and Miniati, 1993). Anthocyanins are present in many fruits, vegetables, flowers, leaves, roots and other storage organisms of plants. Six anthocyanidins are widespread and commonly contribute to the pigmentation of fruits. Cyanidin is the most common and, in terms of frequency of occurrence, is followed in decreasing order by delphinidin, peonidin, pelargonidin, petunidin and malvidin (Figure 12).

Flavonol Kaempferol 3,5,7,4 = OHQuercetin 3,5,7,3,4 = OHMyricetin 3,5,7,3,4,5 = OHMorin 3,5,7,2,4= OH

Flavonol Kaempferol 3,5,7,4 = OHQuercetin 3,5,7,3,4 = OHMyricetin 3,5,7,3,4,5 = OHMorin 3,5,7,2,4= OH

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47

Figure 12. Structures of the anthocynidins (Source: Shahidi and Nackz, 2004).

Chemically, anthocyanins are glycosylated derivatives of the

3,5,7,3-tetrahydroxy-flavylium cation. The aglycon moeities are collectively named anthocyanidins. These are highly reactive and they do not occur naturally in the free aglycon form. A wide range of anthocyanins exist. They differ in the nature and number of sugars attached to the aglycon, as well as in the position of the attachment and in the nature and number of aliphatic or cinamic acids attached to the sugar residues. The presence of several hydroxyl groups on the anthocyanidins as well as one or several sugar molecules, make these compounds quite soluble in water, ethanol and methanol (Escribano-Bailn et al., 2004).

The most commonly found sugars are xylose, arabinose and rhamnose among pentoses and galactose and glucose among hexoses. They are linked to the aglycon by a or linkage of the free hydroxyl. Di- and trisaccharide functional groups are also common, the most usual ones being rutinose (6-O--rhamnosyl-D-glucopyranose) and sophorose (2-O--


48

glucopyranosyl-D-glucopyranose). Sugar moieties are always found attached to the hydroxyl group at position 3 of the anthocyanidin. When additional sugar groups exist, they occupy positions 5 and/or 7, and less frequently 3 and 5. Sugars may be substituted by cinnamic acids (e.g. caffeic, p-coumaric, ferulic, - hydroxybenzoic or sinapic acid) and/or aliphatic acids (e.g. acetic, malic, malonic, oxalic or succinic acid) (Delgado-Vargas et al., 2000). These acyl substituents are commonly bound to the C-3 sugar through an ester bond with the 6-OH or less frequently with the 4-OH group of the sugar (Clifford, 2000). II.2.2. Phenolic compounds as antioxidants A polyphenol substance can be defined as antioxidant only if it fulfils two conditions: firstly, when present at low concentration relative to the substrate to be oxidized, it can delay or prevent its oxidation; secondly, the resulting radicals formed after scavenging must be stable (Kaur and Kappor, 2001). Andersen and Markhan (2006) mentioned that there are approximately 10000 known plant phenolics and model studies have demonstrated that many have antioxidant capacity (Robards et al., 1999). However, there is a wide degree of variation among different phenolic compounds in their efectiveness as antioxidants. Furthermore, there are a number of different mechanisms by which phenolics may act as antioxidants: via free radical scavenging, singlet oxygen quenching, metal-ion chelation (Robak and Gryglewski, 1988; Hamilton et al., 1997; Harborne and Williams, 2000), or inhibition of oxidative enzymes (Francis, 1989). Additional mechanisms may be involved in vivo where phenolics may protect the -tocopherol from oxidation by preferentially oxidise themselves; alternatively, they may regenerate -tocopherol by donating an hydrogen atom to the -tocopherol radical (Miura et al., 1994).

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49

Cook and Samman (1996) stated that flavonoids could act at any of the three stages of the radical-mediated oxidation. Flavonoids could block initiation by scavenging primary radicals such as superoxide. Flavonoids could also react with peroxy radicals to slow down propagation. In addition, the flavonoid radical intermediates, formed after reacting with peroxy radicals, can react with the other radicals formed during propagation, accelerating the termination process. Robards et al. (1999) indicated that phenolic antioxidants function primarily as terminators of the free radical reactions, through their ability to interfere with the chain propagation reactions by rapid donation of hydrogen or electrons to lipid radicals.

Rice-Evans et al. (1997) and Fukumoto and Mazza (2000)

mentioned that the reactivity of phenolic compounds in relation to the radical species follows a mechanism of exchange of reducing equivalents. Besides, the reactivity of phenolic compounds to face radicals depends on the configuration as well as the position and number of hydroxyl groups in their molecules. Thus, the radical scavenging capability of flavonoids has been related to their structural groups: the o-dihydroxyl structure of the B ring (catechol ring), the 2,3-double bond in conjunction with the 4-oxo function, and the additional presence of both 3-and 5-hydroxyl groups (Figure 13) (Bors et al., 1990; Rice-Evans et al., 1996; Rice-Evans et al., 1997; Lien et al., 1999; Heim et al., 2002). For this reason flavanoids containing a catechol moiety (3- and 4-OH) in B-ring or an AC-ring with three OH groups (3-, 5-, and 7-OH) are potent scavengers.


50

Figure 13. Representation of three important structures for neutralizing free radicals by flavonoids (Source: Adapted of Rice-Evans et al., 1996; Rice-Evans et al., 1997).

Heim et al. (2002) found that multiple hydroxyl groups conferred substantial antioxidant, chelating and, in some cases, pro-oxidant activity to the molecule. Methoxy groups introduce unfavorable steric effects, but presence of a double bond and a carbonyl functionality in the C ring increases the activity by affording a more stable flavanoid radical through conjugation and electron delocalization (Heim et al., 2002). The degree of glycosylation with monosaccharides or disaccharides significantly affects the antioxidant properties of the compound: for example, aglycones of quercetin and myricetin were found more active than their glycosides (Shahidi et al., 1992). In vitro assays have demonstrated that the increase of the polymerization degree of flavan-3-ols enhances their effectiveness against a variety of radical species (Heim et al., 2002; Counet and Collin, 2003). Extensive conjugation between 3-OH and B-ring catechol groups, together with the abundant 48 linkages, endow a polymer with significantly higher radical scavenging properties by increasing the stability of its radical (Castillo et al., 2000). The presence of hydroxyl groups at C-3 and C-3 positions is essential for flavonoids to show a strong superoxide-scavenging activity (Shi and Noguchi, 2001).

The antioxidant capacity of phenolic acids and their esters depends

on the number of hydroxy groups in the molecule. Hydroxycinnamic acids

Chapter II ____________________________________________________________________

51

have been found to be more effective than their benzoic acid counterparts. Hydroxycinnamic acids are known as primary antioxidants and act as free radical acceptors and chain breakers (Robbins, 2003; Shahidi and Nackz, 2004).

Another way, flavonoids may prevent radical formation is by

chelation of transition metals. These metals are essential to certain physiological functions, such as the transfer of oxygen in the human body, the stabilization of the three-dimensional structure of proteins, or the catalytic activity of several types of enzymes. They are however also involved in the formation of free radicals. By consequence, metals, such as iron and copper (Fe+2, Cu+) play an important role in the initiation and propagation steps of lipid oxidation. On one hand, the presence of a transition metal can accelerate the initiation step through the removal of an hydrogen from an unsaturated lipid to form a lipid radical.

RH + M+n R + H+ + M+(n-1)

On another hand, metals can also decompose hydroperoxides to form alkoxyl and peroxyl radicals, accelerating the lipid oxidation (Lee et al., 2004).

Metals are also responsible of hydroxyl radical formation, according to the Fentons reaction (Favier, 2003):

They are also implicated in the singlet oxygen formation:

Fe3+(Cu2+) + ROOH Fe2+ (Cu+) + ROO + H+

Fe2+ (Cu+) + ROOH Fe3+ (Cu2+) + RO + OH-

Fe2+(Cu+) + 2O2 Fe+3(Cu+2) + 1O2 + O2

Fe2+(Cu+) + H2O2 Fe+3(Cu+2) + OH + OH


52

Many flavonoid structures have the chemical properties to chelate these metals in such a way that radical generation is inhibited. Chelation of metal ions renders them catalytically inactive. The formation of stable and inert metal complexes is thus a potential mechanism of antioxidant action (Lee et al., 2004). Flavanoids are known to chelate metal ions at the 3-hydroxy-4-keto group and/or 5-hydroxy-4-keto group (when the A-ring is hydroxylated at the fifth position). An o-quinol group at the B-ring also bears a metal chelating activity (Figure 14) (Brown et al., 1998).

Figure 14. Flavanoids and their proposed sites for chelation of metal ions (Men+) (Source: Brown et al., 1998). II.2.3. Biological properties of phenolic compounds

Phenolic compounds have been reported to have a wide diversity of

beneficial effects on human health. Thus, phenolic compounds have received attention as protective and preventive molecules against chronic diseases, such as atherosclerosis and cardiovascular diseases, anti-inflammatory process, cancer, osteoporosis (Middleton et al., 2000; Arts and Hollmanm 2005; Sarni-Manchado and Cheynier, 2006).

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53

It is very important to indicate that in vivo properties of polyphenols depend on their bioavailability (Scalbert and Williamson, 2000). Human and animal studies on the absorption, distribution, metabolism and elimination of dietary phenolics are key steps to know the conditions and quantity of phenolic compounds that are effectively used in the body. These results are relevant in order to further explain the systemic positive effects as well as the nutritional quality that are attributed to polyphenols (Martin and Andriantsitohaina, 2002), and also to correlate their consumption with health beneficial properties. Bioavailability varies considerably from one polyphenol to the other (Yang et al., 2001).

In most countries, a high intake of saturated fats is strongly

correlated with high mortality from coronary heart disease (CHD). But exceptions occur. The best example is the so-called French paradox, where populations of the South of France, in spite of a high average body mass index and a high fat intake are less exposed to cardiovascular diseases. This behavior has been attributed to the regular intake of red wine in the diet (Halliwell, 2000). Frankel et al. (1993b) have concluded that epicatechin and quercetin are the most important wine constituents in reducing CHD. They also supported a previous suggestion that it is the specific combination of antioxidant phenolics in wine that protects against atherogenesis. Frankel et al. (1993b) suggested that the beneficial effects of red wine may be explained by the inhibition of oxidation of LDL by wine phenolics. The oxidative damage to human LDL (particularly to the apolipoprotein B) is considered to be an important step in the development of atherosclerosis. It is a prerequisite for macrophage uptake and cellular accumulation of cholesterol leading to the formation of the atheromal fatty streaks (Esterbauer et al., 1992; Arts and Hollman, 2005).

A wide range of phenolic compounds have been reported to protect

human LDL against oxidative damage (Arts and Hollman, 2005; Kuriyama et al., 2006). Satu-Gracia et al. (1999) reported that phenolics present in Spanish sparkling wines (cavas) inhibit oxidation of LDL. This activity


54

correlates positively with the total phenolic content, as well as the content of quercetin 3-glucuronide, trans-caffeic acid, protocatechuic acid, and coumaric acid. Phenolic acids present in the human diet, such as caffeic and chlorogenic acids, ellagic acid and protocatechuic acid, all protect isolated LDL against oxidative damage (Laranjinha et al., 1994; Robbins, 2003; Wen et al., 2005). The protective effects of anthocyanins against LDL oxidation have been demonstrated in different studies (Ghiselli et al., 1998; Khknen and Heinonen, 2003; Chang et al., 2006). Also, grape seed extracts rich in procyanidins have been shown to have antiatherosclerotic activities (Shrikhande, 2000; Corder et al., 2006). The protection of phenolic compounds against LDL oxidation could be related to the capacity of phenolic compounds to scavenge free radicals (Zhu et al, 2002), to chelate metals and/or to interact with lipid-rich structures (Teissdre et al, 1996; Liao and Ying, 2000; Dai et al., 2006). The partition coefficient of phenolic compounds determines their interaction with these structures and influences their antioxidant capacity performance at this level (Liao and Yin, 2000). As an example, it is well known that the flavonoids and related polymers have different types of interactions with lipid-rich structures. The more hydrophobic flavonoids can partition in the hydrophobic core of these structures, leading to a direct inhibition of lipid oxidation. The more hydrophilic flavonoids interact by hydrogen bonding with the polar head groups at the lipid-water interface of the lipid-rich structures. This type of interaction may provide a direct or indirect protection from external and internal aggressors (Oteiza et al., 2005).

In addition to their cardiovascular protective effect, phenolic

compounds appear to bear antitumor activities (Middleton et al., 2000; Yang et al., 2001; Carnesechi et al., 2002; Ferguson et al., 2004). The epigallocatechin-3-gallate, a polyphenolic component of green tea, has been found to reduce the incidence of spontaneous and chemically-induced tumors in experimental animals, as observed for tumors of liver, stomach, skin, lung and esophagus (Huang et al., 1992). The ellagic, protocatechuic and chlorogenic acids present in fruits and vegetables have been found to

Chapter II ____________________________________________________________________

55

serve as chemopreventive agents against several carcinogens (Nakamura et al., 2001).

Phenolic compounds have also been studied for their anti-inflammatory activities (Middleton et al., 2000; Crouvezier et al., 2007). Cyclooxygenase and lipoxygenase play an important role as inflammatory mediators. They catalyse the first steps of the production of pro-inflammatory eicosanoids from arachidonic acid (Nijveldt et al., 2001). Selected phenolic compounds were shown to inhibit both cyclooxygenase and 5-lipoxygenase pathways, through an inhibiton of the release of arachidonic acid (Kim et al., 1998; Havsteen, 2002).

Isoflavones exert a broad spectrum of biological activities. Some

isoflavone derivatives have a chemical structure resembling that of estrogens. They are thus able to bind to estrogen receptors. By consequence, they bear hormonal agonistic or antagonistic properties depending on the organ and on the age of the consumer. Other flavonoids such as flavones, flavonols and flavanones have also shown affinity for the estrogen receptor (Harborne and Williams, 2000). Besides estrogenic activities, isoflavones protect against several chronic diseases. Results of epidemiological studies indicate that consumption of soybean isoflavones lowers the incidence of breast, prostate, urinary tract and colon cancers. These compounds also provide protection against coronary heart diseases and osteoporosis (Moyad, 1999; Su et al., 2000).

II.2.4. Methods for quantifying phenolic compounds

Numerous methods for quantifying phenolic compounds exist in the

literature, some being more used than others. The most common techniques are based on spectrophotometric and chromatographic measurements.


56

II.2.4.1. Spectrophotometric assays

Folin-Ciocalteu assay. This assay is commomly known as the total phenolic assay (Singleton and Rossi, 1965). The basic mechanism is an oxidation/reduction reaction and, as such, can be considered another method to evaluate the antioxidant capacity (Prior et al., 2005). Reduction of phosphomolybdic-phosphotungstic acid complexes (Folin-Ciocalteus reagent, chromogen) to a blue-colored complex in an alkaline solution (pH ~ 10) occurs in the presence of phenolic compounds. The color development is due to the transfer of electrons to reduce the chromogens. Huang et al. (2005) mentioned that, in essence, the molybdenum (Mo) is more easily reduced in the complex and the electron-transfer reaction occurs between reducing compound and Mo (VI) (Mo (VI) + e Mo (V)). The reaction occurs progressively and the absorbance increases during 30 minutes at wavelengths between 725 and 765 nm.

The Folin-Ciocalteu reagent is not specific and detects all phenolic

groups found in extracts, including those found in the extractable proteins. Another disadvantage of this assay is the interference of reducing substances such as ascorbic acid and sugars. In general, the content in phenolics is expressed as gallic acid equivalents, but others acids (e.g. chlorogenic acid) or even flavonoids (e.g. catechin) can be used, depending on the major phenolic compounds present in the sample.

DMACA (4-(dimethylamino)-cinnamaldehyde) assay. This

DMACA assay is used for the quantification of flavanoids that contain meta-oriented di- or tri-hydroxy substituted benzene rings, with a single bond at the 2,3 position of the C-ring, such as flavan-3-ols, flavan-4-ols, flavan-3,4-diols, flavanones and derivatives. Delcour and Janssens de Varebeke (1985) and Treutter (1989) demonstrated that DMACA does not react with a wide range of flavonoids including dihydrochalcones, flavones and flavonols, and phenolic acids.

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In the DMACA assay, a condensation reaction occurs between the flavanoid molecule and the DMACA in acidic medium, producing the formation of a green chromophore. The DMACA colorimetric assay consists in measuring the absorbance of the reaction medium at 635-640 nm, after aproximately 15 min at room temperature. An appropriate blank must be included. DMACA reacts only with the terminal groups of condensed tannins (Figure 15). The results are usually expressed as mg of catechin equivalents.

CH3

NCH3

CH3

O

OH

OH

OH

OH

OH

N+CH3 CH3

O

OH

OH

OH

OH

OH

+ H2O

Figure 15. Reaction between flavan-3-ols and DMACA in an acidic medium (Source: Mcmurrough and Baert, 1994).

Anthocyanins assay. Anthocyanins are colored pigments and can be quantified by means of colorimetric measurements. The pH differential method (Giusti and Worlstad, 2001) is the most widely used assay to quantify monomeric anthocyanins. Anthocyanin pigments undergo reversible structural transformation with a change in pH. These changes are associated to strikingly different absorbance spectra. The colored flavylium cation form predominates at pH 1.0 while the colorless hemiketal form is the major form at pH 4.5 (Figure 16). For each pH value, the measurement is performed at two wavelengths. To determine the total anthocyanin content, the absorbance at pH 1.0 and 4.5 is measured at the wavelength of visible-maximum and at 700 nm, which allows for haze correction (due to the possible presence of sediments). When the predominant anthocyanin in the


58

Figure 16. Structural transformation of anthocyanins in function of pH (Source: Malien-Aubert et al., 2001).

sample is not known, the wavelength maximum to choose is 520 nm. This corresponds to the wavelength of cyanidin-3-glucoside, which is the most widely found anthocyanin in colored vegetables. The difference in

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59

absorbance is then calculated by subtracting the reading at pH 4.5 from that at pH 1.0 ([Avis-max A700]pH 1.0 [A vis-max A700]pH 4.5), The result is divided by the average extinction coefficients for the four major anthocyanins or by the extinction coefficient of the principal anthocyanin of the sample in order to yield the total anthocyanin content.

II.2.4.2. Chromatographic assays

Various chromatographic techniques have been employed for the separation, preparative isolation, purification, identification and quantification of phenolic compounds such as the high performance liquid chromatography (HPLC) technique, high-speed countercurrent chromatography, thin layer chromatography (Merken and Beecher, 2000; Shahidi and Nackz, 2004).

HPLC techniques are now the most widely used for the separation

and quantitation of phenolic compounds. The chromatographic conditions of the HPLC methods include generally a reversed-phase C18 column, a UV-Vis diode array detector, and a binary solvent system containing acidified water and a polar organic solvent (Tsao and Yang, 2003). Phenolic compounds are identified and quantified by comparing their retention times and UV-visible spectral data to known previously injected standards. The advantages of this technique are 1) the possibility to quantify individual phenolic compounds belonging to different phenolic families thanks to the reverse phase conditions; 2) its versatility, high sensitivity, exactitude and precision (Robards, 2003; Parejo et al., 2004). The main disavantage is its high cost.

Prior to phenolic analysis by HPLC, several steps are usually employed to favour a good performance during the run. These steps comprise liquid-liquid extraction, solid-phase extraction (SPE) or the fractionation of phenolics from crude extracts (Rodriguez-Saona and Wrolstad, 2001; Robards, 2003; Prior et al., 2004). Several works have been


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published on the application of the HPLC methodology for the analysis of phenolics (Robards and Antolovitch, 1997; Merken and Beecher, 2000; Mattila and Kumpulainen, 2002; Tsao and Yang, 2003; Wu and Prior, 2005). On the other hand, normal phase conditions using a fluorometric detector have been successfully applied for proanthocyanidin analysis (Gu et al., 2002). Mass spectrometry (MS) detectors coupled to HPLC (HPLCMS) have been widely employed for the structural characterization of phenolics (Shahidi and Nackz, 2004). More information about this technique is given in section II.3.4.2.

II.3. Extraction, purification and identification processes for phenolic compounds

Prior to the identification of phenolic compounds, several processes

including the extraction of plant materials and a purification step are needed. Today, many methodologies, equipments and solvents are available to achieve an adequate extraction and purification of phenolics. The adequate choice and successfully sequence of operations can warranty an efficient recovery of phenolics from different materials to be analyzed.

The following sections describe the extraction and purification

processes that have been used in the present thesis for analytical purposes to identify phenolic compounds.

II.3.1. Extraction processes Extraction is the first step in the isolation and later identification of

phenolic compounds from botanicals. Due to the chemical nature of phenolic compounds, no satisfactory solvent extraction system is suitable for the isolation of all classes of phenolics. By contrast, different techniques of

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phenolic extraction are used in different studies. Escribano-Bailn and Santos-Buelga (2003) listed the three major techniques that may be used in phenolics extraction are: a) extraction using solvents, b) solid-phase extraction (SPE) and c) supercritical extraction. The extraction using solvents can be divided in solid-liquid extraction and liquid-liquid extraction. In the present section, more information about solid-liquid extraction will be given because solid matrices are more frequently used as source of phenolics than liquid matrices.

Gertenbach (2002) defined a solid-liquid extraction as the use of a

solvent to dissolve and remove a soluble fraction (called the solute, such as a phenolic compound) from an insoluble, permeable solid matrix (plant tissue). In this process the ground plant material is added to the solvent in varying solvent to solid ratios. The mixture is then constantly stirred until an uniform slurry is obtained. If a large contact area is obtained between the two phases, the extraction occurs rapidly. After an appropriate amount of time, the solvent is centrifugated or filtered out of the slurry and the final extract is obtained (Shi et al., 2005).

The main factors that contribute to the efficiency of solvent

extraction are: type of solvent, pH, temperature and time of extraction, number of extraction steps, solvent/material ratio, size of particle.

Type of solvent. The choice of solvent can significantly change both

the type and amount of phenolics that are extracted into the liquid, as well as the rate at which the phenolics are extracted. The solubility of phenolic compounds is governed by the type of solvent (polarity) used. In phenolic extractions, highly polar solvents, such as water, or highly apolar solvents, such as chloroform or hexane, do not usually give good recoveries (Liu et al., 2000). Water solvents produce extracts with a lot of impurities (soluble compounds with no phenolic nature), which make the later purification process more difficult. When the extraction of many components from a solid is required, mixtures of solvents are used, resulting in a solution with


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medium polarity, allowing satisfactory constituent yields for compounds with distinct polarities (Liu et al., 2000).

The most widely used solvent for extracting phenolic substances for

analytical purposes are methanol and methanol/water (Escribano-Bailn and Santos-Buelga, 2003). Other solvents such as acetone, ethanol, ethyl acetate and, to a lesser extent, propanol, and their combinations have also been utilized for the extraction of phenolics (Nackz et al., 1992; Antolovich et al., 2000; Ju and Howard, 2003).

Glycosylated phenolic compounds (more water soluble) are

generally extracted using combinations of water with methanol, ethanol or acetone (Rice-Evans et al., 1997). In contrast, less polar phenolics (aglycones) as isoflavones, flavanones and highly methoxylated flavones and flavonols tend to be more soluble in rather hydrophobic solvents (Bradshaw et al., 2001). Acetone and methanol are employed for extraction of flavan-3-ol compounds but with distinct specificities in the extraction of these substances. Thus, methanol is the best solvent for catechin extraction (flavan-3-ol monomer), whereas acetone presents a better yield for procyanidins (Escribano-Bailn and Santos-Buelga, 2003).

pH of the extraction medium. The addition of acid in the extraction

solvent determines the stability of phenolic compounds such as anthocyanins (Rodriguez-Saona and Wrolstad, 2001), influences the possible solubilization of the hydrolysable polymers, such as lignins, hydroxycinnamic acids and procyanidins (Nackz and Shahidi, 2004; Tsao and Deng, 2004) and finally may improve the desintegration of cell walls, facilitating the solubilization of phenolic compounds.

Anthocyanins are usually extracted with an acidified organic solvent,

most commonly methanol and acetone (Rodriguez-Saona and Wrolstad, 2001). This solvent system destroys the cell membranes and simultaneously dissolves the anthocyanins. The acid also provides favorable conditions for

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the formation of the flavylium chloride salt, thus stabilizing the anthocyanins. The most commonly used acids for anthocyanin extraction are: HCl (stronger acid), tartaric and citric acids (weaker acids), or trifluoroacetic acid (volatile stronger acid). Escribano-Bailn and Santos-Buelga (2003) indicated that the use of methanol containing 0.1% HCl does not cause a significant degradation of the most usual monoacylated anthocyanins.

Tannins from sorghum and dry beans have been extracted using 1%

HCl in methanol (Shahidi and Nackz, 2004). Temperature of extraction. Cacace and Mazza (2003) and Liyana-

Pathiirana and Shahidi (2005) mentioned that the polyphenol yield is increased with increasing temperature. High temperatures improve the efficiency of the extraction since heat renders the cell walls permeable, increases the solubility and diffusion coefficients of the compounds to be extracted and decreases the viscosity of the solvent, thus facilitating its passage through the solid mass (Escribano-Bailn and Santos-Buelga, 2003).

Temperature is limited by the boiling point of the solvent. In addition, many phytochemicals are quite unstable, and the extraction temperature needs to be controlled to prevent thermal decomposition (Gertenbach, 2002). In general, temperatures ranging from 50 to 60C are recommended to extract polyphenols with good extraction recoveries (Pinelo et al., 2004; Silva et al., 2007).

Liquid-solid ratio. The recovery of polyphenols from vegetable

products is also influenced by the ratio of sample to solvent (Shahidi and Nackz, 2004; Silva et al., 2007). A higher liquid-to-solid ratio lowers the concentration of dissolved phytochemicals at the surface of the solid particle, thus providing a higher concentration gradient between the concentrations inside and at the surface of the particles. This higher concentration gradient gives a higher extraction rate. However, a more dilute extract requires a more extensive downstream processing (Gertenbach, 2002). Liquid-solid


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ratios varying from 1/10 to 1/60 are frequently reported in polyphenolic extractions (Gertenbach, 2002; Silva et al., 2007).

Time of extraction. The extraction period is another factor that affects

the recovery of the polyphenolics. Extraction periods varying from 1 min to 24 h have been reported (Shahidi and Nackz, 2004). Longer extraction times increase the chance of oxidation of phenolics unless reducing agents are added to the solvent system (Naczk and Shahidi, 2004).

Particle size of the sample. The rate of extraction increases with

decreasing the particle size (Gertenbach, 2002). The rate-controlling step for extraction is the migration of the solute through the pores of the particles to the particle surface. A smaller particle will lead a shorter path for the solute to reach the surface. A shorter diffusion path equates to a faster extraction rate. Milling can enhance the extraction kinetics (Gertenbach, 2002). High phenolic recoveries have been achieved with particle sizes that ranged from 0.6 to 0.8 mm (Pinelo et al., 2004; Silva et al., 2007).

II.3.2. Purification processes

Extracts rich in phenolic compounds often require purification steps

to eliminate substances that would otherwise interfere with the identification analysis of phenolics. The purification process leads to the removal of interferences but also to the concentration of the analyte(s) of interest and to the improvement of the analytical performances.

A first step in the purification process consists usually in the removal

of lipids from vaccum concentrated phenolic extracts through the use of petroleum ether, hexane or chloroform. Next to this preliminary step, purification methods are applied. The adoption of one or more of these methods will depend on the nature of the phenolics to purify/isolate. These methods can be divided into in solid-liquid and liquid-liquid phase procedures (Tsao and Deng, 2004).

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II.3.2.1. Solid-liquid phase procedures

The solid-liquid phase procedures consist in the migration of a liquid extract through a solid matrix. The solid matrix retains the compounds of interest and the undesirable compounds are eliminated. The compounds of interest are then recovered by means of an elution. The retention of desirable compounds in the matrix depends on their affinity for the matrix. This affinity is governed by specific characteristics such as polarity, ionic forces, molecular weight. Besides, the recovery of the compounds of interest depends on their affinity for the elution solvent. With regard to this characteristic, a selective recovery can be performed. Due to this, in solid-liquid procedures many systems that comprise matrix (adsorbent) and solvent (eluent) differences have been proposed for the purification of phenolic compounds.

One simple method of phenolic purification using SPE consists in the passage of an aqueous solution rich in phenolics through a C18 cartridge (matrix of cartridge is composed of C18 chains bound to silica), previously conditioned with methanol and acidified water (pH ~ 2). The loaded cartridge is washed with acidified water to eliminate interfering substances and the retained phenolic compounds are eluted with a strong solvent such as methanol (Vinha et al., 2005).

Rodrguez-Saona and Worlstad (2001) have proposed a purification

method to purify anthocyanins using SPE. The authors mentioned that this method allows the removal of several interfering compounds present in the crude extracts. After having been preconditioned with low acidic conditions, these columns retain phenolic compounds (e.g., anthocyanins, flanovols, flavan-3-ols), while allowing matrix interferences, such as sugars and acids, to pass through and be discarded as waste. Then washing the retained pigments with ethyl acetate will further remove phenolic compounds other than anthocyanins, these last ones are recovered by means of a fast elution with acidified methanol.


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Column chromatography has also been employed for the fractionation of phenolic extracts. Column chromatography using Sephadex LH-20 gel has been used in various studies to separate the fraction of proanthocyanins from others phenolics in different plant materials and beverages (Kantz and Singleton, 1990; Prior et al., 2004; Sanchez-Moreno et al., 2003). Sephadex LH-20 gel is made from hydroxypropylated dextran beads that have been cross-linked to yield a polysaccharide network (Figure17). The most usual solvents used in this system are ethanol, methanol and acetone and their mixtures with water. The separation is based on the establishment of hydrogen bonds between phenolic hydrogens or carboxylic groups and H-bond acceptors in the gel. The strength of the adsorption depends on the number of phenolic hydrogens per molecule, polymeric polyphenols, such as condensed tannins, are adsorbed more readily than monomers, such as cathechins. Acetone is a better desorbent, since carbonyl oxygen acts as a strong acceptor regarding hydrogen bonding and it is capable of displacing polymeric polyphenols (Kantz and Singleton, 1990).

Figure 17. Partial structure of Sephadex LH-20 (Amersham Bioscience, 2002).

Sanchez-Moreno et al. (2003) used SPE with Sephadex LH-20 to

remove phenolic acids, flavonoids and anthocyanins of red wines to obtain

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procyanidins. The authors utilized 20-mL columns containing 5 g of Sephadex LH-20. Wine samples were passed through the column previously equilibrated with methanol. The column was then eluted with different elution media: 25 mL of 20% (v/v) methanol/water to remove phenolic acids, 40 mL of 60% (v/v) methanol/water to elute the flavonols and anthocyanins and finally 90 mL of pure methanol for the elution of the proanthocyanidins. A similar procedure was used by Prior et al., (2004) to obtain anthocyanins and procyanidins from blueberries and cranberries. Kantz and Singleton (1990) isolated non-polymeric and polymeric phenols from grape extracts using a Sephadex LH-20 column (150 x 50 mm i.d.). Non-polymeric compounds were eluted from the gel with 60% methanol and polymeric phenols were recovered with 60% acetone. Goffman and Bergman (2004) obtained low-molecular-weight phenolics and tannins of rice kernels using small columns of Sephadex LH-20. Absolute ethanol was first used to elute the low-molecular-weight phenolics fraction and then 70% acetone was used to elute the high-molecular-weight phenolics or tannins of rice kernels.

II.3.2.2. Liquid-liquid phase procedures

Liquid-liquid procedures are used for the extraction of phenolics

from liquid samples (Krasteva et al., 2004), but also as a technique of purification and fractionation of groups of phenolics from a sample. The separation of compounds is based on their partition between two immiscible liquids (organic and aqueous phase). The efficiency of recovery depends on the solute affinity for extraction solvent, the ratio between the two immiscible liquids and the number of

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