phenolic compounds (nhom hop chat phenol)

In the present review, the most recent articles dealing with polyphenols in fruits are reviewed, focusing on their occurrence, main methods of extraction, quantification and antioxidant a

Invited review

Phenolic compounds in fruits – an overview

Charles W I Haminiuk,1* Giselle M Maciel,2Manuel S V Plata-Oviedo1& Rosane M Peralta2

1 Programa de Po´s-Graduac¸a˜o em Tecnologia de Alimentos (PPGTA), Universidade Tecnolo´gica Federal do Parana´, Campus Campo Moura˜o, Parana´, Brasil

2 Departamento de Bioquı´mica, Laborato´rio de Bioquı´mica de Microorganismos, Universidade Estadual de Maringa´, Maringa´, Parana´, Brasil

(Received 23 January 2012; Accepted in revised form 2 April 2012)

phenolic acids The growing interest in these substances is mainly because of their antioxidant potential andthe association between their consumption and the prevention of some diseases The health benefits of thesephytochemicals are directly linked to a regular intake and their bioavailability Studies have shown theimportance of the regular consumption of fruits, especially for preventing diseases associated with oxidativestress In the present review, the most recent articles dealing with polyphenols in fruits are reviewed, focusing

on their occurrence, main methods of extraction, quantification and antioxidant assays In addition, thehealth benefits and bioaccessibility⁄ bioavailability of phenolic compounds in fruits are addressed

com-pounds.

Introduction

Phenolic compounds comprise a diverse group of

molecules classified as secondary metabolites in plants

that have a large range of structures and functions They

can be classified into water-soluble compounds

(pheno-lic acids, phenylpropanoids, flavonoids and quinones)

and water-insoluble compounds (condensed tannins,

lignins and cell-wall bound hydroxycinammic acids)

(Rispail et al., 2005) Phenolic compounds have been

considered the most important, numerous and

ubiqui-tous groups of compounds in the plant kingdom (Naczk

& Shahidi, 2004) These substances are synthesised

during the normal development of the plant, as well as

in response to different situations, such as stress and UV

radiation, among others (Naczk & Shahidi, 2004)

Phenolic compounds have an aromatic ring bearing

one or more hydroxyl groups and their structure may

vary from that of a simple phenolic molecule to that of a

complex high-molecular mass polymer (Balasundram

et al., 2006) These antioxidant compounds donate an

electron to the free radical and convert it into an

innocuous molecule

Oxidative stress can cause a series of degenerative

illnesses in humans, such as cancer, multiple sclerosis,

autoimmune disease and Parkinson’s disease, to name a

few (Theriault et al., 2006) Studies have suggested that

a diet rich in phenolic compounds could avoid theoxidative damage that leads to ageing and age-relateddiseases by scavenging the free radicals from cellmetabolism (Kurosumi et al., 2007) Regarding theirbiological effects (antioxidant, antiviral, antimicrobial,anti-tumour and antibacterial activities), polyphenolsare known to participate in protection against theharmful actions of reactive oxygen species (Luo et al.,2011)

The antioxidants present in fruits, such as phenolicacids, flavonoids, anthocyanins and tannins, amongothers, have been frequently associated with health ben-efits (Fu et al., 2011) Historically, ‘fruits have beenconsidered a rich source of some essential dietarymicronutrients and fibres, and more recently they wererecognised as being an important source of a wide array

of phytochemicals that individually, or in combinationmay benefit health’ (Yahia, 2009)

This review focuses on presenting recent studies aboutphenolic compounds in fruits A brief overview of theresearch on polyphenols in fruits is presented Theiroccurrence, their main methods of analyses and anevaluation of their antioxidant properties using different

in vitroand in vivo techniques are addressed Finally, thehealth benefits and the bioavailability⁄ bioaccessibility ofthe phenolic compounds in the human body arediscussed

*Correspondent: Fax: +554435181405; e-mail: haminiuk@utfpr.edu.br

Research on phenolic compounds in fruits

The research on phenolic compounds in fruits has

evolved considerably over the last 20 years (Fig 1) The

role of phenolic compounds in foods, especially in fruits,

has drawn the attention of researchers all over the

world, and a large number of reviews and books have

been published about this topic (Robards et al., 1999;

Kaur & Kapoor, 2001; Balasundram et al., 2006;

Andre´s-Lacueva et al., 2009; Belitz et al., 2009; El

Gharras, 2009; Ignat et al., 2011) Approximately,

3244 articles on the topic of phenolic compounds in

fruits were published from 1991 to 2011

The progressive increase in the number of publications

(40-fold from 1991 to 2011) evaluating polyphenols in

fruits as the main topic mostly reflects the great interest

in studying these compounds worldwide The United

States, Spain, Italy, China and Brazil stand out as the

top five countries around the world where research on

polyphenols in fruits has become prominent (Fig 2)

The research is mainly focused on: (i) determination of

total phenolic, flavonoid and anthocyanin contents, (ii)

evaluation of different types of extraction (liquid–liquid,

solid–liquid and supercritical fluid, (iii) investigation of

the biological activity of polyphenols against some key

diseases and microorganisms, (iv) evaluation of their

total antioxidant capacity using several different

chem-ical methods: (1,1-diphenyl-2-picrylhydrazyl radchem-ical

(DPPHd), oxygen radical absorbance capacity (ORAC),

2,2¢-Azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)

(ABTS), ferric reducing antioxidant power (FRAP),

trolox equivalent antioxidant capacity (TEAC),

b-caro-tene⁄ linoleic acid, etc.), (v) the quantification and

identification of polyphenols by spectrophotometric

methods and high efficiency liquid chromatography

(HPLC) using different detectors (UV⁄ Vis, MS, ELSD,

etc.), and (iv) study of bioaccessibility and bioavailability

of polyphenols

Classification and chemical structure of phenolic

compounds

Phenolic compounds make up a large and fascinating

family of substances (Vermerris & Nicholson, 2006)

Fruits contain considerable levels of bioactive

com-pounds that impart health benefits beyond basic

nutri-tion (Kaur & Kapoor, 2001) The amount of phenolic

compounds in fruits is strongly dependent on the degree

of ripeness, variety, climate, soil composition, geographic

location and storage conditions, among other factors

(Belitz et al., 2009) They are mainly classified according

to the number of phenol rings they contain (phenolic

acids, stilbenes, flavonoids, lignans and tannins) All

these substances have one or more hydroxyl groups

directly linked to an aromatic ring characterising thus the

phenolic structure (Vermerris & Nicholson, 2006)

In the case of the flavonoids group, when they arelinked to one or more sugar molecules they are known

as flavonoid glycosides, and when they are not nected to a sugar molecule they are called aglycones(Williamson, 2004) The degree of glycosylation directlyaffects the antioxidant capacity of flavonoids Usually,the aglycone forms of myricetin and quercetin are moreactive than the glycoside form (Hopia & Heinonen,1999; Kaur & Kapoor, 2001) The flavonoids are themain bioactive compounds found in fruits and are

in fruits produced in two decades (database searched 20 February 2012) Data Source: certain data included herein are derived from the Web of Science  prepared by Thomson Reuters  , Inc (Thomson  ), Philadelphia, PA, USA:  Copyright Thomson Reuters  2012 All rights reserved (out of a total of 3244 articles).

among the top ten countries to publish such studies (database searched

20 February 2012) Data Source: certain data included herein are derived from the Web of Scienceprepared by Thomson Reuters, Inc (Thomson), Philadelphia, PA, USA:Copyright Thomson Reuters2012 All rights reserved.

distributed into six subclasses: flavonols, flavanones,

isoflavones, flavan-3-ols, flavones and anthocyanins

Flavonoids account for approximately two-thirds of

the dietary phenols (Robbins, 2003) and they are mostly

present as glycosides, and partly as esters, rather than as

free compounds (Vermerris & Nicholson, 2006; Belitz

et al., 2009) The per capita consumption of the six

flavonoid subclasses in the United States is estimated to

be 189.7 mg day)1, being mainly flavan-3-ols (Chun

et al., 2007)

Flavonoids or bioflavonoids are widely distributed in

fruits and they are recognised as being natural

antioxi-dants; over 5000 flavonoids have been identified to date

(Lampila et al., 2009) These substances have apparent

roles in plant stress defence, such as in the protection

against damage caused by pathogens, wounding or excess

UV light (Winkel-Shirley, 2002) The term flavonoid is

usually used to describe a broad collection of natural

products that include a C6-C3-C6carbon framework or,

more precisely, phenylbenzopyran functionality (Marais

et al., 2006), and they constitute most of the yellow, red

and blue colours in fruits (Lampila et al., 2009) These

phytochemicals were found to be very effective scavengers

of free radicals in in vitro tests and they are important

antioxidants because of their high redox potential and

their ability to chelate metals (Tsao & Yang, 2003; El

Gharras, 2009; Ignat et al., 2011)

The second most important group of phytochemicals

comprises the phenolic acids, which account for almost

the remaining third of the dietary polyphenols, and

which are present in fruits in a bound form These

substances are divided into two subgroups:

hydroxy-benzoic and hydroxycinnamic acids In contrast to other

phenolic compounds, the hydroxybenzoic and

hydroxy-cinnamic acids present an acidic character owing to the

presence of one carboxylic group in the molecule (Annie

& Jean-Jacques, 2003) Hydroxycinnamic acid

com-pounds are mainly present as derivatives, having a C6

-C3 skeleton Ferulic acid, p-coumaric acid and caffeic

acid are some examples of this class Hydroxybenzoic

acids (C6-C1) are found in various fruits and mostly

occur as esters The most common phenolic acids found

in fruits in this category are gallic, vanillic, ellagic and

syringic acids

Tannins are the third class of polyphenols that are

found in fruits and are mostly present as phenolic

polymers Tannins are astringent and bitter substances

of different molecular weights, and some of them,

especially the hydrolysable tannins, are soluble in water

They are a group of polyhydroxy-flavan-3-ol oligomers

and polymers with carbon–carbon linkages between

flavanol subunits (Schofield et al., 2001) Tannins have

the ability to precipitate proteins The two main types of

tannins are condensed and hydrolysable Gallotannin or

tannic acid is a type of hydrolysable tannin found in fruits

Condensed tannins (proanthocyanidins) are the major

phenolic compounds found in grapes dins, when in contact with salivary proteins, are respon-sible for the astringency of fruits (El Gharras, 2009)

Proanthocyani-Stilbenes are a group of phenylpropanoid-derivedcompounds characterised by a 1,2-diphenylethylenebackbone (C6-C2-C6) (Goyal et al., 2012) Low quanti-ties of stilbenes are present in the human diet, and theirmain representative is resveratrol, mostly in the gly-cosylated form (Delmas et al., 2006; Ignat et al., 2011).Resveratrol is a phytoalexin This substance is mainlyproduced in grapevines in response to injury and fungalinfection (Atanackovic´ et al., 2012), and the maindietary source of resveratrol in fruits is found in redgrape skins Several studies have indicated that resvera-trol has the ability to prevent cancer and coronary,neurological and degenerative diseases (Anekonda,2006; Saiko et al., 2008; Das & Das, 2010; Gresele

et al., 2011) Resveratrol present in red wine is directlylinked to the French paradox, in which Frenchpeople suffer a relatively low incidence of coronaryheart disease even though they have a diet relativelyrich in saturated fats (Ferrie`res, 2004) Furthermore, theincidence of heart infarction in France is about 40%lower than in the rest of Europe (Renaud & Delorgeril,1992; Saiko et al., 2008) It is believed that the contin-uous and moderated ingestion of grape-derived prod-ucts, especially red wine, plays a key role in preventingheart disease

The fifth group of polyphenols comprises the lignans,

a large variety of individual structures mostly consisting

of two phenylpropanoid moieties connected via theirside chain C8 carbons (Davin & Lewis, 2003; Aehle

et al., 2011), usually occurring as glycosides Lignans areone of the major classes of phytoestrogens, which areoestrogen-like chemicals In the gastrointestinal tract,these molecules are converted into compounds (entero-diol and enterolactone) that have both oestrogenic andanti-oestrogenic properties (Meagher & Beecher, 2000).Fruits are not the main dietary source of lignans in foodand low concentrations are found in strawberries andcranberries (Meagher & Beecher, 2000) The highestamount of these chemical compounds is found inflaxseed According to data from the Food CompositionPanel of the Spanish Ministry of Environment andRural and Marine Affairs, the average intake of lignansfrom fruits and vegetables is estimated to be233.6 lg day)1(Moreno-Franco et al., 2011)

The composition of phenolic compounds in fruitsvaries considerably Fruits are a particularly rich source

of flavonoids (especially flavonols, flavan-3-ols andanthocyanins) and hydroxycinnamic and hydroxyben-zoic acids As previously stated, a large amount ofscientific evidence shows that the regular consumption

of fruit is directly linked to the prevention of variousdiseases, and the majority of polyphenols that mightaccount for this are shown in Table 1

Table 1 Main dietary source of polyphenols in fruits

Phenolic

acids

Hydroxycinnamic acids

Passion fruit, Peach

Gavrilova et al (2011) and Fu

et al (2011)

Papaya, Pineapple

Poovarodom et al.(2010), Vidal

et al (2006), Medina et al (2011) and Fu et al (2011)

Hydroxybenzoic acids

Table 1 (Continued)

Medoua & Oldewage-Theron (2011), Rinaldo et al (2010) and Lako et al (2007)

Goulas & Manganaris (2012) and Zhang et al (2011)

Medina et al (2011)

& Selli (2011b)

Miean & Mohamed (2001)

Extraction of polyphenols

The extraction of phenolic compounds is influenced by

several parameters, and the initial step of a preliminary

experiment is to select the most appropriate extraction

conditions Sample preparation plays an important role

in the quantification of phytochemicals from plant

material, and it is the first and usually the most

important process, which greatly influences the

repeat-ability and accuracy of the analysis (Zhao et al., 2011)

To achieve maximum extraction, it is recommended that

several different parameters are tested, such as the

solvent, agitation, extraction time, solute⁄ solvent ratio,

temperature, efficiency of mass transfer and particle size,

for example (Luthria, 2008; Hurtado-Fernandez et al.,

2010; Haminiuk et al., 2011; Yang et al., 2011) Ideally,

the extraction of polyphenols should be performed using

fresh fruit samples However, because of seasonality,

perishability, shelf-life and quality, many researchers

have used freezing and drying processes to preserve theplant material These processes are often a necessarystep for preserving the fruit sample against differenttypes of degradation, for avoiding microorganisms andfor concentrating the antioxidant compounds Someauthors have reported that the freezing process andlong-term frozen storage cause important losses in theamount of phenolic compounds and vitamins found infruits (de Ancos et al., 2000; Chaovanalikit & Wrolstad,2004; Turkben et al., 2010)

Different techniques are available to preserve plantmaterial, and in the last few years, lyophilisation hasbeen widely used Lyophilisation is a process thatremoves the water from materials such as foods, drugsand biological samples, and it has been recognised as animportant and well-established technique for improvingthe long-term stability of a product (Kasper & Friess,2011) In the literature, several authors have reportedthe use of lyophilisation as a drying technique for

Bakowska-Barczak & Kolodziejczyk (2011)

preserving fruit samples (Marques et al., 2007, 2009;

Hurtado-Fernandez et al., 2010; de Torres et al., 2010;

Ignat et al., 2011; Kotikova et al., 2011; Rockenbach

et al., 2011)

The utilisation of finely powdered plant material

enhances the extraction of phenolic compounds by

increasing the surface area of the sample and promoting

disruption of the plant cell wall (Kim & Lee, 2001)

When the fruit sample is obtained (fresh, frozen, dried

or lyophilised), the next stage is extraction, per se The

most commonly used step for extracting phenolic

compounds in fruits is the use of organic solvents The

choice of the most appropriate solvent depends on its

selectivity, miscibility, density, recovery, price, vapour

pressure, viscosity and chemical and thermal stability

Various solvents and conditions have been used to

achieve optimal extraction The application of acidified

methanol, ethanol, acetone and ethyl acetate, and the

combination of these solvents with water, has been

widely reported in the literature (Luthria &

Pastor-Corrales, 2006; Durling et al., 2007; Luthria, 2008;

Wang et al., 2008a; Russell et al., 2009a; Annegowda

et al., 2012; Haminiuk et al., 2011; Prasad et al., 2011;

Ramful et al., 2011) Solid–liquid extraction (SLE) is an

important, simple and efficient technique of mass

transfer used for the recovery of polyphenols from fruit

tissues It allows soluble components to be removed

from the plant matrix and these compounds migrate

into the solvent up to the point of equilibrium (Corrales

et al., 2009) This process can be improved by changing

the concentration gradients, the boundary layer and

diffusion coefficients (Corrales et al., 2009; Ignat et al.,

2011) In addition, to enhance the extraction process,

some authors have combined the SLE technique with

ultrasound This method has been successfully used to

extract bioactive compounds (Herrera & de Castro,

2005; Ma et al., 2009; Casazza et al., 2010; Prasad et al.,

2010; Wang & Zuo, 2011)

Ultrasound-assisted extraction is a quick and efficient

method for extracting phenolic compounds from fruits

(Kim & Lee, 2001), being an effective way of extracting

analytes from different matrices in a shorter time than

other extraction techniques (Herrera & de Castro, 2005)

The increase in polyphenol extraction by this technique

is because of the disruption of cell walls, a reduction in

particle size and enhancement of the mass transfer of cell

contents to the solvent, caused by the collapse of the

bubbles produced by cavitation (Paniwnyk et al., 2001;

Rodrigues & Pinto, 2007) One of the disadvantages of

the solid-extraction process is the co-extraction of

substances such as proteins, sugars and organic acids

(Ignat et al., 2011) that might interfere in the

quantifi-cation of polyphenols, especially with the

Folin-Ciocal-teu method To remove these interferences, the use of

C18 Sep-Pak cartridges is highly recommended This

purification technique separates the non-polyphenolic

substances from the polyphenolic extract of the fruit,producing more accurate results

In the last decade, the use of supercritical fluidextraction (SFE) of bioactive compounds in fruits hasbeen investigated and is gaining popularity Carbondioxide is the most commonly used fluid in SFE(Quitain et al., 2006) This type of extraction is consid-ered an emerging technology (Casas et al., 2009) and itpresents some advantages over classic solvent extractionmethods, because it is a more selective and less toxictechnique (Alpendurada, 2003) Other advantages ofSFE include free-solvent products and the prevention ofoxidation during processing (Vatai et al., 2009; Herrero

et al., 2010) One disadvantage of this technique is theneed for expensive equipment and high pressures, whichincrease the costs compared with conventional liquidextraction Therefore, SFE will only be used whensignificant advantages overcome these disadvantages

Vatai et al (2009) showed that the pre-treatment offruit samples with carbon dioxide (CO2) removed thenon-polar substances, making the polar polyphenolsmore accessible in grapes and elderberries However, theamount of extracted anthocyanins in these fruits was notsignificantly influenced by SFE In another study, SFEwith CO2⁄ EtOH showed similar results for the totalphenolic compounds (TPC) in guava seeds comparedwith the Soxhlet SLE method (Castro-Vargas et al.,2010) Supercritical carbon dioxide was successfullyused to extract resveratrol from grape pomace by Casas

et al (2010) In this study, SFE with CO2⁄ EtOH(400 bar and 35C) presented the highest resveratrolrecovery (49.10 mg per 100 g of dry sample) whencompared with conventional extraction [methanol⁄ HCl(0.1%) for 30 min in an ultrasonic bath], where 3.10 mg

of resveratrol per 100 g of dry sample was obtained

Quantification of phenolic compounds byspectrophotometric techniques

Fruits are an important source of polyphenols in thehuman diet and the proper quantification of thesesubstances is of fundamental importance The mainmethodologies used to quantify the bioactive com-pounds in fruits, which are widely described in theliterature, are the colorimetric method of Folin-Ciocal-teu that estimates the total polyphenols (TPC), thealuminium chloride colorimetric assay that quantifiesthe total flavonoids (TF) and total anthocyanins (TA)estimated by the pH differential method, which is based

on the structural change of the anthocyanin phore between pH values of 1.0 and 4.5 (Granato et al.,2010; Haminiuk et al., 2011) Table 2 shows a briefsummary of the total polyphenols, total flavonoids and

chromo-TA in different fruits

The quantification of phenolic compounds is mainlycarried out by spectrophotometric analysis Generally,

the visible region of the spectrum is used to quantify

total phenolics, flavonoids and tannins, among other

substances

The most common and widespread methodology used

to quantify the total phenolic compounds in foodstuffs

originated from the methodology developed in 1927 by

Otto Folin and Vintila Ciocalteu for the measurement of

tyrosine (Folin & Ciocalteu, 1927; Everette et al., 2010),

and it was adapted in 1965 by Vernon Singleton and

Joseph Rossi for the evaluation of total phenolics in

wine (Singleton & Rossi, 1965) This methodology is

based on chemical reduction by a mixture of tungsten

and molybdenum oxides (Waterhouse, 2001) Upon

reaction with phenols, a blue colour is produced, which

absorbs light at 765 nm (Everette et al., 2010) The

intensity of light absorption at this wavelength is

proportional to the concentration of phenols

(Water-house, 2001) It is noteworthy that this reagent does not

only measure total phenols but it will react with any

reducing substance For this reason, the total reducing

capacity of a sample will be quantified and not only the

level of phenolic compounds (Ikawa et al., 2003) The

average time for this test is 2 h; however, this time can

be reduced by heating the sample The main

disadvan-tage of heating is that it affects the reproducibility of the

assay because heating causes instability of the blue

colour over time (colour loss)

Several articles reporting the total phenolic

composi-tion of fruits estimated using the Folin-Ciocalteu

method (Singleton & Rossi, 1965; Singleton et al.,

1999) are available in the scientific literature: kiwifruit

(Sun-Waterhouse et al., 2009), grape (Yang et al., 2009),mango (Prasad et al., 2011), pomegranate (Cristofori

et al., 2011), cranberry (Coˆte´ et al., 2011), banana,guava, peach and pear (Contreras-Calderon et al.,2011), lemon, orange, passion fruit, pitaya, plum,avocado and papaya (Fu et al., 2011) and apple (Zheng

et al., 2012) In a recent study, a wide variation in thecontents of the total phenolic compounds of sixty-twoChinese fruits was found, where the total phenoliccompounds ranged from 11.88 to 585.52 mg gallic acidequivalent (GAE) per 100 g, with a difference of 49-foldand a mean value of 71.80 mg GAE per 100 g (Fu et al.,2011) Pear (honey) and Chinese date presented thelowest and the highest amounts of total phenoliccompounds, respectively (Fu et al., 2011) High contents

of TPC can be found in fruits: 1063 mg GAE per 100 g

of fresh weight (FW) in acerola (Rufino et al., 2010),

1365 mg GAE per 100 g of FW in cambuci (Haminiuk

et al., 2011), 1797 mg GAE per 100 g of FW in camu (Genovese et al., 2008) and 2167 mg GAE per

camu-100 g of FW in Andean blackberry (Vasco et al., 2008).Flavonoids are mainly accumulated in the outer tissue

of fruits because their synthesis is stimulated by sunlight(Manach et al., 2004; Rosa et al., 2010) The totalflavonoid content in fruits is mainly estimated using thecolorimetric method with aluminium chloride In thismethodology, the fruit extract is added in a methanolicsolution of aluminium chloride at a determined concen-tration In some methodologies, the extract is mixedwith NaNO2before mixing with AlCl3(Sun et al., 2011).After a short period of time, the absorbance is measured

Wang & Lin (2000)

Wang & Lin (2000)

14.60 d

and compared with a flavonoid standard (catechin,

quercetin or rutin) The disadvantage of this

methodol-ogy is that it only gives an estimation of the total

flavonoid content (Ignat et al., 2011) Nevertheless, this

is a simple and efficient methodology for quantifying the

total content of flavonoids in fruits and it can be very

useful when more advanced equipment, such as HPLC,

is not available The flavonoid concentration in fruits

varies by many orders of magnitude and in most of

fruits these compounds are present in the skin Berries

are in the group that presents one of the highest values

of total flavonoids among fruit (USDA, 2011)

Blue-berry, among another nineteen Bulgarian fruits, was

found to contain the highest amount of total flavonoids

with 190.3 mg of catechin equivalent (CE) per 100 g of

FW, followed by sour cherry with 138.6 mg CE per

100 g of FW (Marinova et al., 2005)

Anthocyanins are water-soluble glycosides of

antho-cyanidins (aglycones) (Vermerris & Nicholson, 2006)

These compounds are benzopyrylium and flavylium salts

(Belitz et al., 2009) They are divided into anthocyanidin

aglycones (sugar free) and anthocyanin glycosides These

substances are particularly evident in flowers and fruit

tissues Anthocyanin is derived from two Greek words,

anthos and kyanos, meaning flower and dark blue,

respectively (He & Giusti, 2010) This class of phenolic

compounds is recognised as being the most important

group of pigments in nature and they contribute to the

attractive colours of fruits Anthocyanins are pigments

dissolved in the vacuolar sap of the epidermal tissues of

fruits, to which they impart a pink, red, blue or purple

colour, and they exist in different chemical forms, both

coloured and non-coloured, according to the pH (Mouly

et al., 1994; Manach et al., 2004) Over 635 anthocyanins

have been identified in plants to date (Wallace, 2008; He

& Giusti, 2010), although only six are commonly found:

delphinidin, cyanidin, pelargonidin, malvidin, petunidin

and peonidin

The pH differential method is the most commonly and

widely used assay for quantifying monomeric

anthocy-anins This methodology measures the absorbance at

two different pH values, and it is based on the structural

transformation of the anthocyanin chromophore as a

function of pH (Giusti & Wrolstad, 2001) It was initially

developed to evaluate pigments in strawberry jam

(Sondheimer & Kertesz, 1948) The methodology has

undergone some modifications over time (Fuleki &

Francis, 1968; Wrolstad et al., 1982; Giusti & Wrolstad,

2001) In this methodology, basically the pigments

reversibly change colour with a change in pH The

samples are diluted with aqueous pH 1.0 and 4.5 buffers

and absorbance measurements are made at the

wave-length of the maximum absorbance of the pH 1.0

solution (Wrolstad et al., 2005) The pH differential

method is based on this reaction and the difference in the

absorbance of the pigments at 520 nm is proportional to

the pigment concentration The results are mainlyexpressed on a cyanidin-3-glucoside basis

The group of cherries and berries has been extensivelyreported in literature as containing the fruits with thehighest levels of anthocyanins per serving Berries withred, blue or purple colours constitute one of the mostimportant sources of these phytochemicals (Kahkonen

et al., 2003) Grapes are one of the major dietary sources

of anthocyanins (Munoz-Espada et al., 2004), beingwidely obtained from wine, juices and jams, for exam-ple Some hybrid grape cultivars can reach up to 603 mg

of anthocyanins per 100 g of FW (Mazza, 1995; Yang

et al., 2009) Raspberry, blackberry, blueberry, berry and bilberry, among others, are very rich inanthocyanins Plum and elderberry are also among therichest sources of anthocyanins in fruits (USDA, 2011)

choke-Identification and quantification of phenoliccompounds by HPLC

Liquid chromatography is an important physical ration technique carried out in the liquid phase where amixture of compounds can be easily and rapidlyseparated Reverse-phase high-performance liquid chro-matography (RP-HPLC) is the main method used forthe separation of phenolic compounds in plant-foodmaterial, in which the stationary phase is less polar thanthe mobile phase The stationary phase is generallymade up of hydrophobic alkyl chains, where there arethree common chain lengths: C4, C8and C18(Guzzeta,2011) Silica-bonded C18 columns are widely used toseparate phenolic compounds In RP-HPLC, the reten-tion time of phenolic compounds is higher for sub-stances that are less polar (myricetin, quercetin,kaempferol); meanwhile, polar molecules are elutedmore easily (gallic acid, protocatechuic acid, epigallo-catechin)

sepa-Owing to their chemical complexity and similarity,polyphenols in fruits are usually identified and quanti-fied by RP-HPLC using a gradient elution instead of theisocratic mode, where the mobile phase is generally abinary system (Merken & Beecher, 2000; Kim & Lee,2001) Usually, gradient elution is carried out with highquality ultrapure acidified water (phosphoric, acetic,formic acids) as the polar solvent; meanwhile, acetoni-trile and methanol are usually used as less polar solvents(Kim & Lee, 2001) A small quantity of acid is added tothe solvent system to suppress the ionisation of phenolicand carboxylic groups, which will improve certainparameters such as retention time and resolution(Ha¨kkinen, 2000)

Polyphenols have a maximum absorbance (kmax) ineither the ultraviolet or visible regions and, thus, deter-mination of the optimum absorbance for each substanceplays an important role in the identification, quantifica-tion and accuracy of the analysis The ultraviolet

spectrum in the region of maximum absorbance of gallic

acid, resveratrol and quercetin is shown in Fig 3

High-performance liquid chromatography systems

can be equipped with a wide range of detectors

(refractive index, fluorescence, electrochemical,

light-scattering, mass spectrometric and UV⁄ Vis) (Thompson

& LoBrutto, 2006), which can be used to detect and

quantify polyphenols with and without chromophore

groups, depending on the methodology used

Among the different detectors available, UV⁄ Vis with

a photodiode array is one of the most widely used to

elucidate polyphenols in plant-based materials The

main advantage of diode array detectors (DAD) is that

several results can be obtained from a single run, mainly

because of its collection of UV⁄ Vis spectra (Kim & Lee,

2001), thereby increasing the throughput of the HPLC

In addition, it is possible to determine the correct

wavelength in one run, to detect multiple wavelengths

and to evaluate peak purity, among others (Dionex,

2003)

Sophisticated systems of liquid chromatography

coupled with modern detectors such as

UPLC-DAD-MS⁄ MS (ultra-performance liquid

chromatography-DAD-tandem mass spectrometry), UPLC-DAD⁄ ESI-MS

(ultra-performance liquid chromatography-DAD and

electrospray ionisation-mass spectrometry),

HPLC-PDA-MS⁄ ELSD (HPLC-photodiode array-mass

spec-trometry and evaporative light-scattering detector),

UHPLC-MS⁄ MS (ultra- HPLC–tandem mass

spec-trometry) and HPLC-ESI-TOF⁄ MS

(HPLC–electro-spray ionisation-time of flight-mass spectrometry) arecurrently available, which are able to determine thechemical structure of a wide range of compounds.However, these systems are still expensive, whichgenerally limits access to these types of equipment bymost researchers In this context, the system of liquidchromatography coupled with DAD-UV⁄ Vis is the mostutilised, especially in the identification of phenoliccompounds, because of its low cost, sensitivity, separa-tion efficiency, flexibility and identification potential Acompilation of recent papers published about theseparation and identification of phenolic compounds

in fruits using RP-HPLC-DAD⁄ UV-Vis is summarised

in Table 3

The methods of extraction, separation and the ysis of phenolic compounds in plant-food material wererecently reviewed (Ignat et al., 2011) The applicationsand advances in the liquid chromatography analysis ofpolyphenols have evolved considerably over the years(Kalili & de Villiers, 2011); however, these advances arenot considered in the present review

anal-Antioxidant activity of fruits

Many methods have been used to evaluate and comparethe antioxidant activity of fruits owing to the complexity

of the substrate analysed (Kaur & Kapoor, 2001; Szabo

et al., 2007) The antioxidant capacity is mainly ated through chemical tests and more recently through acell antioxidant test The antioxidant activity using

(c)