C H A P T E R

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Polyphenol Antioxidants from Natural Sourcesand Contribution to Health Promotion

Juliana Maria de Mello Andrade and Daniel FasoloFaculty of Pharmacy, Federal University of Rio Grande do Sul at Porto Alegre, Rio Grande do Sul State, Brazil

1. INTRODUCTION

Biomolecules in fruits and vegetables have attracteda great deal of attention mainly concentrated on theirrole in preventing diseases. Epidemiological studieshave shown that there is a clear significant positiveassociation between the intake of these natural foodproducts, consumed regularly as part of the diet, and areduced rate of heart disease mortalities, common can-cers, and other degenerative diseases.1

Polyphenols are a group of secondary metabolitesthat are widely distributed throughout the plant king-dom, presenting a high variety of molecules that includeat least one aromatic ring substituted by one or morehydroxyl groups.2 In general, they have hydroxyl, meth-oxyl, and/or glycosyl groups in their structures. Fromthis, these natural compounds are structurally diverseand vary from single molecules such as phenolic acids tohighly polymerized structures like tannins. Most fruitsand vegetables are well known as a potential source ofpolyphenolic compounds and thus have been usedworldwide as a nutritional antioxidant.

Polyphenolic compounds display remarkablemedicinal properties including an antioxidant effect.3

The quantities and distribution of these phenols infruits differ depending on the cultivar, the stage ofripeness, and harvest and post-harvest conditions.4,5

One representative of the polyphenols group, the fla-vonoids, are remarkable antioxidant agents.

Dietary polyphenols are potent antioxidants, able toscavenge and intercept free radicals, preventing damageto cellular molecules.6 Antioxidant action, however, isnot limited to reactive oxygen species (ROS) scavenging,and includes the upregulation of antioxidant and detoxi-fication enzymes, modulation of cell signaling and gene

expression, and other cellular effects.7 Additionally, thepotential synergy between the various antioxidant path-ways may enhance the potential antioxidant protection,and must be considered.

Extensive research into natural antioxidants hasrevealed that fruits and vegetables, seeds, cereals, ber-ries, wine, tea, onion bulbs, olive oil and aromaticplants are some of the sources of these substances.Attempts have also been made to identify and evaluateantioxidants in agricultural by-products, ethnic andtraditional products, herbal teas, cold pressed seedoils, exudates resins, hydrolysis products, not evalu-ated fruits and edible leaves, and other raw materialsrich in antioxidant phenols with important nutritionalfunctions and/or their potential application in healthpromotion and prevention against damages caused byfree radicals (reviewed in Dimitrios8).

2. PLANT MATERIAL CONTAININGPOLYPHENOLS—OUR EXPERIENCE

Polyphenols are the most abundant antioxidants inthe diet. The main dietary sources of these compoundsare fruits and plant-derived beverages such as fruitjuices, tea, coffee, and red wine. Vegetables, cereals,chocolate, and dry legumes also contribute to the totalpolyphenol intake.9 In Oliveira and colleagues’ reviewwork,10 many sources of antioxidant polyphenols aredescribed, as well as those already cited above andalso seeds, drinks, etc. Therefore, a description here ofall studies that demonstrate the occurrence of polyphe-nols in diverse sources is not the objective. Thus, justas examples, we will comment below on the presence

253Polyphenols in Human Health and Disease.

DOI: http://dx.doi.org/10.1016/B978-0-12-398456-2.00020-7 © 2014 Elsevier Inc. All rights reserved.

of polyphenols in fruits, as presented in our recentlypublished studies.

In our work entitled: “Phenolic composition in dif-ferent genotypes of guabiju fruits (Myrcianthes pungens)and their potential as antioxidant and antichemotacticagents,”11 we evaluate two different genotypes (PL1and PL2) and a wild type (GB) of this fruit regardingthe total pholyphenols, flavonoids and anthocyanins,as well as the DPPH (1,1-diphenyl-2-picrylhydrazyl)radical scavenging activity and the antichemotacticactivity against polymorphonuclear cells.

The genotypes of this tree are part of the collection ofnative fruits held by Embrapa Clima Temperado (RSBrazil). The fruit of the wild type (GB) are a mixture offruits from different plants. The genotype PL1 is a seed-ling plant producing very dark-skinned fruits ofmedium size (1.5 to 1.8 cm in diameter). In general, thefruits are darker than the ones produced by the PL2genotype, which is generally more productive, althoughits fruits do not differ very much from those of PL1.Samples were collected at random from wild-type fruits(GB) and from two genotypes (PL1 and PL2).

The total polyphenol, flavonoid and anthocyanincontents are presented in Table 20.1, which demon-strates that contents of total polyphenolics in the sam-ples of Myrcianthes pungens ranged from 2438.3 to4613.4 mg/100 g. The average total flavonoids contentamong guabiju ranged from 79.8 to 154.3 mg/100 g,and the anthocyanin content in guabiju fruit rangedfrom 33.4 to 53.1 mg/100 g. The PL1 sample presentedthe lowest concentration of total anthocyanins(334 mg/100 g dwt), while GB exhibited the highestcontent of these substances (531 mg/100 g dwt).

As we discussed in the article: “Levels of plantmetabolites are markedly affected by genetic and envi-ronmental factors, and also by transportation and stor-age conditions. Factors that influence growth, such aslight, temperature, humidity, type of soil, applicationof fertilizers, damage caused by microorganisms andinsects, stress induced by UV radiation, exposure to

heavy metals, and pesticide use all alter the metabolitecomposition of plants.”12,13

For the isolation of anthocyanidins, the total extractof GB was hydrolyzed and fractionated by medium-pressure liquid chromatography (MPLC). Five antho-cyanidins were identified: delphinidin, cyanidin, petu-nidin, peonidin, and malvidin, with cyanidin being themajor component. The radical scavenging activity ofthe compounds was quantified by using two methods:DPPH radical assay14 and the measurement of inhibi-tion production of •OH2 assay.15 All three guabijuextracts exhibited potent activity when compared toTrolox. The GB and PL2 extracts exhibited the highestactivities at lower concentrations (0.25 mg/mL). Theseresults were similar with those found for the inhibitionof •OH2 production.

The chemotaxis assay was performed using themethod described by Boyden,16 with minor modifica-tions introduced by Dresch and colleagues.17 For thetotal extracts, GB, PL1, and PL2 showed significantinhibitory activity ranging from 83.32 to 92.95%. Aninhibitory effect was observed with extract concentra-tions as low as 1 μg/mL. In comparison with the totalextracts, the hydrolyzed sample presented greateractivity even at the lowest dose of 1 μg/mL.

The results of this work demonstrated that high totalpolyphenols, flavonoids, and anthocyanin contentswere found for guabiju fruits. Potent antioxidant capac-ity was observed for the extracts, being compared tothat of other micronutrients, such as vitamin E. Also,notable antichemotactic activities were verified andwere strongly related to their polyphenol contents.Compilation of these data suggest that the consumptionof the M. pungens fruit, rich in polyphenols, may be aninteresting source of equilibrate and healthful dietarycomponents. Also, further studies will be carried out inorder to associate these antioxidant and anti-inflammatory properties with other body diseases.

In another study entitled: “Comparative analysis ofthe chemical composition and antioxidant activity ofred (Psidium cattleianum) and yellow (Psidium cattleia-num var. lucidum) strawberry guava fruit,” we aimedto quantify total polyphenolic compounds, determinetheir qualitative profile, and also to determine thein vitro antioxidant capacity of methanol extracts andthe essential and fixed oil composition.18 Strawberryguava fruits and seeds were collected in EmbrapaClima Temperado.

The results of this study demonstrated that red straw-berry guava exhibited a higher polyphenolic (501.3360.02 mg/100 g) and flavonoid (100.206 0.07 mg/100 g)content compared with yellow strawberry guava(292.036 0.03 mg/100 g, 35.126 0.13 mg/100 g, respec-tively) values (Table 20.2). Some of these observationsare in accordance with the consulted literature; on the

TABLE 20.1 Total Anthocyanins, Flavonoids, and Polyphenolsin Guabiju Fruits

Samples Total

Polyphenols

(g%6SD)

Total Flavonoids

(g%6SD)

Total

Anthocyanins

(g%6SD)

GB 4.4196 0.162a 0.14906 0.0148a 0.5316 0.027a

PL1 2.43836 0.037b 0.07986 0.0046b 0.3346 0.002b

PL2 4.61346 0.214a 0.15436 0.0115a 0.3436 0.016b

Total anthocyanins were expressed as cyanidin-3-glucoside. Polyphenol and

flavonoid concentrations based upon gallic acid or quercetin, respectively,

expressed per 100 gram of lyophilized fruit. Values are averages of triplicate

analysis. Means with the same letters are not significantly different at p, 0.05.

254 20. POLYPHENOL ANTIOXIDANTS FROM NATURAL SOURCES AND CONTRIBUTION TO HEALTH PROMOTION

3. OXIDATION AND ANTIOXIDANT ACTIVITY OF POLYPHENOLS

other hand, differences in the total content of polyphe-nols and flavonoids, mainly for yellow strawberryguava, were observed. These differences may be due tovariations in the maturity, harvest conditions, location ofcollection, among other factors.

The fruits of both cultivars presented quercetin gly-cosides such as hyperoside and isoquercetrin, beingthe first one of the main compounds of the extracts.One anthocyanin was found only in the red skin culti-var extract, and identified as cyanidin by comparisonwith a pure commercial standard. In this study, thevalues of antioxidant activity from red and yellowstrawberry guava demonstrated a dose-dependentresponse. Strong antioxidant effects were verified atconcentrations of 5 and 10 μg/mL. The values of TEAC(Trolox equivalent antioxidant capacity) obtained forP. cattleianum (156 μM/g) and P. cattleianum var. luci-dum (177 μM/g) found are in accordance with resultsfrom other research groups.

Faced with these results, it can be concluded thatstrawberry guava varieties are rich in phenolic com-pounds. These plant metabolites present importantbiological activities, such as the antioxidant capacity—as we demonstrate in this work—highlighting theinclusion of these fruits among functional foods. Thus,the consumption of strawberry guava fruits, whichpresent an important antioxidant capacity, can contrib-ute to supporting a healthy population.

A large number of epidemiological studies provideconvincing evidence of the beneficial roles of fruitsand vegetables in the general diet for maintenance ofhealth and prevention of disease.19 More recently,studies in several fields of science, including epidemi-ology, human medicine, and nutrition, suggest thatfruit and vegetable antioxidants play an important rolein reducing the risk of degenerative diseases such ascardiovascular disease, various cancers, and neurologi-cal diseases. The main compounds responsible for thisactivity are phenolics and ascorbate. Fruits are goodsources of both compounds, and since fruits are often

consumed fresh, antioxidant capacity is not lost due toany adverse effects of heat and oxidation duringprocessing.20

3. ANTIOXIDANT CAPACITY OFNATURAL SOURCES RICH IN

POLYPHENOLS, AND ISOLATEDPHENOLIC COMPOUNDS

The biological activity of medicinal plants has beenthe subject of intense scientific research. The discoveryof promising activities in plants and/or natural pro-ducts is of extreme interest, since herbs are used invarious health areas as alternative forms of treatmentof various diseases and natural products are, in mostcases, the starting point for the development of newdrugs.

Several studies found very good correlationsbetween the total phenol content and antioxidant activ-ity. For example, Aronia (chokeberry) samples wereassayed to determine the antioxidant capacity, mea-sured by different methods, and in all cases the corre-lation was better than that of anthocyanins andantioxidant activity, showing that all polyphenols inchokeberry determine antioxidant properties ratherthan only the dominating anthocyanins.21�23

The antioxidant and cytoprotective activities ofAustralian fruit polyphenols were evaluated by Tanand colleagues.24 Four native Australian fruits,Podocarpus elatus (illawarra plum), Terminalia ferdi-nandiana (kakadu plum), Kunzea pomifera (muntries)and Acrotriche depressa (native currant) were examinedfor antioxidant activity, by the ferric ion reducing anti-oxidant power (FRAP) assay, the oxygen radical absor-bance capacity (ORAC-H) assay for hydrophiliccompounds, and the cellular antioxidant activity(CAA) assay. Additionally, cellular protective activitywas evaluated, using cellular protection against H2O2-induced cell death, through the MTT assay.

Australian commercially grown blueberry wasincluded as a reference sample, due to the recogni-tion of the high antioxidant potential and relatedhealth benefits of blueberries.25 The results of theFRAP assay revealed that kakadu plum exhibited thegreatest antioxidant capacity (1333% of that of blue-berry), followed by native currant (369%). Similarly,the ORAC assay revealed larger oxygen radicalscavenging capacities for all four native fruits com-pared to the blueberry reference. Kakadu plumexhibited 13.3-fold and 2.4-fold greater activity thanblueberry in the ferric ion reducing antioxidantpower (FRAP) and oxygen radical absorbance capac-ity (ORAC-H) assays, respectively.

TABLE 20.2 Total Polyphenols and Flavonoids in Red andYellow Strawberry Guava Fruits

Fruits Total Polyphenols(mg%6SD)

Total Flavonoids(mg%6SD)

P. cattleianum 501.336 0.0168a 100.206 0.0716a

P. cattleianum var.lucidum

292.036 0.0300b 35.126 0.1270b

The polyphenol concentrations were expressed as milligrams of gallic acid per

100 gram of fresh weight. Flavonoid concentrations, based upon quercetin as

standard, were expressed per 100 gram of fresh weight. Different superscripts

on the same column are significantly different (p, 0.05), based on triplicate

analysis.

2553. ANTIOXIDANT CAPACITY OF NATURAL SOURCES RICH IN POLYPHENOLS, AND ISOLATED PHENOLIC COMPOUNDS

3. OXIDATION AND ANTIOXIDANT ACTIVITY OF POLYPHENOLS

Additionally, the results of the CAA assay revealedthat the purified polyphenolic extract of kakadu plumexhibited the greatest cellular antioxidant activity withan EC50 value (μg/mL) of 153.06 24.5, which was sig-nificantly lower than all the other samples. The HPLC-DAD analysis revealed that this plant extract containedthe highest level of total phenolic compounds(815.86 84.4 μg GAEs/mg DW).

On the other hand, the highest level of anthocyaninswas recorded for illawarra plum (447.36 19.6 μg CEs/mg DW), cyanidin 3-glucoside being the main com-pound in its chromatogram. Polyphenolic-rich extractsof kakadu plum and muntries (but not illawarra plumand native currant) extracts efficiently protected RAW264.7 cells against hydrogen peroxide induced apopto-sis in a dose-dependent manner.

The authors concluded that the results suggest thatkakadu plum exhibits the greatest antioxidant potential,exerting antioxidant activity through free radical scav-enging and affecting two downstream transcription fac-tors. Polyphenols from this plant material mayrepresent a significant potential for further develop-ment as a functional food for protection against oxida-tive stress and additional beneficial health applications.

These findings are in accordance with previousresults that demonstrated, except for native currant,strong antioxidant activity, in a range of reagent-basedantioxidant activity assays, for many of the nativeAustralian fruits. And this interesting potential can beassociated with the high levels of polyphenols in thesenatural sources.26�28 Thus, previous workers foundthat specific polyphenols, such as luteolin, myricetin (1)and quercetin-3-β-D-glucoside, have also been demon-strated to have significant cellular antioxidantactivity.29

Cheel and colleagues30 evaluated the correlationbetween polyphenolic, flavonoid and anthocyanin con-tents with antioxidant capacity of achenes and thala-mus from Fragaria chiloensis ssp. chiloensis, F. vesca andF. x ananassa cv. Chandler. The antioxidant activity ofacetone and aqueous extracts was measured by thesuperoxide anion and DPPH discoloration assays, bothusing a spectophotometric-based method. For polyphe-nolic content, F. vesca had the highest value, with268.1 mg gallic acid equivalents/100 g fresh fruit. In allstudied plants, the achenes demonstrated higher phe-nolic, flavonoid and anthocyanin contents, comparedwith whole plant and thalamus. The achene fractionconstitutes a low proportion of the whole fruit.

With regard to antioxidant capacity, F. x ananassacv. Chandler whole fruits showed the highest free rad-ical scavenging activity in the DPPH assay (86%). Theauthors showed that a high and positive correlationexists between flavonoid content in the whole fruitand DPPH activity (r5 0.879, p ,0.05). According to

Hakkinen and colleagues31 the flavonoids in strawber-ries represent 11% of all phenolic compounds withquercetin (2) as the major flavonoid, kaempferol (3)and myricetin (1) as minor flavonoids. As noted intheir study, the DPPH activity of strawberries could beassociated with the anthocyanin content. However,anthocyanins were not relevant as superoxide anionscavengers. In conclusion, phenolics can determine thefree radical scavenging activity of these fruits.

Various structural characteristics of phenolic com-pounds are responsible for determining different levelsof antioxidant activity, like the number and arrange-ment of the hydroxyl groups, and the presence of a3-OH group and ortho-dihydroxy substitution in theB-ring of flavonoids (Figure 20.1); also a double bondbetween C2 and C3 on the C-ring is a prerequisite forgood antioxidant activity, as well as others.32,33 Amongthe polyphenols, quercetin (2) possesses many of thestructural characteristics necessary for potent antioxi-dant activity, and it is one of the most efficient antioxi-dant in the plant kingdom. For example, among theAronia monomer phenolics, quercetin (2) is the mostactive antioxidant, followed by cyanidin glycosidesand chlorogenic acid.

Flavonoids are well known as antioxidant agents.The probable mechanism for the direct scavenging offree radicals is based on the hypothesis that these com-pounds are oxidized by radicals, resulting in a morestable, less-reactive radical species. Because of the highreactivity of the hydroxyl group of the flavonoids,radicals are made inactive.34

In this way, Hatano and colleagues35 studied thepolyphenol composition of cacao liquor and their anti-oxidant effects. The polyphenols, except for (2)-epica-techin 8-C-galactoside (4), had inhibitory effects on thelipid peroxidation in rat liver microsomes with IC50

values of 12�68 mM, indicating that these compoundscontribute to the antioxidant activity of the polypheno-lic fraction of cacao. The most active compounds wereprocyanidin B2 (5) and procyanidin B5 (6), both withIC505 12.0 μg/mL.

Taking into account the inhibitory effects of thesepolyphenols on the autoxidation of linoleic acid, valuesof IC50 ranged from 0.62 μg/mL for (2)-epicatechin (7)to 9.5 μg/mL for (2)-epicatechin 8-C-galactopyranoside.

8

7

6

5 43

2

1'

2'

A C

OB

3'

4'

5'

6'

FIGURE 20.1 Flavonoids basic skeleton.

256 20. POLYPHENOL ANTIOXIDANTS FROM NATURAL SOURCES AND CONTRIBUTION TO HEALTH PROMOTION

3. OXIDATION AND ANTIOXIDANT ACTIVITY OF POLYPHENOLS

The authors also evaluated the antioxidant capacity ofpolyphenols from cacao liquor by the DPPH radicalscavenging activity, finding that procyanidin B2 (5)exhibited the highest activity, with an EC50 value of1.4 μM. The values ranged between 1.4 and 6.2 μM. Theeffects found were attributed to the radical scavengingactivity in the peroxidation chain reactions, based onthe findings that the cacao polyphenols effectively scav-enged the 1,1-diphenyl-2-picrylhydrazyl radical.

Generally, a larger number of hydroxyl groups pro-vide a higher activity as hydrogen and electron donoragents. Monohydroxylated flavonoids present very lowactivity, for example, 5-hydroxy-flavone has non-detectable antioxidant activity.36 Among the dihy-droxylated flavonoids it is possible to highlight thosewith the catechol system (30,40-dihydroxy) in the B-ring.Flavonoids with multiple hydroxyl groups, such asquercetin (2), kaempferol (3), luteolin, eriodictyol andtaxifolin, have strong antioxidant activity when com-pared to α-tocopherol, ascorbic acid, and β-carotene.37

A great deal of research confirms this activity. Asan example, Da Rosa and colleagues38 studied the fla-vonoid composition and antioxidant properties of thecrude methanolic extract and fractions of leaves fromPalicourea rigida. The results demonstrated a higherphenolic concentration in the ethyl acetate fraction(EAF) (933.25 μg/mL), presenting too the higher anti-oxidant activity (IC505 192.77 μg/mL). From the EAFwere isolated and identified three flavonoids: querce-tin 3-O-β-D-glucoside, isorhamnetin-3-glucoside andquercetin-3-O-sophoroside.

Although it is not possible to accurately concludewhich compounds are responsible for the EAF antioxi-dant activity, the authors suggested that flavonoids mustbe, at least, partly responsible for this activity consider-ing the several studies reporting different displayedactivities by this class of compounds, which are predom-inantly described as potent antioxidant agents.39,40

Some time ago, the antioxidant potential of querce-tin-3-O-sophoroside had already been reported byPlumb and colleagues.41 Recently, Razavi and collea-gues42 showed that quercetin 3-O-β-D-glucoside pre-sented a high antioxidant and phytotoxic activity.In other investigations concerning Salicornia herbacea,the results indicated that isorhamnetin-3-glucoside is asuitable compound for developing a new drug for dia-betes treatment or prevention.43 These findings sup-port the hypothesis that flavonoids in the EAF ofP. rigida could be responsible for observed antioxidantpotential.

With regard to flavonoids with interesting antioxi-dant properties, Da Silva and colleagues44 evaluatedthe phenolic and flavonoid contents, as well as theantioxidant capacity, of Lacistema pubescens leaves.The hydromethanolic fraction showed the best

antioxidant activity (IC505 1.8 μg/mL), by DPPHassay, and also the highest content of phenolic sub-stances (283.61 mg/g extract, as tannic acid equiva-lents). These compounds, therefore, appear to beresponsible for the activity found, demonstrating apositive correlation between phenols and antioxidantpotential.

Also, Souza and colleagues45 analyzed the total phe-nolic content and antioxidant activity of five medicinalplants, concluding that three species (Terminalia brasi-liensis, Cenostigma macrophyllu, and Copernicia cerifera)showed a positive correlation between the phenoliccontent and antioxidant capacity, assessed by theDPPH method.

Pure polyphenols were investigated by Ozgovaand colleagues46 who confirmed their antioxidantproperties by different assays. Analyzing the results,the order of flavonoid efficiency in the inhibition ofthe lipid peroxidation-Fe dependent system was: res-veratrol (8).. kaempferol (3).epicatechin.morin (9).catechin (10).quercetin (2).myricetin (1).fisetin(11). In contrast, their efficiency in the NADPH systemdecreased in the order: quercetin (2).trans-resveratrol.fisetin (11).myricetin (1).morin (9).kaempferol(3).epicatechin. catechin (10). Phenolic acids wereless active than the above substances in both assays. Itcan be observed that flavonols were more effectivethan flavanols.

The positive effect of these compounds againstlipid peroxidation is based on the fact that they candonate a hydrogen or an electron, and stabilize thepolyphenol radical formed during its antioxidantaction. However, comparing the efficiency of flavo-noids to inhibit lipid peroxidation in the NADPH-and Fe-dependent systems, it could be shown thatdifferent structures may be essential in the individualsystems. The authors concluded that the high antioxi-dant capacity of many polyphenols depends onseveral modes of interaction and they may participateto differing degrees, depending on the pro-oxidantsystem used. The consequence is a different responseunder variable biochemical conditions in theorganism.

Seo and colleagues47 determined the polyphenoliccomposition of Lonicera japonica extracts, by HPLCwith mass spectroscopy (MS) detector, and the antioxi-dant activity of the flavonoid mixture in the leaf, stemand flowers of this plant. The 25 identified phenolsbelong mainly to the hydroxycinnamic acid and flavo-noids group. The antioxidant activity was highest forthe leaf, followed in order by the flower and stem. Theauthors concluded that the trend of the antioxidantactivities depended principally on the concentration ofthe flavonoid contents. In fact, flavonoids with theC2�C3 unsaturation on the C-ring (structure box)

2573. ANTIOXIDANT CAPACITY OF NATURAL SOURCES RICH IN POLYPHENOLS, AND ISOLATED PHENOLIC COMPOUNDS

3. OXIDATION AND ANTIOXIDANT ACTIVITY OF POLYPHENOLS

were shown to be much more effective antioxidantagents than the other phenolic compounds, as the longconjugation extending from the B-ring to the carbonyloxygen at the C-ring can readily accommodate the oddelectron radical.48

Eight active polyphenols were isolated from longanseeds in the studies of Zheng and colleagues.49 Theyhave tested these compounds against DPPH and super-oxide anion radicals, demonstrating that in both assays,all the purified polyphenols showed radical scavengingactivities in a dose-dependent manner but in differentintensities. Gallic acid, ethyl gallate, 1-O-galloyl-β-D-glucopyranoside, methyl brevifolin carboxylate, brevifo-lin, corilagin, ellagic acid, and 4-O-α-L-rhamnopyrano-syl-ellagic acid were found in this plant.

In the DPPH assay, the scavenging activity (SC50)values of the isolated compounds ranged from 0.80to 5.91 μg/mL, in a decreasing antioxidant order of:gallic acid. ethyl gallate.methyl brevifolin carbox-ylate. corilagin. ellagic acid. brevifolin. 1-O-galloyl-β-D-glucopyranoside. 4-O-α-L-rhamnopyranosyl-ellagicacid. Similarly, the superoxide anion radical scavengingactivity (SC50) values were between 1.04 and 7.03 μg/mL,in the order: gallic acid. ethyl gallate. corilagin.ellagic acid.methyl brevifolin carboxylate. 1-O-gal-loyl-β-D-glucopyranoside. brevifolin. 4-O-α-L-rhamno-pyranosyl-ellagic acid). Gallic acid and ethyl gallatewere, thus, the most active compounds in both assays.

4. BENEFITS OF POLYPHENOLSANTIOXIDANT PROPERTIES IN HUMAN

DISEASES—HEALTH PROMOTION

Oxidative stress is an imbalance in the redox statusof a cell, between the production of ROS and antioxi-dant defense mechanisms, leading to damage, poten-tial mutations and ultimately the formation ofcancer.50 A great deal of research has led to evidencethat oxidative stress, resulting in reactive oxygen spe-cies generation, either through an enzyme or metalcatalyzed process, plays a decisive role in clinical dis-orders.50,51 Defense against oxidative stress is, there-fore, an important factor in preventing thedevelopment of many diseases. A wide definition ofantioxidants describes them as any substance, presentin low concentrations when compared to the oxidiz-able substrate, capable of effectively delaying or inhi-biting the substrate oxidation.52

Current evidence strongly supports a contributionof polyphenols in the prevention of cardiovascular dis-eases, cancers, osteoporosis, suggesting a role in theprevention of neurodegenerative diseases and diabetesmellitus.53 And numerous studies have been

performed to elucidate intrinsic mechanisms to dis-cover new effective antioxidants from natural sources.

4.1 Polyphenols and Inflammation

In this way, Kostyuk and colleagues54 investigatedthe antioxidant property and signal modulation capac-ity of three polyphenols: quercetin (2), resveratrol (8),and verbascoside, from a natural source, in controllingvascular inflammation. This study was proposed dueto the fact that oxidized low-density lipoproteins playa critical role in the initiation of atherosclerosisthrough inflammatory signaling activation.

A significant decrease in intracellular nitric oxidelevels and superoxide overproduction was found inhuman umbilical vein endothelial cells treated withoxidized LDL (oxLDL), but not with LDL. The redoximbalance was prevented by the addition of quercetin(2) or resveratrol (8). Resveratrol and verbacosidemodulated the inflammatory response in humanumbilical vein endothelial cell proliferation, decreas-ing, at least partially, the overexpression of chemo-kines and adhesion molecules after treatment withoxLDL.

The data indicate that plant polyphenols may affectvascular inflammation not only as antioxidants butalso as modulators of inflammatory redox signalingpathways. The authors suggest that verbascoside andresveratrol (8) could be potent candidates as anti-atherogenic agents because they combine antioxidantand anti-inflammatory properties; while quercetin (2),despite its effectiveness in preventing LDL oxidationand cellular redox imbalance, can potentiate pro-inflammatory signaling by oxLDL and consequentlymay cause contradictory effects on early atherogenesis.It is necessary for more studies to be made in order toconfirm these results.

Several studies suggest a correlation between theantioxidant and anti-inflammatory activities, leading tothe conclusion that some plant extracts rich in antioxi-dant compounds, such as polyphenols, can reduceinflammation by a number of mechanisms, includingsuperoxide anion elimination, which is known to par-ticipate in polymorphonuclear cells recruitment ininflamed tissues.55

A growing amount of evidence indicates that theconsumption of plant foods is correlated with a lowerrisk of the development of arteriosclerosis and oxida-tive stress-related diseases.56 Antioxidants mayreduce atherogenesis and improve vascular functionby the inhibition of oxidative modification. Whenthey are present in the cells of the vascular wall, anti-oxidants decrease cellular production of reactive oxy-gen species, thereby preventing endothelialdysfunction.57 Thus, they are capable of improving

258 20. POLYPHENOL ANTIOXIDANTS FROM NATURAL SOURCES AND CONTRIBUTION TO HEALTH PROMOTION

3. OXIDATION AND ANTIOXIDANT ACTIVITY OF POLYPHENOLS

Structures box. Chemical structures of some polyphenols cited in the text. (1) Myricetin; (2) Quercetin; (3) Kaempferol; (4) (2 )-Epicatechin8-C-galactoside; (5) Procyanidin B2; (6) Procyanidin B5; (7) (2 )-Epicatechin; (8) Resveratrol; (9) Morin; (10) Catechin; (11) Fisetin;(12) Epigallocatechin; (13) Epicatechin-3-gallate.

2594. BENEFITS OF POLYPHENOLS ANTIOXIDANT PROPERTIES IN HUMAN DISEASES—HEALTH PROMOTION

3. OXIDATION AND ANTIOXIDANT ACTIVITY OF POLYPHENOLS

symptoms of atherosclerosis, a chronic inflammatorydisease.

An interesting study evaluated the relatively short-term (14 days) ingestion of purple grape juice(7.76 1.2 mL/kg/day), rich in polyphenols, and itsimpact on endothelium vasodilatation. An associationbetween the ingestion of grape juice and significantimprovement in the endothelium-dependent vasodila-tion in men has been observed.58 Furthermore, a regu-lar ingestion of five cups/day of black tea, for 4weeks, by 21 subjects has been shown to result in asignificant increase in endothelium-dependentvasodilatation.59

Also, the intragastric administration of resveratrol(8) (3 mg/kg/day), red wine (4 mL/kg/day) or evendealcoholized red wine (4 mL/kg/day) for 12 weeks tohypercholesterolemic rabbits improved the endothelialfunction, reduced plasma endothelin-1 levels andinduced a significant elevation in nitric oxid (NO)levels.60 The study developed by Fisher and collea-gues61 demonstrated that, in healthy humans, a regularintake of flavanol-rich cocoa for 4 days (821 mg of fla-vanols/day) induced a prominent peripheral vasodila-tion via activation of the NO pathway. All the studiesdemonstrated here and, also those found in the litera-ture, strongly support the view that a polyphenol-richdiet can improve endothelium function.

In addition, considering the isolated compoundsevaluated in the studies, different structure-effectrelationships have been found, depending on theproposed effect. For instance, according to thereview study of Stoclet and colleagues,62 amongstructurally close anthocyanins, delphinidin (but notcyanidin or malvidin) is able to induce NO forma-tion in endothelial cells. By contrast, cyanidin anddelphinidin (but not malvidin), are able to inhibit,for example, vascular endothelial growth factor for-mation in smooth muscle cells.

4.2 Polyphenols in Cardiovascular Diseases

Significant progress is observed in the field of car-diovascular diseases as well, it being well establishedthat some polyphenols, administered as supplementsor in combination with food, can improve health sta-tus, as indicated by several biomarkers associated withcardiovascular risk.63 Epidemiologic studies tend toconfirm the protective effects of polyphenol consump-tion against cardiovascular diseases.64

Among the studied mechanisms by which polyphe-nols may confer cardiovascular protection are theimprovement of endothelial function, the inhibition ofangiogenesis and cell migration, and the proliferationin blood vessels. The beneficial effects of dietary

polyphenols on vascular ischemic obstruction eventsmight also be related to the prevention of thrombosisresulting from the inhibition of platelet activation,65,66

or from a decreased expression of pro-thrombotic andpro-atherosclerotic molecules.67,68

4.3 Polyphenols and Diabetes

Antioxidants are important in diabetes, since it hasbeen demonstrated that low levels of plasma antioxi-dants are implicated as a risk factor for the develop-ment of this disease69 and circulating levels of radicalscavengers are impaired throughout the progression ofdiabetes.70 Many of the complications of diabetes,which lead to the mortality of the patient, have beenlinked to oxidative stress, so antioxidants are impor-tant candidates in the treatment of this disease.71,72

Mechanisms that contribute to increased oxidativestress in diabetes include non-enzymatic glycosylation,auto-oxidative glycosylation and metabolic stress. Therecognized benefits of antioxidants in the preventionof the complications of diabetes support and validatethe use of some traditional medicines, like those testedin the study of McCune and Johns.73 Thirty-five plantspecies were selected from the boreal forest in Canada,and it is well known that most of these plants presentpolyphenols in their composition. Three antioxidantassays were tested and it was verified that the majorityof the species (63 and 97%, respectively) had scaveng-ing activities similar to ascorbic acid in the superoxideand peroxyl radical scavenging assays.

In this way, fruits of Terminalia chebula, T. belerica,and Emblica officinalis, were studied in order to estab-lish an association between the antioxidant activity oftheir extracts and diabetes.74 The authors found thatsamples scavenged the superoxides generated by pho-toreduction of riboflavin. The concentrations of plantextracts needed for 50% scavenging of superoxideswere 20.5, 40.5 and 6.5 μg/mL, respectively. In thehydroxyl radical scavenging system, the extracts’ IC50

were 165.5, 71, and 155 μg/mL, respectively. The IC50

concentrations to inhibit lipid peroxidation by theseplants were 85.5, 27 and 74 μg/mL, respectively.

Hydroxyl radicals, as well as lipid peroxidation,were better inhibited by T. belerica as compared withthe other two extracts. Administration of these plantextracts was found to reduce serum glucose levels innormal rats both in single and multidose studies. Atthe second hour of the study, T. belerica extract was themost active (52.74%), followed by T. chebula (50.98%),and E. officinalis (29.52%). In the multidose study, fromthe 7th to the 11th day, all the extracts significantlyreduced the serum glucose level. The fasting bloodglucose levels in alloxan-induced diabetic rats were

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also tested, demonstrating values ranging from 340 to400 mg/100 mL.

Therefore, T. belerica was the most active plant inboth antioxidant and antidiabetic experiments. Themajor ingredients in this plant are ellagic and gallicacids. The authors suggest that it is possible that theseextracts reduce the effect of inflammatory cytokinerelease during diabetes which, as previously described,may be one of the causative agents for the tissue dis-truction and insulin resistance in this pathology.75

4.4 Role of Phenols in Cancer

The origins and causes of various cancers are notyet very clear. It is well known, however, that themain carcinogenesis inducing agent groups are repre-sented by reactive oxygen and nitrogen species. Or byother free radicals and lipid peroxidation products thatinduce various cellular and nuclear injuries.76,77 In thiscontext, several studies have demonstrated, without adoubt, that there is a strong inverse association (ornegative) between the consumption of fruits andvegetables and the risk of several cancer types andother morbidity and mortality causes.78,79 Thus, forseveral decades researchers have been concerned withisolating the compounds found in plant foods to testthem as potential anticarcinogenic agents.

The main carcinogenesis inhibitor groups are repre-sented by antioxidants, free radical blockers.Polyphenols are included in this group, since it isdescribed that quercetin (2), rutin, luteolin and glyco-side derivatives, myricetin (1), rosmarinic acid and cat-echin (10) protect the DNA of reactive oxygen specieslesions.80,81 Another important candidate is resveratrol(8), which presents potent anticarcinogenic effects.This compound is found in grape skins, with higherconcentrations in red wines, and show antitumorcapacity by inducing neoplastic cell deaths.82 Also,there is strong evidence demonstrating that tea cate-chins (epigallocatechin (12), catechin (10), epicatechin,etc.), especially green tea catechins, have importantanticancer effects.83

4.5 Polyphenols in NeurodegenerativeDisorders

The benefits of the polyphenols antioxidant proper-ties were also evaluated in the central nervous system(CNS). It is well known that free radicals induce lipidperoxidation in cell membranes and initiate neuronaldysfunction and death. Furthermore, after injury, themembrane peroxidation cascade proceeds automati-cally and induces edema, infarction and neuronal dys-function in the brain. In addition, oxidative stress has

been shown to contribute to neuronal dysfunction anddeath in a focal brain ischemia model.84

Itoh and colleagues85 investigated the effect of(2)-epigallocatechin gallate (EGCG), the prevalentgreen tea polyphenol, on neural stem cell proliferationaround the damaged area following traumatic braininjury. This compound presents phenolic hydroxylgroups on its aromatic rings, which confer antioxidantand iron chelating activities to the molecule. In thisin vivo study, rats were treated with water containing0.1% (w/v) EGCG for 10 weeks. Analysis of immuno-histochemistry revealed that EGCG treatment beforeand after traumatic brain injury eliminated and/orabsorbed free radicals (such as O2

2 and •OH) inducedby the injury. Apoptosis and cell death of neuronalcells and neuronal stem cells (NSC) induced by freeradical production were inhibited, thus protectingnestin-positive cells, including NSC, around the dam-aged area in the early phase following traumatic braininjury.

Antioxidants and anti-inflammatory moleculeshave been associated with some neurodegenerativediseases, like Alzheimer and Parkinson pathologies,as demonstrated by Silveira and colleagues.86 Thisresearch work evidenced that the alcoholic extracts ofMyrcianthes pungens fruits presented at least four dif-ferent substances capable of inhibiting AChE inunripe and ripe fruits of this species, by an in vitrobioautographic assay using Fast Blue Salt B reagent.The superexpression of this enzyme is responsible forthe clinical effects on Alzheimer’s disease (AD).Thus, fruits like guabiju, that present compoundscapable of preventing oxidative damage and an anti-inflammatory process, could be interesting sources ofactive anti-degenerative molecules.

Oxidative stress is an important characteristic of ADas determined by increased oxidative stress markersincluding DNA, RNA, lipid, and protein oxidation inAD and mild cognitive impairment.87,88 When ROS isnot removed efficiently, it would be detrimental toneurons exacerbating neurodegeneration.

Although few studies in this area have been pub-lished, it is demonstrated that AD patients presentdecreased levels of plasma antioxidants and totalplasma antioxidant activity.89,90 Additionally, theseelevated markers for oxidative stress precede amyloiddeposition and neurofibrillar tangles (pathologicalcharacteristic of this neurodegeneration), suggestingoxidative stress is an early event involved in ADpathogenesis.91,92

Catechins and polyphenols from green tea exerttheir antioxidative action by chelating metal ions,such as iron and copper, and can also prevent thegeneration of hydroxyl radical via the Fenton reac-tion.93 It was found that (2)-epigallocatechin-3-gallate

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is the main polyphenolic constituent of green tea, fol-lowed by (2)-epigallocatechin (12), (2)-epicatechin(7), and (2)-epicatechin-3-gallate (13). Lee and collea-gues94 demonstrated that the first compound directlyprevents fibril formation of amyloid β, and is capableof rescuing memory impairment, induced by amyloidβ peptide. Furthermore, amyloid β increases the levelof lipid peroxidation markers such as malondialde-hyde in cells, and (2)-epigallocatechin-3-gallateattenuates lipid peroxidation.95

Assuncao and colleagues96 developed an in vivostudy with green tea, and demonstrated that the treat-ment protected lipids and proteins against oxidation,and prevented the increase of lipofuscin deposition inhippocampal neurons of elderly rats, as compared toage-matched controls. Thus, despite the deficiency insystematic clinical trials with tea polyphenols forneurodegenerative diseases, animal and some humanepidemiological studies support the inverse correlationbetween tea consumption and incidence of dementiaand AD.

It was shown that natural polyphenols significantlyattenuated cognitive impairments and amyloid-betaburden.97,98 Also, epidemiological studies suggest thatdietary habit and antioxidant intake may impact theincidence of neurodegenerative disease such as AD.99

Therefore, polyphenols might be a potential pharmaco-logical strategy for prevention or treatment of AD.

Other polyphenols plant sources are blueberries, thatare rich in flavonoids, with catechin (10) the main com-pound, followed by epicatechin and anthocyanins. Inseveral in vitro studies, blueberry extract has shown neu-roprotection via antioxidant and anti-inflammatoryproperties.100 Blueberry extract inhibitedlipopolysaccharide-induced inflammatory responses asevidenced by decreased levels of NO and otherinflammatory markers.101 None of the research describeswhich polyphenols isolated are responsible for improv-ing cognitive function, although Joseph and colleagues102

showed that whole extract and anthocyanin fractionhave the greatest neuroprotection, whereas chlorogenicacid demonstrates the lowest protection.

In this way, the neuroprotective effects of Merlot redwine and its isolated polyphenols were evaluated in anoxidative stress model induced by the Fenton reactionand hydrogen peroxide in the human astrocytoma cellline, by Martın and colleagues.103 The authors usedastrocytes because they are the major glial cells andpresent a variety of crucial roles in the nervous system,providing protection to neurons against oxidative dam-age induced by free radical compounds.

The pre-incubation with Merlot red wine for 1 daycaused a significant increase in cell viability, reducingthe ROS production, in all concentrations assayed (6.8,10.2 and 13.6 mL/L). Twenty-four compounds were

identified in Merlot red wine, the most abundant poly-phenols found belonged to the flavonoid class, such ascatechin (10), epicatechin, quercetin (2), and procyani-dins; gallic acid and tyrosol were also detected.

As a conclusion, the authors indicated that Merlotred wine is a possibly useful candidate in the preven-tion or therapy of neurodegenerative disease, and thatthe neuroprotective activity of the red wine polyphe-nols studied may be extensible to those polyphenols infruits and vegetables.

The potential protective role of these polyphenolswhen isolated demonstrates that they decrease reactiveoxygen species generation, and increase the activityand protein expression of catalase, superoxide dismu-tase, glutathione reductase, and glutathione peroxidaseantioxidant enzymes. These results reveal a high corre-lation between polyphenol content and antioxidantactivity. Of the isolated polyphenols, quercetin (2) andprocyanidins showed the highest neuroprotectiveeffect.

In this research area some controversies areobserved, demonstrating that unequivocal evidenceabout the efficacy, bioavailability, safety and appropri-ate antioxidant dosage in relation to chronic diseases isnecessary. Bioavailability of the natural compounds,for example, is a key factor for new drug discovery.Even though there is a lack of pharmacokinetic dataon polyphenolics, several studies have demonstratedthat polyphenolics could readily cross the blood-brainbarrier and exhibit pharmacological effects in the tar-get regions of the brain.

Although no scientific definitive evidence appearsto exist, it is prudent and advisable in terms of publichealth to increase the consumption of vegetable foods,many of them rich in polyphenol antioxidant sub-stances, and follow a diet similar to the so-called“Mediterranean diet,”104 rich in fruits, vegetables, fish,minerals, etc., in order to ensure a healthy life andlongevity.

5. CONCLUSION

The literature provides important evidence of thepolyphenols richness, mainly from natural sources,and their structural diversity. This chemical diversityleads to different levels of antioxidant activity fromthese compounds, given by different mechanisms andallowing that antioxidants may act in different dis-eases related to the generation and accumulation offree radicals. Therefore, polyphenols may contributeto the maintenance of life quality and/or to theimprovement of symptoms from various diseases,exerting an important detoxification role in the humanbody.

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Cardiovascular, neurodegenerative, cancer andinflammatory diseases, as well as diabetes, amongothers, are examples of pathologies where antioxidantsdemonstrate valuable and interesting applications.However, as a perspective, researchers should be con-centrating on pharmacokinetics research, includingbioavailability analysis of interesting polyphenols,assessment of appropriate dosage, and toxic effects ofhigh dosage, in order to ensure safety and effective-ness in the administration of these compounds for thetreatment and/or prevention of diseases.

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3. OXIDATION AND ANTIOXIDANT ACTIVITY OF POLYPHENOLS