Research Article Antioxidant Properties of Brazilian Tropical … · 2019. 7. 31. ·...

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 132759 8 pageshttpdxdoiorg1011552013132759

Research ArticleAntioxidant Properties of Brazilian Tropical Fruits byCorrelation between Different Assays

Elena Gregoris1 Giuseppina Pace Pereira Lima2 Sabrina Fabris1 Mariangela Bertelle1

Michela Sicari1 and Roberto Stevanato1

1 Department of Molecular Sciences and Nanosystems University Carsquo Foscari of Venice Dorsoduro 2137 30123 Venice Italy2 Institute of Biosciences UNESP University Campus of Botucatu CP 510 18618-000 Botucatu SA Brazil

Correspondence should be addressed to Roberto Stevanato rstevuniveit

Received 16 April 2013 Revised 16 July 2013 Accepted 23 July 2013

Academic Editor Filippo De Simone

Copyright copy 2013 Elena Gregoris et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Four different assays (the Folin-Ciocalteu DPPH enzymatic method and inhibitory activity on lipid peroxidation) based onradically different physicochemical principles and normally used to determine the antioxidant activity of food have been confrontedand utilized to investigate the antioxidant activity of fruits originated from Brazil with particular attention to more exotic andless-studied species (jurubeba Solanum paniculatum pequi Caryocar brasiliense pitaya Hylocereus undatus siriguela Spondiaspurpurea umbu Spondias tuberosa) in order to (i) verify the correlations between results obtained by the different assays with thefinal purpose to obtain more reliable results avoiding possible measuring-method linked mistakes and (ii) individuate the moreactive fruit species As expected the different methods give different responses depending on the specific assay reaction Anyhowall results indicate high antioxidant properties for siriguela and jurubeba and poor values for pitaya umbu and pequi Consideringthat no marked difference of ascorbic acid content has been detected among the different fruits experimental data suggest thatantioxidant activities of the investigated Brazilian fruits are poorly correlated with this molecule principally depending on theirtotal polyphenolic content

1 Introduction

It is known that the consumption of fruit and vegetablereduces the incidence of cardiovascular and cerebrovasculardiseases stroke cancer and ageing related disorders [1ndash3]This effect is attributed to the presence in fruit and vegetablesof antioxidants able to preserve the correct balance oxi-dantsantioxidants in which upset due to an overproductionof oxygen reactive species (ROS) can lead to the so-calledldquooxidative stressrdquo [4ndash6]

Substantial damages have been observedwhenROS inter-act withDNAmembrane lipids and proteins [7ndash10] ROS areinvolved in the carcinogenic stages of initiation promotionand progression [11] they play an important role in the devel-opment of cardiovascular diseases such as ischemic injuryarteriosclerosis hypertension cardiomyopathies congenitalheart diseases and stroke they may be a causal factor ofneurological disorders such as Alzheimerrsquos and Parkinsonrsquosdiseases [12]

Antioxidant substances represent one of the most impor-tant defense mechanisms against free radicals but theonly endogenous antioxidant molecules cannot be effectiveenough to counteract the injuries caused by ROS particularlyin the current times where lifestyles based on smoke drugsalcohol unbalanced diet pollution incorrect exposure tosolar radiation and so forth can facilitate free radicalsformation For this reason increasing the intake of dietaryantioxidant is of great importance to enjoy good health asevidenced by studies on food characterized by high antioxi-dants content [13]

Unfortunately no reliable biomarker of antioxidant activ-ity is available up to now [14 15] because ROS injuries aremediated by different radical and nonradical species whichshow different physicochemical characteristics and reactionmechanism affecting reactivity selectivity partition in aque-ous and lipid phase and so forth [16] In literature manyexperimental methods are reported to determine a generic

2 BioMed Research International

antioxidant activity of a compound but results obtained bydifferent investigations are frequently contradictory [17]

The aim of this work is to compare of the results obtainedby four different methods usually employed to measureantioxidant properties that is reducing capacity by the Folin-Ciocalteu assay radical scavenging ability towards 221015840-diphenyl-1-picrylhydrazyl (DPPHmethod) inhibitory abilityon peroxidation of linoleic acid (LA) and total phenolicdetermination by the enzymatic method [18] in order to (i)verify possible correlations between the results obtained and(ii) obtain more reliable results avoiding possible measuring-method linked mistakes

These assays were applied to a series of Brazil fruits withparticular interest in the more exotic and less studied speciesIn fact information on the nutritional values of the mostexotic species of tropical fruits are limited some studies [19ndash22] provide evidence for the high antioxidant capacity andsignificant amounts of flavonoids and vitamin C for the mostcommon Brazilian fruits as mango [23] starfruit [19] andavocado [24] but no data are reported for more exotic fruitslike pitaya jurubeba siriguela and pequi some of whichnative peoples utilize in popular medicine

The results obtained by these measurements were com-pared with each other and with those obtained by Italiansoft fruits known for their antioxidant activity [25 26]Furthermore to discriminate possible interferences due toascorbic acid and anthocyanins the content of these reducingmolecules in all fruits was also carried out

Similitude and differences were discussed on the light ofthe chemical characteristics of the assay reactions

2 Materials and Methods

21 Chemicals All chemicals of the highest available qualitywere obtained from Sigma Chemical Co (St Louis USA)ABIP (221015840-azobis[21015840-(2-imidazolin-2-yl)propane] dihydro-chloride) was obtained from Wako Chemicals (Germany)The aqueous solutions were prepared with quality milli-Qwater Each experiment was in triplicate

22 UV-VIS and ElectrochemicalMeasurements Spectropho-tometric measurements were recorded on a UV-VIS Shi-madzu UV-1800 instrument equipped with a temperaturecontrolled quartz cell The measures of oxygen consumptionwere performed with a potentiostat Amel 559 equipped withan oxygen microelectrode (MI-730 Microelectrodes)

23 Fruits and Sample Pretreatments Table 1 reports com-mon and scientific names of all studied fruits Mango avo-cado carambola and pitaya were from Sao Paulo state whilejurubeba umbu graviola pequi siriguela and tamarindwerefrom tropical Brazil soft fruits were from Italy After cleaningwith distilled water edible fruits portions were grated andcentrifuged by a Krups centrifuge under nitrogen flux toavoid the oxidation of the natural components and the juicewas immediately analysed

24 Inhibition of Lipid Peroxidation (ILP) The antioxidantactivity of fruits to prevent linoleic acid (LA) peroxidation

Table 1 Selected fruits and their abbreviation

Scientific name Common name AbbreviationPersea americana Avocado AvAnnona muricata Graviola GrSolanum paniculatum Jurubeba JuMangifera indica Mango Haden MHMangifera indica Mango Palmer MPMangifera indica Mango Tommy Atkins MTCaryocar brasiliense Pequi PeHylocereus undatus Pitaya PiSpondias purpurea Purple mombin (siriguela) SiAverrhoa carambola Starfruit (carambola) StTamarindus indica Tamarind TaSpondias tuberosa Umbu UmRubus ulmifolius Blackberry BaVaccinium cyanococcus Blueberry BuRubus idaeligus Raspberry RaRibes rubrum Redcurrant ReFragaria Strawberry Sw

was determined in sodium dodecyl sulfate (SDS)micelles Aspreviously reported [27] the fruitrsquos antioxidant capacity wascalculated as the juice concentration (ppm) halves the rate ofoxygen consumption due to the peroxidation process and itis expressed as inhibitory concentration IC

50

25 22-Diphenyl-1-picrylhydrazyl (DPPH)Radical ScavengingCapacity Assay This method is based on the capacity of anantioxidant to scavenge the stable free radicalDPPH [28]Theprocedure is reported in Stevanato et al [18] the results areexpressed as catechin equivalent concentration (CE)

26 Folin-Ciocalteu Assay and Total Phenolics Content (TPC)by Enzymatic Method The Folin-Ciocalteu assay and theTotal Phenolic Content were determined spectrophotomet-rically according to the procedures previously reported [18]and the results were expressed as catechin equivalent (CE)

27 Total Hydroxycinnamic Acid Content (HCA) Hydrox-ycinnamic acid content was determined according toZaporozhets et al [29] The complex of hydroxycinnamicacids with aluminium (III) was measured at 365 nm andcaffeic acid was used as a standard the results were expressedas milligramsliter of caffeic acid equivalents

28 Total Anthocyanin Content (TAC) The TAC wasdetermined according the pH-differential method [30]Absorbance at 510 and 700 nm of juice buffered at pH 45 e 10was calculatedThe anthocyanin concentrationwas expressedas milligramsliter of cyanidin-3-glucoside equivalents

BioMed Research International 3

Table 2 Results obtained by ILP DPPH TPC Folin HCA and TAA assays of selected fruits

Fruit Abbreviation ILPIC50 (ppm)

DPPHCE (mM)

TPCCE (mM)

FolinCE (mM)

HCA(mgL)

TAA(mM)

Avocado Av 240 plusmn 20 01 plusmn 001 106 plusmn 005 199 plusmn 004 56 plusmn 6 31 plusmn 02

Graviola Gr 87 plusmn 9 24 plusmn 06 37 plusmn 05 86 plusmn 04 42 plusmn 3 47 plusmn 02

Jurubeba Ju 60 plusmn 8 09 plusmn 03 78 plusmn 05 36 plusmn 2 3242 plusmn 20 34 plusmn 02

Mango Haden MH 200 plusmn 20 031 plusmn 005 075 plusmn 009 57 plusmn 02 184 plusmn 12 86 plusmn 08

Mango Palmer MP 240 plusmn 20 089 plusmn 001 121 plusmn 005 45 plusmn 03 80 plusmn 9 5 plusmn 1

Mango Tommy Atkins MT 300 plusmn 20 050 plusmn 007 015 plusmn 003 14 plusmn 01 90 plusmn 10 37 plusmn 06

Pequi Pe 500 plusmn 50 01 plusmn 001 05 plusmn 01 79 plusmn 02 66 plusmn 7 24 plusmn 03

Pitaya Pi 1000 plusmn 100 01 plusmn 001 16 plusmn 02 21 plusmn 02 152 plusmn 12 26 plusmn 02

Siriguela Si 44 plusmn 4 8 plusmn 1 32 plusmn 01 34 plusmn 5 264 plusmn 23 47 plusmn 03

Carambola St 70 plusmn 7 25 plusmn 01 54 plusmn 04 105 plusmn 01 164 plusmn 14 42 plusmn 03

Tamarind Ta 100 plusmn 20 24 plusmn 03 29 plusmn 01 185 plusmn 08 168 plusmn 15 7 plusmn 1

Umbu Um 500 plusmn 30 067 plusmn 005 14 plusmn 02 42 plusmn 01 46 plusmn 3 15 plusmn 02

Blackberry Ba 109 plusmn 6 30 plusmn 02 266 plusmn 006 84 plusmn 01 203 plusmn 19 44 plusmn 06

Blueberry Bu 41 plusmn 7 34 plusmn 01 28 plusmn 01 78 plusmn 05 350 plusmn 25 30 plusmn 01

Raspberry Ra 77 plusmn 9 42 plusmn 01 27 plusmn 01 21 plusmn 1 101 plusmn 9 37 plusmn 03

Redcurrant Re 56 plusmn 2 34 plusmn 04 39 plusmn 04 100 plusmn 01 180 plusmn 12 4 plusmn 04

Strawberry Sw 38 plusmn 4 41 plusmn 03 304 plusmn 009 10 plusmn 3 190 plusmn 13 6 plusmn 1

29 Total Ascorbic Acid (TAA) The TAA is assayed as pre-viously described [31] with minor modifications A 20mMoxalic acid solution containing the sample 0186mM 26-dichlorophenol-indophenol (DCFI) 10mM dinitrophenyl-hydrazine (DNPH) and 13mM thiourea were incubated ina boiling water bath for 15 minutes Once cooled an equalvolume of 85 sulfuric acid was added to the solution andthe absorbance at 520 nm was measured 15 minutes laterThe same procedure was repeated without the sample andthe blank value was subtracted from the absorbance of thesample

In Table 2 where the results obtained by applying ILPFolin DPPH and TPC enzymatic methods are reported itappears that jurubeba and siriguela show very low IC

50values

(ie high antioxidant activity) in the range of those found forthe more active Italian soft fruits (blueberry redcurrant andraspberry) For the same fruits DPPH TPC and Folin assaysgive very high values of CE if compared with the average ofother fruits indicating an univocal high antioxidant activityof these two fruits

On the basis of their IC50values the investigated Brazil-

ian tropical fruits can be roughly divided into three groupscharacterized approximately by good medium and poorantioxidant properties respectively (Figure 1) (1) fruits withlog(IC

50) le 2 (IC

50le 100 ppm) graviola jurubeba siriguela

carambola and tamarind (2) fruits with 2 lt log(IC50) le 25

(IC50ranging from 100 to 316 ppm) avocado and mango (3)

fruits with log(IC50) gt 25 (IC

50gt 316 ppm) pequi umbu

and pitayaIn Figure 2 correlations between data obtained by ILP

expressed as 1IC50and other adopted methods expressed as

catechin equivalent amount (CE) are reported

The comparison of the data obtained by ILP versusDPPH scavenging methods (Figure 2(a)) points out a goodcorrelation (119877 = 079) in fact only few points referred to thatstrawberry blueberry jurubeba and in less amount siriguelascatter from the linear relationship

Analogous graph created for comparison of ILP withenzymatic or the Folinmethods (Figures 2(b) and 2(c)) showsless good correlations (119877 = 060 and 030 resp) but alsoin this case strawberry blueberry jurubeba and in partsiriguela appear to worsen the correlation coefficient

TAC measurements showed the absence of anthocyaninsin analyzed Brazilian fruits while as regards the hydroxycin-namic acid content the values of HCA equivalents obtainedfor the studied fruits and reported in Table 2 show a very highvalue of HCAeq for jurubeba

No correlation appears comparing TAA values with thedata obtained by the other analytical methods (data are notshowed)

3 Discussion

31 On the Assay Methods Several methods are proposed toevaluate the antioxidant activity of molecules or food [26ndash28 32ndash35] Each assaymeasures a specific chemical or physic-ochemical parameter which can be correlated with the com-plex and in part unknownmechanisms related to ROS injuryIt follows that the results obtained are partial and sometimeare affected by other variables not strictly correlated to theantioxidant activity In this work we chose four differentassays which significantly represent the main methods ofmeasuring the antioxidant properties of a substance

The Folin-Ciocalteu is a very aged and largely used assaybased on the absorbance changes due to the oxidation of any


Av Gr Ju MH MP MT Pe Pi Si St Ta Um Ba Bu Ra Re Sw15

20

25

30

Med

ium

Goo

dPo

orA

ntio

xida

nt p

rope

rty

Soft fruitsBrazilian fruits

Log(1

IC50)

Figure 1 Classification of studied fruits on the base of their logarithm IC50values

reduced compounds by a phosphomolybdate and phospho-tungstate solution It is a nonspecific method of measuringthe reducing capacity of all the components of the sampleother than polyphenols such as ascorbate [18 36] In fact toavoid an overestimated evaluation of the antioxidant capacitylaborious pretreatments of the sample are suggested [37]

TPC enzymaticmethod on the contrary being ameasureof the total phenolic content of fruit due to the specificityof peroxidase-catalyzed reaction towards phenolic structuresis an indirect evaluation of the antioxidant power whichactually depends not only on the measured total phenoliccontent but also on the chemical structure of each phenoliccomponent [14]

DPPH method is a measure of the electronic transferfrom the phenolic structure to the stable free radical DPPHbut this reaction presents the following disadvantages whichcan underestimate the antioxidant capacity

(i) it may react slowly or be inert to many antioxidants[38]

(ii) reaction kinetic with antioxidants appears not linearto DPPH concentrations [36]

(iii) reaction of DPPH with some phenolic structurescould not go to completion reaching an equilibriumstate as found for eugenol [36]

4 Results

By a physicochemical point of view ILP technique appears tobetter reproduce the in vivo action of antioxidant substancesagainst radical-induced lipid peroxidation of unsaturatedfatty acids residues of biological membranes measuring in

vitro the slowdown due to an antioxidant of the oxygenconsumption in linoleic acid containing SDS micelles Inthis case the influence due to the different lipophilicity ofthe antioxidant molecules is taken in account too Moreoverin this work only clear juices have been analyzed and asa consequence only water soluble antioxidants have beenassessed

Anyway in order to be certain of the data reliability and togive a wider outlook of the problems related to the definitionof the antioxidant activity of foods the same samples werestudied by the above cited four analytical assays and theresults were compared to put in light possible correlations Infact good correlations between results obtained by differentassays can guarantee the best evaluation of the antioxidantproperties of a sample

41 On the Antioxidant Characteristics of Brazilian FruitsSiriguela jurubeba carambola graviola and tamarind showhigh antioxidant activity similar to that of soft fruits [25 26]This result appears very important considering that for someof these fruits no information in literature is reported inparticular about their antioxidant properties [39] Moreoverthe widespread use for curative actions into local populationsof some of these fruits in particular jurubeba and siriguelasuggests further investigations for their possible nutraceuticalproperties

With reference to the scattering from the linear correla-tion of the data referred to strawberry blueberry jurubebaand siriguela as it results in all three graphs of Figure 2plots of correlation of the data obtained by DPPH Folinand enzymatic methods are graphed in order to verify if thisdeviation could be due to a limit of the ILP assay (Figure 3)


0 1 2 3 4 5 6 7 8 90000

0005

0010

0015

0020

0025

0030

Group A

Group B

Av

PePi

MH

MT

Um

MP

Gr

Ta

St

ReJu

Ba

Ra

BuSw

Si

DPPH CE (mM)

R = 079

y = 00042x

1IC

50

(1p

pm)

(a)

0000

0005

0010

0015

0020

0025

0030

0 1 2 3 4 5 6 7 8

Group B

Group AMT

Pe

MH

MPAv

Um

Pi

Ra

Ta

BuSi

Sw

Re

Gr

St

Ju

TPC CE (mM)

R = 060

y = 00037x

1IC

50

(1p

pm)

(b)

0000

0005

0010

0015

0020

0025

0030

0 5 10 15 20 25 30 35 40

Av

Gr

Ju

MHMP

MTPe

Pi

Si

St

Ta

Um

Ba

Bu

Ra

Re

Sw

Group A

Group B

Folin CE (mM)

R = 030

1IC

50

(1p

pm)1IC

50

(1p

pm)

y = 738 lowast 10minus4x

(c)

Figure 2 Correlation between ILP and (a) DPPH (b) enzymatic and (c) Folin assay

0 1 2 3 4 5 6 7 8 90

1

2

3

4

5

6

7

8

Group A

Group B

MP

Um

MTMHPe

PiAv

Ra

Sw

Ba

Ta

ReGr

Bu

St

Si

Ju

DPPH CE (mM)

TPC

CE (m

M)

R = 034

y = 07897x

(a)

0

1

2

3

4

5

6

7

8

TPC

CE (m

M)

0 5 10 15 20 25 30 35 40

Av

Gr

Ju

MHMP

MTPe

Pi

Si

St

Ta

Um

BaBu Ra

Re

Sw

Group A

Group B

Folin CE (mM)

R = 069

y = 01867x

(b)

0

1

2

3

4

5

6

7

8

DPP

H C

E (m

M)

0 5 10 15 20 25 30 35 40Folin CE (mM)

Av

Gr

Ju

MH

MPMT

PePi

Si

St Ta

Um

BaBu

Ra

Re

Sw

Group A

Group B

R = 058

y = 01595x

(c)

Figure 3 Correlation between (a) enzymatic and DPPH (b) enzymatic and Folin (c) DPPH and Folin assays



100

200

300

400

0000

0005

0010

0015

0020

0025

00303243

1IC

50

(1p

pm)

1IC50

HCAeq

HCA

eq(m

gL)

Figure 4 Correlation between ILP and HCA equivalents

Also in these cases the data of the above-mentioned fruitsappear considerably out of the correlation straight lineindicating that the chemical compounds that are responsibleof the antioxidant activity are differently recorded by thedifferent analytical methods

Jurubeba and siriguela are two striking examples of howdifferent assays may assign different rankings to antioxidantmolecules as it appears in Figure 3(a) while the antioxidantactivity of jurubeba is high when evaluated by the enzymaticmethod and low when evaluated by DPPH in the case ofsiriguela the DPPH method assigns it excellent antioxidantproperties which are not confirmed by the enzymatic assayThe result of the first case can be due to the high contentin jurubeba of polyphenols characterized by a low tendencyto undergo monoelectronic transfer to DPPH as recentlyverified for different flavonoids [14] Further investigations toclarify this contrasting behaviour are necessary in any case

The better correlation results from the comparison ofthe ILP and DPPH data (Figure 2(a)) In fact both theanalytical methods are based on the redox potentials of themonoelectronic transfer and they appear in some way asa direct measure of the radicals stopping power [28 39] ofthe antioxidant substances in the fruit Moreover the jointdata obtained by IC

50andDPPHexperiments are particularly

efficient for separating poor antioxidants from good onesIC50values that are lower than 100 ppm andor CE values that

are higher than 2mM could be assumed as a reasonable rulefor discriminating very good antioxidants

Even if there is a bad correlation between DPPH andenzymatic data (Figure 3(a)) most of the fruit can be roughlyseparated in two groups (A and B) with different degreesof antioxidant activity suggesting the hypothesis that fruit

of the same group could have quite similar compositions ofantioxidant constituents or molecules which react in similarway to the analytical methods

Anthocyanins are not contained in examined Brazilianfruits while hydroxycinnamic acids are detected their cor-relation with ILP is practically absent as shown in Figure 4For this reason antioxidant property must depend on otherparameters

Table 2 indicates that in general Brazilian fruits haveascorbic acid content comparable to that of soft fruits amongthem two varieties ofmango and tamarind havemeaningfullyhigh TAA content and umbu have the lowest one

No evident relationship between the antioxidant activityof fruit and the content of ascorbic acid is observed siriguelaand jurubeba have the highest antioxidant activity but theyexhibit lower values of vitamin C than mango which is not agood antioxidant instead (Table 2) It follows that antioxidantactivity of the majority of fruits is due to compoundsdifferent fromvitaminC like polyphenolsmainly flavonoidsaccording to results reported for other species of fruit [11 22]

5 Conclusion

Brazilian fruits were used as arbitrary alimentary products tocompare four different assays normally utilised to determineantioxidant activity of food

The better correlation was found between the inhibitionof lipid peroxidation and DPPH method Both these assaysare based on monoelectronic transfer and in our opinionthey mime more than others the efficacy of an antioxidantcompound to prevent oxidative damage on cell membranedespite all the limitations of the DPPH assay above reported


and taking into account the laboriousness of the ILPmethodFrom data obtained by these two methods siriguela andjurubeba show the higher antioxidant activity

The antioxidant activity of themajority of the studied fruitis due to compounds different from vitamin C like flavon-oids because no evident relationship between the antioxidantactivity of fruit and the content of ascorbic acid was observed

Conflict of Interests

This work is free from any conflict of interests

References

[1] Willet and W C Eat Drink and Be Healthy The HarvardMedical School Guide to Healthy Eating McGraw-Hill NewYork NY USA 2001

[2] Z Juranic and K Z Ziza ldquoBiological activities of berries fromantioxidant capacity to anti-cancer effectsrdquo BioFactors vol 23no 4 pp 207ndash211 2005

[3] S Zafra-Stone T YasminM Bagchi AChatterjee J AVinsonand D Bagchi ldquoBerry anthocyanins as novel antioxidants inhuman health and disease preventionrdquoMolecular Nutrition andFood Research vol 51 no 6 pp 675ndash683 2007

[4] A R Ness and J W Powles ldquoFruit and vegetables andcardiovascular disease a reviewrdquo International Journal of Epi-demiology vol 26 no 1 pp 1ndash13 1997

[5] H Esterbauer M Dieber-Rotheneder G Striegl and G WaegldquoRole of vitamin E in preventing the oxidation of low-densitylipoproteinrdquo American Journal of Clinical Nutrition vol 53 pp314sndash321s 1991

[6] M A Eastwood ldquoInteraction of dietary antioxidants in vivohow fruit and vegetables prevent diseaserdquo Quarterly Journal ofMedicine vol 92 no 9 pp 527ndash530 1999

[7] L J Marnett ldquoOxyradicals and DNA damagerdquo Carcinogenesisvol 21 no 3 pp 361ndash370 2000

[8] B Halliwell and J M C Gutteridge Free Radical Biology andMedicine Oxford University Press Oxford UK 3rd edition1999

[9] H Esterbauer and P Ramos ldquoChemistry and pathophysiologyof oxidation of LDLrdquo Reviews of Physiology Biochemistry andPharmacology vol 127 pp 31ndash64 1996

[10] HWang G Cao and R L Prior ldquoTotal antioxidant capacity offruitsrdquo Journal of Agricultural and Food Chemistry vol 44 no3 pp 701ndash705 1996

[11] M Valko C J Rhodes J Moncol M Izakovic and M MazurldquoFree radicals metals and antioxidants in oxidative stress-induced cancerrdquo Chemico-Biological Interactions vol 160 no 1pp 1ndash40 2006

[12] M Valko D Leibfritz J Moncol M T D Cronin M Mazurand J Telser ldquoFree radicals and antioxidants in normal physi-ological functions and human diseaserdquo International Journal ofBiochemistry and Cell Biology vol 39 no 1 pp 44ndash84 2007

[13] M Antolovich P D Prenzler E Patsalides S McDonald andK Robards ldquoMethods for testing antioxidant activityrdquo Analystvol 127 no 1 pp 183ndash198 2002

[14] E Gregoris and R Stevanato ldquoCorrelations between polyphe-nolic composition and antioxidant activity of Venetian propo-lisrdquoFood andChemical Toxicology vol 48 no 1 pp 76ndash82 2009

[15] G Chiva-Blanch and F Visioli ldquoPolyphenols and health mov-ing beyond antioxidantsrdquo Journal of Berry Research vol 2 pp63ndash71 2012

[16] M Takashima M Horie M Shichiri Y Hagihara Y Yoshidaand E Niki ldquoAssessment of antioxidant capacity for scavengingfree radicals in vitro a rational basis and practical applicationrdquoFree Radical Biology and Medicine vol 52 no 7 pp 1242ndash12522012

[17] E Niki ldquoAntioxidant capacity which capacity and how to assessitrdquo Journal of Berry Research vol 1 pp 169ndash176 2011

[18] R Stevanato S Fabris and F Momo ldquoNew enzymatic methodfor the determination of total phenolic content in tea and winerdquoJournal of Agricultural and Food Chemistry vol 52 no 20 pp6287ndash6293 2004

[19] K Mahattanatawee J A Manthey G Luzio S T Talcott KGoodner and E A Baldwin ldquoTotal antioxidant activity andfiber content of select Florida-grown tropical fruitsrdquo Journal ofAgricultural and Food Chemistry vol 54 no 19 pp 7355ndash73632006

[20] S A de Assis J C R Vellosa I L Brunetti et al ldquoAntioxidantactivity ascorbic acid and total phenol of exotic fruits occurringin Brazilrdquo International Journal of Food Sciences and Nutritionvol 60 no 5 pp 439ndash448 2009

[21] N M A Hassimotto ldquoAntioxidant capacity of Brazilian fruitvegetables and commercially-frozen fruit pulpsrdquo Journal of FoodComposition and Analysis vol 22 no 5 pp 394ndash396 2009

[22] M I Genovese M D Pinto A E D Goncalves and FM Lajolo ldquoBioactive compounds and antioxidant capacity ofexotic fruits and commercial frozen pulps from Brazilrdquo FoodScience and Technology International vol 14 no 3 pp 207ndash2412008

[23] J A Manthey and P Perkins-Veazie ldquoInfluences of harvest dateand location on the levels of 120573-carotene ascorbic acid totalphenols the in vitro antioxidant capacity and phenolic profilesof five commercial varieties of mango (Mangifera indica L)rdquoJournal of Agricultural and Food Chemistry vol 57 no 22 pp10825ndash10830 2009

[24] L Plaza C Sanchez-Moreno S de Pascual-Teresa B de Ancosand M P Cano ldquoFatty acids sterols and antioxidant activityin minimally processed avocados during refrigerated storagerdquoJournal of Agricultural and Food Chemistry vol 57 no 8 pp3204ndash3209 2009

[25] E Sariburun S Sahin C Demir C Turkben and V UylaserldquoPhenolic content and antioxidant activity of raspberry andblackberry cultivarsrdquo Journal of Food Science vol 75 no 4 ppC328ndashC335 2010

[26] S Y Wang and H Lin ldquoAntioxidant activity in fruits and leavesof blackberry raspberry and strawberry varies with cultivarand developmental stagerdquo Journal of Agricultural and FoodChemistry vol 48 no 2 pp 140ndash146 2000

[27] S Fabris FMomoG Ravagnan andR Stevanato ldquoAntioxidantproperties of resveratrol and piceid on lipid peroxidation inmicelles and monolamellar liposomesrdquo Biophysical Chemistryvol 135 no 1ndash3 pp 76ndash83 2008

[28] M S Blois ldquoAntioxidant determinations by the use of a stablefree radicalrdquo Nature vol 181 no 4617 pp 1199ndash1200 1958

[29] O A Zaporozhets E A Krushinsksya V N BarvinchenkoN A Lipkovskaya and V K Pogorelyi ldquoSpectrophotometricdetermination of hydroxycynnamic acid and related com-pounds in Echinacea preparationsrdquo Pharmaceutical ChemistryJournal vol 37 no 12 pp 632ndash636 2003


[30] M M Giusti and R E Worsltad ldquoCharacterization andmeasurement of anthocyanins by UV-visible spectroscopyrdquo inCurrent Protocols in Food Analytical Chemistry pp F1 2 1ndashF12 13 John Wiley amp Sons New York NY USA 2001

[31] J H Roe and C A Kuether ldquoThe determination of ascobicacid in whole blood and urine through the 2 4-dintrophenyl-hydrazine derivative of dehydroascorbic acidrdquo The Journal ofBiological Chemistry vol 147 pp 399ndash407 1943

[32] R Re N Pellegrini A Proteggente A PannalaM Yang andCRice-Evans ldquoAntioxidant activity applying an improved ABTSradical cation decolorization assayrdquo Free Radical Biology andMedicine vol 26 no 9-10 pp 1231ndash1237 1999

[33] W A Pryor J A Cornicelli L J Devall et al ldquoA rapid screeningtest to determine the antioxidant potencies of natural andsynthetic antioxidantsrdquo Journal of Organic Chemistry vol 58no 13 pp 3521ndash3532 1993

[34] G Cao H M Alessio and R G Cutler ldquoOxygen-radicalabsorbance capacity assay for antioxidantsrdquo Free Radical Biologyand Medicine vol 14 no 3 pp 303ndash311 1993

[35] R Stevanato S Fabris M Bertelle E Gregoris and F MomoldquoPhenolic content and antioxidant properties of fermentingmusts and fruit and vegetable fresh juicesrdquo Acta Alimentariavol 38 no 2 pp 193ndash203 2009

[36] D Huang O U Boxin and R L Prior ldquoThe chemistry behindantioxidant capacity assaysrdquo Journal of Agricultural and FoodChemistry vol 53 no 6 pp 1841ndash1856 2005

[37] V L Singleton R Orthofer and R M Lamuela-RaventosldquoAnalysis of total phenols and other oxidation substrates andantioxidants by means of folin-ciocalteu reagentrdquo Methods inEnzymology vol 299 pp 152ndash178 1999

[38] F Nanjo K Goto R Seto M Suzuki M Sakai and Y HaraldquoScavenging effects of tea catechins and their derivatives on11- diphenyl-2-picrylhydrazyl radicalrdquo Free Radical Biology andMedicine vol 21 no 6 pp 895ndash902 1996

[39] H Lorenzi S F Sartori L B Bacher and M T C de LacerdaFrutas Brasileiras Instituto Planetarium de Estudos da FloraLTDA Sao Paulo Brazil 2006

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MEDIATORSINFLAMMATION

of


antioxidant activity of a compound but results obtained bydifferent investigations are frequently contradictory [17]

The aim of this work is to compare of the results obtainedby four different methods usually employed to measureantioxidant properties that is reducing capacity by the Folin-Ciocalteu assay radical scavenging ability towards 221015840-diphenyl-1-picrylhydrazyl (DPPHmethod) inhibitory abilityon peroxidation of linoleic acid (LA) and total phenolicdetermination by the enzymatic method [18] in order to (i)verify possible correlations between the results obtained and(ii) obtain more reliable results avoiding possible measuring-method linked mistakes

These assays were applied to a series of Brazil fruits withparticular interest in the more exotic and less studied speciesIn fact information on the nutritional values of the mostexotic species of tropical fruits are limited some studies [19ndash22] provide evidence for the high antioxidant capacity andsignificant amounts of flavonoids and vitamin C for the mostcommon Brazilian fruits as mango [23] starfruit [19] andavocado [24] but no data are reported for more exotic fruitslike pitaya jurubeba siriguela and pequi some of whichnative peoples utilize in popular medicine

The results obtained by these measurements were com-pared with each other and with those obtained by Italiansoft fruits known for their antioxidant activity [25 26]Furthermore to discriminate possible interferences due toascorbic acid and anthocyanins the content of these reducingmolecules in all fruits was also carried out

Similitude and differences were discussed on the light ofthe chemical characteristics of the assay reactions

2 Materials and Methods

21 Chemicals All chemicals of the highest available qualitywere obtained from Sigma Chemical Co (St Louis USA)ABIP (221015840-azobis[21015840-(2-imidazolin-2-yl)propane] dihydro-chloride) was obtained from Wako Chemicals (Germany)The aqueous solutions were prepared with quality milli-Qwater Each experiment was in triplicate

22 UV-VIS and ElectrochemicalMeasurements Spectropho-tometric measurements were recorded on a UV-VIS Shi-madzu UV-1800 instrument equipped with a temperaturecontrolled quartz cell The measures of oxygen consumptionwere performed with a potentiostat Amel 559 equipped withan oxygen microelectrode (MI-730 Microelectrodes)

23 Fruits and Sample Pretreatments Table 1 reports com-mon and scientific names of all studied fruits Mango avo-cado carambola and pitaya were from Sao Paulo state whilejurubeba umbu graviola pequi siriguela and tamarindwerefrom tropical Brazil soft fruits were from Italy After cleaningwith distilled water edible fruits portions were grated andcentrifuged by a Krups centrifuge under nitrogen flux toavoid the oxidation of the natural components and the juicewas immediately analysed

24 Inhibition of Lipid Peroxidation (ILP) The antioxidantactivity of fruits to prevent linoleic acid (LA) peroxidation

Table 1 Selected fruits and their abbreviation

Scientific name Common name AbbreviationPersea americana Avocado AvAnnona muricata Graviola GrSolanum paniculatum Jurubeba JuMangifera indica Mango Haden MHMangifera indica Mango Palmer MPMangifera indica Mango Tommy Atkins MTCaryocar brasiliense Pequi PeHylocereus undatus Pitaya PiSpondias purpurea Purple mombin (siriguela) SiAverrhoa carambola Starfruit (carambola) StTamarindus indica Tamarind TaSpondias tuberosa Umbu UmRubus ulmifolius Blackberry BaVaccinium cyanococcus Blueberry BuRubus idaeligus Raspberry RaRibes rubrum Redcurrant ReFragaria Strawberry Sw

was determined in sodium dodecyl sulfate (SDS)micelles Aspreviously reported [27] the fruitrsquos antioxidant capacity wascalculated as the juice concentration (ppm) halves the rate ofoxygen consumption due to the peroxidation process and itis expressed as inhibitory concentration IC

50

25 22-Diphenyl-1-picrylhydrazyl (DPPH)Radical ScavengingCapacity Assay This method is based on the capacity of anantioxidant to scavenge the stable free radicalDPPH [28]Theprocedure is reported in Stevanato et al [18] the results areexpressed as catechin equivalent concentration (CE)

26 Folin-Ciocalteu Assay and Total Phenolics Content (TPC)by Enzymatic Method The Folin-Ciocalteu assay and theTotal Phenolic Content were determined spectrophotomet-rically according to the procedures previously reported [18]and the results were expressed as catechin equivalent (CE)

27 Total Hydroxycinnamic Acid Content (HCA) Hydrox-ycinnamic acid content was determined according toZaporozhets et al [29] The complex of hydroxycinnamicacids with aluminium (III) was measured at 365 nm andcaffeic acid was used as a standard the results were expressedas milligramsliter of caffeic acid equivalents

28 Total Anthocyanin Content (TAC) The TAC wasdetermined according the pH-differential method [30]Absorbance at 510 and 700 nm of juice buffered at pH 45 e 10was calculatedThe anthocyanin concentrationwas expressedas milligramsliter of cyanidin-3-glucoside equivalents




DPPHCE (mM)

TPCCE (mM)

FolinCE (mM)

HCA(mgL)

TAA(mM)




















50values




50) le 2 (IC













3 Discussion





20

25

30

Med

ium

Goo

dPo

orA

ntio

xida

nt p

rope

rty


Log(1

IC50)








4 Results







0 1 2 3 4 5 6 7 8 90000

0005

0010

0015

0020

0025

0030

Group A

Group B

Av

PePi

MH

MT

Um

MP

Gr

Ta

St

ReJu

Ba

Ra

BuSw

Si

DPPH CE (mM)

R = 079

y = 00042x

1IC

50

(1p

pm)

(a)

0000

0005

0010

0015

0020

0025

0030

0 1 2 3 4 5 6 7 8

Group B

Group AMT

Pe

MH

MPAv

Um

Pi

Ra

Ta

BuSi

Sw

Re

Gr

St

Ju

TPC CE (mM)

R = 060

y = 00037x

1IC

50

(1p

pm)

(b)

0000

0005

0010

0015

0020

0025

0030

0 5 10 15 20 25 30 35 40

Av

Gr

Ju

MHMP

MTPe

Pi

Si

St

Ta

Um

Ba

Bu

Ra

Re

Sw

Group A

Group B

Folin CE (mM)

R = 030

1IC

50

(1p

pm)1IC

50

(1p

pm)


(c)


0 1 2 3 4 5 6 7 8 90

1

2

3

4

5

6

7

8

Group A

Group B

MP

Um

MTMHPe

PiAv

Ra

Sw

Ba

Ta

ReGr

Bu

St

Si

Ju

DPPH CE (mM)

TPC

CE (m

M)

R = 034

y = 07897x

(a)

0

1

2

3

4

5

6

7

8

TPC

CE (m

M)

0 5 10 15 20 25 30 35 40

Av

Gr

Ju

MHMP

MTPe

Pi

Si

St

Ta

Um

BaBu Ra

Re

Sw

Group A

Group B

Folin CE (mM)

R = 069

y = 01867x

(b)

0

1

2

3

4

5

6

7

8

DPP

H C

E (m

M)

0 5 10 15 20 25 30 35 40Folin CE (mM)

Av

Gr

Ju

MH

MPMT

PePi

Si

St Ta

Um

BaBu

Ra

Re

Sw

Group A

Group B

R = 058

y = 01595x

(c)




100

200

300

400

0000

0005

0010

0015

0020

0025

00303243

1IC

50

(1p

pm)

1IC50

HCAeq

HCA

eq(m

gL)













5 Conclusion








References













































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of




DPPHCE (mM)

TPCCE (mM)

FolinCE (mM)

HCA(mgL)

TAA(mM)




















50values




50) le 2 (IC













3 Discussion





20

25

30

Med

ium

Goo

dPo

orA

ntio

xida

nt p

rope

rty


Log(1

IC50)








4 Results







0 1 2 3 4 5 6 7 8 90000

0005

0010

0015

0020

0025

0030

Group A

Group B

Av

PePi

MH

MT

Um

MP

Gr

Ta

St

ReJu

Ba

Ra

BuSw

Si

DPPH CE (mM)

R = 079

y = 00042x

1IC

50

(1p

pm)

(a)

0000

0005

0010

0015

0020

0025

0030

0 1 2 3 4 5 6 7 8

Group B

Group AMT

Pe

MH

MPAv

Um

Pi

Ra

Ta

BuSi

Sw

Re

Gr

St

Ju

TPC CE (mM)

R = 060

y = 00037x

1IC

50

(1p

pm)

(b)

0000

0005

0010

0015

0020

0025

0030

0 5 10 15 20 25 30 35 40

Av

Gr

Ju

MHMP

MTPe

Pi

Si

St

Ta

Um

Ba

Bu

Ra

Re

Sw

Group A

Group B

Folin CE (mM)

R = 030

1IC

50

(1p

pm)1IC

50

(1p

pm)


(c)


0 1 2 3 4 5 6 7 8 90

1

2

3

4

5

6

7

8

Group A

Group B

MP

Um

MTMHPe

PiAv

Ra

Sw

Ba

Ta

ReGr

Bu

St

Si

Ju

DPPH CE (mM)

TPC

CE (m

M)

R = 034

y = 07897x

(a)

0

1

2

3

4

5

6

7

8

TPC

CE (m

M)

0 5 10 15 20 25 30 35 40

Av

Gr

Ju

MHMP

MTPe

Pi

Si

St

Ta

Um

BaBu Ra

Re

Sw

Group A

Group B

Folin CE (mM)

R = 069

y = 01867x

(b)

0

1

2

3

4

5

6

7

8

DPP

H C

E (m

M)

0 5 10 15 20 25 30 35 40Folin CE (mM)

Av

Gr

Ju

MH

MPMT

PePi

Si

St Ta

Um

BaBu

Ra

Re

Sw

Group A

Group B

R = 058

y = 01595x

(c)




100

200

300

400

0000

0005

0010

0015

0020

0025

00303243

1IC

50

(1p

pm)

1IC50

HCAeq

HCA

eq(m

gL)













5 Conclusion








References













































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



20

25

30

Med

ium

Goo

dPo

orA

ntio

xida

nt p

rope

rty


Log(1

IC50)








4 Results







0 1 2 3 4 5 6 7 8 90000

0005

0010

0015

0020

0025

0030

Group A

Group B

Av

PePi

MH

MT

Um

MP

Gr

Ta

St

ReJu

Ba

Ra

BuSw

Si

DPPH CE (mM)

R = 079

y = 00042x

1IC

50

(1p

pm)

(a)

0000

0005

0010

0015

0020

0025

0030

0 1 2 3 4 5 6 7 8

Group B

Group AMT

Pe

MH

MPAv

Um

Pi

Ra

Ta

BuSi

Sw

Re

Gr

St

Ju

TPC CE (mM)

R = 060

y = 00037x

1IC

50

(1p

pm)

(b)

0000

0005

0010

0015

0020

0025

0030

0 5 10 15 20 25 30 35 40

Av

Gr

Ju

MHMP

MTPe

Pi

Si

St

Ta

Um

Ba

Bu

Ra

Re

Sw

Group A

Group B

Folin CE (mM)

R = 030

1IC

50

(1p

pm)1IC

50

(1p

pm)


(c)


0 1 2 3 4 5 6 7 8 90

1

2

3

4

5

6

7

8

Group A

Group B

MP

Um

MTMHPe

PiAv

Ra

Sw

Ba

Ta

ReGr

Bu

St

Si

Ju

DPPH CE (mM)

TPC

CE (m

M)

R = 034

y = 07897x

(a)

0

1

2

3

4

5

6

7

8

TPC

CE (m

M)

0 5 10 15 20 25 30 35 40

Av

Gr

Ju

MHMP

MTPe

Pi

Si

St

Ta

Um

BaBu Ra

Re

Sw

Group A

Group B

Folin CE (mM)

R = 069

y = 01867x

(b)

0

1

2

3

4

5

6

7

8

DPP

H C

E (m

M)

0 5 10 15 20 25 30 35 40Folin CE (mM)

Av

Gr

Ju

MH

MPMT

PePi

Si

St Ta

Um

BaBu

Ra

Re

Sw

Group A

Group B

R = 058

y = 01595x

(c)




100

200

300

400

0000

0005

0010

0015

0020

0025

00303243

1IC

50

(1p

pm)

1IC50

HCAeq

HCA

eq(m

gL)













5 Conclusion








References













































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


0 1 2 3 4 5 6 7 8 90000

0005

0010

0015

0020

0025

0030

Group A

Group B

Av

PePi

MH

MT

Um

MP

Gr

Ta

St

ReJu

Ba

Ra

BuSw

Si

DPPH CE (mM)

R = 079

y = 00042x

1IC

50

(1p

pm)

(a)

0000

0005

0010

0015

0020

0025

0030

0 1 2 3 4 5 6 7 8

Group B

Group AMT

Pe

MH

MPAv

Um

Pi

Ra

Ta

BuSi

Sw

Re

Gr

St

Ju

TPC CE (mM)

R = 060

y = 00037x

1IC

50

(1p

pm)

(b)

0000

0005

0010

0015

0020

0025

0030

0 5 10 15 20 25 30 35 40

Av

Gr

Ju

MHMP

MTPe

Pi

Si

St

Ta

Um

Ba

Bu

Ra

Re

Sw

Group A

Group B

Folin CE (mM)

R = 030

1IC

50

(1p

pm)1IC

50

(1p

pm)


(c)


0 1 2 3 4 5 6 7 8 90

1

2

3

4

5

6

7

8

Group A

Group B

MP

Um

MTMHPe

PiAv

Ra

Sw

Ba

Ta

ReGr

Bu

St

Si

Ju

DPPH CE (mM)

TPC

CE (m

M)

R = 034

y = 07897x

(a)

0

1

2

3

4

5

6

7

8

TPC

CE (m

M)

0 5 10 15 20 25 30 35 40

Av

Gr

Ju

MHMP

MTPe

Pi

Si

St

Ta

Um

BaBu Ra

Re

Sw

Group A

Group B

Folin CE (mM)

R = 069

y = 01867x

(b)

0

1

2

3

4

5

6

7

8

DPP

H C

E (m

M)

0 5 10 15 20 25 30 35 40Folin CE (mM)

Av

Gr

Ju

MH

MPMT

PePi

Si

St Ta

Um

BaBu

Ra

Re

Sw

Group A

Group B

R = 058

y = 01595x

(c)




100

200

300

400

0000

0005

0010

0015

0020

0025

00303243

1IC

50

(1p

pm)

1IC50

HCAeq

HCA

eq(m

gL)













5 Conclusion








References













































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



100

200

300

400

0000

0005

0010

0015

0020

0025

00303243

1IC

50

(1p

pm)

1IC50

HCAeq

HCA

eq(m

gL)













5 Conclusion








References













































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of






References













































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of
















Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of





Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

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