This article was downloaded by: [University of Tennessee, Knoxville]On: 04 May 2013, At: 09:51Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House,37-41 Mortimer Street, London W1T 3JH, UK

Separation Science and TechnologyPublication details, including instructions for authors and subscription information:http://www.tandfonline.com/loi/lsst20

Substrate-free Determination of the Radical ScavengingActivity of Phenolic Compounds by PhotochemicalGeneration of Hydroxyl Radicals and HPLC-UVDetectionLeandro M. de Carvalho a b , José M. Monserrat c , Fritz Scholz d , Fernanda O. Lima a ,Marcelo B. da Rosa a b , Maurício Hilgemann a , Larrisa S. Müller b , Henrique Faccin a ,Patrícia B. Ramos c & Carine Viana ba Departamento de Química, Universidade Federal de Santa Maria, Santa Maria-RS, Brazilb Graduate Program in Pharmaceutical Sciences, Federal University of Santa Maria (UFSM),Santa Maria-RS, Brazilc Instituto de Ciências Biológicas, Universidade Federal do Rio Grande (FURG), Rio Grande-RS, Brazild Universität Greifswald, Institut für Biochemie, Greifswald, GermanyAccepted author version posted online: 12 Dec 2012.Published online: 27 Mar 2013.

To cite this article: Leandro M. de Carvalho , José M. Monserrat , Fritz Scholz , Fernanda O. Lima , Marcelo B. da Rosa ,Maurício Hilgemann , Larrisa S. Müller , Henrique Faccin , Patrícia B. Ramos & Carine Viana (2013): Substrate-freeDetermination of the Radical Scavenging Activity of Phenolic Compounds by Photochemical Generation of Hydroxyl Radicalsand HPLC-UV Detection, Separation Science and Technology, 48:7, 1123-1131

To link to this article: http://dx.doi.org/10.1080/01496395.2012.724139

PLEASE SCROLL DOWN FOR ARTICLE

Full terms and conditions of use: http://www.tandfonline.com/page/terms-and-conditions

This article may be used for research, teaching, and private study purposes. Any substantial or systematicreproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form toanyone is expressly forbidden.

The publisher does not give any warranty express or implied or make any representation that the contentswill be complete or accurate or up to date. The accuracy of any instructions, formulae, and drug doses shouldbe independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims,proceedings, demand, or costs or damages whatsoever or howsoever caused arising directly or indirectly inconnection with or arising out of the use of this material.

Substrate-free Determination of the Radical ScavengingActivity of Phenolic Compounds by PhotochemicalGeneration of Hydroxyl Radicals and HPLC-UV Detection

Leandro M. de Carvalho,1,2 Jose M. Monserrat,3 Fritz Scholz,4 Fernanda O. Lima,1

Marcelo B. da Rosa,1,2 Maurıcio Hilgemann,1 Larrisa S. Muller,2 Henrique Faccin,1

Patrıcia B. Ramos,3 and Carine Viana21Departamento de Quımica, Universidade Federal de Santa Maria, Santa Maria-RS, Brazil2Graduate Program in Pharmaceutical Sciences, Federal University of Santa Maria (UFSM),Santa Maria-RS, Brazil3Instituto de Ciencias Biologicas, Universidade Federal do Rio Grande (FURG),Rio Grande-RS, Brazil4Universitat Greifswald, Institut fur Biochemie, Greifswald, Germany

This paper describes the study of the radical scavenging activityof the natural antioxidants rutin, quercetin, caffeic acid, ferulic acid,and resveratrol by using a substrate-free chromatographic methodbased on the generation of .OH radicals by the photolysis of H2O2

in a photochemical reactor. The comparative studies permitted theclassification of some phenolic compounds regarding their in vitroradical scavenging properties (rutin> caffeic acid� quercetin>ferulic acid> resveratrol). The results obtained by the proposedHPLC-UV/H2O2 photolysis method could be well corroborated bythe other methods, which employ the generation of free radicals withdifferent reactivity (ROO., O��

2 , and DPPH.). Furthermore, thereverse-phase chromatographic separation of the antioxidants fromtheir sub-products generated after attacking with HO. radicalsimproved advantageously the selectivity of the measured signal usedfor calculating the radical scavenging activity.

Keywords antioxidants; HO. radicals; HPLC-UV; in vitromethods; radical scavenging activity

INTRODUCTION

The interest in the antioxidant activity of natural compoundshas increased in the last years, mainly due to the concern fordisease prevention and biological damage caused by free radi-cals, such as cancer, cardiovascular diseases, and cerebral dys-functions (1–4). In living organisms, most of the free radicalsare reactive oxygen species (ROS), the hydroxyl radical (HO.)being the most reactive. Furthermore, humans do not have anenzymatic system specialized for its degradation.

The determination of the radical scavenging activity byin vitro methods involves normally the use of a source of freeradicals and a specific substrate, which should be attacked bythe artificially generated free radicals. After oxidizing thesubstrate, the extent or rate of the free-radical controlled oxi-dation can be measured by different instrumental methods(5). In this context, several analytical methods have beendeveloped, which use different reagents and physicalmeasurement principles for monitoring the reaction at adefined time point. Thus, it might explain the contradictoryresults normally obtained by independent methods makinguse of different substrates, ROS and detection principles.

Considering the large variety of compounds with antiox-idant properties, several in vitro methods have beendeveloped for evaluating their radical scavenging activity.These methods normally make use of stable free radicalspecies, and monitor the reaction by UV absorbance orfluorescence measurements. Among the most frequentlyused optical methods for the determination of antioxidantactivity are those based on radical scavenging by usingDPPH (2,2-diphenyl-1-picrylhydrazine) and ABTS [2,20-azyno-bis(3-ethylbenztiazoline) 6-sulfonic acid] reagents(6,7). The determination of hydroxyl radical scavengingactivity by using HPLC and capillary electrophoretic (CE)methods has been described for some antioxidant com-pounds (8–10). The methods are normally based on theuse of salicylate anion as a substrate and the detection ofthe subproducts generated after attacking it with thefree radicals. Many other in vitro methods are alsobased on the use of substrates that react with the freeradicals, followed by the detection of the substrate and=oritssubproducts (5,11,12). Additionally, an alternativeelectrochemical method has been described by Scholz and

Received 26 December 2011; accepted 21 August 2012.Address correspondence to Leandro M. de Carvalho, Departa-

mento de Quımica, Universidade Federal de Santa Maria, CaixaPostal 5051, CEP 97110-970, Santa Maria-RS, Brazil. E-mail:lemacarvalho@gmail.com

Separation Science and Technology, 48: 1123–1131, 2013

Copyright # Taylor & Francis Group, LLC

ISSN: 0149-6395 print=1520-5754 online

DOI: 10.1080/01496395.2012.724139

1123

Dow

nloa

ded

by [

Uni

vers

ity o

f T

enne

ssee

, Kno

xvill

e] a

t 09:

51 0

4 M

ay 2

013

co-workers (13) for the determination of the hydroxylradical scavenging activity. This method uses a modifiedelectrode with an alkyl-thiol self-assembled monolayer(SAM), which interacts with HO. radicals generated bythe Fenton reaction. The method was applied for the deter-mination of the radical scavenging activity of medicinalplant extracts (14).

The determination of radical scavenging by alkyl peroxylradicals (ROO.) has also been described in the literatureapplying gas chromatography and fluorimetric methods(15–17). The fluorimetric methods are based on the gener-ation of alkyl peroxyl radicals by thermal decomposition of2,20-azobis-(2-methylamidinepropane) chloride (ABAP)and its reaction with 20,70-dichlorofluorescein diacetate(H2DCF-DA) as a fluorescent probe. Here, the reagentH2DCF-DA is oxidized by ROO. radicals following an alka-line de-acetylating reaction, generating a fluorescent product.The determination of radical scavenging by anion superoxideradical (O��

2 ) can be performed by using the HPX=XODsystem, which is based on the enzymatic generation of O��

2

species from the oxidation of hypoxanthine to xanthineand uric acid (18). The produced free radical reduces theabsorbing probe nitroblue tetrazolium (NBT) to formazanat pH 7.4, this color change being monitored at 560nm.

This work describes a systematic study of the radicalscavenging activity of some phenolic antioxidant com-pounds by using a novel method based on the generationof .OH radicals by controlled photolysis of H2O2 and itsdirect reaction with the phenolic compounds. After the pho-tolysis process, the remaining phenolic compounds weredetermined by HPLC-UV. The substrate-free proposedmethod was compared with other in vitro methods, whichwere based on the generation of ROO., HO., O��

2 , andDPPH. radicals. Finally, the studies aimed to show thepossibility of using a simple substrate-free HPLC methodto rank the antioxidant compounds regarding their realcontribution to the total antioxidant activity against HO.

radicals in a selective way after the separation of the reactionsubproducts. The intracellular concentrations of reactiveoxygen species in hepatocytes of zebra fish (Danio rerio)were also evaluated to confirm the classification of the anti-oxidants concerning their radical scavenging activity.

MATERIALS AND METHODS

Apparatus

The high performance liquid chromatograms wererecorded with a P680 chromatographic system (Dionex)and an UV detector (UVD-170, Dionex). For the separ-ation a C18 column (4.6mm� 150mm� 5 mm, Dionex)was used and the chromatograms were evaluated with thesoftware Chromeleon 6.70.

The UV photolysis was accomplished using a homemadeUV system with a high-pressure mercury lamp of 80W. The

mercury lamp was surrounded by 12 quartz tubes(Metrohm), which were inserted in a cubic wooden box(21.5 cm edge) with the inner walls covered by aluminumfoil. The whole system was cooled by air circulation usinga ventilator (Ruilian Science Technology) in the top of thereactor. All UV photolysis experiments were performed at30� 2�C.

The spectrophotometric measurements were performedusing an HP 8453 (Hewlett Packard) diode array spectro-photometer with 10-mm quartz cells.

A Victor 2 spectrofluorometer (Perkin Elmer) wasutilized for the fluorimetric measurements.

Materials and Reagents

All chemicals were of analytical grade purity. Water waspurified by a Milli-Q Ultra Pure Water System (Millipore,Bedford, MA, USA). Hydrogen peroxide 30% (v=v) wasobtained from Vetec. Rutin, quercetin, resveratrol, caffeicacid, ferulic acid, H2DCF-DA, ABAP, hypoxanthine(HPX), xanthine oxidase 25 UN (XOD), nitrotetrazoliumblue chloride (NBT), and DPPH were products of Sigma-Aldrich.

In all HPLC experiments, a 60:40 (v=v) binary solutionof acetonitrile-water was used as the mobile phase. TheHPLC mobile phase was filtered through a cellulose acetatemembrane having a pore size of 0.45 mm (Sartorius) andthen degassed in an ultrasonic bath USC 2800A (Ultra-sonic Cleaner Unique) before being fed into the column.All the chromatographic separations were carried out byusing a mobile phase flow rate of 1.0mLmin�1.

Experimental Procedures

Determination of the HO. Radical Scavenging Activity byHPLC-UV Detection after H2O2 Photolysis

The method is based on the kinetic monitoring of thereaction between the photochemically generated HO.

radical and the individual phenolic compounds. After thetemporal attacks (0� 60min) of each phenolic compoundby HO. radicals generated in situ in the photochemical reac-tor, a sample of the solution (20 mL) was immediatelyinjected into the chromatographic system (HPLC-UV) forseparation and monitoring of the main compound peakarea by UV detection at 360 nm for rutin, 375 nm for quer-cetin, 323 nm for caffeic acid, 321 nm for ferulic acid and307 nm for resveratrol. A fixed 0.29mM H2O2 concen-tration was used in all the UV irradiation experiments, inwhich the 10mL quartz tube containing the antioxidant sol-ution (25, 50, and 75 mM) was removed from the UV reactorfor the HPLC-UV measurement every 5min. All the solu-tions were also UV irradiated and measured every 5minin the absence of H2O2 in order to assay the contributionof the direct UV photolysis to the chromatographic peakdecay of the phenolic compounds. This contribution should

1124 L. M. DE CARVALHO

Dow

nloa

ded

by [

Uni

vers

ity o

f T

enne

ssee

, Kno

xvill

e] a

t 09:

51 0

4 M

ay 2

013

be considered in the final calculation of the scavengingactivity (control measurement). The radical scavengingactivity can be expressed as the percentage of the remainingsignal of each compound measured by HPLC-UV after30min reaction with HO. radicals, according to the follow-ing equation:

Radical scavenging activityð%Þ

¼ 1�AUV � AUVþH2O2Þ

AUV

� �� �� 100

where AUV is the peak area of the phenolic compoundmeasured after the direct UV photolysis (without H2O2)and AUVþH2O2 is the peak area of the phenolic compoundmeasured after UV photolysis in the presence of H2O2

(0.29mM). The higher the remaining chromatographic sig-nal for each antioxidant compound after the attack by HO.

radicals, the higher the radical scavenging activity will be.

Comparative Determination of the Radical ScavengingActivity by Direct Fluorimetric and Photometric Detection

The radical scavenging activity against ROO., and HO.

radicals was evaluated employing the fluorescent probeH2DCF-DA in a final concentration of 40 mM, accordingto the methodology employed by Amado et al. (16). Alkylperoxyl radicals (ROO.) were produced by thermaldecomposition of ABAP 4mM at 37�C. The hydroxyl radi-cals (HO.) were produced by the Fenton reaction (Fe2þ=H2O2, 100:500 mM). The radical scavenging activity againstO��

2 radicals was evaluated employing the hypoxanthine=xanthine oxidase (HPX=XOD) system according to Zhaoet al. (18). The radical scavenging activity against DPPH.

radicals was evaluated employing the absorbing probeDDPH according to Huang et al. (19).

Comparative Determination of the Radical ScavengingActivity by Bioassay with Hepatocytes FromZebra Fish (Danio Rerio)

In the comparative bioassays, hepatocytes (ZFL line)from zebra fishDanio rerio (American Type Culture Collec-tion) were employed. ZFL cells were maintained in a RPMI1640 (Gibco) medium containing 0.2 g L�1, sodium bicar-bonate, 0.3 gL�1 L-glutamine, 25mM HEPES, 50 mMmercaptoethanol, 10% (w=v) fetal bovine serum (Gibco),1% (v=v) of an antibiotic solution containing penicillin(100U=mL), 100 mgmL�1 streptomycin (both productsfrom Gibco), and 0.25 mgmL�1 antimycotic (Sigma). Theculture was maintained in disposable plastic flasks at28�C. For assays of antioxidants exposure, cells weredetached from flasks with 0.125% (w=v) trypsin and dis-posed in 24-well culture plates, in which they were allowedto attach during 24 h, reaching a final concentration of6� 107 cells mL�1. Then hepatocytes were exposed for 4 h

to the antioxidants rutin, quercetin, caffeic acid, ferulicacid, and resveratrol at the same concentration employedin the in vitro radical scavenging measurements (50 mM).Stock solutions of each antioxidant were prepared usingdimethyl sulfoxide (DMSO). The final concentration ofDMSO during exposure was 1% (v=v) and does not affectcell viability and intracellular ROS levels. At the end ofthe experiment, the cells were detached, separated in differ-ent aliquots, centrifuged, and washed with sterile phosphatebuffer solution (PBS). An aliquot of each cell suspensionwas re-suspended with PBS plus 40 mM H2DCF-DA toanalyze the intracellular ROS concentration as describedelsewhere (16). Another aliquot of each cell suspensionwas employed for the determination of viability using3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium(MTT,Sigma) to count the total cell number. A third aliquot wasused to count cell number using a Neubauer camara undermicroscope (100 X). The total cell number was employed tostandardize ROS concentration and to express viability interms of total viable cells.

RESULTS AND DISCUSSION

The methods based on the direct reaction ofantioxidants with artificially generated free radicals areadvantageous, considering that they do not include thepossibility of any parallel or secondary reaction in themethod at all. Although it is stated that the substrate-freemethods, such as those based on the DPPH. and ABTS.þ

assays, are not able to mimic the process in natural orbiological systems, they can be very useful for ranking pureantioxidant species and also medicinal plant extracts con-cerning their radical scavenging activity. Additionally, theinformation obtained by in vitro measurements is relevantas a screening tool for studies involving natural antioxi-dants present in biological systems and foodstuffs (20).Considering the current scenario of in vitro methods exist-ing for this purpose, this work shows the possibility ofusing a simple substrate-free HPLC-UV method precededby the photolytic generation of HO. radicals for the quan-titative ranking of a group of antioxidant species (Fig. 1),which are found in plant species.

Determination of the HO. Radical Scavenging Activityby HPLC-UV Separation/Detection Preceded by H2O2

Photolysis

It is well known that the artificial generation of HO.

radicals can be performed by the photodecomposition ofH2O2 in UV irradiation devices, which employ mostly mer-cury lamps as a radiation source (21–24). Once generated inthe medium, the HO. radicals react very quickly with theantioxidant (AH) species, thereby forming a free radicalof higher stability:

HO� þAH ! H2OþA�

RADICAL SCAVENGING ACTIVITY OF THE PHENOLIC COMPOUNDS 1125

Dow

nloa

ded

by [

Uni

vers

ity o

f T

enne

ssee

, Kno

xvill

e] a

t 09:

51 0

4 M

ay 2

013

This reaction rate can be monitored by determining theconsumption of the antioxidant species in the course ofthe reaction. Here, one possibility to monitor the reactionis the determination of the remaining AH concentrationby using optical (e.g., UV-Visible spectrophotometry andfluorimetry) or electrochemical (e.g., voltammetry, ampero-metry) methods. The AH consumption rate can also bemonitored following a chromatographic separation of thedecomposition products, which may improve the selectivityof the measurement of the AH species after attacking themwith free radical species.

The generation of HO. radicals can be controlled in areproducible way by the photolysis of H2O2 by using anUV radiation source with a relatively low power (e.g.,80W). The AH peak decay can be monitored at differentconcentrations, as to calculate the radical scavengingactivity of any antioxidant species. Even though the H2O2

photolysis does not generate a constant concentration ofHO. radicals throughout the reaction time, the procedureis highly reproducible, depending on the purity of chemicalsand temperature control. In the irradiation systemdescribed in this work, the time-dependent decay of pureantioxidants due to direct photolysis is very slow comparedto the decay in the presence of HO. radicals. This shows thatthe studied phenolic compounds are photolytically stableunder the experimental conditions. Figure 2 depicts thechromatograms obtained for the studied antioxidants afterreacting with HO. radicals generated in the described UVirradiation system.

The appearance of new chromatographic signals forresveratrol and phenolic acids at retention times differentfrom the main chromatographic peaks suggests the forma-tion of, at least, one decomposition product after reactionwith HO. radicals, what could not be observed in case ofrutin and quercetin under the same irradiation conditions.In this context, the higher the remaining chromatographicsignal for each compound after the attack by HO. radicals,the higher the radical scavenging activity will be. The rad-ical scavenging activity can be also conveniently followed,if the chromatographic data are plotted as the exponentialdecay related to the reference absorbance signal (A0) mea-sured after the chromatographic separation (Fig. 3). Byplotting ln (A=A0) versus time of the HO. radical attack,the respective decay refers directly to the given radical scav-enging activity of the phenolic compounds. As can be seenin case of resveratrol, there are two distinct linear linesrelated to its bi-exponential decay after reaction with HO.

radicals, being the first one from 0 to 15 minutes (fast decay)and the second one from 15 to 40 minutes (slow decay).Considering the case of caffeic acid, it can be seen that itsmain chromatographic peak completely disappeared after40min reaction with HO. radicals.

From these results, it can be concluded that the higherthe decay of the main chromatographic peak until 30minreaction the lower the scavenging activity of the phenoliccompound. Thus, the measurement of the remainingchromatographic peak gives indirectly the information onthe amount of antioxidant consumed by a fixed concen-tration of HO. radical. Thus, the higher the remained

FIG. 2. Chromatograms showing the behavior of rutin, quercetin,

resveratrol, caffeic acid, and ferulic acid (50mM each) before (solid line)

and after (dashed lines) the reaction with .OH radicals generated by

photolysis of H2O2 (0.29mM). Experimental conditions: UV irradiation

at 30� 2�C; Mobile-phase: 60:40 (v=v) acetonitrile-water solution; flow

rate: 1.0mlmin�1. Other conditions are described in section Experimental

section.FIG. 1. Molecular structure of the phenolic studied antioxidants.

1126 L. M. DE CARVALHO

Dow

nloa

ded

by [

Uni

vers

ity o

f T

enne

ssee

, Kno

xvill

e] a

t 09:

51 0

4 M

ay 2

013

chromatographic peak for any antioxidant the smaller theconcentration of antioxidant consumed by HO. radicals.Similar information has been given by the DPPH method,when calculating the EC50 for pure antioxidants, that is,the amount of antioxidant needed to decrease the freeradical concentration by 50% (20). Here, the lower theEC50 the higher the antioxidant activity.

Corroborating these observations based on the chroma-tographic profile of each compound, Table 1 shows theradical scavenging activities calculated for the antioxidantsusing the proposed method and the equation described inexperimental section. As can be seen, some clear differencesare observed for the radical scavenging activity probedwith HO. formed by maximal 30min photolysis: the high-est radical scavenging activity has rutin, followed by caffeicacid, quercetin, ferulic acid and resveratrol. The samebehavior was also observed for 25 and 75 mM concentra-tions of the studied phenolic compounds.

The results presented in Table 1 were also compared withthose obtained by other independent in vitro methods forthe determination of radical scavenging activity of the phe-nolic compounds at a fixed concentration (50 mM). One ofthe methods was based on the fluorimetric detection ofradicals ROO. and HO. radicals after reacting these specieswith the substrate H2DCF, which generates the fluorescentcompound DCF as a reaction product. The modified meth-odology proposed by Amado et al. (16) includes a systemfor radical generation that greatly augments H2DCFoxidation and fluorescence unless the antioxidants presentin the biological samples degrade and=or intercept thegenerated free radicals. As shown in Table 1, for both kindsof radicals (HO. and ROO.), important differences in the

radical scavenging activity was observed for the differentantioxidants assayed: the highest activity was observed forquercetin and caffeic acid, whereas resveratrol showed noactivity at all. A second substrate-based method used forcomparative results was based on the enzymatic generationof O��

2 radicals. As can be seen, the same differencesobserved for the radical scavenging activity probed withHO. in the proposed HPLC-UV=photolysis method werealso observed for O��

2 radicals.The results obtained with the proposed substrate-free

and the substrate-based fluorimetric=photometric methodswere also compared with the substrate-free DPPH method,which makes use of a relatively stable radical species(DPPH.) in comparison to the radicals used in the formermethods. As can be seen in Table 1, the highest radicalscavenging activity by the DPPH method was observedfor rutin and caffeic acid, followed by quercetin, ferulicacid, and resveratrol.

Comparative Radical Scavenging Activity of PhenolicCompounds by HPLC-UV/H2O2 Photolysis andOther In Vitro Methods

It is well known that the determination of the radicalscavenging activity by independent methods can lead todifferent and sometimes contradictory results. Also, thedifferent mechanisms of antioxidant action may explainthe different results obtained by independent in vitro meth-ods, mainly if compared to in vivo experiments. Themechanisms of antioxidant action can include:

1. the suppression of ROS generation either by inhibitionof enzymes or chelating of trace elements involved inthe production of free radicals,

2. the sequestration of ROS, and3. the protection of antioxidant defenses (25).

In this context, phenolic compounds, such as flavo-noides, have been described as antioxidant species thatfulfill these criteria. Therefore, it is very important to high-light the relevance of characterizing antioxidant propertiesof radical scavenging species by proper in vitro methods forfurther application to medical and nutritional purposes.

In Table 2, a comparison of the different analyticalmethodologies employed for the measurement of the radicalscavenging activity is presented for the studied phenoliccompounds at the same concentration (50 mM). It can beobserved that some differences exist among the most anti-oxidant species (rutin, quercetin, and caffeic acid), althoughthe proposed substrate-free HPLC method and all othercomparative methods were consistent in classifying thesame antioxidants with the lowest radical scavengingcapacity (ferulic acid and resveratrol). The differencesobserved for the antioxidants rutin, quercetin, and caffeicacid by the independent in vitro methods can be explainedconsidering the following aspects. First, the reactivity of

FIG. 3. Logarithmic plot of the exponential decay of the absorbance

signal measured the attack with .OH radicals generated by photolysis of

H2O2 (0.29mM) followed of the HPLC separation. Other conditions are

described in Experimental section.

RADICAL SCAVENGING ACTIVITY OF THE PHENOLIC COMPOUNDS 1127

Dow

nloa

ded

by [

Uni

vers

ity o

f T

enne

ssee

, Kno

xvill

e] a

t 09:

51 0

4 M

ay 2

013

the free radicals employed by each method is different,considering that the HO. radical has a standard reductionpotential of þ2.30V (25), O��

2 radical a reduction potentialof þ0.94V (25), and ROO. radical a standard reductionpotentials that can range from þ0.71 to þ1.44V (25).Secondly, the measurement mechanisms and the detectionprinciples of the compared methodologies are considerablydifferent. Finally, it should be emphasized that the detec-tion of any antioxidant species after a chromatographicseparation in the absence of a substrate advantageouslyimproves the selectivity of the signal measurement that isformerly used for calculating the radical scavenging

activity, since the unpredictable sub-products (Fig. 2) donot interfere on the measured UV absorbance anymore.Thus, the different results observed here and in previousstudies employing other methodologies may also rely onthese aspects.

Considering the results described by the independentin vitro methods, we proposed here a classification of anti-oxidants in groups from highest to lowest radical scaveng-ing activities (Table 2).

The results obtained from the proposed substrate-freemethod, which quantitatively ranks the antioxidants fromhighest to lowest activity, can be also supported by other

TABLE 2Comparative overview of the antioxidant activity of phenolic compounds (50 mM) by the proposed HPLC-UV and other

in vitro methods

Method

Generatedfree

radical Detection methodAntioxidant activity

of phenolic compounds

HPLC-UV=H2O2

photolysisHO. UV absorption

(k¼ 307, 321, 323,360, and 375 nm)

RUT>CAF�QUER>FERUL>RESV

Photometry=DPPH DPPH. Vis absorption(k¼ 515 nm)

RUT�CAF>QUER>FERUL>RESV

Photometry=HPX-XOD O��2 Vis absorption

(k¼ 560 nm)RUT>CAF>QUER>FERUL>RESV

Fluorimetry=ROO-ABAP ROO. Fluorescence emission(exc. k¼ 485 nm; emiss.k¼ 530 nm)

QUER>CAF�RUT>FERUL>RESV

Fluorimetry=OH-Fenton HO. Fluorescence emission(exc. k¼ 485 nm; emiss.k¼ 530 nm)

CAF�QUER >RUT >FERUL>RESV

RUT: Rutin; CAF: Caffeic acid; QUER: Quercetin; FERUL: Ferulic acid; RESV: Resveratrol.

TABLE 1Radical scavenging activity of phenolic compounds (50 mM) determined by HPLC-UV and other in vitro

methods (RSD: relative standard deviation)

AntioxidantRadical scavenging activity (%)

In vitro method

HPLC-UV=HO� a Photometry=DPPH�b Photometry=O��c2 Fluorimetry=

HO�-FentondFluorimetry=

ROO� e

Rutin 84.51 96.64 98.87 43.20 38.80Quercetin 53.24 70.04 50.69 80.10 59.90Caffeic acid 57.46 94.49 92.50 48.40 60.80Ferulic acid 34.35 53.90 41.00 22.60 19.40Resveratrol 22.62 47.48 0.83 0.00 0.00

aRSD (n¼ 3): 3.9–6.8%.bRSD (n¼ 3): 1.0–7.5%.cRSD (n¼ 3): 0.5–2.1%.dRSD (n¼ 3): 2.0–6.2%.eRSD (n¼ 3): 2.1–7.8%.

1128 L. M. DE CARVALHO

Dow

nloa

ded

by [

Uni

vers

ity o

f T

enne

ssee

, Kno

xvill

e] a

t 09:

51 0

4 M

ay 2

013

substrate-free and substrate-based methods (9,11,20,25–28). These works have also shown that the flavonoids rutin,quercetin, or caffeic acid demonstrate a high antioxidantactivity compared to other phenolic antioxidants. Thegreater antioxidant activity of quercetin and caffeic acidcompared to ferulic acid was also observed by in vitromethods using amperometric assays (29).

However, considering the discrepancies of these methodswhen ranking some of the antioxidants studied in thiswork, we decided to compare our results with another inde-pendent method based on the generation of free radicals bycells (hepatocytes from zebra fish). The determination of theradical scavenging activity by the method involving bioas-says showed also differences among the studied antioxi-dants. Here, it was observed that the formazan generationwas enhanced (þ227.6%) after quercetin treatment andinhibited (�41.4%) in cells exposed to ferulic acid whencompared to the control group (p< 0.05). The number ofviable cells was statistically augmented (þ39.7%) only incells exposed to quercetin. Additionally, ROS concentra-tions in cells pre-treated with resveratrol or caffeic acid wereintermediate between the group formed by the control,rutin and ferulic acid on one side and quercetin on theother. When compared to control group, only quercetintreatment induced a significant reduction (�74%) of intra-cellular ROS concentration when compared to controlgroup (p< 0.05). So these results showed some importantdifferences compared to the in vitro results, particularlywith the antioxidant resveratrol.

The structure-activity relationships (SAR) of the studiedcompounds may also partially confirm the observed differ-ences in the radical scavenging activity. As expected forphenolic antioxidants, both the configuration and numberof hydroxyl groups influence on the mechanisms of antiox-idant activity (30). Among the studied antioxidants, thecompounds containing more hydroxyl groups, that is, rutinand quercetin, were found to have a high ability to scavengeHO., ROO., O��

2 , and DPPH. free radicals. Concerning theSAR for the studied phenolic acids, it is expected that caf-feic acid presents a higher antioxidant activity than ferulicacid due to the number of hydroxyl groups in the molecularstructure (30,31). However, the slightly higher antioxidantactivity against HO., O��

2 , and DPPH. radicals observedfor caffeic acid in comparison to quercetin may explain thatnot only the number of hydroxyl groups, but also the con-figuration of the molecule are linked to a low or high abilityto scavenge free radicals. Furthermore, it proves also thatnot only SAR approaches, but also other mechanisms areinvolved in the antioxidant process, as observed for resver-atrol in our ex vivo assays. It is also important to note thatdiscrepancies between in vitro and in vivo results can berelated to the amplification of the antioxidant systems thatsome molecules can induce through the control of geneexpression. In fact, there is evidence that polyphenols and

flavonoids, such as quercetin, modulate glutathione- relatedgene expression (32–34). This finding means that thereare important differences between the direct antioxidantproperties of some molecules (that can be determinedin vitro) and their indirect antioxidant properties, whichwere registered here after in vivo experiments. However, itseems to be a feature of all in vitro methods, regardless ofwhether they use a substrate for interacting with the freeradicals or not. Although the results obtained from bioas-says showed clear differences compared to all the in vitromethods, it corroborated the result that quercetin and caf-feic acid have a higher antioxidant activity compared tothe other phenolic compounds. The principle of the methodcan be also applied for studying the contribution ofdifferent antioxidant classes in medicinal plants, such astriterpenes, coumarins, tannins, and esteroids, so that themajor contribution of antioxidant classes in natural pro-ducts can be investigated. Furthermore, specific antioxidantclasses can be used as markers of a high antioxidant activityin plant species by using in vitro tests, once these results arecompared and corroborated by ex vivo or in vivo tests.

CONCLUSIONS

The paper described the determination of the radicalscavenging activity of some phenolic antioxidants by usinga substrate-free chromatographic in vitro method and otherindependent comparative methods based on the generationof different reactive free radicals (HO., ROO., O��

2 , andDPPH.). The proposed in vitro HPLC method correlatedwell with other in vitro methods concerning the radicalscavenging activities of rutin, quercetin, caffeic acid, ferulicacid, and resveratrol. Additionally, the comparison of theresults obtained by the independent in vitro methods per-mitted us to classify some phenolic compounds regardingtheir in vitro radical scavenging properties. Namely, thephenolic compounds rutin, quercetin and caffeic acid(group 1) presented the highest in vitro antioxidant proper-ties. If the in vitro responses of these antioxidants are com-pared with the in vivo antioxidant responses using cellbioassays, it can be concluded that quercetin has a relativelyhigh and ferulic acid a relatively low radical scavengingactivity according to all in vitro and in vivo assays.

As a final conclusion, it is clear that in vitro assays withdifferent radical species need to be correlated with inde-pendent methods to validate the results. In this work, allthe results obtained by the proposed HPLC-UV=H2O2 pho-tolysis method could be well corroborated by the othermethods, mainly regarding the high antioxidant activity ofquercetin and caffeic acid as well as the low antioxidantactivity of ferulic acid. Furthermore, the chromatographicseparation of the antioxidants from their sub-productsmay advantageously improve the selectivity of the mea-sured signal and lead, consequently, to different resultsfor the calculated radical scavenging activity. The method

RADICAL SCAVENGING ACTIVITY OF THE PHENOLIC COMPOUNDS 1129

Dow

nloa

ded

by [

Uni

vers

ity o

f T

enne

ssee

, Kno

xvill

e] a

t 09:

51 0

4 M

ay 2

013

can be applied to other antioxidant classes for classifyingthe groups in a comprehensive way. Also, plant extractscan be investigated and compared regarding the presenceof molecules with higher antioxidant activity (e.g., quercetinor caffeic acid). Additionally, the method can applied foranalyzing the contribution of specific antioxidant speciesin medicinal and nutraceutical plants considering theirin vitro reactivity against HO. radicals. Thus, this studyhas also practical implications for investigating plantextracts normally used in the phytotherapy, where thedetermination of biomarkers should be performed inorder to assure the medicinal efficacy of natural products.Considering that the monitoring of specific antioxidantsin plant extracts can be used for the selection of natural pro-ducts for pharmaceutical or nutraceutical purposes, theinformation obtained from the proposed in vitro studiescan be applied as a screening tool for medicinal plants orfoodstuffs.

ACKNOWLEDGEMENTS

The authors wish to acknowledge the financial supportgiven by the Brazilian and German foundations CNPq,FAPERGS, CAPES (PROCAD 098=2007), and DAAD.F. Scholz was supported by funds from the Ernst-Moritz-Universitat Greifswald (Germany). L.M Carvalho andJ.M. Monserrat are productivity research fellows fromCNPq.

REFERENCES

1. Havesteen, B.H. (2002) The biochemistry and medical significance of

the flavonoids. Pharmacol. Ther., 96: 67.

2. Valko, M.; Leibfritz, D.; Moncol, J.; Cronin, M.T.D.; Mazur, M.;

Telser, J. (2007) Free radicals and antioxidants in normal

physiological functions and human disease. Int. J. Biochem. Cell Biol.,

39: 44.

3. Simoes, C.M.O. (2004) Farmacognosia: da planta ao medicamento,

5th Ed.; Editora da UFRGS=Editora da UFSC: Porto Alegre=

Florianopolis, Brazil.

4. Pietta, P.G. (2000) Flavonoids as antioxidants. J. Nat. Prod., 63: 1035.

5. Antolovich, M.; Prenzler, P.D.; Patsalides, E.; McDonald, S.;

Robards, K. (2002) Methods for testing antioxidant activity. Analyst,

127: 183.

6. Sanches-Moreno, C. (2002) Review: Methods used to evaluate the free

radicals scavenging activity in foods and biological systems. Food Sci.

Technol. Int., 8: 121.

7. Peres-Jimenez, J.; Saura-Calixto, F. (2008) Anti-oxidant capacity of

dietary polyphenols determined by ABTS assay: A kinetic expression

of the results. Int. J. Food Sci. Technol., 43: 185.

8. Bektasoglu, B.; Ozyurek, M.; Guclu, K.; Apak, R. (2008) Hydroxyl

radical detection with a salicylate probe using modified CUPRAC

spectrophotometry and HPLC. Talanta, 77: 90.

9. Ozyurek, M.; Bektasoglu, B.; Guclu, K.; Apak, R. (2008) Hydroxyl

radical scavenging assay of phenolics and flavonoids with a modified

cupric reducing antioxidant capacity (CUPRAC) method using

catalase for hydrogen peroxide degradation. Anal. Chim. Acta, 616:

196.

10. Wang, Q.; Ding, F.; Zhu, N.; Li, H.; He, P.; Fang, Y. (2003) Determi-

nation of hydroxyl radical by capillary zone electrophoresis with

amperometric detection. J. Chromatogr. A, 1016: 123.

11. Helmja, K.; Vaher, M.; Pussa, T.; Kaljurand, M. (2009) Analysis of

the stable free radical scavenging capability of artificial polyphenol

mixtures and plant extracts by capillary electrophoresis and liquid

chromatography-diode array detection-tandem mass spectrometry.

J. Chromatogr. A, 1216: 2417.

12. Endo, N.; Oowada, S.; Sueishi, Y.; Shimmei, M.; Makino, K.; Fujii,

H.; Kotake, Y. (2009) Serum hydroxyl radical scavenging capacity

as quantified with iron-free hydroxyl radical source. J. Clin. Biochem.

Nutr., 45: 193.

13. Scholz, F.; Gonzalez, G.L.L.; Carvalho, L.M.; Hilgemann, M.;

Brainina, K.Z.; Kahlert, H.; Jack, R.S.; Minh, D.T. (2007) Indirect

electrochemical sensing of radicals and radical scavengers in biological

matrices. Angew. Chem. Int. Ed., 46: 8079.

14. Carvalho, L.M.; Hilgemann, M.; Scholz, F.; Kahlert, H.; Rosa,

M.B.; Wuster, M.; Lindequist, U.; Nascimento, P.C.; Bohrer,

D. (2010) Electrochemical assay to quantify the hydroxyl radical

scavenging activity of medicinal plant extracts. Electroanal.,

22: 406.

15. Winston, G.W.; Regoli, F.; Dugas Jr, A.J.; Fong, J.H.; Blanchard,

K.A. (1998) A rapid gas chromatographic assay for determining

oxyradical scavenging capacity of antioxidants and biological fluids.

Free Rad. Biol. Med., 24: 480.

16. Amado, L.L.; Garcia, M.L.; Ramos, P.B.; Freitas, R.F.; Zafalon, B.;

Ferreira, J.L.R.; Yunes, J.S.; Monserrat, J.M. (2009) A method to

measure total antioxidant capacity against peroxyl radicals in aquatic

organisms: application to evaluate microcystins toxicity. Sci. Tot.

Environ., 407: 2115.

17. Regoli, F.; Winston, G.W. (1999) Quantification of total oxidant scav-

enging capacity of antioxidants for peroxynitrite, peroxyl radicals,

and hydroxyl radicals. Toxicol. Appl. Pharmacol., 156: 96.

18. Zhao, H.; Dong, J.; Lu, J.; Chen, J.; Li, Y.; Shan, L.; Lin, Y.; Fan, W.;

Gu, G. (2006) Effects of extraction solvent mixtures on antioxidant

activity evaluation and their extraction capacity and selectivity for free

phenolic compounds in Barley (Hordeum vulgare L.). J Agric. Food

Chem., 54: 7277.

19. Huang, D.; Ou, B.; Prior, R.L. (2005) The chemistry behind antioxi-

dant capacity assays. J. Agric. Food Chem., 53: 1841.

20. Villano, D.; Fernandez-Pachon, M.S.; Moya, M.L.; Troncoso,

A.M.; Garcıa-Parrilla, M.C. (2007) Radical scavenging ability of

polyphenolic compounds towards DPPH free radical. Talanta,

71: 230.

21. Carvalho, L.M.; Spengler, C.; Garmatz, J.C.; Nascimento, P.C.;

Bohrer, D.; Del- Fabro, L.; Radis, G.; Bolli, A.A.; Garcia, S.C.;

Moro, A.M.; Rosa, M.B. (2008) Voltammetric determination of

metals in waters and biological fluids using sample mineralization with

ultraviolet radiation. Quım. Nova, 31: 1336.

22. Golimowski, J.; Golimowska, K. (1996) UV-photooxidation as

pretreatment step in inorganic analysis of environmental samples.

Anal. Chim. Acta, 325: 111.

23. Yuan, F.; Hu, C.; Hu, X.; Qu, J.; Yang, M. (2009) Degradation of

selected pharmaceuticals in aqueous solution with UV and UV=

H2O2. Water Res., 43: 1766.

24. De, A.K.; Chaudhuri, B.; Bhattacharjee, S.; Dutta, B.K. (1999)

Estimation of OH radical reaction rate constants for phenol and

chlorinated phenols using UV=H2O2 photo-oxidation. J. Haz. Mat.

B, 64: 91.

25. Halliwell, B.; Gutteridge, J.C.M. (2007) Free Radicals in Biology and

Medicine, 4th Ed.; Oxford University Press: New York.

26. Tsimogiannis, D.I.; Oreopoulou, V. (2004) Free radical scavenging

and antioxidant activity of 5,7,30,40-hydroxy-substituted flavonoids.

Innov. Food Sci. Em. Technol., 5: 523.

1130 L. M. DE CARVALHO

Dow

nloa

ded

by [

Uni

vers

ity o

f T

enne

ssee

, Kno

xvill

e] a

t 09:

51 0

4 M

ay 2

013

27. McDonald, S.; Prenzler, P.D.; Antolovich, M.; Robards, K. (2001) Phe-

nolic content and antioxidant activity of olive extracts. Food Chem., 73: 73.

28. Prochazkova, D.; Bousova, I.; Wilhelmova, N. (2011) Antioxidant and

prooxidant properties of flavonoids. Fitoterapia, 82: 513.

29. Milardovic, S.; Ivekovic, D.; Grabaric, B.S. (2006) A novel ampero-

metric method for antioxidant activity determination using DPPH

free radical. Bioelectrochem., 68: 175.

30. Cai, Y.-Z.; Sun, M.; Xing, J.; Luo, Q.; Corke, H. (2006) Structure-

radical scavenging activity relationships of phenolic compounds from

traditional Chinese medicinal plants. Life Sci., 78: 2872.

31. Heim, K.E.; Tagliaferro, A.R.; Bobilya, D.J. (2002) Flavonoid antiox-

idants: chemistry, metabolism and structure-activity relationships. J.

Nutr. Biochem., 13: 572.

32. Arredondo, F.; Echeverry, C.; Abin-Carriquiry, J.A.; Blasina, F.;

Antunez, K.; Jones, D.P.; Go, Y-M.; Liang, Y-L.; Dajas, F.

(2010) After cellular internalization, quercetin causes Nrf2

nuclear translocation, increases glutathione levels, and prevents

neuronal death against an oxidative insult. Free Rad. Biol.

Med., 49: 738.

33. Masella, R.; Di Benedetto, R.; Varı, R.; Filesi, C.; Giovannini, C.

(2005) Novel mechanisms of natural antioxidant compounds in bio-

logical systems: Involvement of glutathione and glutathione-related

enzymes. J. Nutr. Biochem., 16: 577.

34. Moskaug, J.O.; Carlsen, H.; Myhrstad, M.; Blomhoff, R. (2004)

Molecular imaging of the biological effects of quercetin and

quercetin-rich foods. Mech. Ageing Develop., 125: 315.

RADICAL SCAVENGING ACTIVITY OF THE PHENOLIC COMPOUNDS 1131

Dow

nloa

ded

by [

Uni

vers

ity o

f T

enne

ssee

, Kno

xvill

e] a

t 09:

51 0

4 M

ay 2

013