Talanta 77 (2008) 304–313

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Combined HPLC-CUPRAC (cupric ion reducing antioxidant capacity) assay ofparsley, celery leaves, and nettle

Leyla Yıldız, Kevser Sözgen Baskan, Esma Tütem ∗, Resat ApakIstanbul University, Faculty of Engineering, Department of Chemistry, Avcilar 34320, Istanbul, Turkey

a r t i c l e i n f o

Article history:Received 4 April 2008Received in revised form 9 June 2008Accepted 18 June 2008Available online 27 June 2008

Keywords:High performance liquid chromatography(HPLC)Cupric ion reducing antioxidant capacity(CUPRAC) assayParsleyCeleryNettlePlant phenolics

a b s t r a c t

This study aims to identify the essential antioxidant compounds present in parsley (Petroselinum sativum)and celery (Apium graveolens) leaves belonging to the Umbelliferae (Apiaceae) family, and in stingingnettle (Urtica dioica) belonging to Urticaceae family, to measure the total antioxidant capacity (TAC) ofthese compounds with CUPRAC (cupric ion reducing antioxidant capacity) and ABTS spectrophotomet-ric methods, and to correlate the TAC with high performance liquid chromatography (HPLC) findings.The CUPRAC spectrophotometric method of TAC assay using copper(II)-neocuproine (2,9-dimethyl-1,10-phenanthroline) as the chromogenic oxidant was developed in our laboratories. The individual antioxidantconstituents of plant extracts were identified and quantified by HPLC on a C18 column using a modi-fied mobile phase of gradient elution comprised of MeOH–0.2% o-phosphoric acid and UV detection forpolyphenols at 280 nm. The TAC values of HPLC-quantified antioxidant constituents were found, and com-pared for the first time with those found by CUPRAC. The TAC of HPLC-quantified compounds accountedfor a relatively high percentage of the observed CUPRAC capacities of plant extracts, namely 81% of nettle,60–77% of parsley (in different hydrolyzates of extract and solid sample), and 41–57% of celery leaves (indifferent hydrolyzates). The CUPRAC total capacities of the 70% MeOH extracts of studied plants (in theunits of mmol trolox g−1 plant) were in the order: celery leaves > nettle > parsley. The TAC calculated with

the aid of HPLC-spectrophotometry did not compensate for 100% of the CUPRAC total capacities, becauseall flavonoid glycosides subjected to hydrolysis were either not detectable with HPLC, or not convertedto the corresponding aglycons (i.e., easily detectable and quantifiable with HPLC) during the hydrolysis

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. Introduction

Reactive oxygen species (ROS) that emerge as a result of theespirative cycle of oxidative phosphorylation may attack biolog-cal macromolecules like cellular DNA, giving rise to single- andouble-strand breaks that may eventually cause cell ageing, cardio-ascular diseases, mutagenic changes and cancerous tumor growth.ntioxidants can react with ROS and quench free radicals givingise to restriction of radicalic chain propagation, eventually pre-enting tissue damage. When natural antioxidant defences of therganism (of enzymatic, non-enzymatic or dietary origin) are over-helmed by an excessive generation of reactive oxygen species, a

ituation of oxidative stress occurs, in which cellular and extracel-ular macromolecules (proteins, lipids and nucleic acids) can sufferxidative damage, resulting in tissue injury followed by oxidativetress-originated diseases [1]. Antioxidants, when present at low

∗ Corresponding author. Tel.: +90 212 4737034; fax: +90 212 4737180.E-mail address: etutem@istanbul.edu.tr (E. Tütem).

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oncentrations in food or organism, can significantly inhibit oretard oxidative degradation reactions [2].

Polyphenols are one of the most diverse classes of phyto-hemicals widely distributed in plants [3]. Polyphenols are strongntioxidants, and their antioxidant activities are dependent onheir structural properties [4–6]. Plant polyphenols are multi-unctional, i.e., acting as hydrogen atom donor, singlet oxygencavenger, and electron donor (reducing agent) [3]. On the otherand, some polyphenols owe their antioxidant properties to theiretal chelating abilities, as traces of transition metal ions may

atalyze oxidative degradation reactions [7]. Phenolic compoundsan be classified into three broad categories of flavonoids, phenoliccids, and phenolic polymers (tannins).

The widest class of plant phenolics, with over 4000 identifiedpecies in the leaf, seed, bark, and flower parts of plants, is the

avonoid family having the diphenylpropane (C6–C3–C6) skeleton

n which the two aromatic rings (rings A and B) are linked byhree carbons cyclized with oxygen (ring C) (Fig. 1). The phenolicydroxyl groups attached to these rings are largely responsibleor the antioxidant activity of the flavonoid. Flavonoids existing

L. Yıldız et al. / Talanta 77

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n different varieties of plants or even in different parts of theame plant may exhibit significant structural differences [3].hese compounds are present in plants generally as flavonoidlycosides having one or more hydroxyl groups bound to sugars byn acid-labile hemiacetal bond through certain positions (such ashe 7-hydroxyl in flavones and isoflavones) [8].

As a part of alternative disease prevention strategies, folk medic-nal plants have become important for the preservation of humanealth. These plants are cheaply available from local markets, andan be consumed in their simple or processed forms for healtheneficial purposes. Various parts of medicinal plants, includinghe roots, leaves, flowers, seeds, berries or bark, depending onhe solubility of the active constituents, may comprise a largeariety of polyphenolics, namely cinnamic acids, benzoic acids,avonoids, proanthocyanidins, stilbenes, tannins, coumarins, lig-ans and lignins. These compounds have many advantageousiological effects, including antioxidant activity [9].

The Umbelliferae family has a large variety of member plantsnd a wide spreading area. In many laboratories, diverse researchtudies covering morphology, anatomy, cytology, and plant chem-stry are being carried out on this family. This family is very richn terms of secondary metabolites. Compounds like coumarins,avonoids, acetylenic compounds, sesquiterpenic lactones, andolatile oils are extracted from various species of the family, andnd commercial use in biology and medicine. Certain members ofhe Umbelliferae family are folk medicinal plants. Angelica speciesre used as vegetable greens and food ingredients [10]. Species inhis family are rich in vitamins (A, B2, C, E) and minerals (copper,inc, iron, selenium) that may act as immunomodulators. In addi-ion, they contain a wide variety of bioactive phytochemicals likeavonoids and coumarins that show curative, disease preventivend nutritive effects [11].

As a member of Urticaceae family, stinging nettle (Urtica dioica)ich in minerals (especially Fe), vitamin C and pro-vitamin As a medicinal plant and diet food ingredient that finds widese as hypoglycemic, anti-diabetes, diuretic, anti-inflammatory,nti-rheumatic, and hypotensive agent; it also serves to tackleith circulatory problems, prostate complaints, and skin diseases.lthough nettle is essentially consumed as food or food ingredient,

ts medicinal use covers the use of its leaves as herbal tea [12]. Apakt al. measured the total antioxidant capacity (TAC) of tea bags pre-ared from stinging nettle (i.e., from a hot water infusion of thery herb) as 0.18, 0.15, and 0.19 mmol trolox equivalent g−1, usinghe CUPRAC (cupric ion reducing antioxidant capacity), ABTS, andolin methods of TAC assay, respectively [13].

Various antioxidant assay methods have been developed forlant antioxidants (vitamins and polyphenolics) that are impor-ant for human health [14–21]. In this study, the CUPRAC assayecently developed in our laboratories has been used for the foodlants. The chromogenic oxidizing reagent of the CUPRAC assay,

is(neocuproine 2,9-dimethyl-1,10-phenanthroline)copper(II),as previously used for the assay of biologically important

eductants [22], cysteine [23], vitamin E [24], vitamin C [25], androteins [26]. The CUPRAC assay is simple, diversely applicable tooth hydrophilic and lipophilic antioxidants, and its reagents are

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(2008) 304–313 305

table and easily available at low cost [27]. This method has beensed for the TAC assay of herbal teas and apricots [13,28], and ofuman serum [29]. The TAC of food plants subject to this studyas comparatively assayed with the CUPRAC and the widely usedBTS [30] methods.

Many research groups dealing with antioxidant chemistry haveither used or evaluated the CUPRAC method. In a comprehensiveeview by Prior et al. [31], the authors classify CUPRAC as one ofhe electron transfer-based methods, and summarize the superi-rities of the CUPRAC method over other antioxidant assays. Theytate that due to the lower redox potential of the CUPRAC reagent,educing sugars and citric acid – which are not true antioxidantsut oxidizable substrates in other similar assays – are not oxi-ized with the CUPRAC reagent. Gorinstein et al. [32] state thats an advantage to other electron transfer-based assays as ABTSnd Folin, CUPRAC values were acceptable in regard to its realisticH close to the physiological pH. Gorinstein’s research group hassed CUPRAC in several occasions: garlic extract, where CUPRACorrelated well with ABTS/TEAC results [32]; kiwifruit [33] andthylene-treated kiwifruit [34], where CUPRAC gave the highestorrelation (r = 0.97–0.98) among the tested antioxidant assaysith Folin polyphenols content; cereal and pseudocereal methanol

xtracts [35], where there was again a high correlation (r = 0.96)etween CUPRAC and Folin. Mazor et al. [36] applied CUPRAC andRAP simultaneously to a number of –SH compounds, and notedhat CUPRAC but not FRAP was capable of quantitating the onehiol-bearing tripeptide glutathione (GSH) in accordance with the-e reductant behaviour of GSH. The CUPRAC antioxidant capacityf the methanol and chloroform extracts of the stem and root ofhubarb (Rheum ribes) was linear over a relatively wide concen-ration range (up to at least 2.0 absorbance units) [37]. Capanoglut al. [38] stressed that during the processing of tomato fruit toomato paste, CUPRAC antioxidant capacities were the highest val-es for lipophilic extracts, indicating that this is a sensitive assay inrganic solvents (e.g., CUPRAC values broadly followed the trend of

ycopene during processing). The same authors noted that CUPRACas the best antioxidant assay (among five assays) reflecting theecrease in TAC of lipophilic fraction during tomato processingteps [38].

The antioxidant capacities of plants are generally evaluated inerms of their total phenolic contents [39–43]. The speciation analy-is of phenolic compounds [44] and especially their quantificationith high performance liquid chromatography (HPLC) [45–47,19]

re not abundant in literature due to the low resolution in the sep-rations of flavonoids and phenolic acids [48]. Similar to the studiesarried out on food samples, Wojdylo et al. added up the mass quan-ities of the identified phenolics to give the total phenolic content45], which would not be correct on a mole-basis. Thus, total phe-olic contents calculated on a mass basis in such reports would notnable a true comparison of antioxidant capacities, because eachhenolic antioxidant would naturally have a different capacity inhe units of equivalents of a standard reference compound such asrolox.

For rapid separation, identification and quantification of indi-idual antioxidants in the selected plants of this study, the naturef stationary and mobile phases of HPLC analysis were optimizedsing synthetic mixtures of antioxidants; calibration curves wereonstructed for each antioxidant, and the antioxidant compoundsn real mixtures were quantified with the aid of these curves. ThePLC-determined concentration of each antioxidant was multi-

lied with its TEAC (trolox equivalent antioxidant capacity, defineds the mM trolox equivalent concentration of 1 mM solution of theested antioxidant) coefficient, and these products were summedp to yield the theoretical TAC value by virtue of the additiv-

ty of absorbances of constituents in a mixture. In this regard,

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06 L. Yıldız et al. / Tala

he HPLC analysis results enabled the comparison of individualntioxidant constituents of the studied plants together with theirrolox-equivalent antioxidant capacities.

. Experimental

.1. Chemical substances

Gallic acid, catechin, hesperidin, isoquercitrin, ABTS (2,2′-zinobis[3-ethylbenzothiazoline-6-sulfonic acid] diammoniumalt), quercetin, rosmarinic acid (Fluka), chlorogenic acid, narin-enin, hesperetin, rutin, m-phosphoric acid (Sigma), caffeic acid,erulic acid, p-coumaric acid, naringin (Aldrich), myricetin (Acrosrganics), kaempferol (AppliChem), luteolin, apigenin (Alfa Aesar),scorbic acid, neocuproine (2,9-dimethyl-1,10-phenanthroline)Sigma–Aldrich), cupric chloride dihydrate (CuCl2·2H2O),otassium persulfate (K2S2O8), methanol, hydrochloric acid,-phosphoric acid, potassium hydroxide, sodium hydroxideMerck), ammonium acetate, ethanol, acetone (Riedel-de Haën)ere supplied from the indicated sources.

.2. Instrumentation

A Metrohm Herisau E512 pH-meter was used for pH measure-ents. The extraction and equilibration operations were made

sing a Bransonic 221 ultrasonic bath, a HB4 basic KIKA-WERKEater bath, and an Elektro-mag vortex mixer. Plant samplesere lyophilized using a Telstar Cryodos freeze-dryer. Spec-

rophotometric measurements were made with a Cary 1E UV–Vispectrophotometer (Varian Instruments), and chromatographiceparation and identification of plant constituents were performedsing a PerkinElmer HPLC system (comprised of Series 200 UV–Visetector, binary gradient pump, and vacuum degasser). Pure dis-illed water was used throughout, as obtained from Milliporeimpak1 Synergy 185 ultra-pure water system.

.3. Reagent and solutions

Gallic acid, catechin, chlorogenic acid, caffeic acid, feruliccid, p-coumaric acid, naringin, naringenin, hesperetin, rutin, iso-uercitrin, quercetin, myricetin, and luteolin stock solutions wererepared in MeOH; rosmarinic acid and kaempferol in EtOH; api-enin in 0.2 M ethanolic KOH; hesperidin in 0.05 M methanolicOH; ascorbic acid in 1% (w/v) m-phosphoric acid solution. Thentioxidant stock solutions were stable when kept at −20 ◦C for 1onth.For the CUPRAC test of TAC, the following solutions were

repared: CuCl2 solution, 1.0 × 10−2 M, prepared by dissolvinguCl2·2H2O in water; ammonium acetate (NH4Ac) buffer at pH 7.0,.0 M, prepared from NH4Ac in water; neocuproine (Nc) solution,.5 × 10−3 M, prepared daily by dissolving Nc in 96% ethanol. Trolox,.0 × 10−3 M, was prepared in 96% ethanol. For the ABTS test of TAC,he chromogenic radical reagent ABTS, at 7.0 mM concentration,as prepared by dissolving this compound in water and adding2S2O8 to this solution such that the final persulfate concentra-

ion in the mixture be 2.45 mM. The resulting ABTS radical cationolution was left to mature at room temperature in the dark for2–16 h, and then used for ABTS/TEAC assays. The reagent solutionas diluted with EtOH at a volume ratio of 1:10 prior to use.

.4. Preparation of plant samples for analysis

.4.1. Drying of plantsFresh parsley, celery leaves, and nettle samples were supplied

rom the local market, and the leaf parts were freeze-dried at −40 ◦C

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7 (2008) 304–313

or 8–14 h. All plant samples were kept in the dark at room temper-ture in stoppered flasks. They were crushed to fine powder in aorcelain mortar prior to analysis.

.4.2. Extraction of plants

.4.2.1. Extraction of dried plant materials. As possible solvents forxtraction, methanol, ethanol, and acetone at 100, 70, and 50% (v/v)oncentrations, and bidistilled water were used for dried pars-ey leaves; methanol at 70 and 50% and ethanol at 50% for driedelery leaves. Preliminary tests carried out on parsley and celeryeaves showed that 70% MeOH gave the highest extraction yield;n addition, methanol had protective power for phenolic antioxi-ants in subsequent HPLC separations due to its inhibitive effectn phenoloxidase-catalyzed oxidation of phenolics [49]. Thus, net-le was extracted with 70% MeOH. One-gram amount of the driedlant materials was extracted in stoppered flasks placed in an ultra-onic bath first with 15 mL solvent for 45 min, then with addedmL solvent for 45 min, and finally with 5 more mL solvent for5 min, the overall extraction taking 105 min. The plant extractsere first filtered through a filter paper, then through a GF/PET

glass fiber/polyethyleneterephtalate) 1.0/0.45 �m microfilter, andnalyzed.

.4.2.2. Extraction of fresh parsley for vitamin C determination.inely chopped parsley leaves were extracted in an ultrasonic bathith 1% (w/v) aqueous m-phosphoric acid. For this purpose, a 5-g

ample was first extracted with 25 mL acid for 45 min, the extractecanted, and extracted for a second time with 25 mL acid for5 min, the whole extraction taking 90 min [50]. The combinedxtracts were first filtered through a filter paper, then through aicrofilter, and analyzed.

.4.2.3. Nettle infusion. One-gram amount of the dried nettle wasteeped in 50 mL boiling bidistilled water for 5 min. The infusionhus obtained was first filtered through a filter paper, then throughmicrofilter, and analyzed.

.4.3. Hydrolysis process for plant materials

.4.3.1. Hydrolysis of plant extracts. Parsley, celery leaves, and nettleere extracted with 70% MeOH, diluted with water and acidified so

s to finally contain 50% methanol and 1.2 M HCl, and hydrolyzedt 80 ◦C for 4 h [51,52].

.4.3.2. Hydrolysis of dried plant powders. From dried plant mate-ial were taken 0.2 g amounts of samples, and hydrolyzed in a finalolution containing 50% MeOH and 1.2 M HCl at 80 ◦C for 4 h. Theydrolyzates were passed through a microfilter, and analyzed.

.5. Synthetic antioxidant mixtures

The compositions of synthetic mixtures were regulated to con-ain the antioxidants most probably existent in the analyzed plantxtracts.

.5.1. Synthetic mixture 1Prepared to contain in final solution 2.0 × 10−4, 4.0 × 10−5, and

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.5.2. Synthetic mixture 2Prepared to contain in final solution 2.0 × 10−4, 4.0 × 10−5,

.4 × 10−5, and 1.0 × 10−4 M of chlorogenic acid, myricetin, luteolin,nd apigenin, respectively.

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.5.3. Synthetic mixture 3Prepared to contain in final solution equimolar (2.0 × 10−4 M)

oncentrations of catechin and chlorogenic acid.

.6. Methods used in antioxidant assays

.6.1. Spectrophotometric methods

.6.1.1. CUPRAC method. The normal CUPRAC method (CUPRACN),s described by Apak et al. [27], was applied as follows: A mix-ure comprised of 1 mL of 1.0 × 10−2 M copper(II) chloride, 1 mL ofM ammonium acetate buffer at pH 7.0, and 1 mL of 7.5 × 10−3 Meocuproine solution was prepared, x mL sample solution and1 − x) mL distilled water were added, and well mixed (total vol-me: 4.0 mL). This final mixture in a stoppered test tube was leto stand at room temperature for 30 min. At the end of this period,he absorbance at 450 nm was measured against a reagent blank.he ‘incubated CUPRAC method’ (CUPRACI), as described by Apakt al. [27], was applied to antioxidant-containing samples by firstncubating the mixtures at 50 ◦C for 20 min, then measuring thebsorbance at 450 nm.

This method was applied to the extracts, hydrolyzates of all stud-ed plants, steeped infusions of nettle, and synthetic mixtures. Theamples were diluted with 50% MeOH where necessary (i.e., to keephe CUPRAC absorbances within the linear range). The pH of theydrolyzates was first brought to pH 6 with the addition of NaOHolution, and then analysis was performed.

.6.1.2. ABTS method. The ABTS/persulfate method [30] was fol-owed. Briefly, the volumes of (4 − x) mL EtOH and x mL sampleolution were taken. The reagent blank was prepared with 4 mLtOH. One mL amount of 1:10 diluted ABTS radical cation solutionas added to each mixture at 15 s intervals, and well mixed (total

olume: 5.0 mL). The absorbances of all solutions were recorded at34 nm against ethanol at the end of 6th min. The absorbance ofhe reagent blank (A0) diminished in the presence of antioxidants,he absorbance decrease (�A) being proportional to antioxidantoncentration.

This method was also applied to the extracts, hydrolyzates of alltudied plants, steeped infusions of nettle, and synthetic mixtures.he samples were diluted with 50% MeOH where necessary (i.e.,o keep the ABTS absorbance differences within the linear range).ince turbidity was observed in hydrolyzate solutions when the pHas adjusted to pH 6 with NaOH solution addition, samples wereirectly diluted with 50% MeOH.

.6.2. HPLC analysesThe analyses were carried out using a Hamilton HxSil C18

250 mm × 4.6 mm, 5 �m particle size) chromatographic column.wo elution programs were used in the reversed-phase HPLC anal-ses. For polyphenolic compounds, two different solutions of theobile phase, i.e., methanol (A) and 0.2% of o-H3PO4 in bidistilledater (B), were used in gradient elution. The following workingode was adopted for gradient elution (the slope being the rate of

hange of methanol percentage between the indicated time peri-ds):

8 min 7% (A), slope (0.0);8–13 min to 30% (A), slope (−4.0);

13–48 min to 66% (A), slope (1.0);8–55 min to 75% (A), slope (−4.0).

The detection wavelength was 280 nm and the elution rate wasmL min−1. Using the above working mode, the calibration curvesnd linear equations of peak area versus concentration were deter-ined for the phenolic antioxidants of interest. With the aid of

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(2008) 304–313 307

hese calibration curves, plant extracts in 70% MeOH, hydrolyzates,nfusions, and synthetic mixtures were analyzed.

Ascorbic acid determination was performed by using isocraticlution for 8 min, the mobile phase being composed of 7% methanolA) and 93% bidistilled water containing 0.2% of o-H3PO4 (B). Theorking wavelength was 215 nm, and flow rate 1 mL min−1. Using

his elution mode, the calibration curve and linear equation forscorbic acid was determined, and ascorbic acid assay was per-ormed in the m-phosphoric acid extract of fresh parsley.

In both applications (of polyphenolics and ascorbic acid assay),he chromatographic column was washed for 10 min with methanolrior to injection, and equilibrated for 10 min with a solvent mix-ure containing 7% (A) + 93% (B).

. Results and discussion

.1. Choice of solvent, molar absorptivities and TEAC coefficientsf the tested antioxidants

Although it is not an easy task to select a unique solvent for thenalysis of a diverse group of phenolics due to vastly varying chem-cal structures, and to different conditions in isomerization andydrolysis, alcoholic extraction (using MeOH or EtOH) has been thesual approach to handling solid samples [53]. A minimum of 70%ethanol has been reported to be needed to inactivate polyphe-

ol oxidases, which are widely distributed in plants, and to allowaximum recovery of flavonoids, such as monomeric flavan-3-ols

catechin or epicatechin) [54,55]. A few examples where 70% MeOHs solvent proved to be very effective are extraction of flavones andlycosylated flavanones from orange peel [54], anthocyanins fromed and blackcurrant [55], phenolics from E. purpurea roots [56],nd phenolics from Echinacea roots [57]. The extraction efficiencyid not change significantly for Echinacea spp. when MeOH concen-ration in the extracting solvent was increased from 70 to 90% [57].ur extraction yield with 70% MeOH of parsley and celery leavesas highest, confirming literature findings.

The CUPRAC method, using a cupric neocuproine (2,9-dimethyl-,10-phenanthroline) chelate – abbreviated as (Cu(II)-Nc) – as thehromogenic oxidant, is based on the redox reaction with antiox-dants producing the cuprous-neocuproine chelate – abbreviateds (Cu(I)-Nc) – showing maximum light absorption at 450 nm [27].he reaction equation with n-electron reductant antioxidants cane formulated by:

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+ n-e oxidized product + nH+

Antioxidant compounds that are expected to be found in theested plants, namely gallic acid, catechin, chlorogenic acid, caffeiccid, p-coumaric acid, ferulic acid, naringin, naringenin, hesperidin,esperetin, rutin, myricetin, isoquercitrin, luteolin, kaempferol,pigenin, rosmarinic acid, quercetin and ascorbic acid, were usedn standard solutions, and assayed using the normal (at room tem-erature) and incubated (at 50 ◦C) CUPRAC methods [27] againstrolox as the standard reference compound. The same antioxidantolutions were cross-assayed with ABTS/persulfate as the refer-nce spectrophotometric method. The linear calibration equationsf the tested antioxidants (as absorbance in a 1-cm cell versusolar concentration) gave the molar absorption coefficient (ε)

s the slope (Tables 1 and 2); the linear working ranges over

hich Beer’s law was valid are given in the same tables. Theolar absorption coefficient of the tested antioxidant divided by

hat of trolox under the same conditions gave the trolox equiva-ent antioxidant capacity, or TEAC coefficient, of that antioxidantTable 3).

308 L. Yıldız et al. / Talanta 77 (2008) 304–313

Table 1The molar absorption coefficients and linear ranges of the tested antioxidants withrespect to the CUPRAC method (normal and incubated CUPRAC methods)

Antioxidant Molar absorption coefficient, ε(L mol−1 cm−1)

Linear range (mol L−1)

CUPRACN CUPRACI

Trolox 1.7 × 104 1.9 × 104 2.6 × 10−6–7.5 × 10−5

Gallic acid 4.4 × 104 – 1.8 × 10−5–6.7 × 10−5

Catechin 5.2 × 104 6.6 × 104 2.0 × 10−6–1.0 × 10−5

Chlorogenic acid 4.1 × 104 5.1 × 104 4.0 × 10−6–2.0 × 10−5

Caffeic acid 4.8 × 104 5.5 × 104 4.0 × 10−6–2.0 × 10−5

p-Coumaric acid 9.2 × 103 1.9 × 104 1.1 × 10−5–1.5 × 10−4

Ferulic acid 2.0 × 104 2.3 × 104 4.0 × 10−6–2.0 × 10−5

Naringin 3.4 × 102 2.4 × 103 3.0 × 10−4–4.0 × 10−3

Hesperidin 1.6 × 104 2.1 × 104 2.6 × 10−6–7.5 × 10−5

Rutin 4.3 × 104 4.8 × 104 4.0 × 10−6–2.0 × 10−5

Naringenin – 4.2 × 104 1.0 × 10−5–5.0 × 10−5

Hesperetin 1.6 × 104 1.9 × 104 2.6 × 10−6–7.5 × 10−5

Quercetin 9.2 × 104 1.0 × 105 2.0 × 10−6–1.0 × 10−5

Myricetin 7.1 × 104 8.9 × 104 3.7 × 10−6–1.3 × 10−5

Luteolin 4.8 × 104 5.8 × 104 2.3 × 10−6–2.1 × 10−5

Kaempferol 2.5 × 104 3.6 × 104 4.9 × 10−6–4.4 × 10−5

Apigenin 4.6 × 103 1.5 × 104 1.8 × 10−5–6.7 × 10−5

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Table 3The TEAC coefficients of the tested antioxidants with respect to the CUPRAC andABTS spectrophotometric methods

Antioxidant TEACCUPRAC TEACABTS

TEACN TEACI

Myricetin 4.27 4.79 3.43Quercetin 5.49 5.57 3.21Kaempferol 1.47 1.92 0.86Rutin 2.56 2.56 1.15Isoquercitrin 3.14 3.60 1.01Apigenin 0.27 0.82 0.65Luteolin 2.90 3.14 1.01Catechin 3.09 3.56 3.14Naringin 0.02 0.13 0.62Naringenin – 2.28 0.64Hesperidin 0.97 1.11 1.40Hesperetin 0.99 1.05 1.11Gallic acid 2.62 – 3.48Rosmarinic acid 5.65 6.02 2.30Ferulic acid 1.20 1.23 2.16p-Coumaric acid 0.55 1.00 1.63Caffeic acid 2.89 2.96 1.39Chlorogenic acid 2.47 2.72 1.21Ascorbic acid 1.00 1.32 1.15

Table 4HPLC calibration equations of the tested antioxidants

Antioxidant Linear range (mol L−1) Calibration equation (r)

Ascorbic acid 1.0 × 10−4–1.0 × 10−3 y = 3.16 × 109c + 16794 0.9999Gallic acid 4.0 × 10−5–5.0 × 10−4 y = 9.04 × 109c + 73233 0.9997Catechin 4.0 × 10−5–5.0 × 10−4 y = 3.79 × 109c + 4474 0.9997Chlorogenic acid 4.0 × 10−5–5.0 × 10−4 y = 9.00 × 109c + 37950 0.9996Caffeic acid 4.0 × 10−5–5.0 × 10−4 y = 9.44 × 109c + 69934 0.9999p-Coumaric acid 4.0 × 10−5–5.0 × 10−4 y = 1.48 × 1010c + 30571 0.9999Ferulic acid 4.0 × 10−5–5.0 × 10−4 y = 1.05 × 1010c − 24807 0.9998Rosmarinic acid 4.0 × 10−5–5.0 × 10−4 y = 9.49 × 109c + 64341 0.9995Naringin 4.0 × 10−5–5.0 × 10−4 y = 1.69 × 1010c + 9146 0.9999Hesperidin 4.0 × 10−5–5.0 × 10−4 y = 1.65 × 1010c − 44514 0.9988Rutin 4.0 × 10−5–5.0 × 10−4 y = 8.79 × 109c + 3687 0.9999Isoquercitrin 4.0 × 10−5–3.0 × 10−4 y = 6.43 × 109c + 55614 0.9991Myricetin 4.0 × 10−5–5.0 × 10−4 y = 5.93 × 109c − 42007 0.9996

soquercitrin 5.2 × 10 6.7 × 10 1.8 × 10 –1.7 × 10osmarinic acid 9.4 × 104 1.1 × 105 1.8 × 10−6–6.7 × 10−6

scorbic acid 1.7 × 104 2.5 × 104 5.6 × 10−6–6.0 × 10−5

.2. HPLC results of the tested antioxidants

Table 4 shows the linear calibration equations of HPLC analy-is of the tested antioxidants as chromatographic peak area versusoncentration. Fig. 2 gives the chromatogram (obtained with theptimized separation scheme for phenolic antioxidants) of a stan-ard mixture composed of 19 antioxidants (phenolics plus ascorbiccid). As the parametric symbols used in the calibration equationsiven in Table 4, y stands for peak area, c antioxidant molar concen-ration, and r linear correlation coefficient.

In another chromatogram recorded with respect to the opti-ized (isocratic elution) method (figure not shown), the peak of

scorbic acid emerges at about 3.5 min, and the one before that

s due to m-phosphoric acid used in extraction of ascorbic acid.his extractant is useful in preserving ascorbic acid samples duringnalysis.

able 2he molar absorption coefficients and linear ranges of the tested antioxidants withespect to the ABTS/persulfate method

ntioxidant Molar absorption coefficient, ε(L mol−1 cm−1)

Linear range (mol L−1)

rolox 2.6 × 104 5.0 × 10−6–3.0 × 10−5

allic acid 9.1 × 104 2.0 × 10−6–1.0 × 10−5

atechin 8.2 × 104 2.0 × 10−6–1.0 × 10−5

hlorogenic acid 3.2 × 104 4.0 × 10−6–2.0 × 10−5

affeic acid 3.6 × 104 4.0 × 10−6–2.0 × 10−5

-Coumaric acid 4.2 × 104 4.0 × 10−6–2.0 × 10−5

erulic acid 5.6 × 104 4.0 × 10−6–2.0 × 10−5

aringin 1.6 × 104 1.0 × 10−5–5.0 × 10−5

esperidin 3.6 × 104 4.0 × 10−6–2.0 × 10−5

utin 3.0 × 104 4.0 × 10−6–2.0 × 10−5

aringenin 1.7 × 104 1.0 × 10−5–5.0 × 10−5

esperetin 2.9 × 104 3.8 × 10−6–1.9 × 10−5

uercetin 8.3 × 104 2.0 × 10−6–1.0 × 10−5

yricetin 8.9 × 104 2.0 × 10−6–1.0 × 10−5

uteolin 2.6 × 104 3.8 × 10−6–1.9 × 10−5

aempferol 2.3 × 104 8.0 × 10−6–4.0 × 10−5

pigenin 1.7 × 104 1.0 × 10−5–5.0 × 10−5

soquercitrin 2.6 × 104 6.0 × 10−6–3.0 × 10−5

osmarinic acid 6.0 × 104 4.0 × 10−6–2.0 × 10−5

scorbic acid 3.0 × 104 4.0 × 10−6–2.0 × 10−5

Naringenin 4.0 × 10−5–5.0 × 10−4 y = 1.47 × 1010c + 5985 0.9999Hesperetin 4.0 × 10−5–5.0 × 10−4 y = 1.43 × 1010c − 81556 0.9983Quercetin 4.0 × 10−5–5.0 × 10−4 y = 9.12 × 109c + 88516 0.9989Luteolin 4.0 × 10−5–5.0 × 10−4 y = 9.02 × 109c − 79509 0.9990Kaempferol 4.0 × 10−5–5.0 × 10−4 y = 7.23 × 109c − 36137 0.9996Apigenin 4.0 × 10−5–5.0 × 10−4 y = 1.04 × 1010c − 1727 0.9999

Fig. 2. The chromatogram of a synthetic mixture of antioxidants (phenolics plusascorbic acid). (1) Ascorbic acid; (2) gallic acid; (3) catechin; (4) chlorogenic acid;(5) epicatechin; (6) caffeic acid; (7) p-coumaric acid; (8) ferulic acid + sinapic acid;(9) naringin; (10) rosmarinic acid; (11) hesperidin; (12) rutin + isoquercitrin; (13)myricetin; (14) naringenin; (15) hesperetin; (16) quercetin; (17) luteolin; (18)kaempferol; (19) apigenin.

L. Yıldız et al. / Talanta 77 (2008) 304–313 309

3

ic(tiewirtp4es7o

waataT

F(

Fig. 5. The chromatogram of 70% methanolic extract of celery leaves.

Fy

t

T

i=1

Fig. 3. The chromatogram of 70% methanolic extract of parsley.

.3. Results of plant analyses

Plant extracts, extract hydrolyzates (prepared for the purpose ofdentifying complex mixtures containing numerous flavonoid gly-osides by converting to the corresponding aglycons), solid plantdried plant powder) hydrolyzates, and herbal infusions were spec-rophotometrically assayed for TAC with CUPRAC (i.e., normal andncubated CUPRAC methods) and ABTS; the individual antioxidantsxisting in these complex samples were identified and quantifiedith HPLC. In order to confirm qualitative identification, the change

n the peak heights of certain chromatographic bands with fixedetention times were followed with the technique of standard addi-ions. Fig. 3 shows the chromatogram of 70% methanolic extract ofarsley, and Fig. 4 gives the chromatogram of the same extract afterh hydrolysis. Fig. 5 shows the chromatogram of 70% methanolicxtract of celery leaves, and Fig. 6 gives the chromatogram of theame extract after 4 h hydrolysis. Fig. 7 shows the chromatogram of0% methanolic extract of nettle, and Fig. 8 gives the chromatogramf the dried nettle hydrolyzate after 4 h hydrolysis.

The individual antioxidants of plant samples were determinedith the help of calibration curves in HPLC (Table 4). Using the

dditivity property of TAC in a complex sample, the theoretical totalntioxidant capacities of plant samples were calculated by mul-

iplying the concentration with the TEAC value of each identifiedntioxidant, and summing up the products. Thus, the theoreticalAC of the investigated plant material could be estimated using

ig. 4. The chromatogram of 70% methanolic extract of parsley after 4 h hydrolysis.1) p-Coumaric acid; (2) myricetin; (3) apigenin.

wtc

Fc

ig. 6. The chromatogram of 70% methanolic extract of celery leaves after 4 h hydrol-sis. (1) Chlorogenic acid; (2) myricetin; (3) luteolin; (4) apigenin.

he equation:

ACtheoretical =n∑

ci(TEAC)i

here ci is the concentration of antioxidant constituent i found withhe help of HPLC, and (TEAC)i is the trolox equivalent antioxidantapacity (TEAC) coefficient of constituent i with respect to a given

ig. 7. The chromatogram of 70% methanolic extract of nettle. (1) Catechin; (2)hlorogenic acid.

310 L. Yıldız et al. / Talanta 7

FC

siwTiattTbc

3

st2hdca1tw

A(a

cfiwtaseatOeApwTiATsubrbAulcscltueit

TT

S

P

C

N

H

ig. 8. The chromatogram of the dried nettle hydrolyzate after 4 h hydrolysis. (1)hlorogenic acid; (2) caffeic acid.

pectrophotometric method (either CUPRAC or ABTS). The theoret-cal TAC (thus calculated) and experimental TAC (directly measured

ith a given spectrophotometric method) values were compared inable 5. In Table 5, due to the lack of complete species identificationn the chromatograms of the extracts of parsley and celery leaves,nd of information on their exact contributions to the observed TAC,he HPLC-spectrophotometric estimates of TAC were made usinghe information obtained from their hydrolyzate chromatograms.he HPLC-spectrophotometric calculations for nettle were madeased on the identification and quantification of species in thehromatograms of both MeOH extracts and hydrolyzate solutions.

.4. Results of synthetic mixture analyses

The chromatograms of different possible combinations (i.e.,ynthetic mixtures at varying concentrations) of antioxidants iden-ified in the tested plants and of a synthetic mixture hydrolyzed forh were recorded (figures not shown). In the chromatogram of theydrolyzate, it could be clearly seen that, accompanying the largeecrease in magnitude of the chlorogenic acid peak, a new peak of

affeic acid (as a constituent of the former) emerged. Additionally,n intense peak at 3 min, and two small peaks at approximately7 and 29 min appeared. The experimental TAC values of the mix-ures (the compositions of which were described in Section 2.5)ere spectrophotometrically measured by CUPRACN, CUPRACI and

3

s

able 5he theoretical and experimental TAC (total antioxidant capacity) values of plants in the u

ample CUPRACN CUPRACI ABTS

arsley70% MeOH extn. 0.050 0.079 0.04070% MeOH extn. 2 h hydrol. 0.056 0.099 0.01170% MeOH extn. 4 h hydrol. 0.058 0.104 0.012Solid plant, 2 h hyrol. 0.082 0.166 0.022Solid plant, 4 h hydrol. 0.093 0.193 0.021m-Phosphoric acid extn. 0.016 0.023 0.008

elery leaves70% MeOH extn. 0.148 0.180 0.08770% MeOH extn. 4 h hydrol. 0.141 0.169 0.096Solid plant, 4 h hydrol. 0.179 0.229 0.133

ettle70% MeOH extn. 0.076 0.097 0.05870% MeOH extn. 4 h hydrol. 0.077 0.094 0.060Solid plant, 4 h hydrol. 0.186 0.247 0.170Steeped infusion 0.050 0.081 0.036Steeped infusion hydrol. 0.078 0.116 0.027

PLC-spectrophotometric values represent the theoretically found TAC using the additivi

7 (2008) 304–313

BTS assays. The calculated HPLC-spectrophotometric capacitiesTACtheoretical) of the mixtures, along with experimental TAC results,re tabulated in Table 6.

For synthetic mixtures, the experimental CUPRAC and theoreti-al HPLC-CUPRAC results for TAC were very close to each other. Thisnding confirms that if all the antioxidants in a complex mixtureere identified and quantified with the help of HPLC techniques,

heir contribution to the overall capacity could be envisaged,nd the experimentally found TAC values (with the use of thepectrophotometric CUPRAC antioxidant assay) could be correctlystimated by theoretically calculating the TAC using the principle ofdditivity of individual antioxidant capacities (named for the firstime in this study as ‘HPLC-CUPRAC method’ of TAC estimation).n the other hand, if the same comparison was made between thexperimental ABTS TAC values and the theoretically found HPLC-BTS capacities, significant discrepancies were apparent (Table 6),robably showing that there were inherent problems associatedith the TAC additivity in the ABTS method of antioxidant assay.

he ABTS results were also not coherent with those of CUPRAC. Its known from other studies in the literature that when the coloredBTS radical cation was generated with different mechanisms, theEAC values of antioxidants varied significantly [30]. Firstly, it is noturprising that turbidity problems were encountered in this worksing ABTS at pH 6.0, while the ABTS radical reagent is not sta-le above pH 6.5, and therefore Cano et al. [58] had to prepare theeagent solution in glycine–HCl buffer at pH 4.5 to preserve its sta-ility at room temperature. It was shown by Cano et al. [58] that theBTS radical cation is not stable for mixtures containing a ratio ofnreacted ABTS-to-ABTS radical cation (i.e., ABTSunreacted/ABTS*+)

ess than 50, while for most assays, this critical limit in the ratioannot be maintained for maximal stability. Due to the above rea-ons, deviations from additivity of TAC values (of HPLC-analyzedomponents) of antioxidant mixtures might have arisen both in theiterature [59] and in this work, while no problems were encoun-ered in regard to turbidity and additivity of TAC values of mixturessing the CUPRAC method. The data in Table 6 clearly show that thexperimental and theoretical TAC values of antioxidants containedn synthetic mixture solutions 1–3 (in trolox equivalent concentra-ions) were coherent for CUPRAC but not for ABTS.

.5. Evaluation of common results for synthetic and real mixtures

The percentages of experimental antioxidant capacities ofynthetic mixtures and plant extracts as accounted for by

nits of mmol trolox g−1 plant

HPLC-(CUPRACN) HPLC-(CUPRACI) HPLC-(ABTS)

– – –0.043 0.067 0.0510.034 0.062 0.0490.049 0.074 0.0560.035 0.069 0.0540.008 0.011 0.009

– – –0.068 0.097 0.0530.069 0.095 0.049

0.062 0.069 0.0380.043 0.067 0.0510.034 0.062 0.0490.049 0.074 0.0560.035 0.069 0.054

ty principle; extn.: extraction, hydrol.: hydrolysis.

L. Yıldız et al. / Talanta 77 (2008) 304–313 311

Table 6The experimental and theoretical TAC values of synthetic mixture solutions of antioxidants (in the units of mmol trolox equivalents L−1)

Sample CUPRACN CUPRACI ABTS HPLC-(CUPRACN) HPLC-(CUPRACI) HPLC-(ABTS)

Mixture 1 0.32 0.43 0.33 0.29 0.47 0.54M 0M 6M 5

HHc8aoh

ptAHopalpcsrtamif

flcbiail

TTtH

S

P

C

N

fmttMtwehas

otdaTttftemiHmuto

ixture 2 0.80 0.90 0.4ixture 3 1.29 1.41 0.7ixture 3 (2 h hydrol.) 1.32 1.41 0.3

PLC-spectrophotometric estimates are given in Table 7. ThePLC-CUPRAC method was capable of estimating the following per-entages of the experimental CUPRAC capacities of plant samples:1% of nettle extract, 60–77% of different hydrolyzates of parsley,nd 41–57% of different hydrolyzates of celery leaves. The precisionf estimation for the proposed method (i.e., 92–108%) was muchigher for synthetic mixtures.

This study has identified the essential antioxidant compoundsresent in parsley, celery leaves, and stinging nettle, measured theotal antioxidant capacity of these compounds with CUPRAC andBTS spectrophotometric methods, and correlated the TAC withPLC findings. The CUPRAC spectrophotometric method devel-ped in our laboratories was selected for TAC measurement oflant material, because it had low cost, stable and easily avail-ble reagents, and was capable of oxidizing both hydrophilic andipophilic antioxidants relatively rapidly within the protocol timeeriod of the assay. Moreover, the CUPRAC assay results were pre-ise and reproducible, and the TAC of complex mixtures perfectlyhowed the property of additivity. The CUPRAC assay worked at theealistic pH of 7.0 (close to the physiological pH), thereby measuringhe TAC of real samples without exaggeration (e.g., FRAP workingt pH 3.6 and Folin at pH 10 either underestimated or overesti-ated the actual TAC, because phenolic antioxidants were either

n molecular acidic form or in totally dissociated conjugate baseorm, making their oxidation too difficult or too easy, respectively).

Since the basic plant antioxidants comprising numerousavonoids and phenolic acids exist in plants in the form of their gly-osides, esters, or bound to cell walls, their complete identificationy HPLC techniques is almost impossible due to the unavailabil-

ty of the corresponding standards, and therefore these glycosidesre generally converted into their aglycons by acid hydrolysis anddentified in free forms [48]. As it is known that parsley and celeryeaves contain apigenin glycosides, a 4-h hydrolysis was sufficient

able 7he percentages of experimental antioxidant capacities (found by spectropho-ometry) of synthetic mixtures and plant extracts as accounted for byPLC-spectrophotometric estimates

ample CUPRACN (%) CUPRACI (%) ABTS (%)

arsley70% MeOH extd. 2 h hydrol. 77 68 >10070% MeOH extd. 4 h hydrol. 56 60 >100Solid plant, 2 h hydrol. 60 45 >100Solid plant, 4 h hydrol. 37 35 >100m-Phosphoric acid extract 52 46 >100

elery leaves70% MeOH extract hydrol. 48 57 5550% MeOH extract hydrol. 40 51 46Solid plant hydrolyzate 39 41 37

ettle70% MeOH extract 81 71 6670% MeOH extract hydrol. 21 19 14Solid plant hydrolyzate 9 7 5Mixture 1 92 108 >100Mixture 2 98 102 118Mixture 3 92 95 119Mixture 3 (2 h hydrol.) 14 15 53

pastTteiv

aofrmffcpotcmdsda

0.79 0.92 0.471.19 1.34 0.900.18 0.21 0.19

or their conversion. On the other hand, the 4-h hydrolysis producedyricetin almost a half of that converted within 2 h. Thus, within

his relatively long period of hydrolysis, the literature-reported par-ial degradation of myricetin [60–62] was confirmed in this study.

oreover, phenolic acids were observed to largely degrade underhese hydrolysis conditions [60,62,63]. During the investigationsith nettle, the high content of chlorogenic acid in the original plant

xtract could no longer be found in the hydrolyzate. On the otherand, the presence of caffeic acid in the hydrolyzate in spite of itsbsence in the original extract hints to the possible conversion ofome chlorogenic acid into caffeic acid [64].

In a study in which the contribution of phenolic species to thebserved total antioxidant capacity of plum was investigated [59],en polyphenolic compounds including chlorogenic acid, cyani-in, cyanidin glycosides, peonidin, peonidin-3-glycoside, quercetin,nd quercetin glycosides were identified and quantified by HPLC.he HPLC-determined concentrations of individual phenolics mul-iplied by their ascorbic acid-equivalent capacities with respect tohe ABTS method were summed up, and this calculated TAC wasound to be smaller than the experimental TAC measured withhe ABTS method in the same (ascorbic acid) units. This differ-nce between the calculated and actual values was attributed toatrix variations, and to minor antioxidant constituents contained

n the samples such as carotenoids and vitamin C. In this study,PLC-spectrophotometry combinations were carried out to esti-ate the theoretical TAC as trolox equivalents of complex samples

sing the chromatographic standards of the most widely encoun-ered antioxidants. In the hydrolyzates (at 2- and 4-h operations)f synthetic mixtures and plant materials, the pH adjustment toH 6.0 with the addition of NaOH produced turbidity of ABTSssay samples before the 6th min measurement. Therefore, acidicamples were appropriately diluted without pH adjustment prioro ABTS assay measurement. Looking at the results tabulated inable 7, there were great discrepancies between the ABTS spec-rophotometric measurements (experimental TAC) and HPLC-ABTSstimates (theoretical TAC). On the other hand, there was a harmon-cal relationship with the analogic CUPRAC results, arising from thealidity of the principle of additivity of TAC in CUPRAC.

The two-way ANalysis Of VAriance (ANOVA) comparison by theid of F-test of the mean-squares of ‘between-treatments’ (i.e., the-retically expected HPLC-CUPRAC absorbance and experimentallyound CUPRAC absorbance of different samples in Table 6) and ofesiduals [65] for synthetic mixtures 1–3 (excluding the hydrolyzedixture 3) enabled to conclude that there was no significant dif-

erence between treatments. In other words, the experimentallyound CUPRAC results and theoretically expected HPLC-CUPRACalculations were alike at 95% confidence level (using paired com-arisons of either CUPRACN and HPLC-CUPRACN treatment data, orf CUPRACI and HPLC-CUPRACI treatment data). On the other hand,here was significant difference between samples with respect tooncentration of antioxidants (i.e., the residual mean-square was

uch greater than ‘between-sample’ mean-square at 95% confi-

ence level). The ANOVA treatment of relatively limited data alsohowed that the experimental ABTS measurements significantlyiffered from HPLC-ABTS calculations, probably due to the lack ofdditivity of TAC values of synthetic mixture components in the

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irpteicCptTaccetic

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12 L. Yıldız et al. / Tala

BTS method under the employed conditions. To summarize thetatistical test results, the experimental and computational (theo-etical) TAC values of antioxidants contained in synthetic mixtureolutions 1–3 (in trolox equivalent concentrations) were coherentor CUPRAC but not for ABTS.

The HPLC-CUPRAC method was capable of estimating the fol-owing percentages of the experimental CUPRAC capacities of plantamples: 81% of nettle extract, 60–77% of different hydrolyzatesf parsley, and 41–57% of different hydrolyzates of celery leaves.ince the estimation percentages for synthetic mixtures were largerTable 7), it was obvious that any deviations from ideal estimationn the proposed HPLC-CUPRAC procedure resulted from incom-lete identification or conversion of flavonoid glycosides and/orannin polymers in the tested samples. This argument can be fur-her extended to claim that if all the antioxidants in a complex

ixture could be identified and quantified with HPLC, it would beossible to roughly estimate the actual (experimental) TAC of theixture with the proposed procedure thanks to the validity of the

dditivity of TAC in the CUPRAC method.

. Conclusions

This work brings several contributions to food analytical chem-stry (specifically antioxidant assays) briefly summarized as: (i)eporting the trolox equivalent antioxidant capacities of severalhenolic antioxidants – not assayed previously – with respect tohe CUPRAC method; (ii) modifying the mobile phase of gradientlution in HPLC analysis of phenolic antioxidants where resolutionn the separation of phenolic acids and flavonoids is not appre-iably high; (iii) developing a novel method of estimation of theUPRAC total antioxidant capacity of antioxidant mixtures andlant extracts utilizing the principle of additivity of the capaci-ies of individual constituents identified and quantified by HPLC.he sum of the products of the concentrations of HPLC-identifiedntioxidant constituents in plant extracts and their TEAC coeffi-ients (with respect to the CUPRAC method) gave the theoreticallyalculated TAC values, which accounted for a large percentage ofxperimental TAC values measured by CUPRAC spectrophotome-ry. Thus, the ‘HPLC-CUPRAC method’ is proposed for the first timen this study to give a reliable estimate of the actual antioxidantapacity of plant extracts and hydrolyzates.

The proposed HPLC-CUPRAC method enables a realistic compar-son of antioxidant constituents of plant extracts and hydrolyzatesy HPLC analysis, and of their calculated TAC values (without per-orming the actual antioxidant assay) in trolox equivalents. Thisomparison is certainly more meaningful than that of total pheno-ic contents made on a mass basis [45]. Since the additivity propertyf TAC with respect to the proposed method proved to be preciselyalid for synthetic mixtures and approximately valid for real mix-ures of antioxidants, it becomes feasible to roughly estimate thexperimental TAC of complex antioxidant mixtures such as plantxtracts and hydrolyzates on the condition that all the antioxidantsontained therein be properly identified and quantified by HPLC.

The additivity of TAC with HPLC-CUPRAC is approximately validor real mixtures of antioxidants, because real mixtures containavonoids, their glycosides, their many additional configurationalnd conformational isomers as well as their oligomers or polymers,nd it is almost impossible to find the exactly matching standardsor their HPLC analysis. As a result, the chromatographic peaks ofertain isomers may easily overlap, and render their precise HPLC

stimation and quantification almost impossible. Continuouslyeveloping LC/MS/MS techniques for complete characterization oflant polyphenolics face the bottlenecks of insufficiency of datarocessing and available reference compounds. Thus, we cannotpeak of a “one-to-one correspondence” between the experimental

[

[

[

7 (2008) 304–313

UPRAC and computational HPLC-CUPRAC results of real mixtures,nd the proposed methodology of this work may be considered suc-essful if a high percentage of the observed CUPRAC absorbance ofreal mixture can be compensated for by a HPLC-CUPRAC compu-

ational procedure.The CUPRAC total capacities of the 70% MeOH extracts of studied

lants (in the units of mmol trolox g−1 plant) were in the order:elery leaves > nettle > parsley.

cknowledgements

The authors would like to express their gratitude to Researchund of the Istanbul University for the funding of ProjectOP-4/27052004, and to the State Planning Organization ofurkey for the Advanced Research Project of Istanbul University2005K120430). One of the authors, Leyla Yıldız, also wishes toxtend her gratitude to Research Fund of the Istanbul Universityor the support given to her M. Sc. thesis project (T-919/06102006).

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