A comprehensive review of CUPRAC methodology

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Cite this: DOI: 10.1039/c1ay05320e

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A comprehensive review of CUPRAC methodology

Mustafa €Ozy€urek,a Kubilay G€ucl€u,a Esma T€utem,a Kevser S€ozgen Basxkan,a Erol Erca�g,a S. Esin Celik,a

Sefa Baki,a Leyla Yıldız,b Sxeyda Karamanc and Resxat Apak*a

Received 31st May 2011, Accepted 3rd August 2011

DOI: 10.1039/c1ay05320e

Measuring the antioxidant activity/capacity levels of food and biological fluids is carried out for the

meaningful comparison of the antioxidant content of foodstuffs and for the diagnosis and treatment of

oxidative stress-associated diseases in clinical biochemistry. Current literature clearly states that there is

no widely adopted/accepted ‘‘total antioxidant parameter’’ as a nutritional index available for the

labeling of food and biological fluids due to the lack of standardized quantitation methods. The

‘‘parent’’ CUPRAC (CUPric Reducing Antioxidant Capacity) method of antioxidant measurement,

introduced by our research group to world literature, is based on the absorbance measurement of Cu(I)-

neocuproine (Nc) chelate formed as a result of the redox reaction of chain-breaking antioxidants with

the CUPRAC reagent, Cu(II)-Nc, where absorbance is recorded at the maximal light absorption

wavelength of 450 nm; thus this is an electron-transfer (ET)-based method. From the parent CUPRAC

method initially applied to food (apricot, herbal teas, wild edible plants, herby cheese etc.) and

biological fluids (as hydrophilic and lipophilic antioxidants together or in separate fractions), a number

of ‘‘daughter’’ methods have evolved, such as the simultaneous assay of both lipophilic and hydrophilic

antioxidants in acetone-water as methyl-b-cyclodextrin inclusion complexes, determination of ascorbic

acid alone in the presence of flavonoids (with preliminary extraction of flavonoids as their La(III)-

complexes), determination of hydroxyl radical scavenging activity of both water-soluble antioxidants

(using benzoate derivatives and salicylate as hydroxylation probes) and of polyphenols using catalase

aDepartment of Chemistry, Faculty of Engineering, Istanbul University,Avcilar, 34320 Istanbul, Turkey. E-mail: rapak@istanbul.edu.tr; Fax:+90 212 473 7180; Tel: +90 212 473 7028bEmbil Pharmaceutical Co. Ltd, Bomonti, Sxisxli, 34381 Istanbul, Turkey

cDepartment of Chemistry, Fatih University, B. Cekmece, 34500 Istanbul,Turkey

Mustafa €Ozy€urek

Mustafa €Ozy€urek received his

PhD in Analytical Chemistry

from Istanbul University in

2009. Since 2009, he has worked

as an Assistant Professor in the

Analytical Chemistry Division

of the Engineering Faculty of the

University of Istanbul. His areas

of interest include development

of novel antioxidant capacity/

activity assays and of optical

antioxidant sensors, and the

application of antioxidant/anti-

radical activity methods to plant

extracts and biological fluids.

He has authored over 150

research papers and several books and book chapters (28 articles

424 times cited, with an average citation per year of 60.57 and an h-

index of 10).

Kubilay G€uc‚l€u

Kubilay G€ucl€u received his PhD

in Analytical Chemistry from

Istanbul University in 1999.

Since 2001, he has worked as an

Assistant Professor in the

Analytical ChemistryDivision of

the Engineering Faculty of the

University of Istanbul. His areas

of interest include development

of novel antioxidant capacity/

activity assays and of optical

antioxidant sensors, and the

application of antioxidant/anti-

radical activity methods to plant

extracts and biological fluids. He

has authored over 200 research

papers and several books and book chapters (50 papers 697 times

cited, with an average citation per year of 41 and an h-index of 15).

This journal is ª The Royal Society of Chemistry 2011 Anal. Methods

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to stop the Fenton reaction so as to prevent redox cycling of antioxidants, measurement of Cu(II)-

catalyzed hydrogen peroxide scavenging activity and of xanthine oxidase inhibition activity of

polyphenols, TAC measurement of protein thiols in urea buffer, development of a CUPRAC-based

antioxidant sensor on a Nafion cation-exchanger membrane, the off-line HPLC-CUPRAC assay and

finally the on-line HPLC-CUPRAC assay of antioxidants with post-column detection. The current

direction of CUPRAC methodology can be best described as a self-sufficient and integrated train of

measurements providing a useful ‘‘antioxidant and antiradical assay package’’. This review attempts to

unify and summarize various methodologies of main and modified CUPRAC procedures that can

normally be extracted from quite different literature sources.

Esma T€utem

Esma T€utem is a Professor of

Analytical Chemistry at Istan-

bul University, Turkey. She

received her MSc (Chemical

Engineering, 1978) and PhD

(Chemistry, 1985) degrees at

the same university. Her

research interests are focused on

spectrophotometric and deriva-

tive spectrophotometric deter-

minations of biologically and

environmentally important

reductants and metal ions,

spectrophotometric and liquid

chromatographic evaluation of

total antioxidant capacities and

polyphenolic constituents of edible and inedible parts of fruits,

vegetables and medicinal plants. She is the co-author of more than

100 articles and congress presentations (27 of which are published

in SCI-covered journals and have received 583 citations).

S: Esin C‚ elik

Saliha Esin Celik received her

Master’s Degree in Analytical

Chemistry from Istanbul

University in 2005. Since 2005

she has worked as a research

assistant in the Analytical

Chemistry Division of Istanbul

University. She received her

PhD in the same division under

the supervision of Prof. Dr ResxatAPAK in 2011. Her PhD study

was on ‘‘Modified CUPRAC

Antioxidant Capacity Measure-

ments Applicable to Different

Species, Mixtures and Solvent

Media’’. She is the co-author of

16 research papers (published in SCI-covered journals and having

received about 306 citations). Her research interests are antioxi-

dants, development of antioxidant capacity/activity assays, spec-

troscopic and chromatographic applications.

Erol Erc‚a�g

Erol Erca�g received his PhD in

Chemical Sciences (Analytical

Chemistry) in 1995 from Istan-

bul University. He has been an

Associate Professor of Analyt-

ical Chemistry since 2006 at

Istanbul University, and Vice

Institute Director at the Insti-

tute of Marine Sciences and

Management. Current research

includes the application of anti-

oxidant capacity methods to

plant extracts and biological

fluids, and devising analytical

spectroscopic methods for the

determination of energetic materials. He has authored over 24

research papers (published in SCI-covered journals and received

about 125 citations).

Resxat Apak

Resxat Apak received his PhD in

Analytical Chemistry from

Istanbul University in 1982. He

is a full professor of Analytical

Chemistry at Istanbul Univer-

sity and head of the Analytical

Chemistry Division (1990–).

Current research includes the

development of analytical

methods for the determination of

biologically important

compounds; devising analytical

methods and sensors for the total

antioxidant capacity/activity

assay of foodstuffs and human

plasma. He has authored approximately 450 articles (127 of which

are major research articles published in SCI-covered journals), 7

peer-reviewed encyclopedia chapters, and 3 textbooks, and received

about 1773 citations (h-index ¼ 22).

Anal. Methods This journal is ª The Royal Society of Chemistry 2011

Fig. 1 The CUPRAC reaction and chromophore: Bis(neocuproine)

copper(I) chelate cation (Protons liberated in the reaction are neutralized

by the NH4Ac buffer).

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1. Brief overview of antioxidant assays and theoriginal CUPRAC (CUPric Reducing AntioxidantCapacity) assay of total antioxidant capacity

Antioxidants are health-beneficial compounds counterbalancing

an excess of oxidants, comprising reactive oxygen species and

free radicals, that may emerge in the human organism as a result

of ‘oxidative stress’ conditions, described as an imbalance of

oxidants over antioxidants; this imbalance may eventually give

rise to various diseases such as cell ageing, mutagenic changes

and cancer, cardiovascular and neurodegenerative diseases.

Therefore, the determination of total antioxidant capacity

(TAC) is of vital importance to the community of food,

biochemical, and biomedical scientists. During the last fifteen

years, many methods based on free radical scavenging have

been developed to determine the antioxidant potential. The

ABTS/TEAC (2,20-azinobis-(3-ethylbenzothiazoline-6-sulfonicacid/trolox equivalent antioxidant capacity),1 ORAC (oxygen

radical absorbance capacity),2 DPPH (2,2-di(4-tert-octyl-

phenyl)-1-picrylhydrazyl),3 FRAP (ferric reducing antioxidant

power),4 and Folin–Ciocalteau total phenolics5 assays are

conventional test systems used to evaluate the antioxidant

capacity of food samples. The wide variety of antioxidant tests

necessitates the meaningful comparison of results obtained from

these assays. Antioxidant activity (i.e. related to the kinetics of

antioxidant action for quenching reactive species, usually

expressed as reaction rates or scavenging percentages per unit

time) and antioxidant capacity (i.e. thermodynamic conversion

efficiency of reactive species by antioxidants, such as the

number of moles of reactive species scavenged by one mole of

antioxidant during a fixed time period) are both important in

antioxidant research, and care must be exercised to distinguish

between these two terms, which are often used interchangeably

and therefore confused. Antioxidant assays may be classified

with respect to different approaches, such as the type of anti-

oxidant measured (e.g., lipophilic and hydrophilic, enzymatic

and non-enzymatic), character of assay medium (e.g., aqueous

and organic solvent, direct or indirect, in situ and ex situ), type

of assay reagent (e.g., radicalic and non-radicalic), or mecha-

nism of action (such as hydrogen atom transfer (HAT)- and ET-

based assays), the latter being the most favoured classification

approach. Naturally, no single assay is sufficient for reliable

determination of antioxidant activity/capacity, and usually

a train of assays is required to realistically assess the antioxidant

potential of a complex sample. Due to the differences in the

methodology of extraction and measurement of antioxidant

constituents in food matrices, no two tests using different TAC

determination methods, or even the same test with two different

extraction and measurement conditions, may yield identical

results. Thus, it is extremely important to standardize sample

pretreatment and measurement protocols. CUPRAC is

a recently discovered (2004)6 electron transfer ET-based TAC

assay for the overall quantification of all kinds of antioxidants.

ET-based spectrophotometric assays measure the capacity of an

antioxidant by the reduction of a chromogenic oxidant (probe),

which changes colour when reduced. The degree of colour

change (either an increase or decrease of absorbance at a given

wavelength) is correlated with the concentration of antioxidants

in the sample.

This journal is ª The Royal Society of Chemistry 2011

Probe(n) + e� (from antioxidant: AH) / Probe(n�1) + A_+ (1)

ET-based assays include Folin–Ciocalteau total phenolic

content, ferricyanide (hexacyanoferrate(III)/Prussian blue),7

FRAP, CUPRAC, and finally ABTS and DPPH methods (the

latter two being considered as borderline between ET- and HAT-

based assays). As opposed to the first four methods utilizing an

absorbance increase, the latter two methods measure decolor-

ization of the radicalic reagents as a result of reduction with

antioxidants during a fixed time period.

The CUPRAC method is a simple and versatile antioxidant

capacity assay useful for a wide variety of polyphenols, including

phenolic acids, hydroxycinnamic acids, flavonoids, carotenoids,

anthocyanins, as well as for thiols, synthetic antioxidants, and

vitamins C and E. This method was named by our research group

as ‘‘cupric ion reducing antioxidant capacity’’ in 2004, abbrevi-

ated as the CUPRAC method.6 The chromogenic oxidizing

reagent used for the CUPRAC assay is the bis(neocuproine)

copper(II) cation (Cu(II)-Nc) acting as an outer-sphere electron-

transfer agent, and the CUPRAC chromophore, formed by

reduction of this reagent with antioxidants, is bis(neocuproine)

copper(I) cation (Cu(I)-Nc) (Fig. 1). This reagent is useful at

pH 7, and the absorbance of the Cu(I)-chelate formed as a result

of the redox reaction with reducing polyphenols, vitamins C and

E is measured at 450 nm (see Fig. 2, for Cu(I)-Nc spectra

obtained by reacting varying concentrations of quercetin with

the CUPRAC reagent). The orange-yellow color is due to the

Cu(I)-Nc chelate formed. CUPRAC reactions are essentially

complete within 30 min.

The CUPRAC reagent, bis(neocuproine)copper(II) chloride

(Cu(II)-Nc), reacts with n-electron reductant antioxidants (AO)

in the following manner:

n Cu(Nc)22+ + n-electron reductant (AO) 4 n Cu(Nc)2

+ n-electron oxidized product + n H+ (2)

In this reaction, the reactive Ar–OH groups of polyphenolic

antioxidants are essentially oxidized to the corresponding

quinones (Ar]O), and Cu(II)-Nc is reduced to the orange-yellow

coloured Cu(Nc)2+ chelate. Although the concentration of Cu2+

ions is in stoichiometric excess of that of neocuproine (Nc) in the

CUPRAC reagent for driving the redox equilibrium reaction

represented by (eqn (2)) to the right, the actual oxidant is the Cu

(Nc)22+ species and not the sole Cu2+. This is because the standard

Anal. Methods

Fig. 2 Visible spectra of Cu(I)-Nc chelate produced as a result of

CUPRAC reaction with varying concentrations of quercetin.

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redox potential of the Cu(Nc)22+/Cu(Nc)2

+ couple is 0.6 V, much

higher that of the Cu2+/Cu+ couple (0.17 V), because copper(I) is

much more stabilized with neocuproine in a tetrahedral geometry

compared to Cu(II).8 As a result, polyphenols are oxidized much

more rapidly and efficiently with Cu(II)-Nc than with Cu2+, and

the amount of colored product (i.e. Cu(I)-Nc chelate) emerging at

the end of the redox reaction is equivalent to that of reacted Cu

(II)-Nc. The liberated protons are buffered in ammonium acetate

medium at pH 7. In the normal CUPRACmethod (CUPRACN),

the oxidation reactions are essentially complete at room

temperature within 30 min. Flavonoid glycosides require acid

hydrolysis to their corresponding aglycons to fully exhibit their

antioxidant potency. Slow reacting antioxidants may need

slightly elevated temperature incubation so as to complete their

oxidation with the CUPRAC reagent.6,9 The CUPRAC antiox-

idant capacities of a wide range of polyphenolics and flavonoids

were experimentally reported as trolox equivalent antioxidant

capacities (TEAC), defined as the reducing potency—in

Trolox� mM equivalents—of 1 mM antioxidant solution under

investigation. Since the TEAC value is expressed relative to

a reference compound trolox (TR), it is unitless. Experimentally,

the TEAC values were found as the ratio of the molar absorp-

tivity of each compound to that of TR obtained under identical

conditions in the CUPRAC assay.

1.1. Procedures for original CUPRAC assay

1.1.1. Preparation of CUPRAC assay solutions. CuCl2 solu-

tion, 1.0 � 10�2 M Cu(II), is prepared by dissolving 0.4262 g

CuCl2$2H2O in water, and diluting to 250 mL. Ammonium

acetate (NH4Ac) buffer at pH ¼ 7.0, 1.0 M, is prepared by dis-

solving 19.27 g NH4Ac in water and diluting to 250 mL. Neo-

cuproine (Nc) solution, 7.5 � 10�3 M, is prepared daily by

dissolving 0.039 g Nc in 96% ethanol, and diluting to 25 mL with

ethanol. Trolox, 1.0 � 10�3 M, is prepared in 96% ethanol.

1.1.2. Normal (N) sample measurement. To a test tube were

added 1 mL each of Cu(II), Nc, and NH4Ac buffer solutions.

Antioxidant sample (or standard) solution (x mL) and H2O

(1.1 � x) mL were added to the initial mixture so as to make the

final volume 4.1 mL. The tubes were stoppered, and after 0.5 h,

the absorbance at 450 nm (A450) was recorded against a reagent

blank. The UV-Vis spectrophotometer used was Varian CARY

1E, equipped with matched quartz cuvettes. The standard cali-

bration curves of each antioxidant compound were constructed

Anal. Methods

by plotting absorbance versus molar concentration, and the

molar absorptivity of the CUPRACmethod for each antioxidant

was found from the slope of the calibration line concerned.6 The

scheme for normal measurement of hydrophilic antioxidants is

summarized as:

1 mL Cu(II) + 1 mLNc + 1 mL buffer + xmL antioxidant soln. +

(1.1 � x) mL H2O; total vol.¼ 4.1 mL, measure A450 against

a reagent blank after 30 min of reagent addition.

The scheme for normal measurement of lipophilic antioxidants

1 mLCu(II) + 1 mLNc + 1mL buffer + xmL antioxidant soln. in

DCM + (1.1 � x) mL DCM; measure A450 against a reagent

blank after 30 min of reagent addition.

1.1.3. Incubated (I) sample measurement. This measurement

mode is useful for slow reacting antioxidants like naringenin. The

mixture solutions containing sample and reagents were prepared

as described in ‘normal measurement’; the tubes were stoppered

and incubated for 20 min in a water bath at a temperature of

50 �C.6 The tubes were cooled to room temperature under

running water, and their A450 values were measured.

1.1.4. Hydrolyzed (H) sample measurement. This measure-

ment mode is useful for flavonoid glycosides exhibiting enhanced

TAC when hydrolyzed. A suitable mass of the polyphenol

standard was weighed such that the final antioxidant concen-

tration of the methanolic solution would be 1 mM. Each stan-

dard was dissolved in a suitable volume of 50% MeOH. In

a 100 mL flask, sufficient hydrochloric acid was added to each

solution until the final HCl molarity was 1.2 M, and diluted to

the mark with 50% MeOH. This solution was decanted to

a distillation flask into which a few pieces of boiling stone were

added, and refluxed at 80 �C for 2 h. The flask was cooled to

room temperature under running tap water. The hydrolyzate was

neutralized with 1 M NaOH. The neutralized solution was then

subjected to ‘normal (N) sample measurement’.6

1.1.5. Hydrolyzed and incubated (H&I) sample measurement.

The neutralized hydrolyzate was subjected to incubation at 50 �Cin a water bath for 20 min. The A450 of running water-cooled

samples were ‘normally measured’.6

1.1.6. Application of the CUPRAC method to herbal plant

extracts. One tea bag of the commercial herbal teas was dipped

separately into 250 mL of freshly boiled water in a beaker,

occasionally shaken for 2 min, and let to stand in the same

solution for 3 more min, enabling a total stewing time of 5 min.

The herbal tea solution was allowed to cool to room tempera-

ture, and filtration was applied to the sample using a Whatman

black-band filter paper for removing particulates. Steeping was

applied only to herbal tea samples of which the infusions were

measured for antioxidant capacity.10 Other plant extracts and

fruit juices were directly measured after filtration and dilution.

Table 2 Antioxidant capacities of various polyphenolic compounds (inthe units of TEAC: trolox equivalent antioxidant capacity) as measuredby the CUPRAC assay.6,12,13a

Antioxidant

TEACCUPRAC

TEACN TEACI TEACH TEACH&I

FlavonoidsEpicatechin gallate (ECG) 5.32 5.65Epigallocatechingallate (EGCG)

4.89 5.49

Quercetin (QR) 4.38Fisetin (FS) 3.90 4.18Epigallocatechin (EGC) 3.35 3.60Catechin (CT) 3.09 3.56 3.08 3.49Epicatechin (EC) 2.77 2.89Rutin (RT) 2.56 3.80Morin (MR) 1.88 3.32Kaempferol 1.58 1.87Hesperetin (HT) 0.99 1.05 0.85 0.98Hesperidin (HD) 0.97 1.11 0.79 0.95Naringenin (NG) 0.05 2.28 3.03Naringin (N) 0.02 0.13Hydroxycinnamic AcidsRosmarinic acid (RA) 5.65 6.02Caffeic acid (CFA) 2.89 2.96 2.87 3.22Chlorogenic acid (CGA) 2.47 2.72 1.20 1.42Ferulic acid (FRA) 1.20 1.23 1.18 1.34p-Coumaric acid (CMA) 0.55 1.00 0.53 1.15Vitaminsa-tocopherol (TP) 1.10 1.02 0.99 0.87Ascorbic acid (AA) 0.96Benzoic AcidsGallic acid(GA) 2.62Sinapic acid (SNA) 1.24 2.17Vanillic acid (VA) 1.24 1.52 1.32 1.57Syringic Acid (SA) 1.12 1.64 1.13 1.67

a N: Normal measurement; I: Incubated measurement. H: Hydrolyzedmeasurement; H&I: Hydrolyzed and Incubated measurement.

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1.1.7. Calculation of TEAC coefficients. The molar absorp-

tivity of trolox in the CUPRAC method was: 3trolox ¼ 1.67 � 104

L mol�1 cm�1.

The TEACCUPRAC coefficients of phenolic compounds having

linear calibration curves passing through the origin were simply

calculated by dividing the molar absorptivity (3) of the species

under investigation by that of trolox under corresponding

conditions (e.g., the 3 values of normal and incubated solutions

of trolox were 1.67 � 104 and 1.86 � 104 L mol�1 cm�1, respec-

tively (Table 1)).11 For example, the molar absorptivity of cate-

chin was 3 ¼ 5.16 � 104 in the normal CUPRAC method; the

TEAC coefficient of catechin was calculated as 3catechin/3trolox ¼5.16 � 104/1.67 � 104 ¼ 3.09.

The TEAC coefficients of various hydrophilic antioxidant

compounds found with the CUPRAC method are tabulated in

Table 2. The linear calibration curves of the tested antioxidants

as CUPRAC absorbance versus concentration (figures not

shown) generally gave correlation coefficients close to unity

(r $ 0.999) within the useful absorbance range of 0.1–1.1. The

highest antioxidant capacities in the CUPRAC method were

observed for rosmarinic acid, epicatechin gallate, epi-

gallocatechin gallate, quercetin, fisetin, epigallocatechin, cate-

chin, caffeic acid, epicatechin, gallic acid, rutin, and chlorogenic

acid in this order, in accordance with theoretical expectations of

structure–activity relationships, because the number and posi-

tion of the hydroxyl groups as well as the degree of conjugation

of the whole molecule are important for easy electron transfer.

1.1.8. TAC of herbal and food plant extracts. The CUPRAC

method has been utilized in the antioxidant assay of herbal

infusions (of mostly endemic herbs characteristic to Turkey and

the nearby region),10 and the results were compared with the

findings of ABTS/TEAC1 and Folin5 spectrophotometric

methods. The highest TACs were observed for scarlet pimpernel

(Anagallis arvensis), sweet basil (Ocimum basilicum), green tea

(Camellia sinensis), and lemon balm (Melissa officinalis) in this

order (1.63, 1.18, 1.07, and 0.99 mmol trolox equivalent (TE)/g,

respectively). For infusions prepared from ready-to-use tea bags,

the CUPRAC values were highest for Ceylon blended ordinary

tea (4.41), green tea with lemon (1.61), English breakfast ordi-

nary tea (1.26), and green tea (0.94), all of which were manu-

factured types of Camellia sinensis. Standard antioxidant

compounds added in increasing concentrations to the herbal tea

infusions produced linear curves, excluding the possibility of

spectral interference. The CUPRAC capacities of herbal teas

correlated strongly with their Folin phenolics content.10 Further

Table 1 Linear calibration equations of trolox in different solvent mediacalculated with respect to the CUPRAC method

Solvent Linear calibration equation r

100% EtOH y ¼ 1.67 � 104c � 0.033 (N) 0.9999y ¼ 1.86 � 104c + 0.002 (I) 0.9996

100% MeOH y ¼ 1.58 � 104c � 0.010 (N) 0.9995y ¼ 1.60 � 104c � 0.008 (I) 0.9993

MeOH/H2O (4 : 1, v/v) y ¼ 1.50 � 104c � 0.002 (N) 0.9999y ¼ 1.52 � 104c � 0.013 (I) 0.9987

MeOH/H2O (1 : 1, v/v) y ¼ 1.57 � 104c � 0.025 (N) 0.9991y ¼ 1.62 � 104c � 0.030 (I) 0.9993

DCM/EtOH (9 : 1, v/v) y ¼ 1.68 � 104c � 0.020 0.9995

use of the CUPRAC reagent by other researchers for the anti-

oxidant assay of other healing herbs will strengthen efforts for

the classification of herbs with respect to their antioxidant

properties. TACs of nineteen edible wild plants grown in Ayvalik

(Turkey) were assayed by CUPRAC, ABTS, FRAP and Folin

methods. There were good linear correlations among results

obtained with different assays (Fig. 3 and 4).14

1.1.8.1. The technique of standard additions applied to food

plants. In cases where the technique of standard additions was

employed (i.e. increasing amounts of quercetin or other poly-

phenolic standard added to a plant extract or beverage), the real

sample solution was appropriately diluted with water such that

its original CUPRAC absorbance at 450 nm would lie between

0.2–0.4 absorbance units. The standard calibration curves of the

selected polyphenolic standard were redrawn in these real solu-

tions so as to observe the parallelism between the calibration

lines (e.g., of quercetin) individually in water and in real solution.

Such a parallelism indicates the absence of chemical deviations

from Beer’s law that may arise from the interactions between

added antioxidants and plant extract constituents.

1.1.8.2. Calculation of TAC for food plants. If a herbal

infusion (initial volume ¼ Vcup) prepared from (m) grams of dry

Anal. Methods

Fig. 3 The correlation of CUPRAC assay results with those of the

ABTS assay (r ¼ 0.857).

Fig. 4 The correlation of CUPRAC assay results with those of the Folin

assay (r ¼ 0.945).

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matter was diluted (r) times prior to analysis, and a sample

volume of (Vs) was taken for analysis from the diluted extract,

and colour development (after 30 min of reagent addition) took

place in a final volume of (Vf) to yield an absorbance of (Af), then

the mmol ‘‘trolox equivalent’’ (TE) antioxidant capacity per

gram of plant material (as mmol TE g�1) was found using the

equation:

TAC (in mmol TE g�1) ¼ (Af/3TR) (Vf/Vs) r (Vcup/m)

Example calculation:10 1.5465 g of lemon balm (dry herbal tea

material) was weighed, and prepared in a 250 mL infusion; 8 mL

Anal. Methods

of this infusion was diluted to 100 mL prior to analysis (dilution

ratio ¼ r ¼ 12.5). The volume of sample solution taken for

analysis was Vs ¼ 0.2 mL, and the total volume of final solution

(in which colour development took place) in the CUPRAC

method was Vf ¼ 4.1 mL. The final absorbance at 450 nm was

measured as Af ¼ 0.401 in a 1 cm cell. The TAC of lemon balm

using the above equation was (0.401/1.67 � 104) (4.1/0.2) (12.5)

(250/1.5465) ¼ 0.99 mmol TE g�1.

2. Some modifications of the CUPRAC method

2.1. CUPRAC-TAC assay of human serum (by differentiating

lipophilic from hydrophilic antioxidants)

Several methods have been developed to measure the total

antioxidant capacity of biological fluids, such as human serum or

plasma, in view of the difficulties encountered in measuring each

antioxidant component separately, added to the problems caused

by possible interactions between individual antioxidants.9,12,15

Apak et al.9 were able to apply the CUPRAC method to

a complete series of plasma antioxidants for the assay of total

antioxidant capacity of serum, and the resulting absorbance at

450 nm was recorded either directly (e.g., for ascorbic acid,

a-tocopherol, and glutathione) or after incubation at 50 �C for

20 min (e.g., for uric acid, bilirubin and albumin), quantitation

being made by means of a calibration curve. Lipophilic antiox-

idants of serum, i.e. a-tocopherol and b-carotene, were extracted

with n-hexane from an ethanolic solution of serum subjected to

centrifugation, followed by evaporating the hexane phase and

taking up the residue in dichloromethane (DCM) for the final

CUPRAC assay. Hydrophilic antioxidants of serum were

assayed after perchloric acid precipitation of proteins in the

centrifugate. The findings of the CUPRAC method completely

agreed with those of ABTS-persulfate for lipophilic antioxidants

(first extracted with hexane, and subsequent colour development

performed in dichloromethane). As for hydrophilic antioxidants,

a linear correlation existed between the CUPRAC and ABTS

findings for measurements carried out both at room temperature

(r ¼ 0.58) and in 50 �C-incubated solution (r ¼ 0.53). This is also

an advantage of the developed method, as relevant literature

reports that either serum ORAC or serum FRAP does not

correlate at all with serum TEAC. The CUPRAC assay may be

successfully applied to individual antioxidants as well as to their

mixtures and human serum.

The TAC determination of human serum constitutes another

example of the simultaneous assay of lipophilic and hydrophilic

antioxidants by the CUPRAC method. It was a distinct advan-

tage of the developed method that the CUPRAC assay proved to

be efficient for glutathione and thiol-type antioxidants, for which

the FRAP (ferric reducing antioxidant potency) test was basi-

cally nonresponsive. As a distinct advantage over other ET-

based assays (e.g., Folin, FRAP, ABTS, DPPH), CUPRAC is

superior in regard to its realistic pH (close to that of physiolog-

ical pH), favourable redox potential, accessibility and stability of

reagents, and applicability to lipophilic antioxidants as well as

hydrophilic ones. The selected chromogenic redox reagent

(Cu(II)-Nc) for the assay of human serum is easily accessible,

stable, selective, and responsive to all types of biologically

important antioxidants such as ascorbic acid, a-tocopherol,

Fig. 5 Host–guest interaction between antioxidant compounds and M-

b-CD, followed by CUPRAC measurement of the TAC of inclusion

complexes.

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b-carotene, reduced glutathione (GSH), uric acid, and bilirubin,

regardless of chemical type or hydrophilicity.

2.1.1. Procedures for TAC assay of human serum (by sepa-

rately treating lipophilic and hydrophilic antioxidant fractions)

2.1.1.1. CUPRAC assay of lipophilic antioxidants of serum in

DCM solvent. To a test tube were added 1 mL of copper(II)

chloride solution, 1 mL of neocuproine solution, and 1 mL of

NH4Ac buffer solution in this order. A suitable aliquot of the

organic extract (of serum) was added to this tube. To this

mixture, (4� x) mL of DCMwas added, shaken, and the organic

phase was separated from the aqueous phase. An absorbance

reading was taken against a reagent blank at 450 nm. Since the

boiling temperature of DCM is low, the DCM used in the

procedure was cooled to an initial temperature of +4 �C to

prevent evaporation losses. Elevated temperature incubation

tests (as applied to hydrophilic antioxidants in the aqueous

phase) were not carried out with the organic extract.9

Calculation of CUPRAC-TAC values of diluted serum

samples was performed using the equation:

TAC, mM TE ¼ (Af/3TR) � (Vf/Vs) � 103

where Vs is the sample volume taken for analysis from the

organic phase of the serum extract, Af is the A450nm measured

after 30 min of CUPRAC reaction, and Vf is the final volume of

the organic phase (DCM) (4.5 mL), 3TR ¼ 1.67 � 104 L mol�1

cm�1.

2.1.1.2. CUPRAC assay of hydrophilic antioxidants of serum.

To a test tube were added 1 mL of copper(II) chloride solution,

1 mL of neocuproine solution, and 1 mL of NH4Ac buffer

solution in this order. A suitable aliquot of the aqueous extract

(of serum) was added to this tube. If (x) mL of the standard

antioxidant solution was taken, then (0.25 � x) mL H2O was

added to make the final volume 4.75 mL. An absorbance reading

was taken against a reagent blank at 450 nm.9

Calculation of CUPRAC-TAC values of diluted serum

samples was performed using the equation:

TAC, mM TE ¼ (Af/3TR) � (Vf/Vs) � dilution factor � 103

where Vs is the sample volume taken for analysis from the

aqueous phase of the serum extract, Af is the A450nm measured

after 30 min of CUPRAC reaction, Vf is the final CUPRAC

reaction volume (4.75 mL), and dilution factor ¼ (mL of

neutralized aqueous phase of serum extract/mL of serum

sample).

2.2. CUPRAC-TAC assay for simultaneous measurement of

lipophilic and hydrophilic antioxidants

The CUPRAC procedure was applied to both lipophilic and

hydrophilic antioxidants simultaneously, by making use of their

‘host–guest’ complexes with methyl-b-cyclodextrin (M-b-CD),

a cyclic oligosaccharide, in acetonated aqueous medium (see

Fig. 5 for the nature of host–guest interaction between antioxi-

dants and M-b-CD).16 M-b-CD was introduced as the water

solubility enhancer for lipophilic antioxidants. Two percent

M-b-CD (w/v) in a 90% acetone–10% H2O mixture was found to

sufficiently solubilize b-carotene, vitamin E, vitamin C, oil-

soluble synthetic antioxidants, and other phenolic antioxidants.

This method compensates for the wide variability in antioxidant

capabilities of oil- and water-soluble antioxidants showing

different levels of accumulation at the interfaces of oil-in-water

and water-in-oil emulsions, and assigns an objective TEAC

(trolox equivalent antioxidant capacity) value to each antioxi-

dant simply depending on its chemical character (i.e. electron or

H-atom donating ability).

2.2.1. Procedure for simultaneous measurement of lipophilic

and hydrophilic antioxidants. This method enables the simulta-

neous TAC measurement of both lipophilic and hydrophilic

antioxidant fractions of serum in a single water–acetone solution

containing a cyclodextrin-type oligosaccharide capable of

inclusion complex formation with a wide variety of antioxidants.

1 mL CuCl2, 1 mL Nc solution, and 1 mL NH4Ac solution were

added to (x) mL of the M-b-CD-containing final analyte

mixture, followed by (1.1 � x) mL of 2% M-b-CD solution in

1 : 9 (v/v) water–acetone mixture. The absorbance of the final

solution (of 4.1 mL total volume) at 450 nm was read against

a reagent blank after 30 min standing at room temperature.

2.3. Measurement of hydroxyl radical scavenging (HRS)

activity of polyphenolics

The CUPRAC procedure can also detect hydroxyl radicals (_OH),

and measure the activity of _OH scavengers. The hydroxyl radical

is the most reactive of the reactive oxygen species (ROS), but can

be spectrophotometrically detected through its reaction prod-

ucts. A salicylate probe was used to indirectly detect _OHwith the

aid of the hydroxylation products formed, i.e. dihydroxy-

benzoates, which respond positively to the CUPRAC assay.17

This reaction can be used to measure the _OH scavenging activity

of polyphenolics, flavonoids, and other scavenger compounds

(e.g., ascorbic acid, mannitol, glucose). The modified CUPRAC

assay makes use of competition kinetics to simultaneously

incubate the probe with the scavenger under the attack of _OH

generated in a Fenton reaction (comprised of Fe(II)-EDTA

complex +H2O2 as reactants) that is stopped at the end of 10 min

by adding catalase enzyme solution to degrade the remaining

H2O2. Finally, the difference in CUPRAC absorbance of the

probe in the absence and presence of the scavenger is measured,

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because the hydroxylation products of the salicylate probe would

show a higher CUPRAC absorbance alone (i.e. without

scavenger).

2.3.1. Procedures for HRS-CUPRAC assay

2.3.1.1. Preparation of solutions. The salicylate buffer (at

10 mM concentration) was prepared by dissolving 0.160 g of

sodium salicylate in distilled water. Fe(II) at 20 mM concentra-

tion was prepared by dissolving 0.1988 g FeCl2$4H2O in 2 mL of

1 M HCl, and diluting to 50 mL with distilled water. Na2-EDTA

at 20 mM concentration was prepared by dissolving 0.372 g of

the ethylenediaminetetraacetate disodium salt in distilled water

and diluting to 50 mL. Hydrogen peroxide at 10 mM concen-

tration was prepared from a 0.5 M intermediary stock solution,

the latter being prepared from 30% H2O2 and standardized with

permanganate titration. The NaH2PO4–Na2HPO4 buffer solu-

tion (pH ¼ 7.4) at 200 mM was prepared in distilled water.

2.3.1.2. HRS-CUPRAC measurement. To a test tube were

added 1.5 mL of phosphate buffer (pH 7.0), 0.5 mL of 10 mM

sodium salicylate (probe material), 0.25 mL of 20 mM

Na2-EDTA, 0.25 mL of 20 mM FeCl2 solution, (2.0 � x) mL

H2O, (x) mL scavenger sample solution (x varying between 0.1

and 2.0 mL) at a concentration of 1.0 � 10�2 M (glucose and

mannitol) or 2 � 10�5 M (all polyphenolic compounds and

ascorbic acid), and 0.5 mL of 10 mM H2O2 rapidly in this order.

The mixture, with a total volume of 5.0 mL, was incubated for

10 min in a water bath kept at 37 �C. At the end of this period, the

reaction was stopped by adding 0.5 mL of 268 U mL�1 catalase

solution, and vortexed for 30 s. To 0.5 mL of the incubation

solution, the modified CUPRAC method17 was applied in the

following manner:

1 mL Cu(II) + 1 mL Nc + 2 mL NH4Ac buffer + 0.5 mL incu-

bation solution

The absorbance at 450 nm of the final solution at 4.5 mL total

volume was recorded 5 min later against a reagent blank.

The _OH inhibition ratio of herbal extract (%) was calculated

using the following formula:

Inhibition ratio (%) ¼ 100 [(Ao � A)/Ao]

where Ao and A are the CUPRAC absorbances of the system in

the absence and presence of scavenger.

2.4. Measurement of xanthine oxidase inhibition activity of

polyphenolics

This modified CUPRAC method uses xanthine–xanthine oxidase

(X–XO) system for XO inhibitory activity assay of polyphenolics

and ascorbic acid.18 As a part of antioxidant activity assays, XO

activity has usually been determined by following the rate of uric

acid formation from the X–XO system by making use of the

UV-absorbanceat295nmofuric acid formedasa reactionproduct.

Since polyphenolics have strong UV absorption, XO inhibitory

activity of polyphenolics was alternatively determined without

interference by directly measuring the formation of uric acid and

Anal. Methods

hydrogen peroxide using the modified CUPRAC spectrophoto-

metric method at 450 nm. The CUPRAC absorbance of the incu-

bation solution due to the reduction of the Cu(II)-Nc reagent by the

products of the X–XO system decreased in the presence of poly-

phenolics, the difference being proportional to the XO inhibition

ability of the tested compound. The proposed spectrophotometric

method was practical, low-cost, rapid, less open to interferences by

UV-absorbing substances, and could reliably assay uric acid and

hydrogen peroxide in the presence of polyphenols (flavonoids,

simple phenolic acids and hydroxycinnamic acids).

2.4.1. Procedures for xanthine oxidase inhibition activity

2.4.1.1. Preparation of solutions. The xanthine stock solution

was prepared by dissolving 0.0152 g xanthine in 3 mL 1.0 M

NaOH and diluting to 100 mL with distilled water. A working

solution of xanthine was prepared at 5.0 � 10�4 M by taking

25 mL of stock solution, adjusting the pH to pH ¼ 7.8 with the

addition of 0.1 M HCl, and diluting to 50 mL with 0.1 M

phosphate buffer. The original XO solution of initial activity

0.056 U mg�1 solid was diluted with 0.1 M phosphate buffer

(pH ¼ 7.8) to a concentration of 0.04 U mL�1. The perchloric

acid solution at 3.2% concentration (w/v) was prepared in

distilled water. The NaH2PO4–Na2HPO4 buffer solution (pH ¼7.8) at 100 mM was prepared in distilled water. The concentra-

tion of antioxidant solution was set at a low level so as not to give

an initial CUPRAC absorbance but yet to exhibit a measurable

XO inhibitory activity.

2.4.1.2. CUPRAC-XO activity assay. To a test tube were

added 0.5 mL of 0.5 mM xanthine (probe material), (x) mL

antioxidant sample solution (x varying between 0.1 and 0.9 mL)

at a concentration of 1 � 10�5 M (all polyphenolic compounds

and ascorbic acid) or 1.0 � 10�4 M (naringin), (2.0 � x) mL of

1 : 9 EtOH–H2O mixture (v/v), and 0.2 mL of 0.04 U mg�1 XO

rapidly in this order. The mixture in a total volume of 2.8 mL was

incubated for 30 min in a water bath kept at 37 �C. At the end of

this period, the reaction was stopped by adding 0.1 mL of 3.2%

perchloric acid solution, and vortexed for 30 s. To 0.2 mL of the

incubation solution, the modified CUPRAC method (miniatur-

ized method)18 was applied in the following manner:

0.2 mL Cu(II) + 0.2 mL Nc + 0.4 mL NH4Ac buffer + 0.2 mL

incubation solution (Vtotal ¼ 1.0 mL)

The inhibition ratio of food extract (%) was calculated using

the following formula:

Inhibition ratio (%) ¼ 100 [(Ao � A)/Ao]

where Ao and A are the CUPRAC absorbances of the system in

the absence and presence of scavenger, respectively.

2.5. Hydrogen peroxide scavenging (HPS) activity of

polyphenolics

A modified CUPRAC method has been developed to measure

the HPS activity of polyphenolics and ascorbic acid with

Fig. 6 Schematic presentation of the mechanism of CUPRAC antioxi-

dant sensor.

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a simple, low-cost and versatile colorimetric procedure.19 In the

most common UV method used for determination of HPS

activity, scavenging ability is measured depending on the change

of absorbance at 230 nm when H2O2 is consumed by scaven-

gers. The UV method suffers from both the interference of some

phenolics in real samples having strong absorption in the UV-

region and from inefficient degradation of H2O2 with poly-

phenols in the absence of copper or iron salts (i.e. H2O2 is

relatively stable, and not scavenged unless transition metal

compounds are present as catalysts). Thus, HPS activity of

polyphenols was alternatively determined without interference

by incubating the scavenger sample with hydrogen peroxide in

the presence of a Cu(II) catalyst, followed by directly measuring

the concentration of undegraded H2O2 with CUPRAC spec-

trophotometry. The proposed methodology is also superior to

the rather non-specific horseradish peroxidase (HRP)-based

assays, since it is known that some H2O2 scavengers also

interact with HRP, an enzyme which is expensive and unstable

in solution.

2.5.1. Procedures for HPS-CUPRAC assay

2.5.1.1. Preparation of solutions. Hydrogen peroxide at

1.0 mM concentration was prepared from a 0.5 M intermediary

stock solution, the latter being prepared from 30% H2O2 and

standardized with permanganate titration. The NaH2PO4–

Na2HPO4 buffer solution (pH ¼ 7.4) at 0.2 M total phosphate

concentration was prepared in water. The original catalase

solution of initial activity 1340 U mg�1 was diluted with 0.2 M

phosphate buffer (pH ¼ 7.4) to a concentration of 268 U mL�1.

2.5.1.2. HPS-CUPRAC measurement. To a test tube were

added 0.7 mL of phosphate buffer (pH 7.4), 0.4 mL of 1 mM

H2O2, 0.4 mL of 0.1 mM CuCl2$2H2O in this order (hydrogen

peroxide incubation solution, used as reference). To another two

test tubes were added 0.5 mL of phosphate buffer (pH 7.4),

0.4 mL of 1.0 mM H2O2, 0.2 mL scavenger sample solution, and

0.4 mL of 0.1 mM CuCl2$2H2O solution rapidly in this order

(scavenger solutions-I and II). The mixtures in a total volume of

1.5 mL were incubated for 30 min in a water bath kept at 37 �C.At the end of this period, to reference and scavenger solution-I

were added 0.4 mL H2O and to scavenger solution-II was added

0.4 mL of 268 U mL�1 catalase solution, and vortexed for 30 s.

To 1.0 mL of the final incubation solutions, the HPS-CUPRAC

method19 was applied in the following manner:

1 mL Cu(II) + 1 mL Nc + 2 mL NH4Ac buffer + 1.0 mL final

incubation solution

The absorbance at 450 nm of the final solution at 5.0 mL total

volume was recorded 30 min later against a reagent blank.

The HPS activity (%) of polyphenols and real samples were

calculated using the following formula:

HPS ð%Þ ¼ 100��A0 � ðA1 � A2Þ

where A0 is the CUPRAC absorbance of reference hydrogen

peroxide incubation solution, A1 and A2 the CUPRAC absor-

bances of scavenger solutions-I and -II, respectively.

2.6. CUPRAC antioxidant sensor

A low-cost optical sensor was developed using a membrane-

immobilised CUPRAC reagent, Cu(II)-Nc complex, for the

assessment of antioxidant capacity of non-enzymatic antioxi-

dants, their synthetic mixtures, and real samples.20 The Cu(II)-

Nc reagent was immobilized onto a cation-exchange polymer

(Nafion�, a sulfonated tetrafluoroethylene based copolymer)

membrane matrix, and the absorbance changes associated with

the formation of the highly-coloured Cu(I)-Nc chelate as

a result of reaction with antioxidants was measured at 450 nm

(Fig. 6). The TEAC coefficients measured for various antioxi-

dant compounds suggest that the reactivity of the Nafion-

immobilized reagent is comparable to that of the standard

solution-based CUPRAC assay. Testing of synthetic ternary

mixtures of antioxidants yielded the theoretically expected

CUPRAC antioxidant capacities, in accordance with the prin-

ciple of additivity of absorbances of mixture constituents

obeying Beer’s law. This assay was validated through linearity,

additivity, precision and recovery, demonstrating that the

optical sensor is reliable and robust. The sensor was used to

screen TAC of some commercial fruit juices such as orange,

cherry, peach, and apricot juices, and proved to be an effective

tool in measuring the TAC values of food and plant samples

without pretreatment. The optical sensor–based CUPRAC

assay has been shown not to be adversely affected by common

food ingredients such as citrate, oxalate, fruit acids and

reducing sugars, and offers good prospects of providing

a versatile antioxidant sensor in food industries. With new

experimental design for application to human fluids, the sensor

is expected to be useful to biochemical and medicinal chemical

research on oxidative stress.

2.6.1. Procedure for optical sensor-based CUPRAC assay.

The commercial Nafion membrane was sliced into 4.5 � 0.5 cm

pieces, and immersed into a tube containing 2 mL of 2.0 � 10�2

M Cu(II) + 2 mL of 1.5 � 10�2 M Nc + 2 mL of 1 M NH4Ac +

2.2 mL of H2O, and agitated for 30 min in a rotator. The reagent-

impregnated membrane was taken out, and immersed into a tube

containing 8.2 mL of standard antioxidant or real solutions. The

tube was placed in a rotator and agitated for 30 min so as to

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enable colour development. The coloured membrane was taken

out, placed in a 1-mm optical cuvette containing H2O (to prevent

sticking of slices to the walls of the cuvette), and its absorbance at

450 nm was read against a blank membrane prepared under

identical conditions excluding analyte.

2.7. On-line HPLC-CUPRAC method

Efforts directed to individual identification and quantification of

antioxidant compounds in plant matrices may give rise to

problems, because the activities of antioxidant compounds may

decrease during their isolation and purification due to decom-

position. Thus procedures for the separation and quantification

of antioxidants should be performed simultaneously. Recently,

certain assays have been modified for on-line high performance

liquid chromatographic (HPLC) determinations with post-

column detection.21–25 The most widely used assays in post-

column applications are free radical decolorization methods,

based on the scavenging of chromogenic free radicals DPPH24 or

ABTS.25 It is difficult to precisely quantify antioxidant activity

because of the short lifetimes of these radicals. Moreover, reac-

tion kinetics may vary with these radicalic reagents as a function

of phenolic steric effects, solvent composition, and pH. A

method combining separation of components in the complex

matrix and evaluation of antioxidant capacity can provide

significant advantages for such investigations.24

The developed on-line HPLC-CUPRAC method26 combines

chromatographic separation, constituent analysis, and post-

column identification of antioxidants in plant extracts. The

instrumental set-up of the on-line system is given in Fig. 7. In this

system, the separation of polyphenols was performed on a C18

column using gradient elution with two different mobile phase

solutions, i.e. MeOH and 0.2% o-phosphoric acid. The HPLC-

separated antioxidant compounds—first yielding a ‘positive

trace’ chromatogram (Fig. 8)—eventually react with the

Cu(II)-Nc reagent (prepared freshly from the corresponding

solutions of Cu(II), Nc, and NH4Ac) along a reaction coil, and

the reagent is reduced by antioxidants to the yellow-coloured Cu

(I)-Nc complex with an absorption maximum at 450 nm, finally

yielding a ‘negative-trace’ chromatogram (Fig. 8). It was

Fig. 7 Instrumental setup for on-line H

Anal. Methods

observed that the antioxidant capacity of each substance is

reflected by an increase in the area of negative peaks as a function

of increased concentration. The detection limits of polyphenols

at 450 nm (in the range of 0.17–3.46 mM) after post-column

derivatization were comparable to those at 280 nm UV-detection

without derivatization. The developed method was successfully

applied to the identification of antioxidant compounds in crude

extracts of Camellia sinensis, Origanum marjorana, Mentha

(mint) (see Fig. 8 for the on-line chromatogram of mint extract).

The method is rapid, inexpensive, versatile, nonlaborious, uses

stable reagents, and enables the on-line qualitative and quanti-

tative estimation of antioxidant constituents of complex plant

samples. The significant advantages of on-line methods are that

the antioxidant activity of a single compound can be measured

and its contribution to the overall activity of a complex mixture

be calculated, and also the activity of a single compound can be

compared to those of other constituents in the matrix. Moreover,

the simultaneous determination of a substance without antioxi-

dant behaviour can be realized through the absence of a negative

peak at 450 nm as opposed to the presence of a positive peak

detected at 280 nm. For example, caffeine, being a major

constituent of both green and black tea extracts, only appears in

the positive trace chromatogram whereas it is non-existent in the

negative trace of post-column detection, because it lacks the

phenolic hydroxyl groups of reducing character, and conse-

quently does not possess any antioxidant behaviour.

2.7.1. Procedures for on-line CUPRAC assay

2.7.1.1. Chromatographic separation—conventional HPLC

assay (prior to post-column analysis). The analyses were carried

out using a reverse-phase ACE C18 column (4.6 mm � 250 mm,

5 mm particle size) (Milford, MA, USA). Three different HPLC

elution programs were used for tea antioxidants, other poly-

phenolic compounds, and trolox. The mobile phase consisted of

two solvents, i.e. methanol (A) and 0.2% of o-H3PO4 in bidis-

tilled water (B). The following parameters and gradient were

used for the analysis of tea antioxidants:27 (Vsample¼ 20 mL; Flow

rate ¼ 0.8 mL min�1; l ¼ 280 nm): 1 min 0% A � 100% B (slope

1.0); 20 min 70% A � 30% B (slope 1.0); 25 min 0% A � 100% B

(slope 1.0).

PLC-CUPRAC detection system.

Fig. 8 The chromatogram of mint extract showing HPLC (280 nm, positive trace) and on-line HPLC-CUPRAC (450 nm, negative trace) assays.

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The other polyphenolic antioxidants were analyzed using these

parameters and gradient program:28 (Vsample ¼ 20 mL; Flow

1.0); 13 min 30% A � 70% B (slope 1.0); 45 min 66% A � 34% B

(slope 1.0); 50 min 7% A � 93% B (slope 1.0).

Trolox was analyzed using these parameters and gradient

program: (Vsample ¼ 20 mL; Flow rate ¼ 1.0 mL min�1; l ¼280 nm): 5 min 50% A� 50% B (slope 1.0); 13 min 80% A� 20%

B (slope 1.0); 15 min 50% A � 50% B (slope 1.0).

Using the above working modes, the calibration curves were

constructed and linear equations of peak area versus concentra-

tion found for the antioxidants of interest.

2.7.1.2. On-line HPLC-CUPRAC assay with post-column

detection. The on-line HPLC-CUPRAC method exploits the

advantage of rapid detection and capacity determination of

antioxidant compounds in addition to conventional HPLC

separation. In this method, Cu(II)-Nc complex in pH 7 ammo-

nium acetate medium was used as the chromogenic reagent. The

HPLC-separated compounds reacted in a post-column reaction

coil with the CUPRAC reagent, where the detector was set at

a wavelength of 450 nm. CUPRAC reagent was freshly prepared

from the corresponding solutions of Cu(II) : Nc : NH4Ac at

a ratio of 1 : 1 : 1 (v/v/v) prior to analysis, and protected from

daylight. The flow rate of the CUPRAC reagent for post-column

reaction was 0.5 mL min�1.26

2.7.1.3. TAC measurements of synthetic mixture solutions.

Four mixtures of antioxidants were prepared (Cfinal: 0.1 mM),

and the solutions were analyzed for TAC as mM trolox (TR)

equivalents using (i) conventional CUPRAC spectrophotometry,

(ii) conventional HPLC with CUPRAC calculation, and (iii) on-

line HPLC-CUPRAC assay with post-column detection.

Calculations of TAC values according to the three assays are

explained below:

(i) The experimentally found TACCUPRAC (in the units of

mmol TE L�1) of the synthetic mixtures or samples were calcu-

lated by dividing the observed absorbance (A450) by the molar

absorptivity of trolox (A450 ¼ 3TRCTR � 0.01, where 3TR ¼ 1.58

� 104 L mol�1 cm�1 and r ¼ 0.9995) according to the spectro-

photometric CUPRAC assay.

TACCUPRAC ¼ Absorbance ðtotalÞ3TR

� 103 (3)

(ii) TACHPLC values of the synthetic mixtures or samples were

calculated by multiplying the concentration with the TEAC

value of each HPLC-identified antioxidant, and summing the

products.

TACHPLC ¼Xn

ci ðTEACÞi (4)

where TACHPLC is the total antioxidant capacity in mM trolox

(TR)-equivalents, ci is the final concentration of antioxidant

component: i and (TEAC)i is its TEAC coefficient of the

CUPRAC method.

(iii) TAC values of synthetic mixtures or real samples from the

on-line HPLC-CUPRAC method with post-column detection

were calculated by summing the areas of negative peaks of the

individually identified antioxidants and dividing the total peak

Anal. Methods

Fig. 9 The chromatograms of King Luscious (a) and ArapKizi (b) apple

juices.

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area by the slope of the calibration equation of trolox at 450 nm

(y ¼ 1.32 � 1010 CTR + 3.31 � 104 where y ¼ area of negative

peak and CTR ¼ molar concentration of TR):

TACon�line HPLC�CUPRAC ¼Pni¼1

slope� 103 (5)

Binary synthetic mixtures of phenolic antioxidants were

prepared, and TAC values (as mM TR equivalent) were evalu-

ated with detection of constituents at 280 nm and 450 nm. The

calibration equation was found using these TAC values:

TACon-line HPLC-CUPRAC ¼ 0.68 TACHPLC � 1.0 � 10�5

(r ¼ 0.9947)

2.8. Combined HPLC-CUPRAC assay

The combined HPLC-CUPRAC assay (off-line HPLC-

CUPRAC) was developed for the estimation of the TACCUPRAC

of synthetic antioxidant mixtures and complex plant extracts

utilizing the principle of additivity of the capacities of individual

constituents identified and quantified by HPLC.28 This assay is

based on separation, identification and quantification of indi-

vidual antioxidants, especially phenolics in the sample, multi-

plication of the HPLC-determined concentration of each

antioxidant with its TEAC coefficient and summation of these

products to yield the theoretical TAC value by virtue of the

additivity of absorbances of constituents in a mixture. Thus, the

theoretical TAC of the investigated material could be estimated

using eqn (4).

Chun et al. investigated the contribution of phenolic species to

the observed total antioxidant capacity of plum.29 Ten poly-

phenolic compounds including chlorogenic acid, cyanidin, cya-

nidin glycosides, peonidin, peonidin-3-glycoside, quercetin, and

quercetin glycosides were identified and quantified by HPLC.

The HPLC-determined concentrations of individual phenolics

multiplied by their ascorbic acid-equivalent capacities with

respect to the ABTS method were summed up. Miller and Rice-

Evans studied a number of fruit juices in order to establish the

contribution of ascorbic acid and phenolic antioxidants to TAC,

multiplied the ABTS-TEAC coefficients of individual constitu-

ents with their concentrations, and gave a summation of these

products to approximate the TAC of these juices with respect to

the ABTS method.30

Table 3 HPLC calibration equations and linear ranges of the someantioxidants.28,31

Antioxidant Calibration equation

Ascorbic acid y ¼ 3.16 � 109 c + 1.68 � 104

Myricetin y ¼ 5.93 � 109 c � 4.20 � 104

Luteolin y ¼ 9.02 � 109 c � 7.95 � 104

Apigenin y ¼ 1.04 � 1010 c � 1.73 � 103

Chlorogenic acid y ¼ 9.00 � 109 c + 3.79 � 104

Caffeic acid y ¼ 9.44 � 109 c + 6.99 � 104

Catechin y ¼ 3.79 � 109 c + 4.47 � 103

Epicatechin y ¼ 3.71 � 109 c � 3.53 � 103

Phloridzin y ¼ 1.70 � 1010 c + 3.96 � 104

Procyanidin B2 y ¼ 6.70 � 109 c + 6.31 � 104

Anal. Methods

The combined HPLC-CUPRAC assay enables a realistic

comparison of antioxidant constituents of complex samples by

HPLC analysis, and of their calculated TAC values (without

performing the actual antioxidant assay) in trolox equivalents

(e.g., mmol TE L�1). For the calculation of TEAC coefficients

required for theoretical TAC estimation and the comparison of

experimental and theoretical TAC values, CUPRAC methods

{the normal CUPRAC (CUPRACN) and the incubated

CUPRAC (CUPRACI)}6 were applied.

2.8.1. Procedures for HPLC-CUPRAC assay. The chro-

matographic analyses were carried out using a Hamilton Hxsil

C18 (250 mm � 4.6 mm, 5 mm particle size) column. Gradient

elution programs were applied with two different solutions,

(r)Linear range(mM)

0.9999 0.01–1.00.9996 0.04–0.50.9990 0.04–0.50.9999 0.04–0.50.9996 0.04–0.50.9999 0.04–0.50.9997 0.04–0.50.9997 0.04–0.50.9996 0.04–0.50.9995 0.04–0.5

Table 4 The experimental and theoretical TAC values of synthetic mixtures,28 apple juices31 (in the units of mmol TE L�1) and parsley28 (in the units ofmmol TE g�1); analytical performance is indicated by the theoretically calculated percentagesa of experimental TAC valuesc,d

Samples CUPRACN CUPRACI HPLC-(CUPRACN) HPLC-(CUPRACI)

Synthetic MixturesMixture-Ib 0.32 0.43 0.29 (91) 7.16 (109)Mixture-IIc 0.80 0.90 0.79 (98) 4.15 (102)Mixture-IIId 1.29 1.41 1.19 (92) 7.16 (95)Parsley70% MeOH extn. 0.050 0.079 — —70% MeOH extn. (2 h hydrol.) 0.056 0.099 0.043 (77) 0.067 (68)70% MeOH extn. (4 h hydrol.) 0.058 0.104 0.034 (59) 0.062 (60)Solid plant (2 h hydrol.) 0.082 0.166 0.049 (60) 0.074 (45)Solid plant (4 h hydrol.) 0.093 0.193 0.035 (38) 0.069 (36)m-Phosphoric acid extn. 0.016 0.023 0.008 (50) 0.011 (48)ApplesKing Luscious 8.26 11.4 5.83 (70.6) 7.16 (62.9)Arap Kizi 4.70 7.77 3.50 (74.5) 4.15 (53.4)

a Values in parentheses represent the theoretically calculated percentages of the experimental TAC values. b Synthetic mixture-I: 0.2, 0.04, and 0.1 mMof p-coumaric acid, myricetin, and apigenin, respectively. c Synthetic mixture-II: 0.2, 0.04, 0.034, and 0.1 mM of chlorogenic acid, myricetin, luteolin,and apigenin, respectively. d Synthetic mixture-III: Equimolar (0.2 mM) concentrations of catechin and chlorogenic acid.

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methanol (A) and 0.2% of o-H3PO4 (v/v) in bidistilled water (B),

through a C18 column. Naturally, the gradient program was

changed when necessary according to the sample (complex

matrix) and/or antioxidant constituent.

The polyphenolics were analyzed in synthetic antioxidant

mixtures (I–III) and parsley extracts using these parameters and

gradient elution program 128 (Vsample¼ 20 mL; Flow rate¼ 1.0 mL

min�1; l ¼ 280 nm): 8 min 7% A � 93% B (slope 0.0); 8–13 min

30% A � 70% B (slope �4.0); 13–48 min 66% A � 34% B (slope

1.0); 48–55 min 75% A � 25% B (slope �4.0).

Ascorbic acid determination was performed by using isocratic

elution for 8 min, the mobile phase being composed of 7%

methanol (A) and 93% bidistilled water containing 0.2% of o-

H3PO4 (B). The detection wavelength was 215 nm, the elution

rate was the same as for phenolics.

Another gradient elution program (gradient elution program 2)

for phenolic constituents in apple juices31 (Vsample ¼ 20 mL; Flow

0.0); 5–40 min 66% A � 34% B (slope 1.0).

For ascorbic, malic and fumaric acids (gradient elution

program 3) in apple juices31 (Vsample ¼ 20 mL; Flow rate¼ 1.0 mL

min�1; l¼ 215 nm): 3 min 2% A� 98% B (slope 0.0); 3–7 min 7%

A � 93% B (slope 4.0); 7–10 min 7% A � 93% B (slope 0.0).

Using the above working modes, the calibration curves and

linear equations of peak area (y) versus concentration (c) were

determined for the phenolic antioxidants of interest (Table 3).

With the aid of these calibration curves, parsley extracts in 70%

MeOH, hydrolyzates, apple juices (See Fig. 9 for the chro-

matograms of the King Luscious and Arap Kizi apple juices),

and synthetic mixtures were analyzed (Table 4).

In the combined HPLC-CUPRAC assay, firstly, concentra-

tions of individual antioxidants were calculated from identified

peaks in the chromatograms (Fig. 9) using linear equations given

in Table 3. So, individual antioxidant constituents (especially

phenolics and ascorbic acid) in plant food samples can be both

qualitatively and quantitatively analyzed. Secondly, theoretical

TAC values were estimated by multiplying the HPLC-deter-

mined concentrations with the TEAC coefficients given in

Table 2 and summing up the products (eqn (4)) according to the

additivity property of TAC in a complex sample. The theoretical

TAC values and their comparison with the experimental values

were shown in Table 4. The results confirm that if all the anti-

oxidants in a complex mixture were identified and quantified

with the help of HPLC techniques, their contribution to the

overall capacity could be envisaged, and the experimentally

found TAC values (with the use of the spectrophotometric

CUPRAC antioxidant assay) could be correctly estimated by

theoretically calculating the TAC using the principle of additivity

of individual antioxidant capacities by the combined

HPLC-CUPRAC assay. The analytical performance of this

estimation is apparent in the theoretically calculated percentages

of experimental TAC values (Table 4) in synthetic mixtures I–III

(91–109%), while this performance is slightly decreased (36–77%)

in food plant extracts with unidentified antioxidants.

3. Conclusions

The main advantages of the CUPRAC method may be

summarized as follows:6,12,13,32

� The CUPRAC reagent (an outer-sphere electron-transfer

agent) is fast enough to oxidize thiol-type antioxidants, whereas

the FRAP method may only measure with serious negative error

certain thiol-type antioxidants like glutathione (i.e. the major low

molecular-weight thiol compound of the living cell). Redox

potential of GSSG/GSH is the basic indicator of biological

conditions of a cell (CUPRAC gives a direct response to GSH,

and an indirect response to GSSG only after Zn/HCl reduction

and neutralization33), and GSH acts as reconstituent of inter-

cellular ascorbic acid from the dehydroascorbic acid, therefore it

must be measured by a TAC assay claiming versatility.

� The CUPRAC reagent is selective, because it has a lower

redox potential than that of the ferric–ferrous couple in the

presence of o-phenanthroline- or batho-phenanthroline-type

ligands. The standard potential of the Cu(II,I)-Nc redox couple is

0.6 V, close to that of ABTS_+/ABTS (E� ¼ 0.68 V), and FRAP

(E� ¼ 0.70 V). Simple sugars and citric acid—that are not clas-

sified as ‘true’ antioxidants—are not oxidized with the CUPRAC

reagent. On the other hand, the simple ferricyanide (Fe(CN)63�)

Anal. Methods

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reagent has E� ¼ 0.36 V, insufficient to oxidize certain antioxi-

dants having greater redox potentials, while the ferric–ferricya-

nide reagent34 possibly has a potential greater than that of the

ferric–ferrous couple (i.e. E� ¼ 0.77 V), because the reduction

product, Fe(II), is further stabilized by the formation of Prussian

blue: (Fe[Fe(CN)6])� in the presence of excess Fe(CN)6

3�. The

exact chemistry and redox potential of the Folin–Coicalteau

(FC) reagent: phospho-tungsto-molybdate(VI) is unknown35 but

this potential is assumed to be quite high, and therefore FC

reagent may act as a nonspecific oxidant toward certain amino

acids, sugars,36 and simple phenols15 that are not classified under

the title ‘antioxidants’.

� The reagent is much more stable and easily accessible than

the chromogenic radical reagents (e.g., ABTS, DPPH, etc.). The

cupric reducing ability measured for a biological sample may

indirectly but efficiently reflect the total antioxidant power of the

sample even though no radicalic species are involved in the assay.

� The method is easily and diversely applicable in conventional

laboratories using standard colorimeters rather than necessi-

tating sophisticated equipment and highly qualified operators.

The method responds equally well to both hydrophilic and

lipophilic antioxidants.

� The redox reaction giving rise to a coloured chelate of Cu(I)-

Nc is relatively insensitive to a number of parameters adversely

affecting radicalic reagents such as DPPH, e.g., air, sunlight,

humidity, and pH, to a certain extent.

� The CUPRAC reagent can be adsorbed on a cation-

exchange membrane to build a low-cost, linear-response optical

antioxidant sensor.

� The CUPRAC absorbance versus concentration curves are

perfectly linear over a wide concentration range, unlike those of

other methods yielding polynomial curves. The molar absorptivity

for n–e reductants, (8.5� 1.0)� 103 n L mol�1 cm�1, is sufficiently

high to sensitively determine most phenolic antioxidants.

� The TAC values of antioxidants found with CUPRAC are

perfectly additive, i.e. the TAC of a phenolic mixture is equal to

the sum of TAC values of its constituent polyphenols. Additivity

in other antioxidant measurements is not strictly valid.

� Online coupling of HPLC to CUPRAC spectrophotometry

(i.e. HPLC-post column CUPRAC)26 is possible (direct methods

of TAC assay are not suitable for post-column applications

requiring rapid formation or fading of a coloured product).

� The redox reaction producing colored species is carried out

at nearly physiological pH (pH 7 of ammonium acetate buffer)

giving a more realistic simulation of in vivo TAC as opposed to

the unrealistic acidic conditions (pH 3.6) of FRAP or alkaline

conditions (pH 10) of the FC assay. Under conditions more

acidic than physiological pH, the reducing capacity may be

suppressed due to protonation of antioxidant compounds,

whereas under more alkaline conditions, proton dissociation of

phenolics would enhance a sample’s reducing capacity.

� Since the Cu(I) ion emerging as a product of the CUPRAC

redox reaction is in a chelated state (i.e. Cu(Nc)2+), it cannot act

as a prooxidant that may cause oxidative damage to biological

macromolecules in body fluids. The ferric ion-based assays (e.g.,

FRAP)4 were criticized for producing Fe2+, which may act as

a Fenton-type prooxidant to produce _OH radicals as a result of

its reaction with H2O2. It was earlier shown by Tutem et al.8 that

the cuprous neocuproine chromophore does not react with

Anal. Methods

hydrogen peroxide so as to cause redox cycling of Cu(I), but the

reverse reaction, i.e. irreversible oxidation of H2O2 with

Cu(Nc)22+, is possible. The Cu(I)-Nc complex was shown not to

be oxidized by H2O2 at any measurable rate, and the extra

stability of cuprous neocuproine in the presence of oxygen or

hydrogen peroxide was attributed to the steric hindrance in the

four-coordinate cupric complex (which is usually planar), caused

by 2,9-dimethyl substituents of 1,10-phenanthroline in neo-

cuproine.37 Thus, the redox potential: E�Cu(II),Cu(I) is significantly

elevated with Nc, and Cu(I) chelated to Nc may not act as

a prooxidant (i.e. producing reactive oxygen species) toward the

tested antioxidants in a Fenton-type reaction. Although both

bathocuproine disulfonate (BCS) and neocuproine (Nc) prefer-

entially stabilize Cu(I) over Cu(II), and thereby prevent the redox

cycling of Cu(I) formed in the presence of antioxidants, Cu(I)-Nc

is distinctly more hydrophobic than Cu(I)-BCS (the latter being

cell membrane-impermeable due to its higher charge),38 and

therefore, Cu(I)-Nc as the CUPRAC chromophore can be more

useful to the TAC assay of tissue homogenates.

� The original CUPRAC method has recently been imple-

mented in a microplate format (96-well plates) to reduce the

reaction time from 30 to 4 min at 37 �C, and also in an automated

fashion (flow injection analysis: FIA); these high-throughput

methods worked well for human serum and urine samples,

providing TAC values in agreement with those of the end-point

batch (i.e. classical CUPRAC) method.39

� The combined HPLC-CUPRAC method provides an esti-

mation of the TACCUPRAC of antioxidant mixtures and plant

extracts utilizing the principle of additivity of the capacities

of individual constituents identified and quantified by HPLC.

The sum of the products of the concentrations of HPLC-iden-

tified antioxidant constituents in plant extracts and their

TEACCUPRAC coefficients gave the theoretically calculated TAC

values, which accounted for a large percentage of experimental

TAC values measured by CUPRAC spectrophotometry. This

method gives a reliable estimate of the actual antioxidant

capacity of plant extracts and hydrolyzates.

In conclusion, the CUPRAC methodology is evolving into an

‘‘antioxidant measurement package’’ in biochemistry and food

chemistry comprising many assays, and the validated results

seem to have distinct advantages over certain established

methods. By maintaining the CUPRAC reagent and related

chemicals in the laboratory, one can measure ROS (i.e., H2O2,

hydroxyl and superoxide anion radicals) scavenging activity as

well as TAC of antioxidants. However, a battery of measure-

ments are required to adequately assess oxidative stress and

antioxidative defense in biological systems.

Acknowledgements

The authors express their gratitude to Istanbul University

Research Fund, Bilimsel Arastirma Projeleri (BAP) Yurutucu

Sekreterligi for the support given to the Research Projects-2724

and 5096.

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Anal. Methods

A comprehensive review of CUPRAC methodology

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