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A comprehensive review of CUPRAC methodology
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€ urek,a Kubilay G€ Mustafa Ozy€ uc¸l€ u,a Esma T€ utem,a Kevser S€ozgen Bas xkan,a Erol Erc¸ag,a S. Esin C ¸ elik,a a b c a Sefa Baki, Leyla Yıldız, S xeyda Karaman and Res xat Apak* 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
a Department of Chemistry, Faculty of Engineering, Istanbul University, Avcilar, 34320 Istanbul, Turkey. E-mail:
[email protected]; Fax: +90 212 473 7180; Tel: +90 212 473 7028 b Embil Pharmaceutical Co. Ltd, Bomonti, Sxisxli, 34381 Istanbul, Turkey
€ urek received his Mustafa Ozy€ 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€ urek Mustafa Ozy€ 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 hindex of 10). This journal is ª The Royal Society of Chemistry 2011
c Department of Chemistry, Fatih University, B. C ¸ ekmece, 34500 Istanbul, Turkey
Kubilay G€ uc¸l€ u received his PhD in Analytical Chemistry from Istanbul University in 1999. Since 2001, 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/antiKubilay G€uc‚l€u 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).
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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 is a Professor of Analytical Chemistry at Istanbul 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 derivative spectrophotometric determinations of biologically and environmentally important reductants and metal ions, Esma T€ utem 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).
Saliha Esin C ¸ elik 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 Res xat APAK in 2011. Her PhD study was on ‘‘Modified CUPRAC Antioxidant Capacity MeasureS: Esin C ‚ elik 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 antioxidants, development of antioxidant capacity/activity assays, spectroscopic and chromatographic applications.
Erol Erc¸a g received his PhD in Chemical Sciences (Analytical Chemistry) in 1995 from Istanbul University. He has been an Associate Professor of Analytical Chemistry since 2006 at Istanbul University, and Vice Institute Director at the Institute of Marine Sciences and Management. Current research includes the application of antioxidant capacity methods to plant extracts and biological Erol Erc‚a g 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 received his PhD in Analytical Chemistry from Istanbul University in 1982. He is a full professor of Analytical Chemistry at Istanbul University 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 Res xat Apak 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).
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1. Brief overview of antioxidant assays and the original CUPRAC (CUPric Reducing Antioxidant Capacity) 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-sulfonic acid/trolox equivalent antioxidant capacity),1 ORAC (oxygen radical absorbance capacity),2 DPPH (2,2-di(4-tert-octylphenyl)-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 antioxidant 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 mechanism of action (such as hydrogen atom transfer (HAT)- and ETbased 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(n1) + 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 HATbased assays). As opposed to the first four methods utilizing an absorbance increase, the latter two methods measure decolorization 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, abbreviated 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 electrontransfer 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
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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by plotting absorbance versus molar concentration, and the molar absorptivity of the CUPRAC method 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:
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1 mL Cu(II) + 1 mL Nc + 1 mL buffer + x mL 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.
Fig. 2 Visible spectra of Cu(I)-Nc chelate produced as a result of CUPRAC reaction with varying concentrations of quercetin.
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 CUPRAC method (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 antioxidant 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 absorptivity 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 solution, 1.0 102 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 dissolving 19.27 g NH4Ac in water and diluting to 250 mL. Neocuproine (Nc) solution, 7.5 103 M, is prepared daily by dissolving 0.039 g Nc in 96% ethanol, and diluting to 25 mL with ethanol. Trolox, 1.0 103 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 calibration curves of each antioxidant compound were constructed Anal. Methods
The scheme for normal measurement of lipophilic antioxidants was: 1 mL Cu(II) + 1 mL Nc + 1 mL buffer + x mL 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 measurement 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 concentration of the methanolic solution would be 1 mM. Each standard 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 C in 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 temperature, 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. This journal is ª The Royal Society of Chemistry 2011
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1.1.7. Calculation of TEAC coefficients. The molar absorptivity of trolox in the CUPRAC method was: 3trolox ¼ 1.67 104 L mol1 cm1. 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 mol1 cm1, respectively (Table 1)).11 For example, the molar absorptivity of catechin was 3 ¼ 5.16 104 in the normal CUPRAC method; the TEAC coefficient of catechin was calculated as 3catechin/3trolox ¼ 5.16 104/1.67 104 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, epigallocatechin gallate, quercetin, fisetin, epigallocatechin, catechin, 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 position 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 ordinary tea (1.26), and green tea (0.94), all of which were manufactured 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 media calculated with respect to the CUPRAC method Solvent
Linear calibration equation
100% EtOH
y¼ y¼ y¼ y¼ y¼ y¼ y¼ y¼ y¼
100% MeOH MeOH/H2O (4 : 1, v/v) MeOH/H2O (1 : 1, v/v) DCM/EtOH (9 : 1, v/v)
1.67 104c 1.86 104c 1.58 104c 1.60 104c 1.50 104c 1.52 104c 1.57 104c 1.62 104c 1.68 104c
0.033 (N) + 0.002 (I) 0.010 (N) 0.008 (I) 0.002 (N) 0.013 (I) 0.025 (N) 0.030 (I) 0.020
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r 0.9999 0.9996 0.9995 0.9993 0.9999 0.9987 0.9991 0.9993 0.9995
Table 2 Antioxidant capacities of various polyphenolic compounds (in the units of TEAC: trolox equivalent antioxidant capacity) as measured by the CUPRAC assay.6,12,13a TEACCUPRAC Antioxidant Flavonoids Epicatechin gallate (ECG) Epigallocatechin gallate (EGCG) Quercetin (QR) Fisetin (FS) Epigallocatechin (EGC) Catechin (CT) Epicatechin (EC) Rutin (RT) Morin (MR) Kaempferol Hesperetin (HT) Hesperidin (HD) Naringenin (NG) Naringin (N) Hydroxycinnamic Acids Rosmarinic acid (RA) Caffeic acid (CFA) Chlorogenic acid (CGA) Ferulic acid (FRA) p-Coumaric acid (CMA) Vitamins a-tocopherol (TP) Ascorbic acid (AA) Benzoic Acids Gallic acid(GA) Sinapic acid (SNA) Vanillic acid (VA) Syringic Acid (SA)
TEACN
TEACI
5.32 4.89
5.65 5.49
4.38 3.90 3.35 3.09 2.77 2.56 1.88 1.58 0.99 0.97 0.05 0.02
4.18 3.60 3.56 2.89
TEACH
TEACH&I
3.08
3.49 3.80
3.32 1.87 1.05 1.11 2.28 0.13
0.85 0.79
0.98 0.95 3.03
5.65 2.89 2.47 1.20 0.55
6.02 2.96 2.72 1.23 1.00
2.87 1.20 1.18 0.53
3.22 1.42 1.34 1.15
1.10 0.96
1.02
0.99
0.87
2.17 1.52 1.64
1.32 1.13
1.57 1.67
2.62 1.24 1.24 1.12
a N: Normal measurement; I: Incubated measurement. H: Hydrolyzed measurement; H&I: Hydrolyzed and Incubated measurement.
use of the CUPRAC reagent by other researchers for the antioxidant 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 polyphenolic 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 solutions 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
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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 g1.
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2. Some modifications of the CUPRAC method 2.1. CUPRAC-TAC assay of human serum (by differentiating lipophilic from hydrophilic antioxidants)
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).
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 g1) was found using the equation: TAC (in mmol TE g1) 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
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 antioxidants 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 advantage 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 basically nonresponsive. As a distinct advantage over other ETbased assays (e.g., Folin, FRAP, ABTS, DPPH), CUPRAC is superior in regard to its realistic pH (close to that of physiological 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, This journal is ª The Royal Society of Chemistry 2011
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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 separately 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 DCM was 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 mol1 cm1. 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 antioxidants and M-b-CD).16 M-b-CD was introduced as the water solubility enhancer for lipophilic antioxidants. Two percent This journal is ª The Royal Society of Chemistry 2011
Fig. 5 Host–guest interaction between antioxidant compounds and Mb-CD, followed by CUPRAC measurement of the TAC of inclusion complexes.
M-b-CD (w/v) in a 90% acetone–10% H2O mixture was found to sufficiently solubilize b-carotene, vitamin E, vitamin C, oilsoluble 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 antioxidant 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 simultaneous 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 products. A salicylate probe was used to indirectly detect_OH with the aid of the hydroxylation products formed, i.e. dihydroxybenzoates, 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, Anal. Methods
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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 concentration 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 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 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 1.0 102 M (glucose and mannitol) or 2 105 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 mL1 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 incubation 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-absorbance at 295 nm of uric acid formed as a reaction product. 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 spectrophotometric method at 450 nm. The CUPRAC absorbance of the incubation solution due to the reduction of the Cu(II)-Nc reagent by the products of the X–XO system decreased in the presence of polyphenolics, 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 assay 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 5.0 104 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 mg1 solid was diluted with 0.1 M phosphate buffer (pH ¼ 7.8) to a concentration of 0.04 U mL1. The perchloric acid solution at 3.2% concentration (w/v) was prepared in distilled water. The NaH2PO4–Na2HPO4 buffer solution (pH ¼ 7.8) at 100 mM was prepared in distilled water. The concentration 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 1 105 M (all polyphenolic compounds and ascorbic acid) or 1.0 104 M (naringin), (2.0 x) mL of 1 : 9 EtOH–H2O mixture (v/v), and 0.2 mL of 0.04 U mg1 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 (miniaturized 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 This journal is ª The Royal Society of Chemistry 2011
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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 scavengers. The UV method suffers from both the interference of some phenolics in real samples having strong absorption in the UVregion and from inefficient degradation of H2O2 with polyphenols 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 spectrophotometry. 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 mg1 was diluted with 0.2 M phosphate buffer (pH ¼ 7.4) to a concentration of 268 U mL1. 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 mL1 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:
2.6.
CUPRAC antioxidant sensor
A low-cost optical sensor was developed using a membraneimmobilised CUPRAC reagent, Cu(II)-Nc complex, for the assessment of antioxidant capacity of non-enzymatic antioxidants, 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 antioxidant compounds suggest that the reactivity of the Nafionimmobilized 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 principle 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 2.0 102 M Cu(II) + 2 mL of 1.5 102 M Nc + 2 mL of 1 M NH4Ac + 2.2 mL of H2O, and agitated for 30 min in a rotator. The reagentimpregnated 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
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: A0 ðA1 A2 Þ HPS ð%Þ ¼ 100 A0 where A0 is the CUPRAC absorbance of reference hydrogen peroxide incubation solution, A1 and A2 the CUPRAC absorbances of scavenger solutions-I and -II, respectively. This journal is ª The Royal Society of Chemistry 2011
Fig. 6 Schematic presentation of the mechanism of CUPRAC antioxidant sensor.
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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.
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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 decomposition. 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 postcolumn detection.21–25 The most widely used assays in postcolumn 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, reaction 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 postcolumn 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 HPLCseparated 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
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 quantitative 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 antioxidant 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 consequently 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 polyphenolic compounds, and trolox. The mobile phase consisted of two solvents, i.e. methanol (A) and 0.2% of o-H3PO4 in bidistilled water (B). The following parameters and gradient were used for the analysis of tea antioxidants:27 (Vsample ¼ 20 mL; Flow rate ¼ 0.8 mL min1; 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).
Fig. 7 Instrumental setup for on-line HPLC-CUPRAC detection system.
Anal. Methods
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Fig. 8 The chromatogram of mint extract showing HPLC (280 nm, positive trace) and on-line HPLC-CUPRAC (450 nm, negative trace) assays.
The other polyphenolic antioxidants were analyzed using these parameters and gradient program:28 (Vsample ¼ 20 mL; Flow rate ¼ 0.8 mL min1; l ¼ 280 nm): 8 min 7% A 93% B (slope 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 min1; l ¼ 280 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 concentration 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 ammonium 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 min1.26 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) This journal is ª The Royal Society of Chemistry 2011
equivalents using (i) conventional CUPRAC spectrophotometry, (ii) conventional HPLC with CUPRAC calculation, and (iii) online 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 L1) of the synthetic mixtures or samples were calculated by dividing the observed absorbance (A450) by the molar absorptivity of trolox (A450 ¼ 3TRCTR 0.01, where 3TR ¼ 1.58 104 L mol1 cm1 and r ¼ 0.9995) according to the spectrophotometric CUPRAC assay. TACCUPRAC ¼
Absorbance ðtotalÞ 103 3TR
(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 ¼
n X
ci ðTEACÞi
(4)
i¼1
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
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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): n P
TAConline HPLCCUPRAC ¼
yi
i¼1
slope
103
(5)
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Binary synthetic mixtures of phenolic antioxidants were prepared, and TAC values (as mM TR equivalent) were evaluated with detection of constituents at 280 nm and 450 nm. The calibration equation was found using these TAC values: TACon-line
2.8.
¼ 0.68 TACHPLC 1.0 105 (r ¼ 0.9947)
HPLC-CUPRAC
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 L1). 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 chromatographic 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,
Combined HPLC-CUPRAC assay
The combined HPLC-CUPRAC assay (off-line HPLCCUPRAC) 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 individual antioxidants, especially phenolics in the sample, multiplication 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 polyphenolic compounds including chlorogenic acid, cyanidin, cyanidin 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 RiceEvans 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 constituents with their concentrations, and gave a summation of these products to approximate the TAC of these juices with respect to the ABTS method.30
Fig. 9 The chromatograms of King Luscious (a) and Arap Kizi (b) apple juices.
Table 3 HPLC calibration equations and linear ranges of the some antioxidants.28,31
Antioxidant
Calibration equation
Ascorbic acid Myricetin Luteolin Apigenin Chlorogenic acid Caffeic acid Catechin Epicatechin Phloridzin Procyanidin B2
y y y y y y y y y y
Anal. Methods
¼ 3.16 109 c + 1.68 104 ¼ 5.93 109 c 4.20 104 ¼ 9.02 109 c 7.95 104 ¼ 1.04 1010 c 1.73 103 ¼ 9.00 109 c + 3.79 104 ¼ 9.44 109 c + 6.99 104 ¼ 3.79 109 c + 4.47 103 ¼ 3.71 109 c 3.53 103 ¼ 1.70 1010 c + 3.96 104 ¼ 6.70 109 c + 6.31 104
(r)
Linear range (mM)
0.9999 0.9996 0.9990 0.9999 0.9996 0.9999 0.9997 0.9997 0.9996 0.9995
0.01–1.0 0.04–0.5 0.04–0.5 0.04–0.5 0.04–0.5 0.04–0.5 0.04–0.5 0.04–0.5 0.04–0.5 0.04–0.5
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Table 4 The experimental and theoretical TAC values of synthetic mixtures,28 apple juices31 (in the units of mmol TE L1) and parsley28 (in the units of mmol TE g1); analytical performance is indicated by the theoretically calculated percentagesa of experimental TAC valuesc,d
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Samples Synthetic Mixtures Mixture-Ib Mixture-IIc Mixture-IIId Parsley 70% MeOH extn. 70% MeOH extn. (2 h hydrol.) 70% MeOH extn. (4 h hydrol.) Solid plant (2 h hydrol.) Solid plant (4 h hydrol.) m-Phosphoric acid extn. Apples King Luscious Arap Kizi
CUPRACN
CUPRACI
HPLC-(CUPRACN)
HPLC-(CUPRACI)
0.32 0.80 1.29
0.43 0.90 1.41
0.29 (91) 0.79 (98) 1.19 (92)
7.16 (109) 4.15 (102) 7.16 (95)
0.050 0.056 0.058 0.082 0.093 0.016
0.079 0.099 0.104 0.166 0.193 0.023
— 0.043 (77) 0.034 (59) 0.049 (60) 0.035 (38) 0.008 (50)
— 0.067 (68) 0.062 (60) 0.074 (45) 0.069 (36) 0.011 (48)
5.83 (70.6) 3.50 (74.5)
7.16 (62.9) 4.15 (53.4)
8.26 4.70
11.4 7.77
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 mM of 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.
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 (Vsample ¼ 20 mL; Flow rate ¼ 1.0 mL min1; 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 oH3PO4 (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 rate ¼ 1.0 mL min1; l ¼ 280 nm): 5 min 30% A 70% B (slope 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 rate ¼ 1.0 mL min1; l ¼ 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 chromatograms of the King Luscious and Arap Kizi apple juices), and synthetic mixtures were analyzed (Table 4). In the combined HPLC-CUPRAC assay, firstly, concentrations 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-determined concentrations with the TEAC coefficients given in Table 2 and summing up the products (eqn (4)) according to the This journal is ª The Royal Society of Chemistry 2011
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 antioxidants 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 intercellular 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 classified 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 antioxidants having greater redox potentials, while the ferric–ferricyanide 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)63. 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 necessitating 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 cationexchange 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 mol1 cm1, 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 neocuproine.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) preferentially 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 implemented 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 estimation 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-identified 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 measurements 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
