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Development of a Silver Nanoparticle-Based Method for the Antioxidant Capacity Measurement of Polyphenols Mustafa Ö zyürek, Nilay Güngör, Sefa Baki, Kubilay Gücļ ü, and Reşat Apak* Department of Chemistry, Faculty of Engineering, Istanbul University, Avcilar 34320, Istanbul, Turkey S Supporting Information *

ABSTRACT: A sensitive colorimetric method for the detection of polyphenols (i.e., flavonoids, simple phenolic, and hydroxycinnamic acids) was proposed in this research based on the reduction of Ag+ ions by polyphenols in the presence of citrate-stabilized silver seeds. The color of the stable suspension was controlled by varying the concentration of trisodium citrate, silver nitrate, and silver seeds. The reduction of Ag+ to spherical silver nanoparticles (SNPs) by polyphenols in the presence of trisodium citrate and silver seeds produced a very intense surface plasmon resonance (SPR) absorption band of SNPs at 423 nm. The plasmon absorbance of SNPs allows the quantitative spectrophotometric detection of the polyphenols, and the developed method gave a linear response over a wide concentration range of standard polyphenolic compounds. In contrast to other reported NP-based antioxidant assays, it was established in this work that growth but not nucleation of SNPs gave a linear concentration-dependent response. The trolox equivalent antioxidant capacity (TEAC) values of various (hydrophilic and lipophilic) antioxidants using the developed method were comparable to those of the CUPRAC assay. Common food ingredients like oxalate, citrate, fruit acids, amino acids, and reducing sugars did not interfere with the proposed sensing method. This assay was validated through linearity, additivity, precision and recovery, demonstrating that the assay is reliable and robust. The developed method was used to screen total antioxidant capacity (TAC) of some commercial fruit juices and herbal teas without preliminary treatment, and showed a promising potential for the preparation of antioxidant inventories of a wide range of food plants.

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here is considerable interest in antioxidants due to their possible health benefits (i.e., prevention of oxidative stress related diseases such as cancers and cardiovascular and neurodegenerative diseases) and application in nutrition and medicine, making estimation of total content of antioxidants very popular.1 Total antioxidant capacity (TAC), which is a resultant measure of the ability of all antioxidants present in food or biological materials to counteract the oxidation of an indicator by an oxidant or to reduce a probe substance, has been widely employed in the analysis of these matrices.2,3 By means of standardized tests of TAC performed on complex real samples, the antioxidant values of foods, pharmaceuticals, and other commercial products can be meaningfully compared, and variations within or between products can be controlled. A variety of molecular spectroscopic assays to determine TAC of food and plant extracts, beverages, and biological fluids3 have been developed, which may be broadly classified as electrontransfer (ET)-based (CUPRAC, Folin-Ciocalteau, FRAP, ferricyanide), hydrogen atom transfer (HAT)-based (ORAC), and mixed ET/HAT-based (ABTS-persulfate/TEAC, DPPH) assays.4,5 Because of their small size (1−100 nm) and very high specific surface area, silver nanoparticles (SNPs) have attracted much attention and found applications in diverse areas, including medicine, catalysis, textile engineering, biotechnology and bioengineering, water treatment, electronics, and optics.6 © XXXX American Chemical Society

However, the nanomaterial-based methods have rarely been used in food science (specifically antioxidant research), probably because of the substoichiometric character of the concerned reduction reactions by antioxidants leading to NP formation. To the best of our knowledge, the TAC determination of polyphenols and related food and biological samples with a reasonable precision and concentration range for a wide variety of antioxidant compounds using a SNP-based assay has not yet been reported, and this study was intended to fill such a literature gap. SNPs have diverse properties and applications, like electrical conductivity, chemical stability, catalytic and antibacterial activity, DNA sequencing, and surface-enhanced Raman scattering (SERS).7,8 Commonly used reducing agents for production of stable SNPs as colloidal dispersions 9 are sodium borohydride, sodium citrate, ascorbic acid, and elemental hydrogen.7 Initially, the reduction of various Ag+ species leads to the formation of silver atoms (Ag0), which is followed by agglomeration into oligomeric clusters.10 These clusters eventually lead to the formation of colloidal Ag particles.10 Aside from conventional reducing agents, various plant and Received: July 12, 2012 Accepted: August 16, 2012

A

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treatment. Thus, a rapid, simple, and diversely applicable antioxidant capacity assay for dietary polyphenols and antioxidant vitamins was developed by exploiting the extraordinary optical properties of SNPs.

biological extracts have been used as reductants for the synthesis of SNPs.11 However, our preliminary experiments have revealed that use of an antioxidant as a single (seedproducing) reactant for NP formation is not beneficial for TAC assay development, because it does not lead to a linear concentration-dependent response (i.e., of SPR absorbance versus antioxidant concentration). Controlled synthesis of SNPs is based on a two-step reduction process12 involving NaBH4 reduction and citrate stabilization, where secondary nucleation during growth was inhibited by a weak reductant (ascorbate).13 When SNPs are dispersed in liquid media, they exhibit a strong UV−vis absorption band that is not present in the spectrum of the bulk metal.13 This extinction band is attributed to collective excitation of the electron gas in the particles, with a periodic change in electron density at the surface (SPR, surface plasmon resonance absorption).14 The SPR absorption of SNPs has very high molar extinction coefficients (ε ≈ 3 × 1011 M−1 cm−1) and are expected to allow higher sensitivity in optical detection methods than conventional reagents;13 however, this ε is a corrected value calculated on the basis of moles of SNPs,15 and ideal sensitivity in NPbased TAC assays cannot be achieved, because of various factors affecting SPR absorption such as reaction stoichiometry, particle size and shape, and dielectric constants of both the metal and the surrounding medium.16 Since the properties of surface plasmons can be tailored as a result of altering the NP surface and, specifically, the shell thickness, the seed-mediated particle growth technique17 was adopted in this work. Linearity of responses over a reasonable concentration range together with the additivity of TAC values for constituents of complex mixtures are a prerequisite for meaningful comparison of TAC values of different food samples found with the aid of the proposed TAC assay. The SNPs produced in the presence of air oxygen in water/bis(2-ethylhexyl)sulfosuccinate/n-alkane system by the reduction of Ag(I) with quercetin (QR) have been found not to yield perfectly linear calibration curves.18 In the present work, a SNP-based method for the TAC assay of polyphenols, their synthetic mixtures, and real samples was described for the first time. Because the Ag+ ion-reducing ability of polyphenols is measured, the method is named by our research group “Silver NanoParticle Antioxidant Capacity”, abbreviated as the SNPAC method. First, we used relatively monodisperse silver seed particles prepared and stabilized by reduction of Ag+ ions with the weak reductant trisodium citrate, hindering the introduction of new nucleation centers17 and ensuring controlled subsequent growth. Second, we added an antioxidant as the secondary reducing agent for further reduction of Ag+ ions on silver seeds to form a core−shell structure. For core−shell NPs, it is the outermost layerand not the corethat dominates the interaction with incoming light.17 Antioxidant and Ag+ ions diffuse to the surface of the seed particle, where electron transfer takes place, resulting in the growth of SNPs. The novel nanomaterial-based method for TAC estimation relies on the polyphenol-mediated growth of SNPs and optical monitoring of the corresponding plasmon absorption bands. The optical properties of such polyphenolmediated growth of SNPs correlate well with the antioxidant (reducing) capacity of the corresponding phenolic acids, because tailoring of optical properties is mainly dependent on Ag-nanoshell thickness,17 closely related to the stoichiometry of Ag+ reduction reaction with the antioxidant molecule. The SNPAC method was successfully applied without interference to complex samples such as fruit juices without preliminary

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EXPERIMENTAL SECTION Instrumentation and Chemicals. The following chemical substances, of analytical reagent grade, were supplied from the corresponding sources. From Sigma−Aldrich (Steinheim, Germany): neocuproine (2,9-dimethyl-1,10-phenanthroline) (Nc), quercetin (QR), gallic acid (GA), (−)epigallocatechin gallate (EGCG), (−)epicatechin (EC), (−)epicatechin gallate (ECG), (−)epigallocatechin (EGC), chlorogenic acid (CHA), luteolin (LT), rutin (RT), L-ascorbic acid (AA), rosmarinic acid (RA), caffeic acid (CFA), and trolox (TR); from Merck (Darmstadt, Germany): silver nitrate (AgNO3), trisodium citrate-2-hydrate, copper(II) chloride dihydrate, ammonium acetate (NH4Ac), methanol, ethanol, and ammonium chloride; from Fluka (Buchs, Switzerland): (+)catechin (CT), fisetin (FIS), α-tocopherol (TP), and apigenin. The various beverages, herbal teas, and olive oil samples were purchased from local stores. The spectra and absorption measurements were recorded in matched Helma quartz cuvettes using a Varian CARY Bio 100 UV−vis spectrophotometer (Mulgrave, Victoria, Australia). Other related apparatus and accessories were a J.P. Selecta water bath (Barcelona, Spain), and Telstar Cryodos freeze dryer (Terrassa, Spain). SEM images of SNPs synthesized with citrate and/or GA were recorded with the aid of scanning electron microscopy (SEM; FEI Model Quanta 450 FEG, Hillsboro, OR, USA). Preparation of Solutions. The AgNO3 solution (1.0 mM), 1% trisodium citrate solution (w/v), CuCl2 (10.0 mM), and NH4Ac buffer (1.0 M, pH 7) solutions were all prepared in pure distilled water (Millipore Simpak1 Synergy 185, USA), and neocuproine solution (7.5 mM) in absolute ethanol. All antioxidant compounds were freshly prepared in EtOH at 1.0 mM concentration and L-ascorbic acid in distilled water at 1.0 mM concentration prior to measurement. Preparation of SNPs. The SNPs were prepared by using the chemical reduction method that employed trisodium citrate as a stabilizer capped on the SNP surface.19 In this method, 50 mL of 1.0 mM AgNO3 was heated to boiling for 10 min. To this solution, 5 mL of 1% trisodium citrate was added drop by drop. During the process, the solution was mixed vigorously. The solution was heated until its color change was evident (pale yellow). This “initial SNP solution” was then removed from the heating element and stirred until cooled to room temperature. SNPAC Method. The reacting mixture for the SNPAC method contained in a final volume of 2.8 mL the following constituents: 2 mL of initial SNP solution, x mL of antioxidant standard or real sample solution, and (0.8 − x) mL of H2O. Reaction mixtures were incubated at 25 °C for 30 min. During the reaction, the color of the colloidal solution (pale yellow) gradually evolved, which indicated the reduction of Ag+ and the formation of core−shell structure. The absorbance of the reagent blank (A0) increased in the presence of antioxidants, the increment of absorbance (ΔA) being proportional to antioxidant concentration. The increase in absorbance (ΔA) caused by antioxidants, recorded at 423 nm against a reagent blank at the end of 30 min, reflected the SNPAC reactivity and was plotted against the concentration of the antioxidant. The B

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standard calibration curve of each antioxidant compound was constructed in this manner as absorbance versus molar concentration, and the molar absorptivity (ε) of the SNPAC method for each antioxidant was found from the slope of the calibration line concerned. The TEAC value of a given antioxidant represents the ratio of εantioxidant to that of trolox measured under the same conditions of the SNPAC assay. The TEAC coefficient, being a slope ratio, is unitless (as defined in other TAC tests). The scheme for measurement of antioxidants is summarized as

TAC expected = TEAC1 concn1 + TEAC2 concn2 + ... + TEACn concn n TAC found experimentally =

absorbance (total) ± intercept × 103 εtrolox

Interference Studies. The interference effects of 1000-fold (as mol/mol) concomitant species commonly found in fruit juices to the determination of 7.14 × 10−3 mM antioxidant in ethanolic solution using SNPAC method were studied. Scanning Electron Microscopy (SEM) Analysis. The morphology of the SNPs synthesized with citrate and/or GA was determined by SEM. SNPs were placed onto carbon tape mounted on an aluminum stub for SEM image. The samples were freezed using SHUR/freeze cryogen spray and dried under vacuum (6 × 10−2 mbar). The secondary electron images of AgNPs were captured in SEM mode at the desired magnification. Statistical Analysis. Descriptive statistical analyses were performed using Excel software (Microsoft Office 2002) for calculating the means and the standard error of the mean. Results were expressed as the mean ± standard deviation (SD). Using SPSS software for Windows (version 13), the data were evaluated by two-way Analysis Of Variance (ANOVA).21

2 mL of initial SNP soln + x mL of antioxidant soln + (0.8 − x) mL of H 2O; total volume = 2.8 mL, measure A423 against a reagent blank after 30 min at room temperature

CUPRAC Method. The commonly used colorimetric CUPRAC method was applied as the reference method of comparison for determining the antioxidant capacity of polyphenols and real samples. This method, as described by Apak et al.,20 is based on the reduction of a cupric neocuproine complex (Cu(II)-Nc) by antioxidants to the cuprous form (Cu(I)-Nc). To a test tube were added 1 mL each of Cu(II), Nc, and NH4Ac buffer solutions. Antioxidant standard solution or real samples (x mL) and H2O (1.1 − x mL) were added to the initial mixture to make the final volume of 4.1 mL. The tubes were stoppered, and after 1/2 h, the absorbance at 450 nm (A450) was recorded against a reagent blank. The scheme for normal measurement of antioxidants is summarized as

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RESULTS AND DISCUSSION Although Au-NP-based antioxidant assays have been attempted with acceptable success,22 a versatile TAC assay based on SNP formation has not been developed. A SNP-based assay is expected to be more sensitive and selective than its gold-based analogue, because its Mie resonance occurs at energies distinct from any bulk interband transition, enabling a silver colloid to have a stronger and sharper plasmon resonance than gold,17 and because the reduction potential of Ag(I) is lower than that of Au(III), respectively. In the development of a novel antioxidant assay using NPs, one should bear in mind that optical properties are strongly dependent on the method of NP formation and, hence, on the reduction process.23 In the current approach, relatively monodisperse seed particles were prepared by reduction of Ag+ ions with trisodium citrate, because improved monodispersity involving a narrow size distribution of SNPs achieved by citrate capping may ensure the predominance of growth over nucleation,24 thereby affecting the precision of the subsequent TAC determinations. Otherwise, new nucleation and ripening cycles associated with the use of a stronger reducing agent like NaBH4 may give rise to smaller NPs with significant variability in particle size24 and subsequently to shifts in the SPR absorption peak wavelengths and bandwidths (i.e., closely dependent on particle size distribution) adversely affecting the reproducibility of TAC measurements with different antioxidants. In the second step of this process, polyphenols as secondary reductants were added for reduction of Ag+ ions on silver seeds to generate core−shell SNPs. The polyphenols and/or antioxidant vitamins and Ag+ ions diffuse to the surface of the seed particles to enable electron transfer resulting in the deposition of more Ag atoms on the seeds, and the final core−shell SNP structures exhibit the characteristic SPR absorption. The findings of the proposed assay illustrate that the optical properties of such polyphenol (or vitamin)-mediated NPs generation/thickening correlate

1 mL Cu(II) + 1 mL Nc + 1 mL NH4Ac buffer + x mL antioxidant soln + (1.1 − x) mL H 2O; total volume = 4.1 mL, measure A450 against a reagent blank after 30 min of reagent addition

Addition of RT, GA, and CT Standard Solutions to Green Tea and Olive Oil Extract. A 100-μL aliquot of green tea infusion and 150 μL of 0.1 mM RT, 100 μL of 0.1 mM GA, or 130 μL of 0.1 mM CT solution were taken into a tube. A 100-μL aliquot of olive oil extract and 130 μL of 0.1 mM CT solution were taken into a tube. Solutions with RT, GA, and CT added were separately subjected to the SNPAC assay. Measurement of Synthetic Mixture Solutions. Synthetic mixtures of the antioxidants in EtOH were prepared in suitable volume ratios and were subjected to the SNPAC method. The theoretical trolox equivalent TAC of a synthetic mixture solution (expressed in the units of mM TR) was calculated by multiplying the TEAC coefficient of each antioxidant constituting the mixture with its final concentration (in mM TR units) and summing up the products. The experimental trolox equivalent TAC of the same mixture was calculated by dividing the observed absorbance (A423) to the molar absorptivity of TR (εTR being 7.8 × 103 L mol−1 cm−1 under the selected conditions). Then, the theoretically found TAC values were compared to the experimentally observed ones to test the applicability of Beer’s law (i.e., the principle of additivity of individual absorbances of constituents making up a mixture). Validity of Beer’s law for a mixture implies that the observed absorbance is the sum of the individual absorbances of the constituents. C

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well with the antioxidant (reducing) capacity of the corresponding compounds. On the other hand, if such a twostep procedure is not followed (such as SNPs formation with direct reaction of Ag(I) salt with the antioxidants contained in a plant seed or leaf extract),25 both the slowness of reaction and the shifts in SPR absorption maxima due to the formation of heterogeneous particles may not enable a robust assay development. The first step of the SNPAC method involves the preparation of small-sized spherical SNPs (average diameter = 66 nm) by chemical reduction of AgNO3 with trisodium citrate, possibly producing dicarboxyacetone:26 2Ag + + (CH 2COONa)−C(OH)(COONa)−(CH 2COONa) → 2Ag 0 + (CH 2COONa)−CO−(CH 2COONa) + Na + + H+ + CO2 ↑

(1) Figure 1. UV−vis spectra of (a) GA alone and SNPs formed (b) in the absence of GA and (c) in the presence of GA.

In the second step, the reactive Ar−OH groups of polyphenols are oxidized to the corresponding quinones while Ag+ is reduced to the highly colored SNPs (core−shell formation) showing maximum absorption at 423 nm (standard potential, E°, for physiologically important antioxidants: 0.2− 0.6 V, E°Ag(I),Ag = 0.8 V). n Ag + + Ar(OH)n → n Ag 0 + Ar(O)n + nH+

Figure 2 shows the SNPAC reaction kinetics with individual antioxidants measured at room temperature. It is apparent from

(2)

It should be noted that not all phenolic −OH substituents are reduced to the corresponding quinones in the SNPAC method, and the efficiency of this reduction depends on the number and position of the phenolic −OH groups, as well as on the overall conjugation level of the polyphenolic molecule.4 The limitations of the method may be the difficulty of reduction of antioxidants having a standard potential greater than 0.8 V (which is quite rare for food and biological antioxidants), and the spontaneous formation of SNPs from Ag(I) salts with substances other than true antioxidants (e.g., surfactants). Analytical Figures of Merit. Relevant solution conditions, such as the concentration of AgNO3 and trisodium citrate (affecting the stability and growth of SNPs) have been optimized. The findings revealed that all components associated with SNP formation/growth are essential to stimulate the polyphenol-mediated growth, and that the most favorable NP spectral changes correspond to AgNO3 and trisodium citrate concentrations of 1 mM and 1% (w/v), respectively. A pH study performed over the 3.0−7.0 range indicated that the highest absorbance response was observed at pH 6.0 (see Figure S-1 in the Supporting Information). Figure 1 displays the absorption spectra of SNPs formed in the absence (Figure 1b) and presence of (Figure 1c) GA. GA alone at 0.1 mM initial concentration produced a small 423 nmabsorbance (Figure 1a). Adding GA to the SNP solution (containing the citrate capping agent) results in a characteristic sharp plasmon absorption band at 423 nm, indicative of Ag(0) nanoparticles. This band is due to surface plasmon resonance of SNPs and it may be attributed to the collective oscillation of electron gas in the particles with a periodic change in the electronic density at the surface. Such generation of polyphenol mediated-SNPs proceeds with further reduction of Ag(I) to Ag(0) by the polyphenolics (e.g., GA) on the preformed SNP seeds. The antioxidant concentration-dependent response arises from the increase in absorbance between the curves (Figure 1b → Figure 1c).

Figure 2. SNPAC reaction kinetics with GA, CT, CFA, and AA; rate of increase in absorbance at 423 nm with time for an initial concentration of 0.1 mM antioxidant solution at room temperature.

Figure 2 that GA, CT, CFA, and AA showed an initial absorbance increase and then stabilization with time, which determined the optimal time period of measurement (i.e., 30 min after the mixing of reagents with the analyte). The SEM images of the SNPs are presented in Figure 3, revealing that SNPs resulting from the redox reactions appear typically spherical in shape, and are fairly monodisperse and homogeneous particles of a reasonable size distribution. The size of the SNPs in the absence and presence of polyphenols were, on average, 66 and 112 nm, respectively. The SNPs absorbance signals are linearly dependent upon the concentrations of the polyphenolics. For example, Figure 4 shows the UV−vis spectra and color change of SNPs formed with varying concentrations of QR for the proposed SNPAC method. As the concentration of QR increases, the absorbance corresponding to the plasmon resonance of the SNPs is intensified. The plasmon absorbances of SNPs achieved with QR were linear within the concentration range of 1.33−39.8 μM (as final concentrations in solution), and the method D

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polyphenolic antioxidant) and its molar concentration (c) was evaluated using 15 compounds. Table 1 summarizes the linear equations (A = mC + n), correlation coefficients (r), and linear concentration ranges of these pure compounds. As can be seen from Table 1, all antioxidants could be assayed with the proposed SNPAC assay (TEAC coefficients (significantly different) and P = 0.05, Fexp = 1.487, Fcrit (table) = 4.600, Fexp < Fcrit (table)). The TEAC coefficients found by the SNPAC method correlated well (r = 0.936) with those of the CUPRAC method (Table 1). The calibration curves of selected antioxidants, ECG, EGCG, RT, and LT are shown in Figure S-2 in the Supporting Information. The antioxidant polyphenolic compounds listed in Table 1 demonstrate that the highest capacities in the SNPAC method were observed for ECG, RA, EGCG, QR, EGC, CT, and EC, in this order. This is in accordance with theoretical expectations, because the number and position of the hydroxyl groups, as well as the degree of conjugation of the entire molecule, are important in determining TAC.27 The antioxidant potency of flavonoids of similar conjugation level is roughly proportional to the total number of −OH groups and is positively affected by the presence of an o-dihydroxy moiety in the B-ring.28 The decreasing order of antioxidant capacities, using the SNPAC method, were observed for ECG [7 OH; 5.33], EGCG [8 OH; 4.24], QR (5 OH; 3.83), EGC [6 OH; 3.64], CT [5 OH; 3.61], EC [5 OH; 3.46], and GA (3 OH; 2.91) (the values given in parentheses show the number of hydroxyl groups in the molecule and the TEACSNPAC coefficients of these compounds, respectively). The catechin group, also known as “tea antioxidants”, gave a capacity order in accordance with the number and position of their −OH groups, together with the overall extent of conjugation in the molecule.29 The different order of TAC of ECG and EGCG in the proposed assay may be attributed to the steric hindrance caused by the galloyl groups of EGCG during diffusion to the surface of the silver seed particles where the electron transfer takes place. The presence of 5-hydroxy-4-keto group in A and C rings in flavonols, the 2,3-double bond connecting the two ring systems of flavonol via conjugation, and the 3′,4′-dihydroxy substitution of the B

Figure 3. SEM images of SNPs synthesized with (a) trisodium citrate and (b) trisodium citrate + GA.

showed excellent linearity (r = 0.999) over a relatively broad concentration range of the analyte. The linear equation for the calibration graph of TR drawn at the wavelength of 423 nm, with respect to the developed method, was A423 = 7.8 × 103C TR + 0.004

(r = 0.999)

and the molar absorptivity was expressed as ε = 7.8 × 103 L mol−1 cm−1. The limit of detection (LOD) and limit of quantification (LOQ) for TR in the SNPAC assay were calculated using the equations; LOD = 3 sbl/m and LOQ = 10 sbl/m, respectively, where sbl is the standard deviation of a blank and m is the slope of the calibration line. The LOD and LOQ for TR were found to be 0.23 and 0.77 μM, respectively. The precision, which is expressed as the relative standard deviation (RSD, %) in the tested concentration range, was ∼5.2%. SNPAC absorbances of TR were linear within the concentration range of 1.28 × 10−7−1.12 × 10−4 M (as final concentrations in solution), and the method showed excellent linearity over a relatively broad concentration range of analyte. To determine the reproducibility of the method, a ruggedness experiment was performed. The SNPAC procedure for fruit juices gave intra-assay and inter-assay coefficients of variation (CV) of ∼0.96% and 1.21%, respectively. The correlation between the SNPAC absorbance (A) for a given antioxidant compound (ascorbic acid, α-tocopherol, or

Figure 4. UV−vis spectra of SNPs in the presence of different concentrations of QR (μM): (a) 0, (b) 3.57, (c) 7.14, (d) 10.71, (e) 14.28, and (f) 17.85 μM for the proposed SNPAC method. (The color images of the test tubes containing SNPs formed in the presence of different concentrations of QR are shown in the inset figure.) E

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Table 1. Linear Equations, Correlation Coefficients (r), TEAC Coefficients,a and Linear Ranges for Antioxidants, with Respect to the Proposed SNPAC Method antioxidants

TEACSNPAC

TEACCUPRACb

Simple Phenolic Acids 1.67−52.30

2.91

2.62

Hydroxycinnamic Acids 1.02−30.51

5.02

5.30

1.55−61.14

2.47

2.80

1.26−49.79

3.04

2.47

1.33−39.80

3.83

4.38

2.44−43.20

3.62

3.90

+ 0.02

0.72−28.36

5.33

5.30

+ 0.02

0.91−35.65

4.24

4.88

+ 0.03

0.74−43.33

3.46

2.77

+ 0.04

0.35−41.13

3.61

3.09

− 0.01

2.11−42.60

3.64

3.34

0.96−56.25

2.66

2.38

0.70−41.20

3.64

2.56

2.19−62.10

2.47

0.12

0.88−103.00

1.44

0.96

0.96−111.00

1.33

1.10

linear equation and correlation coefficients

gallic acid

A = 2.27 × 104c + 0.01 (r = 0.9991)

rosmarinic acid

A = 3.90 × 104c + 0.01 (r = 0.9977) A = 1.93 × 104c + 0.02 (r = 0.9990) A = 2.37 × 104c + 0.02 (r = 0.9981)

caffeic acid chlorogenic acid

linear range (μM)

Flavonols quercetin fisetin

A = 2.99 × 104c + 0.01 (r = 0.9995) A = 2.82 × 104c − 0.019 (r = 0.9978) Flavan-3-ols

ECG EGCG EC catechin EGC

A = 4.16 × 104c (r = 0.9993) A = 3.31 × 104c (r = 0.9994) A = 2.70 × 104c (r = 0.9988) A = 2.82 × 104c (r = 0.9941) A = 2.84 × 104c (r = 0.9994)

Flavons luteolin rutin apigenin

A = 2.08 × 104c + 0.03 (r = 0.9952) A = 2.84 × 104c + 0.03 (r = 0.9974) A = 1.92 × 104c + 0.01 (r = 0.9973) Others

ascorbic acid α-tocopherol

A = 1.13 × 104c + 0.04 (r = 0.9995) A = 1.04 × 104c + 0.04 (r = 0.9963)

a TEAC coefficients (significantly different) (by exclusion of the values for apigenin with highest TEAC variability; P = 0.05, Fexp = 1.487, Fcrit (table) = 4.600, Fexp < Fcrit (table)). bData taken from refs 2 and 20. TEACCUPRAC = 1.16 TEACSNPAC − 0.782 (r = 0.936).

ring are considered as important structural characteristics for antioxidant potency,30 all three of which are combined in QR. As a result, the TEAC coefficient in the SNPAC assay was highest among flavonols for QR. Generally, one may expect that, when the sugar bond in RT is broken, its antioxidant potency may approach that of the corresponding aglycon (QR). The glycoside RT, initially having a CUPRAC-TEAC of 2.56, when effectively hydrolyzed to the aglycon (QR), showed a TEACCUPRAC value of 4.22 (close to that of QR).20 However, the TEAC coefficients of QR and RT, with respect to the SNPAC method, were 3.83 and 3.64, respectively, without any preliminary treatment such as hydrolysis. Similarly, QR and RT did not exhibit significant TEAC differences in the Folin method of TAC assay, and this result may better reflect in vivo conditions. As for hydroxycinnamic acids, which are almost the most abundant phenolic components in the citrus family and in some other fruits, the TEAC coefficients with respect to the SNPAC

method (and with respect to the CUPRAC assay, as shown in parantheses) were as follows (see Table 1): RA 5.02 (5.30), CHA 3.04 (2.47), and CFA 2.47 (2.80). RA, having the higher conjugation level, showed the highest TEAC in both assays. Phenolic compounds with two or more electron-donating groups have lower anodic peak potentials and higher antioxidant potencies than monosubstituted phenols. Generally, there is a relationship between antioxidant activities and oxidation potentials. Simić et al.31 found that phenolic compounds with low anodic peak potential vs Ag/AgCl (Epa) (