Reaction time and DPPH concentration influence antioxidant activity and kinetic parameters of bioactive molecules and plant extracts in the reaction with the DPPH radical

Journal of Food Composition and Analysis 35 (2014) 112–119

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Original Research Article

Reaction time and DPPH concentration influence antioxidant activity and kinetic parameters of bioactive molecules and plant extracts in the reaction with the DPPH radical Angela Fadda a, Maria Serra b, Maria Giovanna Molinu a, Emanuela Azara b, Antonio Barberis a, Daniele Sanna b,* a b

Institute of Sciences of Food Production, National Research Council, Traversa La Crucca 3, Regione Baldinca, 07040 Li Punti, Sassari, Italy Institute of Biomolecular Chemistry, National Research Council, Traversa La Crucca 3, Regione Baldinca, 07040 Li Punti, Sassari, Italy

A R T I C L E I N F O

A B S T R A C T

Article history: Received 20 November 2013 Received in revised form 24 April 2014 Accepted 2 June 2014 Available online 24 August 2014

The antioxidant activity of lemon and pomegranate juice, infusion of Java green tea, rosemary oil and pure chemicals was studied by the DPPH method. The chemical composition of the samples was determined by liquid chromatography–mass spectrometry (LC–MS). The kinetics of the reaction is different among extracts. The reaction of DPPH with lemon juice is complete in 3 min; 87 and 98 min are necessary for green tea infusion and pomegranate juice, respectively; 283 min are needed for rosemary essential oil. In samples with an intermediate and slow kinetic behavior, the antioxidant activity measured after 2 h of reaction is significantly higher than that measured after 30 min. These differences are 18% for Java green tea infusion, 21% for rosemary essential oil, 23% for pomegranate juice. The results demonstrate that the percentage of remaining DPPH decreases with increasing initial DPPH concentrations (between 25 and 200 mM), while keeping constant antioxidant/DPPH ratio. Moreover the time necessary to reach the steady state is dependent, while keeping the same antioxidant/DPPH ratio, on the initial DPPH concentration, showing that longer time intervals are required when using lower DPPH concentrations (25 mM). These results confirm the necessity of standardizing the method to be able to compare results from different laboratories. ß 2014 Elsevier Inc. All rights reserved.

Keywords: Food analysis Food composition Laboratory harmonization DPPH UV–vis spectroscopy Reaction kinetics Antioxidant classes BHT Essential oil Pomegranate juice Lemon juice Green tea infusion

1. Introduction In biological systems, oxidative damage produced by reactive oxygen species (ROS) is the main cause of several degenerative diseases (Sanders and Greenamyre, 2013; Wang et al., 2013). Antioxidants are involved in the prevention and protection of cells against ROS. Fruits and vegetables are important sources of natural antioxidants. The intake of polyphenols, flavonoids, carotenoids and ascorbic acid through a diet rich in fruits and vegetables has been associated with a reduced risk of cancer and age-related disorders (Lau et al., 2005; Perez-Vizcaino and Duarte, 2010). Studies dealing with free radical scavenging effects of dietary constituents of vegetables, fresh fruits or plant extracts are steadily increasing. Several methodologies have been used to estimate

* Corresponding author. Tel.: +39 0792841207; fax: +39 0792841299. E-mail address: [email protected] (D. Sanna). http://dx.doi.org/10.1016/j.jfca.2014.06.006 0889-1575/ß 2014 Elsevier Inc. All rights reserved.

antioxidant activity (Huang et al., 2005; Karadag et al., 2009; Prior et al., 2005). The DPPH test is one of the oldest and most frequently used methods for the determination of the antioxidant activity of food extracts and single compounds (Brand-Williams et al., 1995; ˜ o et al., 2007). This Ratty et al., 1988; Stratil et al., 2006; Villan method is based on the spectrophotometric or on the electron paramagnetic resonance (EPR) spectroscopy measurement of DPPH depletion during reaction with an antioxidant. The antioxidant activity is frequently expressed as EC50 (Brand-Williams et al., 1995), as percentage of free radical inhibition (Schirra et al., 2007) or as L-ascorbic acid equivalents (Leong and Shui, 2002). The estimation of the antioxidant activity is usually performed at a fixed endpoint, which is extremely variable among laboratories. In most of the DPPH assays reported in the literature, the remaining DPPH concentration is measured after an incubation time of 15 min (Schirra et al., 2007), 30 min (Ahmad et al., 2012) and only rarely after 2 h (Surveswaran et al., 2007). However, the use of a fixed time in the estimation of the antioxidant activity often

A. Fadda et al. / Journal of Food Composition and Analysis 35 (2014) 112–119

does not take into account the kinetic behavior of the antioxidants, and indeed measurements are performed without verifying if the reaction is effectively complete. As a consequence, the use of short reaction times may provide underestimated values of the antioxidant activity. This frequently occurs with isolated compounds or plant extracts that exhibit a slow reaction with DPPH. In order to bypass this problem, some authors have proposed a kinetic approach in the study of the antioxidant activity of citrus juices (Sendra et al., 2006; Sentandreu et al., 2007), sesame extracts (Suja et al., 2004) and red wines (Magalha˜es et al., 2012). Sa´nchez-Moreno et al. (1998), in order to provide information about reaction time with DPPH, introduced another parameter to describe the antioxidant activity: antiradical efficiency (AE). This parameter combines the antiradical power (1/EC50) with the reaction time expressed in terms of TEC50, i.e. the time needed to reach the endpoint of the reaction at antioxidant concentration corresponding to EC50. These authors associated the time at the steady state with the antioxidant concentration; on the other hand, they did not consider the influence of the DPPH concentration on the time needed to reach the endpoint conditions. However, in routine measurements of the antioxidant activity, it is practically impossible to investigate the kinetic behavior of a particular sample, and therefore the time needed to reach the end of the reaction should be previously known. In this work we evaluated the influence of the fruit and plant sample composition on the time necessary to attain the endpoint conditions in the reaction with DPPH. To meet this purpose, the kinetic evolution of the reaction of DPPH with lemon juice, pomegranate juice, green tea infusion and rosemary essential oil was studied. These samples were selected for their different chemical composition as well as their high antioxidant activity. This study demonstrates that the time needed to reach the steady state and the % of DPPH remaining during the time course depends on initial DPPH concentration.

2. Materials and methods 2.1. Reagents and solutions All reagents and solvents were of analytical grade, unless otherwise specified and used without further purification. 2,2-Diphenyl-1-picryhydrazyl radical (DPPH) was purchased from Alfa Aesar (London, UK), ethanol 96% and ethanol absolute from Carlo Erba (Rodano, MI, Italy). L-Ascorbic acid, gallic acid (3,4,5-trihydroxybenzoic acid), ()-epigallocatechin gallate (()cis-2-(3,4,5-trihydroxyphenyl)-3,4-dihydro-1(2H)-benzopyran3,5,7-triol 3-gallate, EGCG), and 2,6-di-tert-butyl-4-methylphenol (BHT), were purchased from Sigma–Aldrich (Milan, Italy). Water was purified with a Milli-Q system from Millipore (Millipore Corporation, Billerica, MA, USA). DPPH stock solutions were prepared in absolute ethanol at 1 mM concentration. Antioxidant stock solutions were prepared in absolute ethanol at the following concentrations: L-ascorbic acid 0.01 M, gallic acid 17 mM, ()-epigallocatechin gallate 4 mM, BHT 1 mM. All stock solutions were stored in the dark at 4 8C and the analyses were performed within 3 days. 2.2. Plant extracts preparation 2.2.1. Java green tea A commercial green tea was purchased from retail outlet. The Java green tea infusion was prepared by pouring 100 mL of distilled water at 80 8C into a beaker with a 2 g bag and left for 5 min according to the producer instructions. The infusion was cooled down and filtered (Whatman 113) before analysis.

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2.2.2. Pomegranate juice Pomegranate juice was obtained from pomegranates (Punica granatum L. cv ‘Wonderful one’) purchased at the local market. The juice was prepared by squeezing the arils of 5 pomegranates per replication (3 replications, approximately 1.5–2 kg each) with a commercial juicer. Before analysis, the juice was centrifuged at 13,000  g for 15 min and the supernatant was used without any further treatment. 2.2.3. Lemon juice Lemons (Citrus limon L. Burm cv ‘Verna’) were harvested at the experimental orchard of the Institute of the Sciences of Food Production located in central western Sardinia (latitude 398550 3100 N, longitude 88350 4300 E). The juice was obtained by squeezing 5 fruits per replication (3 replications, approximately 0.5 kg each) with a commercial juicer. Before analysis, the juice was centrifuged at 13,000  g for 15 min and the supernatant filtered through a 0.45 mm acetate cellulose filter. 2.2.4. Rosemary essential oil Rosemary was collected from wild populations in northern Sardinia. The sampling was done by a randomized collection of about 20 shrubs in an area of nearly 100 m2. Leaves were separated from shoots and three lots of 200 g each were separately hydrodistilled for 2 h in a glass Clevenger-type apparatus using distilled water (1 L) (European Pharmacopeia, 2002). Oils were recovered without using any solvent and stored at 20 8C until analysis. 2.3. Chemical composition of plant extracts 2.3.1. Determination of total flavonoids Total flavonoids were determined according to the colorimetric assay described by Kim et al. (2003). Briefly, 1 mL of the diluted samples (fruit juices or green tea) was mixed, with 4 mL of ultrapure water. Thereafter 0.3 mL of 5% NaNO2, 0.3 mL of 10% AlCl3 and 2 mL of 1 M NaOH were added to the flask, respectively, at time zero and after 5 and 6 min. At 6 min, the mixture was adjusted with water to a final volume of 10 mL and thoroughly mixed. The absorbance was measured at 510 nm. Catechin calibration curve (2.5–20 mg/mL, R2 = 0.999) was used to quantify the total flavonoids content of the samples and the results were expressed as mg of catechin equivalents (CE) per 100 ml of juice. Analyses were performed in triplicate for each sample. 2.3.2. Determination of total phenolic content Total phenolic levels were measured in fruit juices and in green tea infusion using the Folin–Ciocalteu assay (Singleton and Rossi, 1965). Aliquots of the diluted samples were mixed, in a 25 mL volumetric flask, with the Folin–Ciocalteu reagent (1:1) and with 10 mL of sodium carbonate 7.5%. The reaction mixture was incubated for 120 min at room temperature and the absorbance measured at 750 nm. The results were expressed as milligrams of gallic acid equivalents (GAE) per 100 mL of sample by means of a calibration curve of gallic acid (five calibration points, concentration in the range 10–100 mg/L, R2 = 0.989). Samples were analyzed in triplicate. 2.3.3. Determination of total anthocyanin content Total anthocyanin content was determined only in pomegranate juice by the pH-differential method (Lee et al., 2005) using 2 buffer systems: potassium chloride buffer (0.025 M), pH 1, and sodium acetate buffer (0.4 M), pH 4.5. The samples were diluted with the corresponding buffer and the absorbance was measured at 520 and 700 nm. The amount of total anthocyanins was expressed as mg/L of cyanidin-3-glucoside equivalents. All analyses were performed in triplicate.

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2.3.4. L-Ascorbic acid determination An HPLC Agilent 1100 system equipped with a diode array detector (DAD) (Agilent Technologies, Palo Alto, CA, USA) was used for the analysis of L-ascorbic acid. Each sample (10 mL) was analyzed on a Zorbax C18 column (Agilent 250  4.6 mm, particle size 5 mm) using a flow rate of 0.7 mL/min. A 2% KH2PO4 solution was used as mobile phase (adjusted to pH 2.8 with phosphoric acid) (Lee and Coates, 1999). The detector was set at 254 nm. Quantification was carried out by external calibration with Lascorbic acid (five calibration points, concentration in the range 10–100 mg/L. R2 = 0.999). The concentration of L-ascorbic acid was expressed as mg/L. All analyses were performed in triplicate. 2.3.5. Liquid chromatography/mass spectrometry The LC–MS analyses were performed with an Agilent 1100 LC System (Agilent Technologies, Palo Alto, CA, USA) equipped with a binary pump, diode-array detector, column thermostat, degasser and an autosampler mode HTS-PAL. The LC was coupled to a single stage quadrupole mass spectrometer (Agilent G1946 MSD 1100) interfaced with an electrospray atmospheric pressure ionization source. LC–MS analysis was carried out to identify phenolic compounds in Java green tea infusion, flavonoids in lemon juice and anthocyanins in pomegranate juice. All analyses were performed in triplicate and pomegranate juice was dilute ten times before injection. Analytical data were acquired by Agilent ChemStation HP A.10.02. Chromatographic separation of phenolic compounds was carried out according to Wu et al. (2012) with slight modifications. A gradient program was employed using Eluent A (0.2% acetic acid – 0.1% trifluoracetic acid in water) and Eluent B (acetonitrile) with the following linear gradient settings: at 0 min 90% A, at 20 min 80% A, at 38 min 68% A. The flow rate was set at 0.250 mL/min, the run time was 55 min and the column temperature 35 8C. Injection volume was 10 mL. The diode array detector was set at 280 and 320 nm. Chromatographic separation of anthocyanins was carried out with a Luna C8 column (150 mm  2.1 mm, 3 mm, Phenomenex, Torrance, CA, USA) provided with a security guard cartridge (C8, 4  2 mm). The mobile phases were A (0.2% acetic acid – 0.2% trifluoracetic acid in water) and B (acetonitrile). The applied elution conditions were: 0– 20 min, linear gradient from 10% to 20% B; 20–38 min, 32% B. The flow rate was set at 0.3 mL/min and the column temperature was 37 8C. Injection volume was 50 mL. The diode array detector was set at 270 and 520 nm. Mass spectra were acquired using electrospray ionization in the positive (PI) and negative (NI) ionization mode with the following conditions: m/z range 270–800, ion spray voltage 3200 mV and fragmentor 85 eV (PI), ion spray voltage 3400 mV and fragmentor 50 eV (NI). After optimization, heated nebulizer parameter was set as follow: temp. 350 8C, nebulizer pressure 42 psig and flow rate of drying gas 9.8 L/min. Identification of phenol and anthocyanin compounds was carried out by means of their UV spectra, molecular weight and MS fragments. EGCG was quantified with LC-DAD at 280 nm by external calibration with the standard compound. Calibration curve was performed with five concentrations of EGCG in duplicate (5.5–550 mg/L, R2 = 0.999). The results were expressed as mg/ 100 ml of Java green tea infusion. 2.4. Determination of the radical scavenging activity 2.4.1. Radical scavenging activity The radical scavenging activity of fruit juices, essential oil, tea infusion and standard compounds (L-ascorbic acid, gallic acid, BHT and EGCG) was determined spectrophotometrically with the DPPH method according to Brand-Williams et al. (1995). A fixed volume (1.9 mL) of ethanolic solutions of plant samples or standard

compounds, at different concentrations, was mixed to 100 mL of DPPH (1 mM in absolute ethanol) and stored in the dark at room temperature for 30 min and 2 h. UV–vis readings were carried out with a spectrophotometer Perkin-Elmer Lambda 35 at 517 nm. The antioxidant activity was expressed as EC50. All measurements were performed in triplicate. 2.4.2. Dependence of the steady state time by the extract composition The time interval necessary to reach the end of the reaction with DPPH was determined for all the plant samples previously described and compared to pure standard compounds. Five different concentrations (selected in order to have percentages of inhibition, calculated after 30 min, equally distributed between 0 and 100%, that is ca. 17, 33, 50, 66 and 83%) of each plant sample or pure compound were allowed to react with 100 mL of an ethanolic solution of DPPH 1 mM. The mixture was shaken and transferred to a quartz cuvette. The decrease of absorbance at 517 nm was recorded after 3 min from the addition of DPPH and at regular intervals of 3 min for the first 30 min and, subsequently, every 5 min until the steady state of the reaction was achieved. The time at the steady state is the time at which the difference between two subsequent absorbance readings is 0.001 A (that is, the photometric repeatability of the instrument in use). 2.4.3. Effect of DPPH concentration on the DPPH/antioxidant reaction kinetics Systems with increasing DPPH concentration and a constant DPPH/ antioxidant ratio, corresponding to that of EC50, were used to evaluate the effect of DPPH concentration on the DPPH/antioxidant reaction kinetics and on the time at endpoint conditions, i.e. the time necessary to reach the end of the reaction. Java green tea and BHT were used in these experiments. Appropriate volumes of antioxidants were mixed to solutions of DPPH at five different initial concentrations (from 25 mM to 200 mM). The assays were carried out in duplicate, and data points were expressed as the average of the percentage of remaining DPPH, calculated from the following equation:

% of remaining DPPH ¼

As  100; A0

where A0 is the absorbance of the DPPH solution without antioxidant and As is the absorbance of the sample, or in terms of absorbance decrease. The degradation rate (DR) of DPPH was calculated in samples containing Java green tea and BHT; this parameter is defined as DR = ln(ABS0/ABS60)/60, where ABS0 and ABS60 represent the absorbance, measured at 517 nm at time 0 and after 60 min, of solutions containing DPPH and Java green tea, or BHT, with the same DPPH/antioxidant ratio but at different initial DPPH concentrations. 2.5. Statistical analysis Statistical analysis was performed by Statgraphics centurion software (Herndon, VA, USA). Analysis of variance (ANOVA) was carried out according to a single factor, complete randomized block design with three replicates for each sample. Mean separation was carried out by least significant difference (LSD) test application. 3. Results and discussion 3.1. Chemical composition of fruit extracts The concentration of total phenolics, total flavonoids, total anthocyanins and L-ascorbic acid was determined for each sample.

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Table 1 Total phenolics, total flavonoids, total anthocyanin and L-ascorbic acid content in plant samples studied.* Plant samples

Total phenolicsa (mg of GAE/100 mL)

Total flavonoidsb (mg of CE/100 mL)

Total anthocyaninc (mg/L)

L-Ascorbic

Java green tea Pomegranate Lemon

301.01  1.40 200.79  4.82 38.95  1.28

81.03  0.52 20.36  1.93 6.27  0.95

– 498.08  27.07 –

– <0.2 512.26  27.16

* a b c

acid (mg/L)

Data are expressed as means  standard deviation (n = 3). GAE: gallic acid equivalents. CE: cathechin equivalents. Total anthocyanins content is expressed as mg of cyanidin-3-glucoside equivalents/L.

Results are reported in Table 1. The green tea infusion was the sample with the highest total phenolic and flavonoid content (301.01 mg of GAE/100 mL and 81.03 mg of CE/100 mL respectively). These values are consistent with previous works that have identified flavanols, flavandiols and phenolic acids as the main constituents of green tea (Atoui et al., 2005). The constituents of the green tea infusion, identified by comparison with literature data, are listed in Table 2 (Wu et al., 2012). LC–MS analysis showed that the green tea analyzed include catechins, purine alkaloids, flavonol glycosides. Epigallocatechin gallate (EGCG) was the most abundant constituent (59.57 mg/100 ml). Anthocyanins were found in pomegranate fruits at the concentration of 498.08 mg/ L. According to LC–MS data, six anthocyanins were detected in pomegranate juice (see Table 3). Cyanidin-3-glucoside, cyanidin3,5-diglucoside and pelargonidin-3-glucoside were the most abundant, while delphinidin 3,5-diglucoside, delphinidin 3-glucoside and pelargonidin 3,5-diglucoside were the minor peaks. A similar anthocyanin composition has been previously reported by other authors (Fischer et al., 2011). Total phenolics and total flavonoids were also present at concentrations of 200.79 mg of GAE per 100 mL and 20.36 mg of CE per 100 mL, respectively. No Lascorbic acid was found in pomegranate juice. Among the samples analyzed only lemon juice had L-ascorbic acid (512 mg/L). In accordance with literature data, the amount of total phenolics and total flavonoids was 38.95 mg GAE/100 mL and 6.27 mg of CE/100 mL, respectively (Tounsi et al., 2011). The main flavonoids identified with LC–MS are listed in Table 4. A similar flavonoid composition has been previously reported by AbadGarcı´a et al. (2009).

3.2. Influence of reaction time on estimating the radical scavenging activity of plant samples and pure compounds The radical scavenging activity of the standards and extracts analyzed is summarized in Table 5. The radical scavenging activity of BHT, one of the most common reference compounds for antioxidant activity, was also reported for comparison. After 30 min of reaction, EC50 of L-ascorbic acid was 0.81 mmol/ mg DPPH and remained almost unchanged after 2 h. L-Ascorbic acid is considered as an antioxidant of medium strength, but it reacts very rapidly with DPPH (Sendra et al., 2006). Our results show that the reaction of L-ascorbic acid with DPPH was completed within 3 min, thus confirming the fast kinetic behavior of this compound. Lemon juice reacted very rapidly with DPPH. After 30 min of reaction, the EC50 was 121.5 mL/mg DPPH, in accordance with other values obtained for the juice of lemon fruit (Guimara˜es et al., 2010; Sanna et al., 2012). After 2 h of reaction, the DPPH depletion did not significantly differ from that achieved at 30 min. An opposite performance was observed for gallic acid and epigallocatechin gallate. When measured at 30 min, the EC50 of gallic acid was 0.272 mmol/mg DPPH, 7.5% higher than that measured after 2 h. Similarly, in epigallocatechin gallate the measured radical scavenging activity increased slightly after 2 h, as revealed by the lower EC50 value. These differences, even if small, are significant from the statistical point of view. Both these compounds display a slow kinetic behavior toward DPPH (Brand˜ o et al., 2007). The dependence of the Williams et al., 1995; Villan radical scavenging activity upon time was even more evident in some of the samples studied. In pomegranate juice an average

Table 2 Retention time and mass spectral data of main phenolic compounds and purine alkaloids from Java green tea infusion. Peak

Compound

tR (min)

[MH]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

5-Galloylquinic acid Gallic acid Theobromine Gallocatechin Epigallocatechin Catechin Caffeine Epicatechin Epigallocatechin gallate Gallocatechin gallate Vitexin-2-O-rhamnoside Mirycetin-3-O-galactoside Mirycetin-3-O-glucoside Rhamnosylvitexin isomer Quercetin-3-rutinoside Quercetin-3-O-galactoside Epicatechin gallate Kaempferol-3-O-rutinoside Kaempferol-3-O-galactoside Kaempferol-3-O-glucoside

4.97 5.31 6.54 6.95 9.05 10.57 11.04 12.34 12.50 12.94 13.24 13.80 14.00 14.75 15.00 15.82 16.05 16.59 17.11 17.63

343 169 181a 305 305 289 195a 289 457 457 577 479 479 577 609 463 441 593 447 447

Fragment ions (m/z)

a

[M+H]+.

191, 125 261, 261, 245, 138 245, 305, 305, 413, 317 317 413, 301 301 289, 285 285 285

169

219, 139, 125 219, 139, 125 205 205 287, 169, 125 287, 169, 125 293

293

271, 169, 125

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116

Table 3 Retention time and mass spectral data of main anthocyanins detected in pomegranate juice. Peak

Compound

tR (min)

[MH]

Fragment ions (m/z)

1 2 3 4 5 6

Delphinidin 3,5-diglucoside Cyanidin 3,5-diglucoside Pelargonidin 3,5-diglucoside Delphinidin 3-glucoside Cyanidin 3-glucoside Pelargonidin 3-glucoside

19.88 22.96 25.73 26.25 29.18 31.52

627 611 595 466 449 433

303 287 271 303 287 271

increase of about 23% of antioxidant activity was observed after 2 h of reaction as compared to values obtained at 30 min. Similar outcomes were achieved for Java green tea and rosemary essential oil. At 2 h, the EC50 of these two extracts was, respectively, 18 and 21% lower than that measured at 30 min. As revealed by the kinetic analysis, the reaction time intervals of Java green tea and rosemary were longer than 1 h, and this could account for the large difference of values observed between the two measurements at different times. 3.3. Kinetic behavior of plant samples and pure compounds The time interval necessary to complete the reaction strongly depends on the sample. Fig. 1 shows the evolution of the reduction of DPPH by fruit and plant samples. Lemon juice (Fig. 1A) displayed extremely fast antiradical kinetics, regardless of the concentration used. The endpoint conditions were reached within 3 min. A similar kinetic behavior was observed for L-ascorbic acid (Fig. 2A). The relationship between the kinetics of lemon juice and L-ascorbic acid can be explained considering that on non-pigmented citrus fruits, L-ascorbic acid is the major contributor to the total antioxidant capacity, while flavonoids and other phenolic compounds, also present in lemon juice, seem to contribute less to the radical scavenging activity (Gardner et al., 2000). The kinetic analysis of the other plant samples and of the other pure compounds showed different kinetic behaviors toward DPPH. Pomegranate juice (Fig. 1B) and Java green tea (Fig. 1C) displayed an intermediate kinetic behavior. They reached the endpoint conditions after 90 and 80 min of reaction, respectively, and the achievement of the steady state of the reaction was concentrationdependent. When applied at high concentrations, the time evolution of the absorbance curve of pomegranate juice and Java green tea showed a rapid decrease of the DPPH radical, followed by a slow decay of DPPH at longer time intervals. Similar kinetic scans were observed for gallic acid (Fig. 2B) and epigallocatechin gallate ˜ o et al. (2007) ascribed the biphasic behavior of (Fig. 2C). Villan polyphenol compounds to a fast abstraction of phenol H-atom followed by a slow decay due to reactions with the products of dimerization or with the products of reaction of the antioxidants. The polyphenolic compounds present in both pomegranate and Java green tea may explain such a kinetic pattern; however, in complex mixtures of antioxidants, as in plant samples, different mechanisms could be involved in the reaction with DPPH. This is clear when comparing the time interval necessary to complete the Table 4 Retention time and mass spectral data of main flavonoids detected in lemon juice. Peak

Compound

tR (min)

[MH]

Fragment ions (m/z)

1 2 3 4 5

Isorhamnetin-3-O-rutinoside Eriodictyol-7-O-rutinoside Quercetin-3-O-rutinoside Diosmetin-7-O-rutinoside Hesperetin 7-rutinoside

12.45 14.93 17.44 17.75 18.03

623 595 609 607 609

– 287 301 299 –

Table 5 EC50 of plant samples and standards measured after 30 min and after 2 h of reaction with DPPH.* Plant extracts/pure compounds

Units

EC50 30 min

2h

mL/mg DPPH

121.5  1.7 52.9  0.4a 36.1  1.3a 1.50  0.11a

127.9  5.7a 40.5  0.4b 29.5  0.4b 1.19  0.02b

mmol/mg DPPH mmol/mg DPPH mmol/mg DPPH mmol/mg DPPH

0.272  0.003a 6.14  0.15 0.81  0.03 0.365  0.05a

0.251  0.063b

Lemon (juice) Pomegranate (juice) Java green tea (infusion) Rosemary (essential oil)

mL/mg DPPH mL/mg DPPH mL/mg DPPH

Gallic acid BHT** L-Ascorbic acid Epigallocatechin gallate

a

0.353  0.003b

* Data are expressed as means  standard deviation (n = 3); in each row, values not sharing common letters are significantly different (P < 0.05). ** BHT: 2,6-di-tert-butyl-4-methylphenol.

reactions of epigallocatechin gallate and Java green tea with DPPH radical. The first reached the endpoint conditions after 118 min, whereas Java green tea displayed faster antiradical kinetics. The kinetic behavior of rosemary essential oil has been studied in detail for the first time (Fig. 2C). From this figure it is clearly evident that the reaction of rosemary essential oil with DPPH is extremely slow and cannot be considered complete after 280 min. The flattening of the curves is reached only for the higher concentrations used, because at very long time intervals the DPPH is completely depleted from the reaction medium. Since the purpose of this work is not to determine the absolute value of EC50 for rosemary essential oil but only to demonstrate that the kinetics of reaction with DPPH are extremely variable, we have not investigated the reactivity after 280 min. However, the EC50 values measured after 30 min and 2 h are likely overestimated compared to the values measured at the steady state of the reaction (t > 280 min). 3.4. Effect of DPPH concentration on its percentage remaining during the time course The final DPPH concentration is not strictly prescribed in the original method and in the subsequent papers describing improvements of the method itself (Brand-Williams et al., 1995; Mishra et al., 2012). In the present paper the effect of the initial DPPH concentration on the % of remaining DPPH was evaluated. Fig. 3A shows the evolution of the % of remaining DPPH during the reaction between DPPH and BHT, in systems with increasing DPPH concentration and with a constant ratio of DPPH/BHT. A similar experiment was carried on with the infusion of Java green tea, and the results are reported in Fig. 3B. It should be noted that to avoid the effect of pH and solvent type on the kinetics of DPPH scavenging, as previously reported (Dawidowicz and Olszowy, 2012; Dawidowicz et al., 2012), all measurements with BHT were performed using absolute ethanol as BHT and DPPH solvent. Regarding Java green tea infusion, the amount of water in the system was very low (5%) and its effect on the reactivity of DPPH was considered to be negligible (Sanna et al., 2012). The concentrations of the samples (BHT and Java green tea infusion) and DPPH used to measure the EC50 after 30 min, as reported in Table 2, were used as references (Fig. 3, solid line). The decrease of absorbance was followed for 60 min. After that time the highest concentration of remaining DPPH was observed for the lowest initial DPPH concentration used, whereas an increase of initial DPPH concentration decreased the percentage of remaining DPPH. These results clearly demonstrate that an increase of DPPH concentration increases its consumption. As can be seen in Fig. 3, at the end of the reaction time (60 min), the percentage of remaining DPPH, calculated for systems with different DPPH concentrations, sharply increases with increasing DPPH concentrations (it should be

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Fig. 1. Time course evolution of the absorbance, measured at 517 nm, in reaction mixtures with DPPH (5  105 M) and different concentrations of plant samples. (A) Lemon juice: 0.71; 2.09; 3.47; 5 mL/mL, (B) pomegranate juice: 0.28; 0.47; 0.65; 0.83; 1.01 mL/mL; (C) Java green tea: 1.15, 7.68; 14.25; 20.82; 27.35 mg/mL; (D) rosemary essential oil: 8.65; 13.9; 30; 40.75; 51.50 mL/mL.

stressed that in these systems the antioxidant/DPPH ratio was kept constant). Since these are free radical reactions, a change in the initial DPPH concentration may lead to a change in the reactions mechanisms between DPPH and the antioxidants. These results demonstrate that the DPPH concentration affects the calculation of the EC50 value and, as a consequence, the comparison of radical scavenging activities measured in terms of EC50, using different DPPH concentrations, may be untrustworthy. 3.5. Effect of DPPH concentration on the TEC50 (time at the steady state) Brand-Williams et al. (1995) classified the kinetics of reaction of the examined antioxidants with DPPH, as rapid, intermediate or

slow. Sa´nchez-Moreno et al. (1998) on the basis of the TEC50, classified the antioxidants as rapid (<5 min), intermediate (5– 30 min) and slow (>30 min). According to these authors, the time necessary to reach the steady state of the reaction depends on the antioxidant concentration, but no indication is provided about the relationship between the time at endpoint conditions and the DPPH concentration. The influence of the DPPH concentration on the time at the steady state was studied during the reaction of DPPH with BHT or Java green tea. Fig. 4 shows the evolution of absorbance over time during 60 min of DPPH/BHT (Fig. 4A) and DPPH/Java green tea (Fig. 4C) reaction carried out with increasing amounts of DPPH, but maintaining a constant ratio of antioxidant to DPPH. In Fig. 4A and C all measurements were reported to the same DPPH concentration. The figure clearly shows the

Fig. 2. Time course evolution of the absorbance, measured at 517 nm, in reaction mixtures with DPPH (5  105 M) and different concentrations of pure compounds. (A) L-Ascorbic acid: 6.8; 11.7; 16.7; 21.67; 26.04 mM; (B) gallic acid: 1.40; 3.41; 5.4; 7.41; 9.4 mM; (C) epigallocatechin gallate: 1.65; 3.61; 5.6; 7.58; 9.56 mM.

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Fig. 3. Percentage of remaining DPPH during the reaction of DPPH with 2,6-di-tert-butyl-4-methylphenol (BHT) (A) and Java green tea (B). Analyses were performed varying the DPPH concentration in the range 25–200 mM, while keeping the DPPH/antioxidant ratio corresponding to EC50.

dependence of TEC50 on DPPH concentration. When high DPPH concentrations are used, the reaction with the antioxidant is delayed (it is worth stressing that the antioxidant/DPPH ratio was kept constant). This relationship is even more evident with Java green tea samples (Fig. 4C). In this case, when a low concentration of DPPH is used (2.5  105 M), the TEC50 value for Java green tea becomes 28 min, whereas it increases to 87 min when a higher concentration is used (5  105 M). Since all the

samples contain a BHT or Java green tea concentration corresponding to EC50, this means that TEC50 depends on the initial DPPH concentration used in the experiments. In fact if a low DPPH concentration is used, the flattening of the curve will be reached earlier, while using high DPPH concentrations will delay the reaching the steady state plateau. To highlight the dependence of the reaction rate on the DPPH concentration, the degradation rate, as defined in the experimental

Fig. 4. Dependence of the TEC50 on the DPPH concentration when studying the kinetics of reaction of DPPH with 2,6-di-tert-butyl-4-methylphenol (BHT) (A) and Java green tea (C) in samples with the same antioxidant/DPPH ratio but with DPPH concentrations in the range 25–200 mM. For a better comparison, all measurements were reported to the same DPPH concentration. The effect of the DPPH concentration on the degradation rate is depicted for BHT (B) and Java green tea (D).

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section, was calculated for the five samples containing different DPPH concentrations reported in Fig. 3 for both BHT and Java green tea. In the case of BHT, the degradation rate is dependent on the DPPH concentration (see Fig. 4B); the dependence is not linear in the concentration range 25–200 mM, but two distinct slopes can be clearly distinguished with a sharp change at DPPH concentration ca. 80 mM. In the case of Java green tea (see Fig. 4D) at concentrations lower than ca. 60 mM the degradation rate increases with increasing the DPPH concentration, while at higher values stops to increase and becomes almost constant. These changes in the degradation rate affect both the % of DPPH remaining and the value of the time needed to reach the end of the reaction (TEC50). Changes in degradation rate as a function of the DPPH concentration are observed for both BHT and Java green tea despite the first is a pure compound while the second is a mixture of different antioxidants. Therefore changes in degradation rate can be observed irrespective of the chemical composition of the sample. Finally the parameter TEC50 is dependent on the DPPH concentration, and to be used in a meaningful way it has to be correlated not only with the antioxidant concentration but also with the initial amount of DPPH. 4. Conclusions The results presented in this work further confirm, despite its extensive use, the need to standardize the DPPH method. The estimation of the antioxidant activity may be strongly influenced by the kinetic behavior of the samples (extracts or standards). For samples or standards that display a fast reaction with DPPH, like lemon juice and L-ascorbic acid, the estimation of the antioxidant activity is only weakly influenced by the reaction time. By contrast, the choice of a proper reaction time is extremely important for those samples with a slow kinetic behavior, like essential oils. In this paper it was also demonstrated that the kinetics of the DPPH/ antioxidant reaction, and consequently the calculation of the EC50 value, also depend on the initial DPPH concentration. For these reasons, it would be wise to perform some preliminary measurements to estimate the proper conditions in terms of time necessary to reach the end of the reaction and of the DPPH concentration. In conclusion, all the parameters (initial DPPH concentration, reaction time depending on the examined sample) in the protocol of the DPPH assay should be strictly defined in order to have directly comparable results from different laboratories. In fact, simplifications of the method are in some cases counter-productive because while on the one hand they make the experimental work easier, on the other hand they provide experimental results that are not really comparable. References Abad-Garcı´a, B., Berrueta, L.A., Garmo´n-Lobato, S., Gallo, B., Vicente, F., 2009. A general analytical strategy for the characterization of phenolic compounds in fruit juices by high-performance liquid chromatography with diode array detection coupled to electrospray ionization and triple quadrupole mass spectrometry. J. Chromatography A 1216 (28), 5398–5415. Ahmad, N., Fazal, H., Ahmad, I., Abbasi, B.H., 2012. Free radical scavenging (DPPH) potential in nine Mentha species. Toxicol. Ind. Health 28 (1), 83–89. Atoui, A.K., Mansouri, A., Boskou, G., Kefalas, P., 2005. Tea and herbal infusions: their antioxidant activity and phenolic profile. Food Chem. 89 (1), 27–36. Brand-Williams, W., Cuvelier, M.E., Berset, C., 1995. Use of a free radical method to evaluate antioxidant activity. LWT Food Sci. Technol. 28 (1), 25–30. Dawidowicz, A.L., Olszowy, M., 2012. Mechanism change in estimating of antioxidant activity of phenolic compounds. Talanta 97, 312–317. Dawidowicz, A.L., Wianowska, D., Olszowy, M., 2012. On practical problems in estimation of antioxidant activity of compounds by DPPH method (problems in estimation of antioxidant activity). Food Chem. 131 (3), 1037–1043.

119

4th ed.Council of Europe, Strasbourg Cedex, France, pp. 183–184 2.8.12. Fischer, U.A., Carle, R., Kammerer, D.R., 2011. Identification and quantification of phenolic compounds from pomegranate (Punica granatum L.) peel, mesocarp, aril and differently produced juices by HPLC-DAD–ESI/MSn. Food Chem. 127 (2), 807–821. Gardner, P.T., White, T.A.C., McPhail, D.B., Duthie, G.G., 2000. The relative contributions of vitamin C, carotenoids and phenolics to the antioxidant potential of fruit juices. Food Chem. 68 (4), 471–474. Guimara˜es, R., Barros, L., Barreira, J.C.M., Sousa, M.J., Carvalho, A.M., Ferreira, I.C.F.R., 2010. Targeting excessive free radicals with peels and juices of citrus fruits: grapefruit, lemon, lime and orange. Food Chem. Toxicol. 48 (1), 99–106. Huang, D., Ou, B., Prior, R.L., 2005. The chemistry behind antioxidant capacity assays. J. Agric. Food Chem. 53 (6), 1841–1856. Karadag, A., Ozcelik, B., Saner, S., 2009. Review of methods to determine antioxidant capacities. Food Anal. Methods 2 (1), 41–60. Kim, D.-O., Chun, O.K., Kim, Y.J., Moon, H.-Y., Lee, C.Y., 2003. Quantification of polyphenolics and their antioxidant capacity in fresh plums. J. Agric. Food Chem. 51 (22), 6509–6515. Lau, F.C., Shukitt-Hale, B., Joseph, J.A., 2005. The beneficial effects of fruit polyphenols on brain aging. Neurobiol. Aging 26 (Suppl. 1), 128–132. Lee, H.S., Coates, G.A., 1999. Vitamin C in frozen, fresh squeezed, unpasteurized, polyethylene-bottled orange juice: a storage study. Food Chem. 65 (2), 165–168. Lee, J., Durst, R.W., Wrolstad, R.E., 2005. Determination of total monomeric anthocyanin pigment content of fruit juices, beverages, natural colorants, and wines by the pH differential method: collaborative study. J. AOAC Int. 88 (5), 1269–1278. Leong, L.P., Shui, G., 2002. An investigation of antioxidant capacity of fruits in Singapore markets. Food Chem. 76 (1), 69–75. Magalha˜es, L.M., Barreiros, L., Maia, M.A., Reis, S., Segundo, M.A., 2012. Rapid assessment of endpoint antioxidant capacity of red wines through microchemical methods using a kinetic matching approach. Talanta 97 (0), 473–483. Mishra, K., Ojha, H., Chaudhury, N.K., 2012. Estimation of antiradical properties of antioxidants using DPPH assay: a critical review and results. Food Chem. 130 (4), 1036–1043. Perez-Vizcaino, F., Duarte, J., 2010. Flavonols and cardiovascular disease. Mol. Asp. Med. 31 (6), 478–494. Prior, R.L., Wu, X., Schaich, K., 2005. Standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements. J. Agric. Food Chem. 53 (10), 4290–4302. Ratty, A.K., Sunamoto, J., Das, N.P., 1988. Interaction of flavonoids with 1,1-diphenyl-2-picrylhydrazyl free radical, liposomal membranes and soybean lipoxygenase-1. Biochem. Pharmacol. 37 (6), 989–995. Sa´nchez-Moreno, C., Larrauri, J.A., Saura-Calixto, F., 1998. A procedure to measure the antiradical efficiency of polyphenols. J. Sci. Food Agric. 76 (2), 270–276. Sanders, L.H., Greenamyre, J.T., 2013. Oxidative damage to macromolecules in human Parkinson disease and the rotenone model. Free Radic. Biol. Med. 62, 111–120. Sanna, D., Delogu, G., Mulas, M., Schirra, M., Fadda, A., 2012. Determination of free radical scavenging activity of plant extracts through DPPH Assay: an EPR and UV–vis study. Food Anal. Methods 5 (4), 759–766. Schirra, M., Palma, A., D’Aquino, S., Angioni, A., Minello, E.V., Melis, M., Cabras, P., 2007. Influence of postharvest hot water treatment on nutritional and functional properties of Kumquat (Fortunella japonica Lour. Swingle Cv. Ovale) fruit. J. Agric. Food Chem. 56 (2), 455–460. Sendra, J., Sentandreu, E., Navarro, J., 2006. Reduction kinetics of the free stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) for determination of the antiradical activity of citrus juices. Eur. Food Res. Technol. 223 (5), 615–624. Sentandreu, E., Izquierdo, L., Sendra, J., 2007. Total, cumulative fast-kinetics and cumulative slow-kinetics antiradical activities of juices from clementine (Citrus clementina), clementine-hybrids and satsuma (Citrus unshiu) cultivars and their utility as discriminant variables. Eur. Food Res. Technol. 225 (2), 271–278. Singleton, V.L., Rossi, J.A., 1965. Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. Am. J. Enol. Viticul. 16 (3), 144–158. ˇ , V., 2006. Determination of total content of phenolic Stratil, P., Klejdus, B., Kuba´n compounds and their antioxidant activity in vegetables. evaluation of spectrophotometric methods. J. Agric. Food Chem. 54 (3), 607–616. Suja, K.P., Jayalekshmy, A., Arumughan, C., 2004. Free radical scavenging behavior of antioxidant compounds of sesame (Sesamum indicum L.) in DPPH system. J. Agric. Food Chem. 52 (4), 912–915. Surveswaran, S., Cai, Y.-Z., Corke, H., Sun, M., 2007. Systematic evaluation of natural phenolic antioxidants from 133 Indian medicinal plants. Food Chem. 102 (3), 938–953. Tounsi, M.S., Wannes, W.A., Ouerghemmi, I., Jegham, S., Njima, Y.B., Hamdaoui, G., Zemni, H., Marzouk, B., 2011. Juice components and antioxidant capacity of four Tunisian citrus varieties. J. Sci. Food Agric. 91 (1), 142–151. ˜ o, D., Ferna´ndez-Pacho´n, M.S., Moya´, M.L., Troncoso, A.M., Garcı´a-Parrilla, Villan M.C., 2007. Radical scavenging ability of polyphenolic compounds towards DPPH free radical. Talanta 71 (1), 230–235. Wang, C.-H., Wu, S.-B., Wu, Y.-T., Wei, Y.-H., 2013. Oxidative stress response elicited by mitochondrial dysfunction: implication in the pathophysiology of aging. Exp. Biol. Med. 238 (5), 450–460. Wu, C., Xu, H., He´ritier, J., Andlauer, W., 2012. Determination of catechins and flavonol glycosides in Chinese tea varieties. Food Chem. 132 (1), 144–149.