Optimization of Microwave-Assisted Extraction of Polyphenols from

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Optimization of Microwave-Assisted Extraction of Polyphenols from Herbal Teas and Evaluation of Their in Vitro Hypochlorous Acid Scavenging Activity Burcu Bekdeşer, Nazan Durusoy, Mustafa Ö zyürek,* Kubilay Gücļ ü, and Reşat Apak Department of Chemistry, Faculty of Engineering, Istanbul University, Avcilar, 34320 Istanbul, Turkey ABSTRACT: Hypochlorous acid (HOCl) is an important reactive oxygen species (ROS) and non-radical and is taking part in physiological processes concerned with the defense of the organism, but there has been limited information regarding its scavenging by polyphenols. This study was designed to examine the HOCl scavenging activity of several polyphenols and microwave-assisted extracts of herbal teas. HOCl scavenging activity has usually been determined spectrophotometrically by a KI/taurine assay at 350 nm. Because some polyphenols (i.e., apigenin and chrysin) have a strong ultraviolet (UV) absorption in this range, their HOCl scavenging activity was alternatively determined without interference using resorcinol (1,3dihydroxybenzene) as a fluorogenic probe. In the present assay, HOCl induces the chlorination of resorcinol into its nonfluorescent products. Polyphenols as HOCl scavengers inhibit the chlorination of the probe by this species. Thus, the 25% inhibitive concentration (IC25) value of polyphenols was determined using the relative increase in fluorescence intensity of the resorcinol probe. The HOCl scavenging activities of the test compounds decreased in the order: epigallocatechin gallate > quercetin > gallic acid > rutin > catechin > kaempferol. The present study revealed that epigallocatechin gallate (IC25 = 0.1 μM) was the most effective scavenging agent. In addition to polyphenols, four herbal teas were evaluated for their HOCl activity using the resorcinol method. The proposed spectrofluorometric method was practical, rapid, and less open to interferences by absorbing substances in the range of 200−420 nm. The results hint to the possibility of polyphenols having beneficial effects in diseases, such as atherosclerosis, in which HOCl plays a pathogenic role. KEYWORDS: hypochlorous acid scavenging activity, spectrofluorometry, resorcinol probe, polyphenols, flavonoids, herbal teas, microwave-assisted extraction

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the formation of excess TNB.2,8 Because the protein carbonyl assay is affected by ROS other than HOCl,9 this assay cannot be taken as a specific indicator of HOCl. Polyphenols are plant secondary metabolites that exhibit a broad spectrum of biological and pharmacological activities. Flavonoids as simple natural polyphenols represent the most common and important subgroup of polyphenols.11 Flavonoids can effectively quench HOCl, but there is limited data in the literature on their HOCl scavenging activities.12 Therefore, the aim of the present work was to determine HOCl scavenging activities of polyphenols (flavanols, flavones, flavonols, flavanones, etc.) and herbal teas [green tea (Camellia sinensis), sage (Salvia officinalis), yarrow (Achillea millefolium), lady’s mantle (Alchemilla vulgaris)] using the spectrofluorometric resorcinol method. The most abundant phenolics present in green tea are flavon-3-ol derivatives (gallocatechin, epigallocatechin, catechin, epicatechin, epigallocatechingallate, and epicatechingallate), which play an important role in antioxidant activity.13 Sage contains flavonoids and phenolic acids. Among these substances, rosmarinic acid and luteolin, were reported as major phenolic constituents.14 The phenolic acids and flavonoids identified in yarrow were caffeic acid, ferulic acid, luteolin, and apigenin.15 Lady’s mantle contains flavonoids (i.e.,

INTRODUCTION Hypochlorous acid (HOCl) is an important reactive oxygen species (ROS) that causes oxidation and chlorination reactions.1 HOCl is produced by the myeloperoxidase enzymatic system in the presence of H2O2 and Cl− (H2O2 + MPO

Cl− + H+ ⎯⎯⎯⎯→ HOCl + H2O) or when commercial sodium hypochlorite solution is acidified by sulfuric acid to pH 6.2.2 The production of HOCl is part of the host defense mechanism against microorganisms, but under certain conditions, it can also destroy healthy tissues1 and is believed to be an important factor in progression of some diseases, such as atherosclerosis and rheumatoid arthritis.3,4 This non-specific, oxidizing, and chlorinating agent rapidly reacts with several biomolecules, causing damage to cell structures.5 Different analytical methods have been used to detect the HOCl scavenging activity.6 These methods are based on the inhibition of oxidation of the elastase inhibitor 1-antiproteinase,7 the prevention of 5-thio-2-nitrobenzoic acid (TNB) oxidation in the presence of scavengers,8 the generation of carbonyl groups in bovine serum albumin (BSA) by HOCl,9 and the inhibition of the oxidation of human serum albumin (HSA) and measurement of oxidation products by gel permeation high-performance liquid chromatography (HPLC) and polyacrylamide gel electrophoresis.10 However, there are some restrictions on the application of these methods. For instance, free thiol-containing scavengers, such as glutathione and cysteine, cause interference in the TNB assay because of © XXXX American Chemical Society

Received: June 27, 2014 Revised: September 19, 2014 Accepted: November 3, 2014

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dx.doi.org/10.1021/jf503065h | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry

Article

Methanol and 1.0% (v/v) acetic acid were used as the mobile phase for HPLC analysis. MAE of Herbal Teas. The ETHOS-One (Milestone, Shelton, CT) closed vessel oven system equipped with 12 Teflon [polytetrafluoroethylene (PTFE)] vessels and an automatic fiber optic temperature control system were used for MAEs. Extractions were performed at 500 W and 80 °C, and a magnetic stirring rod was added in each vessel. The cut herbal materials were extracted using the following method: a 10 mL ethanol (80%) solution was added to 0.5 g of dried sample powder placed in an inner vessel. The vessels were placed symmetrically in the oven system. The herbal material in EtOH was microwave-irradiated as follows: 3 min for heating to the desired temperature (80 °C), 3 min for balancing at 80 °C, and 5 min for cooling. All herbal extracts were diluted (1:100) with 80% ethanol. The ethanolic extracts were filtered through a filter paper and membrane media (0.45 μm) successively before analysis. Resorcinol Assay. The fluorometric resorcinol assay was performed as described by Ö zyürek et al.,21 with some modifications. The reaction mixture contained polyphenol solution or herbal extract at a proper concentration, 0.5 mL of 0.15 mM HOCl for starting the reaction, and water was added to make the final volume of 4.0 mL. After incubation for 10 min at 37 °C, 1.0 mL of resorcinol (50 μM) was added to the reaction mixture and incubated for 5 min. At the end of the incubation period, the fluorescence intensity of the remaining resorcinol was measured at 304 nm (λex = 277 nm). Maximal fluorescence reduction (i.e., intensity difference) of resorcinol took place under HOCl attack without antioxidants, and this difference was reduced in the presence of polyphenols, enabling the HOCl scavenging activity measurement. The HOCl scavenging activity was calculated with the aid of eq 1, where Io is the fluorescence intensity of the resorcinol probe initially (i.e., prior to HOCl attack) and I1 and I2 are the fluorescence intensities of the resorcinol probe after chlorination in the absence and presence of polyphenolic scavenger, respectively.

luteolin, quercetin, apigenin, rutin, and kaempferol), which are the constituents responsible for its free radical scavenging activity.16 Resorcinol is known as a fluorescent compound (molar fluorescence coefficient, ε = 1.59 × 107 M−1 cm−1), and its fluorescence intensity decreases as a result of its sequential chlorination with HOCl.17 Heasley et al. reported that the nonfluorescent monochloro- to dichloro- and to trichlororesorcinol products are formed.18 The HOCl scavenging activity of the tested polyphenols (as IC25 values) is directly linked to the decrease in resorcinol fluorescence under HOCl attack in the presence of polyphenols. The scavenging activity of the polyphenols was also measured with the KI/taurine assay chosen as a reference method.19 In this study, the HOCl scavenging activities of polyphenols (flavonoids, simple hydroxybenzoic and hydroxycinnamic acids, etc.) individually and in mixtures (e.g., in herbal teas) were determined with the developed method for the first time. In the preparation of herbal teas for analysis, the method of microwave extraction was used, having distinct advantages of reduced cost and time and increased reproducibility over classical extraction methods (such as Soxhlet extraction, liquid− liquid extraction, column chromatography, and preconcentration techniques). The results obtained with the developed method for polyphenolic compounds and herbal teas were statistically compared [using the analysis of variance (ANOVA) test] to those found by the reference methods KI/taurine and HPLC. As a result, the advantages of the optimized sample preparation (microwave extraction) and fluorometric determination (HOCl scavenging activity measurement) procedures were clearly stated.

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inhibition ratio (%) = 100[(I2 − I1)/(I0 − I1)]

MATERIALS AND METHODS

(1)

The IC25 values of the polyphenols were calculated using a plot of inhibition percentage as a function of Cscavenger. Because the fluorescence intensity of the reaction mixture was derived from the resorcinol probe alone, possible interferences from scavenger compounds or real sample constituents were absent. KI/Taurine Assay. HOCl scavenging activity of polyphenols and herbal materials was determined using the spectrophotometric KI/ taurine reference method described by Soobrattee et al.19 The reaction mixture contained 1.0 mL of pH 7.4 phosphate buffer, 0.5 mL of taurine, 0−2.0 mL of scavenger solution at a proper concentration, 0.2 mL of HOCl, and water added to make the final volume of 3.7 mL. After the formation of taurine chloramine, potassium iodide (0.5 mL) was added and absorbance was measured at 350 nm (ε = 2.29 × 104 M−1 s−1).22 The herbal tea extracts were used by diluting at a ratio of 1:100 so as not to yield any absorbance interference at 350 nm. The HOCl scavenging activity was calculated with respect to eq 2, where Ao and A are the absorbances of the reaction mixture in the absence and presence of polyphenol, respectively.

Reagents and Apparatus. The following chemical substances of analytical reagent grade were supplied from the corresponding sources: methanol (MeOH), ethanol (EtOH), quercetin (QR), kaempferol, myricetin, rutin (RT), chlorogenic acid (CHA), naringenin, luteolin (LT), apigenin, gallic acid, and taurine (2-aminoethanesulfonic acid) from Sigma (Taufkirchen, Germany), 4-chlororesorcinol, p-coumaric acid, chrysin, and rosmarinic acid from Aldrich (Taufkirchen, Germany), sodium hypochlorite (NaOCl) from Merck (Darmstadt, Germany), acetic acid and potassium iodide from Riedel-de Haën (Taufkirchen, Germany), and resorcinol, hesperetin, (+)-catechin (C), (−)-epicatechin (EC), (−)-epigallocatechin (EGC), (−)-epigallocatechin gallate (EGCG), and ellagic acid from Fluka (Buchs, Switzerland). Herbal material leaves (green tea, sage, yarrow, and alchemilla) were obtained from a local market. The fluorescence intensity and absorption measurements were performed using a Varian (Mulgrave, Victoria, Australia) Cary Eclipse model spectrofluorometer and a Cary 100 model ultraviolet−visible (UV−vis) spectrophotometer, respectively. The ETHOS-One (Milestone, Shelton, CT) closed vessel oven system was used for microwave-assisted extraction (MAE). Distilled water was supplied from Simpak1 Synergy purification system (Millipore, Billerica, MA). A Waters Breeze 2 model HPLC system (Milford, MA) consisting of a 2998 photodiode array detector and Empower PRO software was used for chromatographic separation, detection, and data acquisition. Preparation of Solutions. The HOCl solution was prepared daily from a commercial solution (0.5%, v/v) by acidification with diluted sulfuric acid to pH 6.2. This stock solution was diluted 10-fold, and its concentration was determined using constant molar absorptivity (ε = 100 L mol−1 cm−1) at 235 nm.20 The fluorogenic resorcinol probe, potassium iodide, and taurine solutions were prepared in distilled water at 50 μM, 2.0 M, and 0.15 M concentrations, respectively. The polyphenolic solutions were freshly prepared in absolute ethanol.

inhibition ratio (%) = 100[(Ao − A)/Ao]

(2)

HPLC Assay. A gradient elution system was used for the determination of the resorcinol probe. The initial composition of the mobile phase (50% A/50% B) was modified using a linear gradient to (slope of 1.0) 60% A/40% B at 5 min, 70% A/30% B at 8 min, 80% A/20% B at 11 min, and a final composition of 90% A/10% B at 14 min. The mobile phases A and B were methanol and 1% (v/v) acetic acid, respectively. The chromatograms were obtained using a reversephase Agilent Eclipse XDB C18 column (4.6 × 250 mm, 5 μm particle size), with the following parameters: Vsample, 20 μL; flow rate, 1.0 mL min−1; and λresorcinol, 275 nm. The retention time of 4-chlororesorcinol, a chlorination product, was found with the aid of a standard. Statistical Analysis. All data were entered in an Excel database and analyzed with SPSS software for Windows. The data obtained B



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from the resorcinol and KI/taurine assays were compared to the aid of the ANOVA test at a selected confidence level of 95%.23

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RESULTS AND DISCUSSION The HOCl scavenging activity of polyphenols (i.e., flavonoids and simple hydroxybenzoic and hydroxycinnamic acids) individually and in mixtures was evaluated by our recently developed resorcinol fluorometric method.21 In the preparation of herbal extracts for analysis, the method of microwave extraction was used, having distinct advantages of reduced cost and time and increased reproducibility over classical extraction methods (such as Soxhlet extraction, liquid−liquid extraction, column chromatography, and preconcentration techniques). While MAE enables the reduction of the extraction time (because of its fast and multiple extraction capability) and solvent volumes, sonication may require a longer time and larger solvent volumes and sometimes repeated extractions.24 The scavenging activity measurements using the resorcinol method were statistically compared to those obtained with KI/ taurine and HPLC. The fluorescence intensity of resorcinol was followed between 0 and 20 min (Figure 1) with and without scavenger

Figure 2. Fluorescence spectra of the resorcinol probe at varying scavenger (QR) concentrations [a, 10 μM resorcinol (alone); b, 8.0 μM QR; c, 6.0 μM QR; d, 4.0 μM QR; e, 2.0 μM QR; and f, 0 μM QR (reference)].

(Figure 3). Green tea was chosen as a representative sample for the optimization MAE technique. The effect of the extraction time (0−10 min), temperature (50−100 °C), type of solvent (ethanol, methanol, or water), composition of solvent (ethanol/water at 20, 40, 60, 80, and 100%), and liquid/solid ratio (10−40) on MAE efficiency were determined. The effect of the extraction temperature and time are shown in Figure 3a. It can be observed that increasing the temperature of the solvent from 50 to 100 °C significantly increased the extraction efficiency up to 3 min, because of the increase in solubility with the temperature.25 The inhibition percentage slightly decreased when the temperature and time were higher than 80 °C and 3 min, respectively. Therefore, 80 °C and 3 min were chosen as the optimal temperature and time for effective MAE. Figure 3b shows that the extraction of green tea was greatly influenced by the ethanol concentration in water. The inhibition percentage of green tea extract increased with an increase in the ethanol concentration up to 80% (v/v), beyond which a decrease in HOCl scavenging activity was observed. The possible reasons are (i) highest solubility of lipophilic and hydrophilic antioxidants at this solvent composition and (ii) expansion of the contact surface area with some amount of water.25 Therefore, 80% (v/v) ethanol concentration in water was chosen as optimal for effective MAE. Figure 3c shows that the maximum extraction efficiency was obtained using methanol among the pure solvents tested. However, when water was added to pure solvents, 80% ethanol (v/v) was the most appropriate leachant solvent for effective MAE, probably because of the maintenance of a lipophilic/ hydrophilic balance of dissolved antioxidants. Figure 3d shows the effect of the liquid/solid ratio on the MAE of green tea. The HOCl scavenging activity of green tea increased with an increase in the liquid/solid ratio up to 20:1 (mL g−1), beyond which the activity decreased because of unnecessary dilution of antioxidants. Therefore, the optimal liquid/solid ratio was determined as 20:1 (mL g−1) to reach a high extraction efficiency. Testing of Incubation Reaction Products with the Resorcinol Assay. It is apparent that neither QR nor chlorination products of QR (QR + HOCl) upon HOCl attack exhibit a fluorescence intensity at 350 nm (spectra e and f of Figure 4), meaning that QR competition with the resorcinol probe for HOCl can be followed simply by observing the changes in the resorcinol probe without interference from QR. In other words, the only constituent giving rise to a change

Figure 1. Fluorescence intensity as a function of time: (◆) resorcinol alone and resorcinol exposed to HOCl in the (▲) absence and (■) presence (3 μM QR) of a scavenger.

(i.e., QR). Standing under incubation conditions, the resorcinol probe did not lose its original fluorescence intensity, while upon HOCl oxidation, this intensity was greatly attenuated because of the formation of chlorination products. This fluorescence attenuation was decreased in the presence of an antioxidant polyphenol, such as QR, because of its competition with resorcinol for HOCl quenching. The optimal incubation time was chosen as 5 min, which was sufficient to reach a plateau in intensity (Figure 1). Figure 2 shows the fluorescence spectra of resorcinol with varying QR concentrations. The maximal decrease in fluorescence intensity of resorcinol was observed under HOCl exposure in the absence of a scavenger as a result of conversion to non-fluorescent products. In comparison to the behavior of the resorcinol probe under HOCl exposure, the fluorescence intensity at 304 nm rose with increasing scavenger concentration (2.0−8.0 μM QR). This fluorescence increase is related to the scavenging activity of the scavenger. HOCl scavenging activity can be calculated using this intensity difference of resorcinol recorded in the presence and absence of a scavenger at varying concentrations. Optimization of MAE Conditions. MAE conditions were optimized by measuring the HOCl scavenging activity (inhibition percentage) with the use of the resorcinol method C



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Figure 3. Effects of MAE conditions on the inhibition percentage of green tea extract: (a) effect of the extraction time and temperature, (b) effect of the ethanol composition, (c) effect of the solvent type, and (d) effect of the liquid/solid ratio (average of N = 3 measurements for each data point).

Table 1. HOCl Scavenging Activity of Polyphenols Determined by the Resorcinol and KI/Taurine Methods (N = 4 or 5)a

HOCl scavenger rosmarinic acid gallic acid 3-coumaric acid ellagic acid chlorogenic acid

Figure 4. Fluorescence spectra of the resorcinol probe: (a) 0.5 mM resorcinol (initial concentration), (b) resorcinol + QR, (c) resorcinol + QR + HOCl, (d) resorcinol + HOCl, (e) QR + HOCl, and (f) QR (λex = 277 nm).

(−)-epigallocatechin gallate quercetin apigenin chrysin (−)-epigallocatechin hesperetin naringenin rutin myricetin (−)-epicatechin (+)-catechin kaempferol luteolin

in fluorescence intensity in the system is the resorcinol probe (spectra a−d of Figure 4). Because QR scavenges some of the HOCl in the incubation system, the intensity increases (relative to that of the chlorinated probe) as a result of this competition with QR (spectra c of Figure 4). Analysis of the Results of Resorcinol and KI/Taurine Methods. The scavenging activity of polyphenols was evaluated by a fluorometric resorcinol assay recently developed in our laboratory.21 This method evaluates the activity of a scavenger to inhibit the hypochlorite-mediated chlorination of resorcinol, producing non-fluorescent products. The diminution of fluorescence intensity is related to the HOCl scavenging activity of the scavenger. The proposed method was compared to the spectrophotometric KI/taurine method in Table 1. Quercetin exhibits a high antioxidant 26 and radical scavenging activity,27 which depend upon the 5-hydroxy-4keto substitution in the A ring, the 2,3 double bond promoting conjugation between the rings, and 3′,4′-dihydroxy catechol substitution in the B ring. Gomes et al.28 reported that

IC25 value with respect to the resorcinol method (μM) Phenolic Acids 1.26 1.44 2.18 2.92 7.20 Flavonoids 0.10 1.19 1.22 1.36 1.41 1.58 1.73 2.05 2.29 2.34 2.52 2.64 3.56

IC25 value with respect to the KI/ taurine method (μM) NDb 2.52 ND 3.27 5.79 0.14 1.64 ND ND 2.58 12.14 12.19 4.69 2.27 2.47 2.49 4.33 ND

a

p = 0.05; Fexp = 3.788; Fcrit(table) = 4.747; and Fexp < Fcrit(table). bND = IC25 value within the studied concentration range could not be determined.

quercetin (IC50 = 1.1 μM) was the most effective scavenger of HOCl, followed by apigenin and chrysin with an equal IC50 (IC50 = 3.9 μM) and luteolin (IC50 = 7.1 μM). The IC25 values found with the fluorometric resorcinol method concur with this order of activities (Table 1). The 5,7-dihydroxylated A ring and C3−OH substitution contribute significantly to the HOCl D



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scavenging activity.28 When quercetin was subjected to HOCl, chlorination occurred only at two sites (C6 and C8) of the A ring. Binsack et al.29 detected the chlorination products of quercetin as 6-chloroquercetin and 6,8-dichloroquercetin by HPLC and nuclear magnetic resonance (NMR) techniques. The hierarchy for HOCl scavenging activity of the flavonol aglycones with respect to the resorcinol assay was quercetin > myricetin > kaempferol (Table 1). Soobrattee et al.19 reported that the antioxidant capacity of these flavonols is also connected to the number of hydroxyl groups present. The presence of a third hydroxyl group in the B ring at the C-5 position does not contribute to an extra effectiveness of myricetin compared to that of quercetin simply having an o-diphenolic structure. Firuzi et al. reported that rutin (quercetin 3-rutinoside; IC25 = 22.7 μM) has lower HOCl scavenging activity than its corresponding aglycone (quercetin; IC25 = 8.8 μM).30 This is supported by data in Table 1 that the glycosylation of flavonoids reduces their HOCl scavenging activity (IC25 values of rutin and quercetin were 2.05 and 1.19 μM, respectively). HOCl scavenging activities of flavan-3-ols using the resorcinol method were observed for epigallocatechin gallate (8 OH; 0.10 μM), epigallocatechin (6 OH; 1.41 μM), epicatechin (5 OH; 2.34 μM), and catechin (5 OH; 2.52 μM) in this order, with the values in parentheses showing the number of hydroxyl groups per molecule and the IC25 value, respectively. An increase of HOCl scavenging activities of flavan-3-ols correlated with the number of hydroxyl groups. Similarly, Soobrattee et al.19 reported that the antioxidant activities of flavan-3-ols decreased with a decrease in hydroxyl groups (EGCG > ECG > EGC > EC > C). Particularly, the three adjacent phenolic −OH groups on both the B ring (at 3′, 4′, and 5′ positions) and the gallate moiety of EGCG give a higher stability to the radical structure emerging from HOCl oxidation by increasing electron delocalization over the whole molecule.31,32 In our study, EGCG ranked as the strongest HOCl scavenger in both the resorcinol and KI/taurine assays (Table 1), but Soobrattee et al. surprisingly reported close HOCl scavenging activities for EGCG and EC using the KI/ taurine assay, despite the fact that epicatechin is devoid of odihydroxy substitution in the B ring.19 It has been reported in the literature that simultaneous light absorption of the probe and scavengers may give rise to problems in the KI/taurine spectrophotometric method. For example, the HOCl scavenging activity of the oxicam group non-steroidal anti-inflammatory drugs could not be determined with KI/taurine because of the strong absorption in the range of 200−420 nm.33 Similarly, because flavones have a strong absorption band (apigenin, 337 nm; chrysin, 313 nm) in this range,34 HOCl scavenging activity of flavones could not be evaluated by the KI/taurine method. The two-way ANOVA test was used for statistical analysis.23 ANOVA showed that the experimentally found results (consisting of 13 HOCl scavengers excluding the values for compounds that could not be evaluated by the KI/taurine method) with both methods were statistically alike at a 95% confidence level (Fexp = 3.788; Fcrit(table) = 4.747; and Fexp < Fcrit(table) at p = 0.05). Thus, the fluorometric resorcinol method was validated against the KI/taurine method for HOCl scavenging activity assessment of polyphenols. Analysis of the Results Obtained by Resorcinol and HPLC Methods. The initial and remaining amounts of resorcinol probe undergoing HOCl attack were determined by fluorometric and HPLC methods in the presence and

absence of a complex scavenger matrix (i.e., green tea extract). The retention times for resorcinol and its chlorination product 4-chlororesorcinol (detected at 275 nm) were 3.10 and 4.60 min, respectively. The chromatograms showed that more resorcinol remained in the reaction medium with increasing volumes of green tea extract because of less conversion to chlorination products. This situation can be clearly observed from the increasing peak heights of the resorcinol probe. For example, approximately 46.5% of resorcinol was converted into its chlorination products in the absence of a scavenger. In the presence of a potent scavenger solution, such as 0.5 and 1.0 mL volumes of green tea extract, the conversion ratio of resorcinol was considerably smaller (27.0 and 7.5%, respectively). Table 2 shows the concentration of the remaining resorcinol probe in the incubation mixture with respect to the resorcinol Table 2. Analysis of Resorcinol before and after Incubation with HOCl in the Presence and Absence of Green Tea Extract (Reference) Using Resorcinol and HPLC Methods (N = 3)a concentration of remaining resorcinol with respect to the resorcinol assay (mM)

mixture reference 0.3 mL of 0.5 mL of 0.8 mL of 1.0 mL of a

green green green green

tea tea tea tea

extract extract extract extract

1.03 1.10 1.38 1.57 1.78

± ± ± ± ±

0.05 0.05 0.03 0.05 0.08

concentration of remaining resorcinol with respect to the HPLC assay (mM) 1.07 1.19 1.46 1.68 1.85

± ± ± ± ±

0.05 0.03 0.02 0.04 0.06

[Resorcinol]FL = 1.03[resorcinol]HPLC + 0.03 (r = 0.9973).

and HPLC assays. The remaining resorcinol concentrations (mM) were calculated with the aid of the corresponding calibration curves and correlated well among themselves (r = 0.9973). Application of the Resorcinol Method to Some Herbal Extracts. The resorcinol assay was applied to herbal extracts, and the results were compared to the KI/taurine assay as inhibition percentage values. The obtained results demonstrate that green tea extract exhibited the highest HOCl scavenging activity by a large difference in both assays. This is not surprising in view of the fact that green tea is distinguished by its remarkable content of polyphenols, especially catechins, such as epicatechin, epigallocatechin, epicatechin gallate, and epigallocatechin gallate,35 all of which exhibited high HOCl scavenging activities with respect of the spectroflorometric resorcinol assay (Table 1). The results are also in accordance with those reported by Valentão et al.,36 indicating that green tea infusion exhibited higher HOCl scavenging activity than small centaury (Centaurium erythraea L.)-lyophilized infusion. The inhibition percentages measured with the resorcinol and KI/taurine methods are shown in a bar diagram (Figure 5). The hierarchy for HOCl scavenging activity of herbal teas with respect to the resorcinol assay was green tea > sage tea > yarrow > lady’s mantle. In comparison of the resorcinol and KI/taurine assays in the HOCl scavenging activity measurement, it was observed that the fluorometric resorcinol assay can be securely used without interference from polyphenols or herbal tea constituents, while real sample components or scavengers may show strong absorption in the 200−400 nm range, thereby potentially interfering with the KI/taurine assay. E



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Notes

The authors declare no competing financial interest.

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(1) Winterbourn, C. C.; Kettle, A. J. Biomarkers of myleperoxidasederived hypochlorous acid. Free Radical Biol. Med. 2000, 29, 403−409. (2) Magalhaes, L. M.; Segundo, M. A.; Reis, S.; Lima, J. L. F. C. Methodological aspects about in vitro evaluation of antioxidant properties. Anal. Chim. Acta 2008, 613, 1−19. (3) Zhang, R.; Brennan, M. L.; Shen, Z.; MacPherson, J. C.; Schmitt, D.; Molenda, C. E.; Hazen, S. L. Myeloperoxidase functions as a major enzymatic catalyst for initiation of lipid peroxidation at sites of inflammation. J. Biol. Chem. 2002, 277, 46116−46122. (4) Hawkins, C. L.; Davies, M. J. Degradation of hyaluronic acid, poly- and mono-saccharides, and model compounds by hypochlorite: evidence for radical intermediates and fragmentation. Free Radical Biol. Med. 1998, 24, 1396−1410. (5) Magalhaes, L. M.; Segundo, M. A.; Reis, S.; Lima, J. L. F. C.; Estela, J. M.; Cerda, V. Automatic in vitro determination of hypochlorous acid scavenging capacity exploiting multisyringe flow injection analysis and chemiluminescence. Anal. Chem. 2007, 79, 3933−3939. (6) Gatto, M. T.; Firuzi, O.; Agostino, R.; Grippa, E.; Borsò, A.; Spinelli, F.; Pavan, L.; Petrolati, M.; Petrucci, R.; Marrosu, G.; Saso, L. Development of a new assay for the screening of the hypochlorous acid scavengers based on reversed-phase high-performance liquid chromatography. Biomed. Chromatogr. 2002, 16, 404−411. (7) Haenen, G. R.; Bast, A. Scavenging of hypochlorous acid by lipoic acid. Biochem. Pharmacol. 1991, 42, 2244−2246. (8) Ching, T.-L.; de Jong, J.; Bast, A. A method for screening hypochlorous acid scavengers by inhibition of the oxidation of 5-thio2-nitrobenzoic acid: Application to anti-asthmatic drugs. Anal. Biochem. 1994, 218, 377−381. (9) Yan, L. J.; Traber, M. G.; Kobuchi, H.; Matsugo, S.; Tritschler, H. J.; Packer, L. Efficacy of hypochlorous acid scavengers in the prevention of protein carbonyl formation. Arch. Biochem. Biophys. 1996, 327, 330−334. (10) Grippa, E.; Pavone, F.; Gatto, M. T.; Petrucci, R.; Marrosu, G.; Silvestrini, B.; Saso, L. In vitro evaluation of antioxidant activity by electrophoresis and high performance liquid chromatography. Biochim. Biophys. Acta 2000, 1524, 171−177. (11) Bravo, L. Polyphenols: Chemistry, dietary sources, metabolism, and nutritional significance. Nutr. Rev. 1998, 56, 317−333. (12) de Groot, H.; Rauen, U. Tissue injury by reactive oxygen species and the protective effects of flavonoids. Fundam. Clin. Pharmacol. 1998, 12, 249−255. (13) Del Rio, D.; Stewart, A. J.; Mullen, W.; Burns, J.; Lean, M. E.; Brighenti, F.; Crozier, A. HPLC−MSn analysis of phenolic compounds and purine alkaloids in green and black tea. J. Agric. Food Chem. 2004, 52, 2807−2815. (14) Zheng, W.; Wang, S. Y. Antioxidant activity and phenolic compounds in selected herbs. J. Agric. Food Chem. 2001, 49, 5165− 5170. (15) Wojdyło, A.; Oszmiański, J.; Czemerys, R. Antioxidant activity and phenolic compounds in 32 selected herbs. Food Chem. 2007, 105, 940−949. (16) Smolyakova, I. M.; Andreeva, V. Yu.; Kalinkina, G. I.; Avdeenko, S. N.; Shchetinin, P. P. Development of extraction techniques and standardization methods for a common lady’s mantle (Alchemilla vulgaris) extract. Pharm. Chem. J. 2012, 45, 675−678. (17) Pistonesi, M. F.; Nezio, M. S. D.; Centurión, M. E.; Palomeque, M. E.; Lista, A. G.; Band, B. S. F. Determination of phenol, resorcinol and hydroquinone in air samples by synchronous fluorescence using partial least-squares (PLS). Talanta 2006, 69, 1265−1268. (18) Heasley, V. L.; Burns, M. D.; Kemalyan, N. A.; McKee, T. C.; Schroeter, H.; Teegarden, B. R.; Whitney, S. E.; Wershaw, R. L. Aqueous chlorination of resorcinol. Environ. Toxicol. Chem. 1989, 8, 1159−1163.

Figure 5. Inhibition percentage of some herbal teas (1:100 diluted extract) calculated with the resorcinol assay in comparison to the KI/ taurine assay.

Possible Advantages and Drawbacks. In principle, the proposed resorcinol assay may be susceptible to interferences, arising from the fact that the excitation and emission wavelengths fall in the UV region. However, no such interference has experimentally been observed in this study, because the possible scavengers, taken for analysis at relatively small concentrations compared to the probe, did not cause any change in the fluorescence intensity of resorcinol. ROS scavenging methods using fluorogenic probes generally provide a superiority of simplicity, selectivity, and sensitivity over other similar spectroscopic methods.37 The existing methods for quantifying HOCl scavenging activity have some limitations, such as possible UV absorption in the KI/taurine assay. The widely used TNB method, on the basis of the prevention of TNB oxidation in the presence of HOCl scavengers, may suffer from the extra capability of thiols to directly reduce 5,5′dithiobis-(2-nitrobenzoic acid) (DTNB) to TNB without involvement of any scavenger. The fluorometric resorcinol assay overcomes all of these disadvantages by rendering the relatively specific detection of HOCl and is reliably applied to scavenging activity estimation of polyphenols and herbal materials. In addition, the resorcinol probe is a right choice for the HOCl assay, because other common oxidants and radical species (except hydroxyl radicals) may not oxidize resorcinol by limitation of their standard redox potentials, thereby supporting selectivity of the proposed assay. This method has been successfully validated against reference assays (KI/taurine and HPLC).

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Telephone: +90-212-473-7070. Fax: +90-212-473-7180. Email: [email protected]. Funding

Nazan Durusoy thanks the Istanbul University Research Fund, Bilimsel Arastirma Projeleri (BAP) Yurutucu Sekreterligi, for the support given to her M.Sc. Thesis Project 29902 and the Institute of Pure and Applied Sciences, Istanbul University (Istanbul University Fen Bilimleri Enstitüsü), for the support given to her M.Sc. thesis work with the title “Development of a Novel Spectrofluorometric Method for the Hypochlorous Acid Scavenging Activity Measurement of Polyphenols”. The authors also extend their gratitude to the Turkish Scientific and Technical Research Council (TUBITAK) for the Research Project 112T372. F



Article

(19) Soobrattee, M. A.; Neergheen, V. S.; Luximon-Ramma, A.; Aruoma, O. I.; Bahorun, T. Phenolics as potential antioxidant therapeutic agents: Mechanism and actions. Mutat. Res. 2005, 579, 200−213. (20) Aruoma, O. I. Scavenging of hypochlorous acid by carvedilol and ebselen in vitro. Gen. Pharmacol. 1997, 28, 269−272. (21) Ö zyürek, M.; Bekdeşer, B.; Gücļ ü, K.; Apak, R. Resorcinol as a spectrofluorometric probe for the hypochlorous acid scavenging activity assay of biological samples. Anal. Chem. 2012, 84, 9529−9536. (22) Gressier, B.; Lebegue, N.; Brunet, C.; Luyckx, M.; Dine, T.; Cazin, M.; Cazin, J. C. Scavenging of reactive oxygen species by letosteine, a molecule with two blocked −SH groups. Pharm. World Sci. 1995, 17, 76−80. (23) Miller, J. C.; Miller, J. N. Statistics for Analytical Chemists, 3rd ed.; Ellis Horwood and Prentice Hall: New York, 1993. (24) Eskilsson, C. S.; Björklund, E. Analytical-scale microwaveassisted extraction. J. Chromatogr. A 2000, 902, 227−250. (25) Hemwimon, S.; Pavasant, P.; Shotipruk, A. Microwave-assisted extraction of antioxidative anthraquinones from roots of Morinda citrifolia. Sep. Purif. Technol. 2007, 54, 44−50. (26) Apak, R.; Gücļ ü, K.; Demirata, B.; Ö zyürek, M.; Ç elik, S. E.; Bektaşoğlu, B.; Berker, K. I.; Ö zyurt, D. Comparative evaluation of total antioxidant capacity assays applied to phenolic compounds, and the CUPRAC assay. Molecules 2007, 12, 1496−1547. (27) Seyoum, A.; Asres, K.; El-Fiky, F. K. Structure−radical scavenging activity relationships of flavonoids. Phytochemistry 2006, 67, 2058−2070. (28) Gomes, A.; Fernandes, E.; Silva, A. M. S.; Santos, C. M. M.; Pinto, D. C. G. A.; Cavaleiro, J. A. S.; Lima, J. L. F. C. 2Styrylchromones: Novel strong scavengers of reactive oxygen and nitrogen species. Bioorg. Med. Chem. 2007, 15, 6027−6036. (29) Binsack, R.; Boersma, B. J.; Patel, R. P.; Kirk, M.; White, C. R.; Darley-Usmar, V.; Barnes, S.; Zhou, F.; Parks, D. A. Enhanced antioxidant activity after chlorination of quercetin by hypochlorous acid. Alcohol.: Clin. Exp. Res. 2001, 25, 434−443. (30) Firuzi, O.; Mladěnka, P.; Petrucci, R.; Marrosu, G.; Saso, L. Hypochlorite scavenging activity of flavonoids. J. Pharm. Pharmacol. 2004, 56, 801−807. (31) Bors, W.; Heller, W.; Michel, C.; Saran, M. Flavonoids as antioxidants: Determination of radical-scavenging efficiencies. Methods Enzymol. 1990, 186, 343−355. (32) Plaza, M.; Pozzo, T.; Liu, J.; Ara, K. Z. G.; Turner, C.; Karlsson, E. N. Substituent effects on in vitro antioxidizing properties, stability, and solubility in flavonoids. J. Agric. Food Chem. 2014, 62, 3321−3333. (33) Van Antwerpen, P.; Dubois, J.; Gelbcke, M.; Neve, J. The reactions of oxicam and sulfoanilide non steroidal anti-inflammatory drugs with hypochlorous acid: Determination of the rate constants with an assay based on the competition with para-aminobenzoic acid chlorination and identification of some oxidation products. Free Radical Res. 2004, 38, 251−258. (34) Rice-Evans, C. A.; Miller, N. J.; Paganga, G. Structure− antioxidant activity relationships of flavonoids and phenolic acids. Free Radical Biol. Med. 1996, 20, 933−956. (35) Graham, H. N. Green tea composition, consumption, and polyphenol chemistry. Prev. Med. 1992, 21, 334−350. (36) Valentão, P.; Fernandes, E.; Carvalho, F.; Andrade, P. B.; Seabra, R. M.; Bastos, M. L. Hydroxyl radical and hypochlorous acid scavenging activity of small centaury (Centaurium erythraea) infusion. A comparative study with green tea (Camellia sinensis). Phytomedicine 2003, 10, 517−522. (37) Gomes, A.; Fernandes, E.; Lima, J. L. F. C. Fluorescence probes used for detection of reactive oxygen species. J. Biochem. Biophys. Methods 2005, 65, 45−80.

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Optimization of Microwave-Assisted Extraction of Polyphenols from

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