Selective Fluorescent Probe - ACS Publications - American Chemical

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Nitrogen Dioxide Absorbance Capacity of Flavanols Quantified by a NO2‑Selective Fluorescent Probe Yan Yan,† Chee Kian Tan,† Haixia Wu,† Suhua Wang,*,‡ and Dejian Huang*,†,§ †

Food Science and Technology Programme, Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore 117543, Republic of Singapore ‡ Institute of Intelligent Machines, Chinese Academy of Sciences, Hefei, Anhui 230031, People’s Republic of China § National University of Singapore (Suzhou) Research Institute, 377 Lin Quan Street, Suzhou Industrial Park, Jiangsu 215123, People’s Republic of China S Supporting Information *

ABSTRACT: Taking advantage of a nitrogen dioxide (NO2)-selective probe reported by our group previously, we have developed a fluorescent assay for quantifying NO2 absorbance capacity of flavanols and related polyphenolic compounds. A nonfluorescent NiII dithiocarbamate complex containing sulforhodamine fluorophore reacted rapidly and selectively with NO2 and turned on the fluorescence of the rhodamine. In the presence of radical scavengers, such as tea catechins, the rates of the fluorescence turning on by NO2 were suppressed in a dose-dependent manner. When a simple kinetic equation of the initial reaction rates is applied, the rate constants of the antioxidant reaction with NO2 can be derived using epicatechin as a reference standard, and the value is comparable to that obtained by the pulse radiolysis method. The scavenging capacity against NO2 of nine common phenolic compounds was evaluated, and their structure−activity relationship was also established. Additionally, the mechanism behind NO2 scavenging by phenolic compounds was determined by liquid chromatography−mass spectrometry and secondary mass, using epicatechin and gallic acid as examples. Our assay serves as the first example for convenient and sensitive quantification of NO2 scavenging activity of antioxidants. KEYWORDS: nitrogen dioxide, flavanols, fluorescent probe, reactive nitrogen species, rate constant

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INTRODUCTION Reactive oxygen/nitrogen species (ROS/RNS) may result in inflammation, cardiovascular disease, cancer, and aging-related disorders.1,2 Dietary antioxidants, including phenolic compounds, vitamins E and C, and carotenoids, are believed to be nutrients effective in the prevention of these oxidative stressrelated diseases by sacrificially scavenging ROS/RNS.3−5 The protective effects of antioxidants have received increasing attention within the general public and biological, medical, nutritional, and agrochemical fields, resulting in the requirement of simple, convenient, and reliable antioxidant capacity determination methods.6 Despite the numerous published methods in measuring antioxidant capacity, no one assay can give quantification of the total antioxidant capacity, because of the highly dynamic and complex reactions between antioxidants and different ROS/RNS.7 Among the RNS, nitrogen dioxide (NO2) is a major air pollutant generated from high-temperature oxidation of molecular nitrogen and oxygen in the combustion process.8 Whereas in a biological system, NO2 is implicated as the root cause of toxic effects of RNS derived from in vivo peroxynitrite decomposition,9 oxidation of nitric oxide (NO), and one electron oxidation of nitrite by horseradish peroxidase (Scheme 1).10 Being a strong lipophilic, air-stable, and persistent radical oxidant, NO2 is known to trigger lipid auto-oxidation, which would cause cell-membrane damage and oxidative nitration of aromatic amino acids, particularly tyrosine.11 Dietary antioxidants can counteract NO2-caused oxidative stress. To assess © 2014 American Chemical Society

Scheme 1

the antioxidant activity in scavenging NO2, a valid assay would thus be a valuable tool to guide selection of diets that are potentially a rich source of NO2 scavengers. However, despite many papers reporting new methods for quantification of antioxidant capacity,12,13 there is no such assay reported. On the other hand, NO scavenging activity of antioxidants has been reported rather frequently in the literature since 1996.14 Because NO converts rapidly to NO2 under aerobic conditions, it is difficult to distinguish which radicals (NO or NO2) the antioxidants actually reacted with without control experiments. Moreover, NO is supposed to have beneficial effects on human health.15 Its removal by antioxidants may lead to detrimental effects to health. On the contrary, NO2 is a strong radical oxidant and known only to cause damage on biomolecules and human health.16 However, there is no report on methods for quantifying NO2 scavenging activity of dietary antioxidants. Received: Revised: Accepted: Published: 5253

January 18, 2014 May 6, 2014 May 15, 2014 May 15, 2014 dx.doi.org/10.1021/jf5001925 | J. Agric. Food Chem. 2014, 62, 5253−5258

Journal of Agricultural and Food Chemistry

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the resulting reaction solution, the fluorescent probe concentration is 3.0 μM, the DEANO concentration is 0.80 mM, the phosphate buffer concentration is 1.0 mM, and the methanol concentration is 80%. Fluorescence intensity of each well was immediately monitored upon addition of DEANO solution every minute for 15 min with excitation wavelength of 540 nm and emission wavelength of 590 nm at 37 °C. Before each reading, the microplate was shaken for 4 s at low intensity to ensure sample homogeneity. Methanol control (20 μL) was conducted in parallel for each of the samples on the same microplate as a blank. Data Processing (NO2 AC Value). With a half life of 2 min, DEANO decomposes to give NO when dissolved in psychological pH medium at 37 °C.18 Under aerobic ambient conditions, NO undergoes autoxidation, generating NO2.9,19,20

Herein, we reported the first fluorometric assay for NO2 absorbance capacity (abbreviated as NO2 AC) of antioxidants by taking advantage of the novel NO2-selective probe reported by our group previously, using diethylamine NONOate (DEANO) as the NO2 source.17 The chemical mechanisms behind NO2 scavenging by phenolic compounds were deduced from the reaction products formed as revealed by liquid chromatography−mass spectrometry (LC−MS) and secondary mass, using epicatechin (EC) and gallic acid as examples. Through measuring the antioxidant capacity of flavanols and related polyphenolic compounds as well as characterization of the reaction products, the structure−activity relationship was established, which provides a rational explanation on the good correlation of peroxyl radical scavenging activity and NO2 scavenging activity.

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2 •NO + O2 → 2 •NO2

(1)

NO2 produced will either react with the fluorescent probe (P) or be scavenged by an antioxidant (AH). There exists competition between them for the constant flux of NO2 (Ri).21

EXPERIMENTAL SECTION

Materials and Instruments. All experiments were performed with reagents of analytical grade or high-performance liquid chromatography (HPLC) grade. Chemicals were purchased from Sigma-Aldrich Chemical Co. (Singapore) and used without further purification. A NO2-selective fluorescent probe, a sulforhodamine-B-containing nickel(II) dithiocarbamate complex, was prepared according to our previous reported method.17 Fluorescence measurements were gathered using a Synergy HT microplate fluorescence reader from Bio-Tek Instruments, Inc. (Winooski, VT), installed with KC4 software. The fluorescence spectrum was obtained on a PerkinElmer LS55 fluorometer with a slit width at 10 nm for both excitation and emission. Ultraviolet−visible (UV−vis) spectroscopic studies were obtained on a Shimadzu UV-1601 UV−vis spectrophotometer accompanied by a quartz cell and installed with UVProbe software. HPLC analysis was performed on a Waters 2695 HPLC system equipped with an Alliance 2659 separation module and a Waters 2996 photodiode array (PDA) detector (Milford, MA) and installed with an Empower program. A C18 reverse-phase column (Waters Atlantis T3, 6 μm, 4.6 × 150 mm) was used throughout this study. The photodiode array (PDA) acquisition wavelength range was set at 210−700 nm. LC−MS spectra were acquired using a Bruker Amazon ion trap mass spectrometer (Billerica, MA) equipped with an electrospray ionization (ESI) source and a Dionex ultimate 3000RS HPLC system (Bannockburn, IL), installed with Chameleon, Hystar, and Trapcontrol software. The heated capillary and spray voltage were maintained at 200 °C and 4.5 kV, respectively. Nitrogen was operated at 80 psi for sheath gas flow rate and 20 psi for auxiliary gas flow rate. The full-scan mass spectra from m/z 70 to 1000 were obtained in negative-ion mode with a scan speed of 1 scan per second. Construction of the Dose Response Curve for DEANO. NO2 probe stock solution was diluted in methanol to a final concentration of 3 μM. A series of concentrations of DEANO (0, 200, 400, 500, 600, 700, 800, 900, and 1000 μM) was obtained by dilution with sodium hydroxide solution and mixed with phosphate buffer and probe solutions in a ratio of 1:1:8 to a final volume of 3.0 mL. The fluorescence intensities were measured 5 min after the addition of DEANO stock solutions on the fluorescent spectrometer (see Figure S2A of the Supporting Information). The data in Figure S2B of the Supporting Information was obtained using the microplate reader. Preparations of reagents followed the above procedure but were mixed to a final volume of 200 μL in each well of the 96-well microplate. Fluorescence intensities were monitored over a period of 10 min. Experimental Procedure for Nitrogen Dioxide Radical Absorbance Capacity Assay (NO2 AC). NO2 probe solution (4.3 μM) was prepared in methanol, and DEANO (8.0 mM) was completely dissolved in sodium hydroxide solution (10 mM, pH 12) and kept in an ice bath. Stock solutions of phenolic compounds were diluted with methanol to 10, 20, 30, and 40 μM working solutions. Fluorescent probe (140 μL), sample (20 μL in methanol), phosphate buffer (10 mM, 20 μL), and DEANO solution (20 μL) were pipetted and mixed in individual wells of a 96-well flat-bottom microplate. In

kp

P + •NO2 → fluorescent products kAH

AH + •NO2 ⎯⎯→ A• + NO2− + H+

(2) (3)

The radical A• can further react with NO2 to form an adduct, as we have shown in the mechanistic study (vide inf ra). The radical coupling reaction is not likely to be a rate-limiting step. For the initial reaction, reaction 3 is dominant because the antioxidant concentration is at its highest. Therefore, the rate of probe oxidation can be expressed by the following equation:

V=−

k p[P]R i d[P] = dt k p[P] + kAH[AH]

(4)

In the absence of antioxidant ([AH] = 0), the uninhibited reaction rate is

V0 = R i

(5)

From eqs 4 and 5, the following relation between the fluorescence increase rates in the absence (V0) and presence (V) of an antioxidant can be drawn

V0 k [AH] = 1 + AH V k p[P]

(6)

The plot of V0/V against [AH] will yield a linear function with an intercept at (0, 1) and a slope of kAH/kp[P]. With reference to Figure 1A, a tangent at the second minute was plotted on each kinetic curve to obtain the initial fluorescence increase rates for each concentration. The V0/V values are plotted against their corresponding [AH], as expressed in Figure 1B. Under the experimental conditions, the probe concentration [P] is constant at the initial reaction stage; thus, kp[P] is a constant. Therefore, the kAH value can be obtained from the slope and is the NO2 AC value as kAH = SAHk p[P]

(7)

where SAH is the slope for the antioxidant (AH). However, to obtain the absolute values for kAH, kp needs to be determined. If a reference standard can be used, there is no need to obtain the absolute kAH values for comparison of relative NO2 scavenging activity. We selected one of the most common flavanols, EC, as a reference standard to compare to samples. Therefore SEC = kEC/(k p[P])

(8)

∴ kEC = SECk p[P]

(9)

Normalizing kAH with kEC, we have 5254

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60 to 0% in 60 min at a flow rate of 0.2 mL/min. The column was equilibrated to 60% A in 10 min prior to the next run. The above was also performed for the analysis of the reaction product from EC and NO. The LC conditions for LC−MS analysis were similar to those mentioned above, except with 5 μL of sample injected and detection wavelengths at 280 and 350 nm. The same procedure was used for analysis of product between gallic acid and NO2.

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RESULTS AND DISCUSSION Fluorescent Approach for Quantification of NO2 Scavenging Activity. Here, we present an accurate and highly efficient assay to assess antioxidant capacity against NO2, monitoring competitive reaction kinetics between an introduced antioxidant and probe. In this assay, we applied our newly developed, NO2-selective “turn on” fluorescent probe containing (dithiocarbamato)2NiII as a fluorescence quencher and NO2 reaction center.17 The fluorescence of the rhodamine fluorophore was quenched by (dithiocarbamato)2NiII via photo-induced electron transfer. The fluorescence “turn on” is caused by oxidation of the complex by NO2 that triggered release of oxidized ligands.17 This probe demonstrated good selectivity because NO2 is the only radical that can turn on the fluorescence, while other common ROS, such as ClO−, H2O2, ONOO − , O 2 − , NO, HO • , and ROO • , could not. 17 Furthermore, it is evident that this probe is photostable and the antioxidants examined will not influence the fluorescence (see Figure S1 of the Supporting Information); therefore, this probe was suitably used for monitoring the NO2 scavenging activity of antioxidants. DEANO was proven to be a good NO2 source in oxygenated solution to establish the dose−response curve for the NO2 fluorescent probe, yielding the identical products as NO2 gas after reacting with the probe.17 There exists a good linear relationship between the fluorescence intensity ratio and the concentration of DEANO (see Figure S2B of the Supporting Information), and 800 μM was selected for use in the assay. It should be pointed out that DEANO or other similar NO donors have been applied in the investigation of NO scavenging activity of polyphenolic compounds. However, we found that NO does not react with EC under air-free conditions. It is likely that the reaction is due to other RNS with polyphenolic compounds. Characterization of the reaction products showed that NO2 is the species responsible for the reaction. Our results highlighted the importance of eliminating oxygen when one intended to measure NO scavenging activity. We took advantage of the sensitive NO2 fluorescent probe as a reporting reaction in a competitive reaction system, where generated NO2 reacts with probe and antioxidants, which protect the probe from being oxidized, as shown by a reduced rate fluorescence intensity change (Figure 1A). The initial fluorescence increase rates in the presence of different amounts of antioxidants were determined from the plot. V0/V versus the antioxidant concentrations yield excellent linear curves (panels B and C of Figure 1). The slopes of the curves for different antioxidants were normalized with that of EC and shown in Table 1. The rate constants of the compounds with NO2 can be readily obtained by multiplying the NO2 AC values with kEC, which is quantified as 1 × 108 M−1 s−1.22 Therefore, our assay provides a rapid and sensitive method to quantify NO2 scavenging activity. Our results are in good agreement with the rate constants of epigallocatechin gallate (EGCG) and gallic acids measured using pulse radiolysis methods, which shown

Figure 1. (A) Increments of fluorescence intensity of the probe (3 μM) against time after the addition of DEANO (800 μM) and EC at different concentrations. (B) V0/V as a function of the concentration of EC. (C) Dose responses of V0/V versus different concentrations of selected antioxidants. (D) Molecular structure of the fluorescent probe that is selective to NO2 detection. NO2 AC value of AH =

SAHk p[P] kAH S = = AH kEC SECk p[P] SEC

(10)

On the basis of eq 10, kAH can be obtained because kEC was measured recently as 1.0 × 108 M−1 s−1 by a pulse radiolysis study.21,22 For convenience sake, epicatechin equivalency (ECE) is used to express the NO2 AC. For single compounds, the concentrations used were the same, and the NO2 AC values for polyphenolic compounds are dimensionless. The NO2 AC value for EC is defined as 1 because it is the reference. For samples that are composed of mixtures of antioxidants, the unit of NO2 AC will be micromoles of ECE per gram (for solid) or micromoles of EC equivalents per liter for liquid samples, such as fruit juices. Preparation of NO2 Gas. NO2 gas was prepared by adding concentrated nitric acid dropwise to copper wire and collected in a dry round-bottom flask with phosphorus pentoxide inside. The flask was sealed and kept in a dark and cool place. NO2 gas at certain concentrations was prepared by diluting pure NO2 with argon. Caution: The highly toxic NO2 gas should be used with extreme caution and handled only in relative small quantities in a wellventilated fume hood. Preparation of NO Gas and Its Reaction with EC. Reaction of sodium nitrite and ascorbic acid was reported to give NO gas.23 Sodium nitrite (2.87 g) was dissolved in deionized water, and ascorbic acid (3.67 g) were accurately weighed. EC (3 mg/mL) was prepared by dissolution in methanol. All reagents were placed in the airtight setup, and then the system was flushed with argon gas for 30 min to ensure an inert environment, so that there would be absence of NO autoxidation. Aqueous NaNO2 solution in the buret was then released in a dropwise manner to react with ascorbic acid placed in the roundbottom flask for generation of NO gas. Approximately 1 L of NO gas was generated and passed through concentrated NaOH (4 M) first to remove acidic gas before NO is bubbled to EC solution. There was no visible color change for the EC solution after 6 min. The resulting EC solution was flushed with argon for 0.5 h to remove any NO in the solution and analyzed by HPLC. HPLC and LC−MS Studies of the Reaction Product. The reaction products between NO2 and EC (2.0 mg/mL) in methanol were analyzed by HPLC with a detection wavelength from 210 to 700 nm. The mobile phases were water (solvent A) and methanol (solvent B). The sample (10 μL) was injected into the HPLC system with the column equilibrated with 60% solvent A. Gradient elution was used and programmed to obtain a linear increase in the solvent A ratio from 5255

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Table 1. NO2 AC Values (EC Equivalents) of Phenolic Compounds phenolic compounds

slope (kAH/kp[P])

EC (standard) ECG EGC EGCG phenol catechol pyrogallol gallic acid EAC

0.36 0.67 0.47 0.85 0.13 0.20 0.22 0.26 0.17

NO2 AC value (SAH/SEC) 1.00 1.83 1.29 2.33 0.35 0.54 0.62 0.73 0.47

± ± ± ± ± ± ± ± ±

0.08 0.10 0.04 0.03 0.03 0.02 0.01 0.02 0.05

that EGCG has a rate constant of 2.3 times larger than that of EC and the rate constant for gallic acid is half that of EC.22,22 Structure and Activity Relationship of NO2 Scavenging Activity of Flavanols. We have quantified the NO2 scavenging activity of five common flavanols, including epiafzelechin (EAC), EC, epigallocatechin (EGC), epicatechin gallate (ECG), and EGCG (Figure 2), and four compounds of

Figure 2. Structure of EGCG, indicating four ring numbers. The OH groups in red are responsible for its NO2 scavenging activity, while those in blue contribute little.

structural relevance to flavanols. They are phenol, catechol, pyrogallol, and gallic acid. From the data, a few observations are remarkable: (1) The scavenging potential of these nine compounds against the NO2 radical are in the order of phenol < EAC < catechol < pyrogallol < gallic acid < EC < EGC < ECG < EGCG. (2) For the flavanols, the one with the most phenolic hydroxyl groups exhibits the greatest scavenging activity. A similar trend is observed for phenol, catechol, and pyrogallol. (3) The flavanols are seen to exert a greater antioxidant effect than their model phenolic compounds, such as catechol and phenol. From these observations, we can conclude that the orthodihydroxyl groups are an important structural feature for high NO2 scavenging activity, while the meta-OH units in the A ring of the flavanols have little contribution in NO2 scavenging activity. The galloyl ester unit in EGC and EGCG further enhance the NO2 scavenging activity in comparison to EC and EGC, yet the addition of one more ortho-OH group in the B ring of flavanol has much less effect on NO2 scavenging activity (by comparing EC to EGC). Reaction Products and Mechanisms of Catechin/ Gallic Acid with NO2. It is commonly known that the B and D rings (galloyl) of the flavanols are responsible for their radical scavenging activity (Figure 2).22,24,25 To shed some light on the NO2 scavenging activity, we characterized the reaction products of NO2 with EC by HPLC and LC−MS/MS. The HPLC chromatograms of the reaction products are shown in Figure 3B. EC (Rt = 18.4 min) reacted with NO2 generated three isomeric products with a retention time at 29.8, 31.8, and

Figure 3. (A) HPLC profile of pure EC, recorded at 280 nm. (B) LC− MS LC profile of EC and its oxidized product with NO2, recorded at 350 nm. The ESI (anionic mode)−MS of the three products has m/z of 334, and they give the same MS2 pattern with major peaks at m/z of 124.8, 164.7, and 287.8. (C) HPLC profile of pure gallic acid, recorded at 280 nm. (D) LC−MS LC profile of gallic acid and its oxidized product with NO2, recorded at 350 nm. The ESI (anionic mode)−MS of the product has m/z of 169.7, and it gives the MS2 pattern with a peak at m/z of 124.2.

42.6 min, respectively, with m/z 334.0 ([M − H]−) and m/z 669.0 ([2M − H]−). Fragmentation of MS2 for m/z 334.0 gave rise to peaks with m/z of 287.8, 124.8, and 164.7. The m/z 287.8 peak corresponds to the loss of NO2 (molecular weight of 46) from the molecular ion, while the m/z 124.8 and m/z 164.7 ions were formed from the characteristic heterocyclic ring fragmentation (see Figure S3 of the Supporting Information).26,27 This fragmentation pattern indicates that NO2 is not attached in the A ring. The phenoxyl radical derived by hydrogen atom abstraction from the B-ring OH groups of EC is delocalized among three resonance structures.28 The combination of the radical with NO2 will lead to three isomers observed by LC−MS (Scheme 2). The nitrated B ring absorbs light at a longer wavelength at the visible light range as the solution color turned from colorless to light yellow, and the UV−vis spectra of the products gave rise to multiple bands centered at 350 nm 5256

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about twice that of EC (1.00 ± 0.08) and EGC (1.29 ± 0.04), respectively. This correlates well with ECG and EGCG having twice the number of catechol/pyrogallol moieties compared to EC and EGC, respectively. These results are in agreement with the notion that the NO2 scavenging effect is attributed to the ortho OH groups in the B and D rings.24 The bimolecular rate constants of NO2 and EC, EGCG, and gallic acid were quantified by a pulse radiolysis method to be 1.0 × 108, 2.3 × 108, and 5.3 × 107 M−1 s−1, respectively.22,21 The NO2 scavenging activity measured using our method is in good agreement with these values. Using reaction rate constants to determine NO2 AC values is accurate but technically complicated. It should be pointed out that the linearity range is not very large between the concentration of antioxidants and the rate changes. This may limit its application to food sample measurements when, particularly, their antioxidant activity is unknown. For application of this method to a large amount of samples, end point readings would be more convenient in providing IC50 for comparison of the NO2 scavenging activity of different samples. Under air-saturated conditions, NO is reported to react with oxygen as the dominant pathway to give NO2. The kinetic analysis of the NO oxidation reaction in the presence of thiols and amine was identical to that obtained for the autoxidation of NO.29 Nitrosylation of thiols only occurs under oxygenated conditions. Therefore, NO2 is responsible for the reaction. In agreement with this pioneering work, we have demonstrated that NO failed to react with EC even with 30 min at room temperature with a large excess of NO. In the presence of oxygen, EC is nitrosylated by NO2. Because of the strong oxidative power of NO2 and its relevancy of NO2 formation as a result of endogenously formed NO, the scavenging activity of NO2 (and not NO) is an important but neglected aspect of antioxidants. Our assay overcomes the technical hurdle to quantify the NO2 scavenging capacity of dietary antioxidants, including flavanols. Given the fact that there are a wide range of ROS/ RNS that are important to biological systems, different antioxidant capacity assays are needed to comprehensively evaluate antioxidant activity. These reactive oxygen species and their related scavenging capacity assays include peroxyl radicals (ROO•) in the ORACFL assay,30 hydroxyl radical (MII/H2O2) averting capacity assay,31 superoxide anion absorbance capacity assay (SORAC assay),32 NO2 AC, peroxynitrite (ONOO−) absorbance capacity assay, hypochlorous acid absorbance capacity assay, and singlet oxygen absorbance capacity assay. Because these ROS/RNS species have different reactivity patterns, it is essential to use different methods to quantify their scavenging activity. This is only possible with validated and convenient assays in place.

Scheme 2. Proposed Mechanism for NO2 Radical Scavenging by EC and Gallic Acida

a

R = moiety of A and C rings of EC.

and stretched to 400 nm (see Figure S4A of the Supporting Information). The reaction between gallic acid and NO2 gave rise to an unusual product with an UV−vis absorption maximum at 350 nm, indicating a NO2 adduct with phenyl (Figure 3D). The mass spectrum shows peaks at m/z 170 and 341, which fit that of nitrogallol (molecular weight of 171) and its dimer (molecular weight of 342). We proposed that the reaction happens via two steps (Scheme 2): first, hydrogen atom abstraction (or electron transfer followed by the loss of a proton) by NO2 resulted in a phenolic radical, and second, NO2 would attack carbon at the para position of the phenol radical to give a ketone intermediate. Subsequent decarboxylation resulted in the observed product, 4-nitrogallol. The molecular weight of the product is only 1 unit more than that of gallic acid and can be deceptive to assign its structure at the first sight. MS2 of gallic acid and that of nitrogallol give rise to the same fragment gallol anion at m/z 125. The structure of nitrogallol requires further confirmation by preparative-scale synthesis, infared spectroscopy, and X-ray crystallographic data. Overall, the mechanistic study using EC and gallic acid validate the conclusion made by several other researchers that the radical scavenging by catechin will produce a phenoxyl radical localized on the B or D ring (represented by gallic acid, a common moiety for the D ring).20,23,24 UV−vis spectroscopic analyses on the products between EC and NO2 and those of EC and DEANO under normal aerobic conditions produced the same spectra (see Figure S4A of the Supporting Information), showing product absorbance peaks from 300 to 400 nm. However, as a NO donor, DEANO does not release NO2 directly but releases NO, which is also a radical species. To figure out whether phenolic compounds scavenge NO2 directly or scavenge NO to prevent NO2 generation, UV− vis spectroscopic analyses for the reaction between EC and NO gas and that of EC with DEANO (under deaerated conditions) were performed. As demonstrated in Figure S4B of the Supporting Information, no change was found, in comparison to the EC spectra. This observation is reaffirmed by HPLC analysis (see Figure S5 of the Supporting Information), which exhibited only a single absorption peak for EC (Rt = 18 min), suggesting that there was no reaction between EC and NO. ECG and EGCG contain an additional gallate moiety (D ring) compared to EC and EGC, respectively. The gallate group enhanced the NO2 scavenging activity of ECG (NO2 AC value of 1.83 ± 0.10) and EGCG (NO2 AC value of 2.33 ± 0.03) by

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ASSOCIATED CONTENT

S Supporting Information *

Fluorescent probe (3 μM) demonstrating excellent photostability and antioxidants examined having little influence on fluorescence (Figure S1), (A) increments of the fluorescence intensity of the fluorescent probe (3 μM) after the addition of DEANO at different concentrations and (B) dose response of the fluorescence intensity of the fluorescent probe (3 μM) obtained 2 min after the addition of DEANO (Figure S2), ESI(−)−MS/MS fragmentation pattern for EC (Figure S3), (A) UV−vis spectrum showing the EC absorption peak at 280 nm and product peaks (300−400 nm) from the reaction of EC 5257

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Journal of Agricultural and Food Chemistry

Article

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with NO2 gas and (B) UV−vis spectrum showing only the EC absorption peak observed at 280 nm for the reaction between EC and NO gas or DEANO in degassed solution (Figure S4), and HPLC profile for the reaction mixture of EC with NO in degassed solution (Figure S5). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *Telephone: 65-6516-8821. Fax: 65-6775-7895. E-mail: [email protected]. Funding

Dejian Huang thanks the Agency of Science, Technology and Research (A*STAR) of Singapore for financial support (Grant 112 177 0036). Dejian Huang and Suhua Wang thank the National Natural Science Foundation of China for a grant (Grant 21228702). Notes

The authors declare no competing financial interest.

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REFERENCES

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dx.doi.org/10.1021/jf5001925 | J. Agric. Food Chem. 2014, 62, 5253−5258