Dietary Flavonoids Scavenge Hypochlorous Acid via Chlorination on

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Functional Structure/Activity Relationships

Dietary Flavonoids Scavenge Hypochlorous Acid via Chlorination on A and C Rings as Primary Reaction Sites: Structure and Reactivity Relationship Xin Yang, Tian Wang, Jinlong Guo, Mingtai Sun, Ming Wah Wong, and Dejian Huang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06689 • Publication Date (Web): 22 Mar 2019 Downloaded from http://pubs.acs.org on March 24, 2019

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Journal of Agricultural and Food Chemistry

Dietary Flavonoids Scavenge Hypochlorous Acid via Chlorination on A and C Rings as Primary Reaction Sites: Structure and Reactivity Relationship

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Xin Yang,† Tian Wang,# Jinlong Guo,# Mingtai Sun,†,‡ Ming Wah Wong,*,# Dejian Huang*,†,§

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†Food

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gapore, 3 Science Drive 3, Singapore 117543, Republic of Singapore

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‡Institute

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#Department

Science and Technology Programme, Department of Chemistry, National University of Sin-

of Intelligent Machines, Chinese Academy of Sciences, Hefei, Anhui 230031, China of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore, 117543,

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Singapore

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§National

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Jiangsu 215123, China

University of Singapore (Suzhou) Research Institute, 377 Linquan Street, Suzhou,

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1 ACS Paragon Plus Environment


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ABSTRACT

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Dietary flavonoids are known as scavengers of reactive oxygen species such as hypochlorous acid.

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In spite of abundant scavenging capacity data reported, few reports have addressed the relation-

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ship between the scavenging capacity and structures of different flavonoids. We characterized the

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reaction products of five flavonoids (apigenin, quercetin, naringenin, ampelopsin and epicatechin)

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with hypochlorous acid and found that primary chlorination reaction occurred on A ring (C6 or

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C8) and/or C rings but not on B rings. Correlation of the hypochlorous acid scavenging capacity

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(IC50 values) and the structural features of flavonoids revealed that the hydroxyl groups in the A

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ring and B ring can enhance the scavenging capacity whereas the C(2)C(3) double bond has nega-

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tive impact to the HClO scavenging capacity. Combining the SAR analysis and chemical study, we

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proposed that reaction mechanism between flavonoids and HClO should be electrophilic substi-

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tution reaction. Density functional theory (DFT) results are in consistent with the selectivity of

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chlorination on the flavonoids. Our findings highlight the importance of considering specific re-

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active oxygen species when measuring radical scavenging capacity of dietary antioxidants.

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KEYWORDS: hypochlorous acid scavenging capacity, flavonoids, antioxidant, density functional theory, structure-activity relationship.


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INTRODUCTION

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Reactive oxygen species (ROS) in aerobic organisms play important roles in normal function of

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organisms yet overproduction of them may lead oxidative stress,1 which is a causative factor of

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several chronic diseases2-7 such as cancer, cardiovascular diseases,8-10 and aging itself.11-13 Among the

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various ROS, hypochlorous acid is a weak acid (pKa 7.5) with HClO and ClO- both present amount

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equal amount at physiological pH and can act as a weapon to deactivate pathogens in vitro and in

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vivo.14 Hypochlorous acid is formed by oxidation of chloride by hydrogen peroxide in the presence

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of myeloperoxidase (MPO) found in white blood cells such as neutrophils,15 monocytes, and macro-

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phages.16 Hypochlorous acid is a non-specific oxidizing agent that can react rapidly with a wide

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range of biomolecules including amino acids,17 proteins,18 carbohydrates,19 lipids,20 and DNA.21

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Phytochemicals that can scavenge reactive oxygen species have believed to be able to provide

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protective effects on oxidative stress and flavonoids are a major class of dietary antioxidants. Struc-

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ture-activity relationship (SAR) for the ROS/RNS scavenging capacity of flavonoids were investi-

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gated and it was suggested that increasing numbers of the phenolic groups enhance the antioxi-

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dant or prooxidant activity in certain cases.22 In addition, C(2), C(3)-double bond in C ring allows

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electron delocalization across three ring skeleton (A, B, C rings) and thus provides stabilization of

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the aryloxyl radicals formed upon reaction with ROS.23 In biological systems, there are wide range

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of reactive oxygen species, which have different reactivity patterns with dietary antioxidants. How-

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ever, there is little study on product characterizations of flavonoids and specific reactive oxygen

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species. Scavenging HClO is of special importance because of its relation to inflammation related

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tissue damage. Binsack and coworkers pioneered the chemical characterization of the reaction

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products of quercetin and rutin with HClO and found that the chlorination occurs at C6 and C8

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positions of A rings. It is remarkable that the chlorinated products are more potent antioxidants 3 ACS Paragon Plus Environment


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than the parent compounds.24 Furthermore, Boersma et al studied the reaction products of isofla-

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vones (genistein, biochanin-A, and daizein) and found that the chlorination occurred at A-ring.25

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To further systematically uncover the structure and reactivity relationship between 24 types of fla-

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vonoids and their scavenging capacity against individual reactive oxygen species. We measured the

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HClO scavenging capacity of characterized the reaction products of flavonoids with different rep-

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resentative structures (apigenin, quercetin, naringenin, ampelopsin and epicatechin) with hypo-

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chlorous acid.

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EXPERIMENTAL SECTION

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Materials and Instruments. Hypochlorous acid (15%) was obtained from Merck & Co., Inc and

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its concentration quantified using UV-VIS spectroscopic method. Fluorescein disodium were ob-

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tained from Aldrich (Singapore, Singapore). Flavonoids (7,8-dihydroxyflavone, baicalein, luteolin,

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scutellarein, wogonin, fisetin, kaempferol, morin, myricetin, quercetin, 3,3',4'-trihydroxyflavone,

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3,5,7,8,3',4'-hexahydroxyflavone, alpinetin, eriodictyol, liquiritigenin, hesperetin, naringenin, pi-

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nocembrin, ampelopsin, taxifolin, catechin, epicatechin, epigallocatechin, genistein) were ob-

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tained from Nanjing Plant Origin Biological Technology Co., Ltd. A Synergy HT microplate fluo-

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rescence reader (Bio-Tek Instruments, Inc., Winooski, VT) was used for measurement of fluores-

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cence with an excitation filter of 485 ± 20 nm and an emission filter of 530 ± 25 nm. The plate reader

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was controlled by software KC4 3.0 (revision 29). Sample dilution was accomplished by a Precision

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X automatic pipetting system managed by precision power software (version 1.0) (Bio-Tek Instru-

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ments, Inc.). The 96-well polystyrene microplates and the covers were purchased from VWR Inter-

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national Inc (Bridgeport, NJ). All aqueous solutions were prepared with 18.2 MΩ·cm ultrapure wa-

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ter obtained by a Millipore water purification system. 1H NMR spectra were measured using a

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Bruker AVANCE I 300 NMR spectrometer, and mass spectrometry was performed on a Thermo 4 ACS Paragon Plus Environment

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Scientific LCQ Fleet ion trap mass spectrometer in ES positive mode. Merck F254 silica gel-60

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plates were chose to do thin-layer chromatography (TLC) and silica gel-60 (230–400 mesh) was

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selected as solid phrases for column chromatography.

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All flavonoids were dissolved in DMF to make 5.0 mM stock solution for storage at freezer (-20

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oC).

They were diluted with 75 mM potassium phosphate buffer (pH 7.4) to 50 µM for measure-

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ment. Hypochlorous acid was prepared by adjusting the pH of a 12-15% (m/v) solution of NaClO

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to 6.2 with dropwise addition of 10% sulfuric acid. The concentration of HClO was further deter-

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mined spectrophotometrically at 235 nm using the molar absorption coefficient of 100 (M−1cm−1).26

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A 0.1 mM stock solution of fluorescein was stored in fridge (4 oC) and diluted to working solutions

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(0.1 μM) immediately before the determination of HClO scavenging capacity.

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Experimental Procedure for Hypochlorous acid Scavenging Assay. The HClO scavenging

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capacity was measured by monitoring the effect of flavonoids on HClO induced bleaching of flu-

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orescein by slide modification of a protocol previously described.27, 28 Reaction mixtures contained

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the following reactants at the indicated final concentrations (final volume of 200 μL): 25 μL sam-

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ples at five different concentrations, 150 μL fluorescein (0.1 μM), and 25 μL HClO (200 μM). The

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fluorimetric assays were performed at 37 °C in the microplate reader, at the emission wavelength

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at 528 ± 20 nm with excitation at 485 ± 20 nm. The mixture in a total volume was incubated for 20

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min in a plate reader kept at 37 °C. The IC50 (50% inhibition concentration) values of the scavengers

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were determined using fluorescein. The HClO scavenging capacity was calculated (Support infor-

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mation Figure S-12 and S-13). The oxidation products of fluorescein was studied in the LC-MS (Fig-

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ure S-14). The effects of flavonoids and flavonoids chlorinated products were discussed using lute-

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olin as an example (Figure S-15).



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Characterizations of Reaction Product by LC-MS. The LC–MS system was equipped with a

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C18 column (Phenomenex, Luna 5µ C18, 250 × 4.6 mm) for analysis of the reaction products be-

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tween flavonoids and HClO. The samples (30 μL) were filtered through 0.2 μm membrane (Merck

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Millipore, USA) before being injected into the HPLC system. The column was equilibrated with

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100% mobile phase A (water) for 10 min before gradient elution of mobile phase A ratio from 100 to

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0% in 30 min in linear increase at a flow rate of 1.0 mL/min, and then remain 100% mobile phase B

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(methanol) for the following 10 min. The Waters 2998 Photodiode Array (PDA) Detector was connected

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to the HPLC system with detection wavelengths from 190 and 800 nm. Bruker AmaZon-X is applied for

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LC-MS and LC-MS2 to characterize unknown compounds. The LC conditions for LC−MS analysis were

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similar to those mentioned above, except detection wavelengths were set at 280 and 350 nm. All mass

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spectra were acquired in both positive and negative ion mode using electrospray ionization. The parent

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ion was selected with a width of ±2.5 Da and fragmented with 50% setting.

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Preparative scale isolation of reaction products of flavonoids and hypochlorous acid.

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Flavonoids (100 mg, luteolin, apigenin, quercetin, naringenin, ampelopsin and epicatechin) were

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dissolved in acetone/water (1:1) solution and reacted with HClO (pH 6.2) in at molecular ratio of 1:

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0.5. After evaporation of the solvent in vacuo, the mixture was subjected to silica column chroma-

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tography to separate the products.

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Density Functional Theory (DFT) Study. A first principles study of flavonoids: luteolin, quer-

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cetin, naringenin, ampelopsin and epicatechin were carried out to investigate their differences in

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the relevant electronic properties. All calculations were performed using Density Functional The-

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ory (DFT) methods implemented in the Gaussian 09 package.29 Both geometry optimization and

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electronic structures are calculated using B3LYP hybrid function with 6-311+g(d,p) basis sets.30-32


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The HOMO (highest occupied molecular orbital) and HOMO-1 (the second highest occupied mo-

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lecular orbital) of all flavonoids were displayed on Gauss View 5.0.33

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The condensed Fukui functions were calculated by taking the finite difference approximations

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from population analysis of atoms in molecules, depending on the direction of electron transfer,

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through the following formula:34

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𝑓𝑘 + = 𝑞𝑘(𝑁 + 1) − 𝑞𝑘(𝑁) [for nucleophilic attack] (1)

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𝑓𝑘 − = 𝑞𝑘(𝑁) − 𝑞𝑘(𝑁 − 1) [for electrophilic attack] (2)

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𝑓𝑘 0 = [𝑞𝑘(𝑁 + 1) − 𝑞𝑘(𝑁 − 1)]/2 [for radical attack] (3)

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where qk is the gross charge of atom k in the molecule.

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Statistical Analysis. Descriptive statistical analyses were performed using Origin 8.0 for calcu-

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lating the means and the standard error of the mean. Results were expressed as the mean ± stand-

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ard deviation (SD).

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RESULTS AND DISCUSSION

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Hypochlorous acid Scavenging Capacity of Phenolic Compounds. This study on the SAR of

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the scavenging of HClO by flavonoids was started by characterizing the scavenging capacity of

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substituted phenols which shared a key group (phenolic moieties) with flavonoids. The concentra-

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tion of phenol that can inhibit half of fluorescein oxidation (IC50) is 18.7 µM (1/IC50 =0.047 ± 0.0035).

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Catechol, resorcinol and hydroquinone were tested to determine the effect of OH-substitution on

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the scavenging capacity of phenol (Figure 1). Substitution position and the number of OH are the

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key factors increases the scavenging capacity (1/IC50) of phenols. Catechol is more active than phe-

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nol, resorcinol shows better scavenging capacity than pyrogallol.



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The phenomenon that hydroxyl substituent at different positions results in dissimilarity in the

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activity could be explained by the electronic effect. As shown in the Figure 2, the electron donating

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or withdrawing effect was described by Hammett σ with negative value and positive value respec-

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tively35. In a conjugated system, hydroxyl group acts as electron donor and enhances the electronic

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density at the ortho and para positions while decrease the electron density at the meta position. As

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such all the potential reaction sites in the phloroglucinol are activated whereas the other phenols,

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some potential reaction sites in the aromatic ring were inactivated due to the electron withdrawing

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effect by OH groups at the meta position. During the chlorination of these phenols, chlorine atom

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in hypochlorous acid acts as electrophilic center and prefers to attack the electron-rich site in the

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aromatic ring (as shown in Scheme 1). This nicely fits with the ranking order of scavenging capacity

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against hypochlorous acid observed for the catechol, pyrogallol, resorcinol and phloroglucinol.

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Thus, nucleophilic substitution reaction is suitable to describe the initial reaction between phenols

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and HClO. With this understanding in mind, we examined the flavonoids structure and hypo-

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chlorous acid scavenging capacity relationship.

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Hypochlorous acid Scavenging Capacity of Flavonoids: Importance of B-ring HO groups as

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shown in Figure 3, luteolin (3,5,7,3’,4’-pentahydroxyflavone), is a very good scavenger of HClO (IC50

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= 2.39 μM). In sharp contrast, the homolog, apigenin (one HO less in B ring) has much lower HClO

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scavenging capacity with IC50 > 9.375 μM). Similar trend is observed in other flavonols (quercetin

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vs kaempferol), flavanones (eriodictyol vs naringenin), flavanonols (ampelopsin vs taxifolin), and

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flavanol (epicatechin vs epigallocatechin). This observation agreed nicely with the theory pro-

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posed by Cao, Sofic and Prior 22 that more OH groups in the aromatic ring result in better antioxi-

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dant activity. 22


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HClO scavenging capacity of flavonoids: importance of HO groups in A ring. As shown in Figure

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4, the extra OH group in the C6 of the A ring result in the increase of scavenging capacity from

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IC50> 9.375 μM of apigenin to 3.99 μM of scutellarein). This trend was observed in flavonols and

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flavanones.

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The loss of the OH group in the C5 position of the A ring results in a significant decrease in the

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scavenging capacity of the flavonoids. Moreover, OH group in the 7 sites also play a positive role in

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increasing the HClO scavenging capacity. Similarly, the loss of the hydroxyl group in the 5 site of

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the naringenin resulting in the increased IC50 from 1.97 μM to 6.55 μM (liquiritigenin). Overall，

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the hydroxyl groups exhibit positive effect both in A-ring and B-ring, then enhance the electron

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density on the aromatic ring, resulting in better attraction of electrophilic attack by hypochlorous

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acid.

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Effects of C(2)C(3) double bonds in C-ring. The C(2)C(3) double bond in the C seems to play

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a negative role in the scavenging capacity as illustrated by two pairs of compounds, luteolin vs

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eridictyol and apigenin vs naringenin (Figure 5), with the latter pair having greater impacts. This

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results were unconventional because C(2)=C(3) allows electron delocalization across the molecule

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for stabilization of the aryloxyl radicals, which has been proposed previously Rice-Evans, et al. 36.

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However, the conjugated system linked by C(2)-C(3) double bond may lead to more electron flow

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into electrophilic center in ketone group of C-ring. In the case of HClO, the reaction does not

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involve radicals and the electron delocalization may play a negative role.

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Characterization of reaction products of flavonoids with HClO. To shed some light on the

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reasons behind the observed trend of IC50, we characterized the primary reaction products of se-

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lected flavonoids and HClO by LC-MS(n). To ensure the reaction only involve HClO, we conducted

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the reaction under weakly acidic conditions (pH 6.20), which flavonoids are not deprotonated. As 9 ACS Paragon Plus Environment


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HClO is a potent oxidant and, if it is in large excess, it further oxidize the primary oxidation prod-

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ucts of the flavonoids. To avoid such complication and better characterize the primary products of

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HClO and flavonoids, we intentionally conduct the reaction with HClO as the limiting reagent.

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The reaction products of the flavone (apigenin), flavonol (quercetin), flavanone (naringenin) and

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flavanonol (ampelopsin) were summarized in the Figure 6A. For apigenin, three chlorination prod-

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ucts were observed, they are AP-1, AP-2 and AP-3 formed at different retention times (27.68, 28.51

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and 32.43 min) with similar UV-VIS spectral characteristics to that of apigenin. These products

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were firstly characterized by their mass spectra (Table S1) and fragments analysis (Figure S7). These

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compounds exhibit mass charge ratio (anionic mode) of 303.68 (AP-1), 337.29 (AP-2), and

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371.51(AP-3) and are respectively mono, di, and trichlorinated apigenin which has m/z of 269.24.

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To confirm the exact structures of the products, they were isolated and analyzed by 1H NMR (Figure

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S1), the NMR spectra shown that monochlorinated apigenin (AP-1) was modified at the C(3) site

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of the C-ring, and dichlorinated apigenin (AP-2) was modified at the C(3) site of the C-ring and

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C(8) site of the A-ring whereas trichlorinated apigenin (AP-3) occurred at C(3) site of the C-ring

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and C(8) as well as C(6) site of the A-ring.

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Similarly, three more lipophilic chlorination products of quercetin, QU-1, QU-2 and QU-3 were

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detected by HPLC at 25.63, 26.86, and 29.12 min following exposure of quercetin to HClO (Figure

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6B). The MS signals of chlorinated products were recorded in the Table S1, and the fragmentation

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analysis was shown in the Figure S8. The chlorinated sites of in the quercetin were revealed by 1H

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NMR at C(8) and C(6) site of the A-ring (Figure S2). This result matched the conclusion proposed

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by Binsack and coworkers.24 It is apparent that C(8) and C(6) are both sites for chlorination the

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isomeric products were observed.


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Similarly, three chlorinated products of naringenin, NA-1, NA-2 and NA-3, were detected by

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HPLC with retention times of 13.88, 14.23 and 16.26 min. Their structures are shown in Figure 6C.

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Moreover, the fragmental analyses of NA-1 or NA-2 and NA-3 (Figure S9) show that the monochlo-

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rination and dichlorination only happened at C(6) or C(8) site. The dichlorinated product NA-3

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was characterized by 1H NMR (Figure S3). Remarkably, C(3) was not chlorinated because, from 1H

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NMR spectrum, the hydrogen in the C(3) site remained. This is in contrast to flavone apigenin.

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The reaction products between flavanonol and HClO was studied using ampelopsin (AM) as an

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example. Three chlorinated products AM-1, AM-2 and AM-3 were observed by HPLC with reten-

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tion times of 20.21, 21.93 and 24.28 min respectively (Figure 7D). LC-MS results shown that the

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molecular ion (M-1, anionic mode) of 353.18, 353.18 and 387.02 respectively. From the 1H NMR spec-

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tra of the monochlorinated product AM-1 and AM-2 (Figure S4), the C(8) or C(6) were the reaction

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sites in the chlorination. The MS fragmentation patterns of AM-1, AM-2, or AM-3 (Figure S10)

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suggested that only A-ring is the reactive sites in the chlorination.

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The reaction between flavanol and HClO seems to be a different pattern. Following addition of

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HClO to epicatechin, HPLC analysis revealed one major product (RT: 13.88 min) that was very close

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to epicatechin (RT: 14.23 min) and photodiode array analysis of the peak identified similar spectral

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characteristics (Figure 7). In addition, two more products appeared at 16.26 min and 18.28 min,

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indicating products of high polarity. Fragment analysis was undertaken to identify the structures

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of these reaction products with mass of 323.71 Da (CA-1), 307.71 Da (CA-2) and 425.61 Da (CA-3)

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respectively. The hydrogen atom in C(6) or C(8) is the most reactive site for chlorination, resulting

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in the formation major reaction product CA-1, whose structure was further confirmed by 1H NMR

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(Figure S5). Then further chlorination happened in the rest potential sites of the A-ring and C-ring

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(CA-3). However, the formation of CA-2 observed in 16.26 min indicated a different mechanism 11 ACS Paragon Plus Environment


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which the OH group in the C-ring was substituted by chloride (protonation of HO group followed

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and nucleophilic substitution by Cl-). The structures of CA-1, CA-2 and CA-3 were confirmed by

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the fragmental analysis in Figure S11.

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Reaction mechanisms of flavonoids with HClO. From the results shown in Figure 6 and 7, it

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is apparent that there is a common features of the reaction between HClO and different types of

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flavonoids. Chlorination happens on A ring and C ring but not on B ring. We propose a reaction

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mechanism, electrophilic aromatic substitution, as described in the Scheme 2. The electrophile (Cl

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atom in HClO) reacts with the electron-rich carbon (A or C ring) of the flavonoids. However it is

239

not obvious, judging from the flavonoid structure, which carbon in the flavonoid ring skeleton is

240

most likely nucleophile. Therefore, we seek DFT calculation to gain some insights.

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As widely-used quantum chemical-descriptors, the HOMO-LUMO energies lay an essential role

242

in quantitative structure-activity and structure-property relationship (QSAR/QSPR) studies of

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wide range of chemical reactions.37,38 The HOMO-LUMO frontier orbital compositions for flavo-

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noids (apigenin, quercetin, naringenin, ampelopsin and epicatechin) calculated at the DFT/6-

245

311G(d,p) level are depicted in Figure 8 (a), (b), (c), (d) and (e) respectively. The highest and second

246

highest occupied MO’s (HOMO and HOMO-1) were executed to describe the orientation of elec-

247

trophilic aromatic substitution and reveal the principle under the reactive site for the reaction be-

248

tween flavonoids and HClO.

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The HOMO electron densities of apigenin are spread mainly over C(3), C(6) and C(8) in the A-

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ring, which means that these sites may be the reactive site for the electrophilic attack, because the

251

electron donating ability is positively related with the HOMO energy level and the higher HOMO

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energy means the stronger electron donation ability. The high density of negative charge distribu-

253

tion in the C(3) accounts for its high reactivity with HClO. As for quercetin, the OH group in the 12 ACS Paragon Plus Environment

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C(3) is not a good leaving group and thus prevent chlorination happening at C(3) site. Instead, C(8)

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and C(6) in the A-ring of the flavonol were the preferred chlorination sites, which is consistent

256

with experimental results.

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The HOMO electron density distribution localizes dominantly on the C(6) and C(8) sites of the

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A-ring, and the low electron density on the C(2)-C(3) may due to the missing of double bond in

259

the pyrone moiety. This hinders electron delocalization between B ring and A ring.39 Therefore,

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C(6) and C(8) at the A-ring are the only possible functional sites for the chlorination.

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From the frontier molecular orbitals (FMO) compositions of ampelopsin and epicatechin, it can

262

be observed that the electron density for HOMO is distributed more in B-ring compared with A

263

and C-rings. But the HOMO-1 orbital of epicatechin and the HOMO-2 orbital of ampelopsin were

264

characterized by a charge distribution mainly on A-ring. This indicated that the C(6) and C(8) sites

265

on the A-ring still have potential to be the electrophilic reaction sites.

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Fukui indices, the derivatives of the electron density of a molecule with respect to the number of

267

electrons,40 can be employed as indicators of chemical reactivity to evaluate the regions of the

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molecule to undergo nucleophilic, electrophilic and radical attack.41 The study of the charge distri-

269

bution was carried out by considering two types of charge analysis, natural population analysis

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(NPA) and electrostatic potential analysis (ESP). The analysis of Fukui indices of flavonoids was

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conducted under the same level B3LYP/6-311+G(d,p), the Fukui indices result was summarized in

272

Table S2 and principal chemical reactivity sites was showed in Figure 9. According to the Fukui

273

indices, the electrophilic attack in apigenin can be presented in the C(3) atom, the preferred site

274

for electrophilic attack in quercetin, naringenin, ampelopsin and epicatechin molecule was the C(6)

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and C(8) of the ring A. The conclusions are also linear with DFT calculation and experimental

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results. 13 ACS Paragon Plus Environment


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Based on our results of HClO scavenging capacity and the structure of flavonoids, it becomes

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apparent that the hydroxyl groups in the carbon skeleton of flavonoids can enhance the reactivity

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by donating the electron and increasing the electron density of the chlorination sites. In addition,

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the double bond in the C(2) and C(3) amplifies the electron-withdrawing effect of ketone in the C-

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ring, resulting in decreasing HClO scavenging capacity. Meanwhile, under conditions of a large

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molar excess of hypochlorite (>10-fold molar excess of HClO), oxidation of flavonoid may also oc-

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cur in addition to chlorination on B ring. 25, 42, 43

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Flavonoids represent major group of polyphenolic antioxidants, which are known radical scaven-

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gers due to their possession of multiple phenolic groups capable of quenching peroxyl radicals in

286

radical chain reactions.44, 45 In contrast, our results are in agreement with previous work,24, 25 which

287

suggest that the flavonoids may react other biologically relevant reactive oxygen species, such as

288

HClO, through the aromatic carbons. It is therefore important to elucidate the reaction products

289

and the mechanisms between different ROS and flavonoids in order to better understand the struc-

290

ture and activity relationship of polyphenolic antioxidants.

291

CONCLUSIONS

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In summary, our results have illustrated that hypochlorous acid was quenched by wide range of

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flavonoids through electrophilic reaction with the flavonoids, particularly at A ring, in which the

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hydroxyl groups can enhance the scavenging capacity of the flavonoids. From our results, fla-

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vanones, particularly eriodictyol (with IC50 of 1.2 µM) have high scavenging activity. The double

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bond in the C ring has negative impact to the scavenging capacity because it helps to channel

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electron towards electron-withdrawing ketone group on C(4). It is unintuitive that the chlorination

298

does not happen on B rings, which are the reaction sites for radicals (such as peroxyl radical). As 14 ACS Paragon Plus Environment

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299

Prior has observed that ROS scavenging activity of fruits and vegetables are dependent on the spe-

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cific ROS and recommended to use multiple ROS scavenging capacity plot (spider web plot) to

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comprehensively characterize the antioxidant capacity of foods.46 Therefore, it is imperative to es-

302

tablish the structure and reactivity relationship between flavonoids and individual biologically rel-

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evant reactive oxygen species in order to understand the chemical reasons of the different ROS

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scavenging capacity of fruits and vegetables and their health promotion property.

305

ASSOCIATED CONTENT

306

Supporting Information

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Additional information as noted in the text (Figures S1−S15) is available free of charge via the Inter-

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net at http://pubs.acs.org.

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Corresponding Author

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

311

ACKNOWLEDGMENT

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Authors thank Singapore Ministry of Education for financial support (grant no: MOE2014-T2-1-

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134).

314

ABBREVIATIONS

315

ROS, reactive oxygen species; RNS, reactive nitrogen species; DFT, density functional theory; MPO,

316

myeloperoxidase; SAR, structure-activity relationship; FMO, frontier molecular orbitals.

317

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Figure 1. Hypochlorous acid scavenging capacity (1/IC50) of phenol, catechol, pyrogallol, resorcinol, phloroglucinol.



Figure 2. The electronic influence (Hammett σ) of substitution of an OH group in an aromatic ring. The more negative the σ is the greater the electron donating effect. The effect at the ortho and para position is electron donating and at the meta position electron withdrawing.


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Scheme 1. The proposed mechanism (nucleophilic substitution reaction) for the reaction between phenol and hypochlorous acid.



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Figure 3. In impact of OH group at B ring on the hypochlorous acid scavenging capacity (IC50) of flavonoids.


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Figure 4. Impact of A-ring OH group on the hypochlorous acid scavenging capacity (IC50) of flavonoids.



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Figure 5. The hypochlorous acid scavenging capacity (IC50) of flavonoids from the different subclasses: flavones, flavonols, flavanones, and flavanonols.


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Figure 6. HPLC and LC-MS analysis of reaction products formed between flavonoids and HClO. All flavonoids (150 μM) was reacted with HClO (50 μM), and the reaction products were separated by reverse-phase HPLC. Analysis of the reaction products of flavonoids with HClO. (A) Analysis of the reaction products of apigenin with HClO. (B) Analysis of the reaction products of quercetin with HClO. (C) Analysis of the reaction products of naringenin with HClO. (D) Analysis of the reaction products of ampelopsin with HClO. AP, apigenin; QU, quercetin; NA, naringenin; AM, ampelopsin.



Figure 7. Analysis of the reaction products of epicatechin with HClO.


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Scheme 2. The proposed mechanism (electrophilic aromatic substitution) for the reaction between flavonoids and hypochlorous acid. A is the reaction between flavones and HClO, B is the reaction between flavonols, monochlorinated flavones and HClO, and C is the reaction between flavanones and HClO.



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Figure 8. Charge distribution of FMOs (HOMO and HOMO-1) in the optimized molecule of flavonoids: (a) apigenin, (b) quercetin, (c) naringenin, (d) ampelopsin and (e) epicatechin, calculated under the B3LYP/6-311+G(d,p) level.


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Figure 9. Preferred sites for flavonoids in electrophilic reaction with HClO according to Fukui induces: (a) apigenin, (b) quercetin, (c) naringenin, (d) ampelopsin and (e) epicatechin.



Table of Contents (TOC)


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Dietary Flavonoids Scavenge Hypochlorous Acid via Chlorination on

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