Development of a Fluorescent Probe for Measurement of

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Food and Beverage Chemistry/Biochemistry

Development of a Fluorescent Probe for Measurement of Singlet Oxygen Scavenging Activity of Flavonoids Darina Pronin, Saarangan Krishnakumar, Michael Rychlik, Haixia Wu, and Dejian Huang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b04025 • Publication Date (Web): 30 Aug 2019 Downloaded from pubs.acs.org on August 30, 2019

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Journal of Agricultural and Food Chemistry

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Development of a Fluorescent Probe for

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Measurement of Singlet Oxygen Scavenging

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Activity of Flavonoids

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Darina Pronin,§ Saarangan Krishnakumar,∥ Michael Rychlik, §Haixia Wu*,†,∥ and Dejian

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Huang*,∥

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†

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University, Hohhot, 010021, People’s Republic of China

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∥

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3, 117543, Singapore.

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Department of Chemistry, College of Chemistry and Chemical Engineering, Inner Mongolia

Department of Food Science and Technology, National University of Singapore, Science Drive

§

Technische Universität München (TUM), Arcisstr, 21, 80333, Munich, Germany

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1 Environment ACS Paragon Plus


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ABSTRACT: A turn-on fluorescent probe, HOCD-RB, for monitoring singlet oxygen (1O2) was

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developed by linking rhodamine B as fluorophore with dimethylhomoocoerdianthrone (HOCD) as

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1

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rhodamine B and HOCD moieties. Upon exposure to 1O2, it rapidly forms endoperoxide with

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HOCD and turns on the fluorescence of rhodamine B by 18-flods. Taking advantage of HOCD-

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RB probe that shows fast response, high sensitivity and selectivity for 1O2, it is applied for imaging

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of endogenous 1O2 in living cells and the fluorometric assay for evaluating 1O2 quenching activity

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of selected common flavonoids found in our daily diets. The results show that the 1O2 scavenging

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activity of flavonoids depends on not only the structure of individual flavonoid, but also the

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competitive interactions between mixed flavonoids. The best antioxidant capacity for individual

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and mixed flavonoids is epigallocatechin gallate and the mixture of catechin gallate with

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kaempferol, respectively. Overall, this work provided a new tool for detection and imaging of

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singlet oxygen activity in biological system as well as an efficient fluorometric assay of 1O2

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scavenging activity.

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KEYWORDS: singlet oxygen, flavonoids, fluorescent probe, fluorometric assay, cell imaging.

O2 reaction site and fluorescence quencher due to the intramolecular energy transfer (ET) between

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INTRODUCTION

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Oxidative stress is correlated with initiation and progression, not only for cardiovascular diseases

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but also for inflammatory diseases, cancer and aging.1 According to the WHO’s report (2014),

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cardiovascular diseases are the number one cause of death globally. As radical scavengers and 1O2

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quenchers, antioxidants including ascorbic acid, carotenoids and flavonoids, are believed to be

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essential to reduce the harmful effects of oxidative stress.2 The flavonoids are natural polyphenolic

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compounds, which widely distributed in fruits, vegetables, roots, flowers tea and wine.3 Because

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of flavonoids, plants are capable of protecting themselves against oxidative damage, even 1O2 can

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form in plant during the photosynthesis.4 So far, there are various radical scavenging methods used

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to assess the antioxidant activity,5 but only a few assays on 1O2 quenching activity of antioxidants

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were reported, including self-luminescence,6 electron spin resonance (ESR),7 and absorbance

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probe towards 1O2.8 However, the luminescence assays are not convenient due to lack of

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quantitative information, while ESR methods are expensive and complicated and not specific,9 and

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absorbance-based method typically has low sensitivity. Lima and co-workers reported a

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fluorometric assay towards to 1O2 based on the dihydrorhodamine (DHR)-123 by measuring the

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thermal decomposition of water-soluble endoperoxide in a buffered solution. However, it is not

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clear whether the DHR-123 probe is a specific redox probe or its oxidation is affected by other

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ROS besides 1O2.10

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Currently attaching anthracene onto fluorophores is a common method for designing turn-on 1O2

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fluorescent probes,11 the endoperoxide formed by anthracene trapped 1O2 will block electron

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transfer between excited fluorophore and anthracene ring and thus enhancing the fluorescence

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signals of fluorophore, such as, thiafulvalene,12 Eu(III) complexes.13 However, those probes with

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limited success either pH sensitivity, photo-bleaching or high background fluorescence. The 3 Environment ACS Paragon Plus


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commercially available probe SOSG can undergo self-photosensitization reaction, which result in

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false positive results when using for detection assay of 1O2.14

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Dimethylhomoocoerdianthrone (HOCD) is an intense blue aromatic compound and consists of

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an anthracene in structure, therefore, HOCD can be used for a new type of 1O2 trap. Moreover,

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HOCD has excellent photo-stable in solution and only 1O2 generated by illuminating other

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photosensitizer can bleach it into corresponding endoperoxide.15,16 Thus, the thermolysis study of

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HOCD-endoperoxide decays into HOCD shows that the half-time is around 170 years at room

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temperature, it indicates that HOCD-endoperoxide is also very photo-stable.17,18 Our previous

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work shows that HOCD derivative named HOCD-SO2Cl is photo-stable with a very broad visible

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absorption spectrum ranging from 500 to 700 nm, and easily link with amino compounds by

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sulfonamidation.19 Herein, we developed the first non-fluorescent probe HOCD-RB based on

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energy transfer (ET) for efficient monitoring 1O2 by linking rhodamine B19 as fluorophore. The

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probe was then applied successfully to evaluate 1O2 quenching activity to flavonoids in vitro and

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in living cells by establishing sensitive fluorometric assay method.

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MATERIALS AND METHODS

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Materials and Instrumentation. 1H and 13C NMR spectra were recorded in deuterated solvents

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with a Bruker spectrometer (Karlsruhe, Germany). High resolution ESI-MS spectra were obtained

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from the microTOF-Q II 1026 mass spectrometer with an ESI source. Fluorescence spectra were

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carried on by using Shimadzu RF-5301PC spectrofluorophotometer with a quartz cell. HPLC

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spectra were acquired on a Waters 2695 HPLC system equipped with an Alliance 2659 separation

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module and Waters 2996 PDA detector (Milford, MA, USA). LC-MS analysis was performed on

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Bruker Amazon ion trap mass spectrometer (Billerica, MA) equipped with an ESI source and a


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Dionex ultimate 3000RS HPLC system (Bannockburn, IL). The irradiation of the samples was

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carried out with the THORLABS OSL1-EC Fiber Illuminator (white lamp, 150 W, 340-800 nm),

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(Newton, NJ, USA). All solvent used were of reagent grade unless otherwise specified. All

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chemicals unless indicated were obtained from Sigma Aldrich and used as received.

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Synthesis of Probe HOCD-RB. Rhodamine B pyrazine19 (9.2 mg, 0.018 mmol) was dissolved

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in dry CH2Cl2 (5 mL) following by adding triethylamine (3 µL, 0.022 mmol) and stirred for 15

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min under argon atmosphere. Then HOCD-SO2Cl (10.2 mg, 0.020 mmol) was added, the reaction

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mixture was continue to stir for 2 hours at r.t. under argon atmosphere. The reaction mixture was

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rotary evaporated to dryness. The crude product was purified with basic aluminum oxide column

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chromatography with chloroform as eluted solvent, and gave pure purple HOCD-RB (11.65 mg,

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yield: 66%). 1H NMR (300 MHz, CDCl3) δ 9.08 (s, 1H), 8.79-8.73 (m, 3H), 8.23 (s, 2H), 8.17 (d,

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J = 9 Hz, 1H), 8.04 (d, J = 6 Hz, 1H), 7.78 (d, J = 9 Hz ,1H), 7.62 (s, 4H), 7.45 (s, 1H), 6.92 (d, J

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= 6 Hz, 2H), 3.52 (t, J = 6, 14H), 3.26-3.14 (m, 5H), 2.54 (s, 3H), 2.53 (s, 3H), 1.25 (m, overlap

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with grease peaks, 12H).13C NMR (126 MHz, CDCl3) δ183.71, 183.67, 168.02, 157.79, 156.24,

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155.76, 134.91, 134.71, 132.28, 131.48, 131.46, 130.36, 129.20, 114.33, 113.87, 96.23, 46.09,

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29.81, 21.43, 12.67, 8.79. HR-MS (ESI): cacld for C62H55N4O6S [M+], 983.3837; found, 983.3834.

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Selectivity Study of HOCD-RB. The mixture solution of HOCD-RB and 1,2-dioleoyl-3-

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trimethylammoniumpropane chloride salt (DOTAP, 20 equiv.) in chloroform was evaporated by

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using a stream of argon, and the resulting lipid film was hydrated with deionized water to give a

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clear stock solution of HOCD-RB-DOTAP, which concentrations were 50 µM and 1.0 mM for

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HOCD-RB and DOTAP, respectively. Then diluted the stock solution of HOCD-RB-DOTAP with

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10 mM PBS (pH 7.4) to get the final concentration of HOCD-RB at 0.1 µM. Potassium superoxide

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was prepared in DMSO. Hydroxyl radical was produced in solution by adding Fenton reagents 5 Environment ACS Paragon Plus


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(H2O2 and FeSO4). Concentrations of sodium hypochlorite and hydrogen peroxide were quantified

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by measuring their absorbance in their UV absorption immediately before use. The final

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concentrations of ROS (or its precursor) were controlled at 10 µM. The reducing agents (ascorbic

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acid and glutathione) were all dissolved in deionized water and the final concentrations were 0.1

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mM. The blank sample was only HOCD-RB in PBS buffer (0.1 µM).

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Fluorometric Assay for 1O2 Quenching Activity of Flavonoids. A series of different

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concentration of each antioxidant standard samples were obtained by mixing different amounts of

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an antioxidant stock solution with the solution of HOCD-RB-DOTAP and rose Bengal in

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deionized water, then diluted with PBS buffer (100 mM, pH 6.8) (Table S1). The final

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concentrations of HOCD-RB and rose Bengal was 1.0 µM and 0.59 µM, respectively. After 20

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min irradiation of samples, the scavenging activity had been observed with a decrease of emission

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intensity at 585 nm (λex = 500 nm).1O2 scavenging activity of samples (with individual antioxidant)

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was calculated using the following equation 1:20

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𝑆𝑐𝑎𝑣𝑒𝑛𝑔𝑖𝑛𝑔 𝑎𝑐𝑡𝑖𝑐𝑖𝑡𝑦 (% 𝑖𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛) =

(𝐸1𝑂2 − 𝐸𝑆𝑎𝑚𝑝𝑙𝑒 ) × 100 𝐸1 𝑂

(1)

2

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Where ESample and 𝐸1 𝑂2 was the emission value of sample with and without antioxidants, respectively.

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To investigate the antioxidant interaction among flavonoids and evaluate their synergistic

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properties, individual flavonoid standards were mixed in pairs (1:1 molar ratio), and the condition

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of fluorometric assay was same as that of individual antioxidant. The experimental quenching

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capacity (EQC) was calculated using the equation 2:21


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%𝐸𝑄𝐶 = 100 −

(𝐸𝑠𝑎𝑚𝑝𝑙𝑒 − 𝐸𝑏𝑙𝑎𝑛𝑘 ) × 100 𝐸1𝑂2

(2)

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Where 𝐸𝑠𝑎𝑚𝑝𝑙𝑒 and 𝐸𝑏𝑙𝑎𝑛𝑘 was the emission value of mixed flavonoids with and without HOCD-

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RB and rose Bengal, respectively. 𝐸1𝑂2 was the emission value of HOCD-RB and rose Bengal with

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no added mixed flavonoids.

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The theoretical quenching capacity (TQC) and the synergistic effect (SE) of mixed flavonoids were calculated using the equation 3 and equation 4, respectively:22

%𝑇𝑄𝐶 = 100 −

[(100 − 𝐸𝑄𝐶𝐴 ) × (100 − 𝐸𝑄𝐶𝐵 )] 100

(3)

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Where 𝐸𝑄𝐶𝐴 and 𝐸𝑄𝐶𝐵 represented the percentage EQC of each individual flavonoid A and B. It

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should be pointed out that such treatment has an underlying assumption that there is a linear dose

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response of EQC with the concentration of respective antioxidants. And the concentrations of

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individual flavonoid A and B were same as that in the sample of mixed flavonoids in order to

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calculate the SE of mixed flavonoids A with B.

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𝑆𝐸 =

𝐸𝑄𝐶 ⁄𝑇𝑄𝐶

(4)

Synergistic effect was considered when SE value was higher than 1 (SE > 1), antagonistic when was lower than 1 (SE < 1) and additive when was approximately 1 (SE ~ 1).

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Cytotoxicity Effects. The cytotoxicity effects of HOCD-RB-DOTAP and DOTAP alone to

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RAW 264.7 cells (ATCC, Manassas, VA, USA) were measured with MTT method. 2×104 cells

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per well was seeded in a 96-well microplate and incubated overnight, then treated with 100 µL

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DMEM (Dulbecco's modified Eagle's medium, GIBCO Grand Island, NY, USA) containing the



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mixture of 5 µM HOCD-RB with 20 equiv. DOTAP or 100 µM DOTAP alone per well for 4 hours.

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After washing with PBS thrice, each well was treated with 100 µL DMEM containing 5 mg/mL

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MTT (Sigma-Aldrich, MO, USA). After 3 hours of incubation, DMEM was removed and 100 µL

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DMSO was added to each well to dissolve the formazan crystals. Then the plates were shaken for

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10 min before the absorbance at 540 nm was read on microplate reader. Wells containing only

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culture medium without any cells were set as blank control, while cells treated with DMEM alone

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and served as negative control. Three replicates were set for each treatment. Cell viability was

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calculated according to the equation 5:23

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𝐶𝑒𝑙𝑙 𝑣𝑖𝑎𝑏𝑖𝑙𝑖𝑡𝑦 (%) =

(𝐴𝑠𝑎𝑚𝑝𝑙𝑒 − 𝐴𝑏𝑙𝑎𝑛𝑘 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 ) × 100 𝐴𝑛𝑒𝑔𝑎𝑡𝑖𝑣𝑒 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 − 𝐴𝑏𝑙𝑎𝑛𝑘 𝑐𝑜𝑛𝑡𝑟𝑜𝑙

(5)

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Imaging of Endogenous 1O2 in Living Cells. RAW 264.7 cells were transferred to chambered

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cover glass (Lab-Tek chambered #1.0 borosilicate cover glass system) at 6x103cells/well and

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incubated for 24 hours. After washing with PBS thrice and treatment with HOCD-RB-DOTAP (5

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µM) for 1 hour, the cells were stimulated with phorbol 12-myristate 13-acetate (PMA) 5 and 10

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µM for 45 min to trigger generation of 1O2. A separate well was maintained without any PMA

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stimulation to serve (HOCD-RB-DOTAP alone) as negative control, whereas the cells pre-

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stimulated with histidine (400 µM), epicagallocatechin gallate (EGCG, 100 µM) and

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epigallocatechin (EGC, 100 µM) followed by PMA stimulation (10 µM) for 45 min. The

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intracellular localization of HOCD-RB was investigated using 2D- imaging and 3D- sequential

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scanning under confocal microscopy (Olympus IX 81, Fluoview FV1000) equipped with a 60X

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water lens. HOCD-RB was excited with a 420 nm Ar laser, and the fluorescent images were

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collected using filter sets selective above 540 nm wavelength. Images were processed in IMARIS

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3.0 (BITPLANE AG) software.


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Detection Products of HOCD-RB Reacted with 1O2 in Living Cells. RAW 264.7 cells were

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transferred to 35 mm petri plate and incubated for 24 hours in CO2 incubator, then stimulated with

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PMA 10 µM for 45 min and washed with PBS thrice. Then HOCD-RB-DOTAP was treated for

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30 min and wash with PBS thrice to remove excess probe. 0.5 ml of 25% cold RIPA lysis buffer

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was added to the plate placed on ice to promote cell lysis. The cells were then scrapped and

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centrifuged at 13500 rpm for 8 min at 4 °C. Ice-cold chloroform was added to extract the products,

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separated and collected the organic layer to further analyze by using ESI-MS.

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HPLC and LC–MS Analyses of Oxidation Products of EGCG and 1O2. The reaction

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products between EGCG (0.098 mg/mL) and 1O2 in methanol were analyzed by HPLC with a Luna

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Sov C18 column using the mobile phases of solvent A [20 mM AcONH4/MeOH (90/10, v/v)] and

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solvent B [20 mM AcONH4/MeOH (20/80, v/v)]. The samples of EGCG alone or with rose Bengal

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irradiated for 5 min. were monitored under the following conditions. A. gradient elution of solvent

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A ratio from 100% to 0% was utilized and decreased in 60 min. at a flow rate of 1.0 mL/min. The

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LC-MS was analyzed with LC/ESI in positive mode and scanned from m/z 200-1000. The heated

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capillary and spray voltage were set at 650 °C and 5.0 kV, respectively. The flow rate of nitrogen

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was operated at 30 psi for sheath gas and 50 psi for auxiliary gas, respectively. The LC conditions

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were carried out under the same gradient conditions and column as the HPLC experiment.

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RESULTS AND DISCUSSION

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Synthesis and Spectroscopic Property of HOCD-RB. When the emission spectrum of

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fluorophore and the absorbance spectrum of HOCD is well overlap, there will be intramolecular

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ET from fluorophore to HOCD moieties, and result in the fluorescence is quenched, while the

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fluorescence will be recovered when HOCD moiety react with 1O2 and lead to form HOCD-



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endoperoxide, which hinder the ET process. With these in mind, we developed an off-on

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fluorescent probe HOCD-RB, which was readily accomplished by one-step sulfonamidation

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reaction in good yield (Scheme S1) and was confirmed by high resolution mass spectrum and

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NMR (Figure S1). The detection mechanism of 1O2 is showed in Scheme 1.

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Scheme 1. Monitoring mechanism of HOCD-RB.

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HOCD-RB is non-fluorescence when excited at 480 nm as the ET, but the fluorescence could

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be rapidly turned on by 1O2, which shows an emission maximum at 576 nm in 10 mM phosphate

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buffered saline solution (pH 7.4, Figure S2). The fluorescence intensity was enhanced around 18-

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folds although the concentration of HOCD-RB was as low as 0.1 µM and reacted with 1O2 only

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for 10 min, which is competitive to other recently reported 1O2 probes.24-26 HOCD-RB responds

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fast (within minutes) to 1O2 due to the high reactivity of HOCD with 1O2. In addition, the

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magnitude of fluorescence enhancement is more than 6 times better than a similar 1O2 fluorescent

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probe with rhodamine B linked to anthracene as a quencher,27 which turned on only around 3-folds

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in buffer at 10 µM probe concentration. Therefore, HOCD is a better fluorescence quencher than

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anthracene for rhodamine B as fluorophore and our probe is more sensitive in detecting 1O2 with

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nanomolar (100 nM) concentration needed for the probe. Finally, the corresponsive endoperoxide

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of probe was verified by ESI-MS (Figure S3), which confirmed that the fluorescence enhancement

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is due to 1O2 not other ROS. 10 Environment ACS Paragon Plus

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Fluorescence Response and Selectivity of HOCD-RB towards 1O2. In Figure 1A, HOCD-RB

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(1 µM) shows a fast response towards 1O2. Based on the kinetic study, a marked fluorescence

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enhancement was observed after adding 1O2 only for 3 min, which increased to around 50%, the

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maximum increment of fluorescence reached at 20 min and levelled off at least 20 min thereafter.

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These results imply that HOCD-RB has a very fast response to 1O2 and the corresponding

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endoperoxide product is photo-stable in 100 mM PBS (pH 6.8) at room temperature.

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The selectivity of HOCD-RB (0.1 µM) towards common biologically relevant small molecules

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in phosphate buffered saline solution was evaluated (Figure 1B). Only 1O2 could increase the

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fluorescence intensity around 18-folds, whereas other reactive oxygen species (ROS, 100 equiv.)

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and reducing agents (1000 equiv.) did not change noticeably. Therefore, HOCD-RB shall be able

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to withstand various common active species in biological system and can highly selectively to 1O2

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with confidence.



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Figure 1. (A) Time courses of HOCD-RB (1 µM) in 100 mM PBS (pH 6.8) at r.t., the

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enhancement of fluorescence intensity at 585 nm was recorded (λex = 500 nm); (B) Selectivity of

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HOCD-RB towards biological ROS and reducing agents. The fluorescence responses were

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obtained upon addition of various biological species to HOCD-RB (0.1 µM) in 10 mM PBS (pH

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7.4) for 10 min at r.t. (λex = 480 nm, λem = 576 nm). Legend: 10 µM (100 equiv.): ClO-, H2O2,

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O2-, HO.; 0.1 mM (1000 equiv.): ascorbic acid (Vc) and Glutathione (GSH). 1O2 is generated by

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photo-irradiation of rose Bengal. Probe was delivered by using 20 equiv. of DOTAP.

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Fluorometric Assay for 1O2 Scavenging Activity of Antioxidants and Interaction between

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Flavonoids. Structurally, flavonoids consist of a benzene ring (A-ring) attached to a heterocycle

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(C-ring), which carries a phenyl group (B-ring) at C2.28 For catechins, EGCG and catechin gallate


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(CG) share a common galloyl group in ring C. For flavonol, myricetin (M), quercetin (Q) and

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kaempferol (K) are different in the number of hydroxyl groups in ring B (Figure 2).

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Figure 2. Structures of flavonoids measured for 1O2 quenching assay

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With highly sensitivity and selectivity HOCD-RB in hand, we developed a fluorometric assay

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based on HOCD-RB (1 µM) in order to measure 1O2 quenching ability of antioxidants. The

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fluorescence enhancement of HOCD-RB reacted with 1O2 would be inhibited in the present of

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antioxidants, therefore, we applied the IC50 (the concentration of antioxidant causing 50%

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fluorescent incensement inhibition) to evaluate the antioxidant quenching activity of individual

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antioxidant towards 1O2, the synergistic effect (SE) of flavonoid standards mixtures are used to

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investigate the antioxidant interactions between flavonoids. Measured the fluorescence of samples

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performed for 20 min and plotted the quenching inhibition curves (Figure S4). The dose response

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curves of caffeic acid, carotene, trolox and vitamin C are very flat, and none of them reach the 50%

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inhibition except vitamin C. For the 40% inhibition activity, only 10 µg/mL caffeic acid is required,

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whereas for ß-carotene, vitamin C and trolox is 69, 55, 52 µg/mL, respectively. These results

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indicate that caffeic acid is the most powerful antioxidant among the present group.



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According to the value of IC50 summarized in Table 1, among catechins, the order of scavenging

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activity is as follows: EGCG >CG > EGC > EC, which suggesting that the more the number of

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hydroxyl groups in structure, the stronger activity of antioxidants and lead to the lower

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concentration of antioxidants needed, so the galloyl group plays a crucial role in enhancing the

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scavenging activity. Same conclusion is also obtained among M, Q and K.29,30 However, M

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(IC50=1.68 µg/mL) has much stronger antioxidant capacity than EGC (IC50 = 2.78 µg/mL), even

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though the number of hydroxyl groups is same in their structure, so do comparing EC (IC50 = 9.17

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µg/mL) to Q (IC50 = 2.11 µg/mL). Therefore, we infer that a double bond at C2-C3 position

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conjugated with a keto group, as well as hydroxyl groups contributes towards scavenging activity,

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so EGCG is the most potent 1O2 quencher among the tested flavonoids, these results are in

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accordance with previous reported assay.31-33

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Table 1. 1O2 quenching capacity of tested flavonoids (n = 3) flavonoids

IC50 (µg/mL)

Epicatechin (EC)

9.17 ±0.085

Epigallocatechin(EGC)

2.78 ±0.017

Catechingallate (GC)

1.42 ±0.034

Epigallocatechin gallate (EGCG)

0.59 ±0.001

Kaempferol (K)

3.99 ±0.003

Quercetin (Q)

2.11 ±0.015

Myricetin (M)

1.68 ±0.028

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The possibility of fluorescent interference from flavonoids themselves and their oxidation

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products were ruled out, because the emission spectra suggest they are all non-fluorescent (Figure

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S5, EGCG as representative of flavonoids). For the antioxidant interactions between flavonoids,

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as shown in Table 2, the SE values obtained for catechin mixtures are all significantly lower than


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1, thus antagonistic interactions are considered for the pairs. The SE value of EGCG+CG (0.20 ±

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0.01) is the lowest in all mixtures, although the individual oxidation abilities of them are quite high,

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so we deduct that there are strong competitive reactions between the galloyl fragment of each other,

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that lead to largely decrease net quenching capacity. In addition, the SE value of EGCG+EGC

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(0.26 ± 0.04) is significantly lower than that of CG+EGC (0.64 ± 0.01), and the SE value of

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EGC+EC (0.57 ± 0.03) is still significantly lower than 1 even no containing the galloyl fragment

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in structure, these results indicate that competitive reaction also existed in ring B due to hydroxyl

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groups. Therefore, we conclude that there is a correlation between the structure and interaction of

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catechins. Individual catechin shows an increasing quenching capacity in the presence of the

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galloyl fragment and three hydroxyl groups in ring B, but for the mixtures, the galloyl fragments

271

in ring C behave competitively and this effect is even extended to ring B, so the effects of catechin

272

mixtures are all antagonistic.

273

For flavonols, the SE values are significantly higher than 1, except for the M+Q (1.18 ± 0.09)

274

pair. The pairs M+K (1.56 ± 0.08) and Q+K (1.68 ± 0.22) provides synergistic effects, whereas

275

the M+Q pair stated as additive. These results can be attributed to the fact that keto group

276

conjugated to the C2-C3 double bond in ring C not competitive among the flavonols mixtures.

277

Furthermore, it is noticeable that the number of hydroxyl groups in ring B also influences the

278

interaction between the pairs, the synergistic effect is lower due to competitive interactions. So it

279

can state that the less hydroxyl groups are involved, the higher of the SE values raise.

280

Table 2. Synergistic effects of individual catechins and flavonols (n = 3) Mixture (1:1)

SE

Effect

EGCG + CG

0.20 ±0.01

Antagonistic

EGCG + EGC

0.26 ±0.04

Antagonistic



EGCG + EC

0.37 ±0.01

Antagonistic

CG+ EGC

0.60 ±0.01

Antagonistic

CG + EC

0.64 ±0.01

Antagonistic

EGC + EC

0.57 ±0.03

Antagonistic

M + Qa

1.18 ±0.09

Additive

M+K

1.56 ±0.08

Synergistic

Q+K

1.68 ±0.22

Synergistic

281

a

282

The SE values of the mixtures of catechins with flavonols (1:1 molar ration) are summarized in

283

table S2 and plotted in Figure 3. Firstly, the SE values for catechin-flavonol mixtures are all

284

significantly higher than that of catechin mixtures by themselves, even though they are still lower

285

than 1 and express as antagonistic effects, these results suggest that the keto group conjugated to

286

the C2-C3 double bond in C ring can weak the competitive reactions of the galloyl fragments and

287

hydroxyl groups, which increase the SE values. Secondly, it is not surprising that the SE value of

288

the pairs CG with flavonoids are higher than that of EGCG with flavonoids, because the total

289

number of hydroxyl groups in pairs of CG with flavonoids are less than that of EGCG mixtures.

290

At last, for GC groups, the same structure in ring B of GC and Q caused the interaction of hydroxyl

291

groups between each other became stronger than that of GC with M, so the SE value of GC+Q is

292

lowest. In summary, the antioxidant capacity of flavonoids depend on their structure, involving

293

galloyl fragment, hydroxyl groups, keto group conjugated to the C2-C3 double bond in ring C and

294

interaction between each other, specially.

No significant difference between experimental and theoretical quenching value, p < 0.01.


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295 296

Figure 3. Synergistic effect values (SE) of mixtures formed catechins with flavonols. The dashed

297

line was the SE value equal to 1.0. (* no significant difference between experimental and

298

theoretical quenching value, p < 0.01)

299

Imaging of Endogenous 1O2 in Living Cells. With encouraging results from chemical systems,

300

we further evaluated the performance of HOCD-RB in detection of endogenous 1O2 in cell line

301

models. The lipophilic probe was uptaken into RAW 264.7 cells by using a vehicle made of

302

DOTAP cationic liposome, which is commonly used for helping compound easily uptaken by

303

cells,34 the final concentration of HOCD-RB in the culture media fixed at 5 µM. The cytotoxicity

304

experiments of HOCD-RB-DOTAP and DOTAP alone in cells were carried out by MTT assay,

305

and the results suggest both of them show high cell viability and low cytotoxicity to live cells

306

(Figure S6). The 3D fluorescent images of the cells were non-stimulated and stimulated with PMA

307

(8 µM) suggest the localization of HOCD-RB throughout the cytoplasm, meanwhile clearly

308

illustrate that 1O2 are generated after PMA stimulation and lead to turn on the fluorescence

309

intensity, which is in agreement with the results in chemical systems. The DIC images clearly

310

display the morphology of the cell is maintained (Figure 4A).

311

The 2D fluorescent images of cells were conducted for evaluating the scavenging activity of 1O2

312

in living cells, and the incensement fluorescent intensities are plotted and showed in Figure 4B.



313

As shown in Figure S7, a dose-dependent fluorescence enhancement of cells after treatment with

314

increased the concentration of PMA from 5 to 10 µM (Figure S7b, 7c). Whereas cells pre-

315

stimulated with histidine (400 µM) followed by PMA (10 µM) stimulation, the images clearly

316

show largely reduced fluorescent intensity when compared to only PMA (10 µM) stimulated cells,

317

these results indicate that histidine has inhibited the PMA assisted 1O2 production in cells (Figure

318

S7g). In addition, to observe their effect on 1O2 quenching in living cells, the cells were pre-treated

319

with EGC (100 µM) and EGCG (100 µM) followed by PMA stimulation (Figure S7h, 7i), as

320

expected, the fluorescence enhancement is comparable to that of the control cells (Figure S7a), the

321

results are in accordance with that of in vitro. Moreover, the corresponsive endoperoxide of

322

HOCD-RB (M.W. = 1015.4) in cells was also verified by ESI-MS, which display that the

323

corresponsive endoperoxide further hydrogenated to dihydroxide and cause the molecular weight

324

to increase 2 (Figure S8).

325 18 Environment ACS Paragon Plus

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326

Figure 4. (A) Imaging of 1O2 in RAW 264.7 cells. HOCD-RB (5 µM) was turned on by 1O2

327

contributed by PMA stimulation, (a) and (b) are 3D fluorescent images of cells non-stimulated

328

and stimulated with PMA (8 µM), (c) and (d) are the corresponding DIC images. (B)

329

Quantitative increase in fluorescent intensity of HOCD-RB (5 µM) following treatments

330

mentioned in Figure S6. Legend: (1) HOCD-RB only, (2) HOCD-RB+PMA (5 µM), (3) HOCD-

331

RB+PMA (10 µM), (4) HOCD-RB+ histidine (400 µM) + PMA (10 µM). (5) HOCD-RB+ EGC

332

(100 µM) +PMA (10 µM). (6) HOCD-RB+EGCG (100 µM) +PMA (10µM). Data point is mean

333

±standard deviation, n = 8.

334

Reaction Products and Mechanisms of EGCG with 1O2. Based on previous reported works

335

and above results, the B ring and galloyl moieties of flavonoids are important for the scavenging

336

activity, and the -R2 group and adjoining -OH group in B ring are the radical target sites.35-37 To

337

shed some light on the 1O2 scavenging activity, the reaction products of 1O2 with EGCG were

338

analyzed by HPLC and LC-MS/MS. After irradiation of rose Bengal with EGCG, some new peaks

339

were generated with the retention time at 5.60, 10.35, 12.18 and 13.78 min, whereas the peak

340

belongs to EGCG (2.82 min) was disappeared (Figure S9), this suggests that EGCG has been

341

consumed during the reaction. The products with retention time at 13.78 min were further

342

investigated by LC-MS (Figure S10). According to previous works, oxidation of EGCG can form

343

theasinensin A, theaflavine-3’-galatte, theaflavine and EGC formed by the cleavage at the ester

344

bond between the gallic acid with EGC moieties.38,39 therefore, the peak at m/z 564.4 was assigned

345

as theaflavine, which was support by the LC-MS spectrum of theaflavine standard (Figure S11).

346

Beecher reported that theaflavine can form by the oxidation of the B ring of EGCG or EGC,

347

loss of CO2 and simultaneous merger with the B ring of second molecule of EGCG or EGC,40

348

therefore, 1O2 may oxidize EGCG to hydrolyze the gallyol fragment and further form theaflavine, 19 Environment ACS Paragon Plus


349

that followed by a loss of -OH group and gave rise to the fragment at m/z 547.3. The fragment

350

with m/z 503.3 was fit loss of C2H4O mass unit from the fragment with the molecular weight of

351

547.3. The signal at m/z 283.2 was another fragment unit as result of the oxidation of theaflavine,

352

which further underwent a loss of 44 and 16, resulted in the fragments at m/z 239.2 and 223.1,

353

respectively. The proposed structures of reaction products formed by EGCG with 1O2 as shown in

354

Scheme 2.

355 356

Scheme 2. Proposed structures of reaction products of EGCG with 1O2.

357

In conclusion, the highly sensitive and selective off-on fluorescent singlet oxygen probe HOCD-

358

RB allows for detection of 1O2 with good sensitivity with only sub micromolar concentration of

359

the probe and high fluorescence enhancement degree (18-folds). Comparing to other turn-on 1O2

360

probes, our probe used of HOCD as the 1O2 reaction site and fluorescence quencher renders

361

superior sensitivity and selectivity towards 1O2. Coupled with its high photo-stability, we are able

362

to detect endogenously produced 1O2 in live cells. Moreover, our probe are successfully applied in

363

the fluorometric assay for evaluation 1O2 quenching activity of dietary flavonoids. Therefore,

364

HOCD-RB is very promising probe that can greatly aid our study on 1O2 activity in photodynamic

365

therapy, effectiveness of photosensitizers, and 1O2 quenching activity of dietary antioxidants. 20 Environment ACS Paragon Plus

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366

ASSOCIATED CONTENT

367

Supporting Information

368 369

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.xxx.

370

Scheme of synthesis of probe HOCD-RB, NMR spectra of HOCD-RB, Emission spectrum in

371

PBS buffer, ESI-MS spectra of endoperoxides in PBS buffer and living cells, Dose response curves

372

of antioxidants, Fluorescence spectra of EGCG and related samples, Cytotoxicity effects and 2D

373

fluorescent images of cells treated with probe and some flavonoid, HPLC and LC-MS of reacted

374

products of EGCG with 1O2, Theaflavine standard LC chromatogram and mass spectrum (PDF).

375

AUTHOR INFORMATION

376

Corresponding Authors

377

*E-mail for Dejian Huang: [email protected]

378

*E-mail for Haixia Wu: [email protected]

379

ORCID

380

Dejian Huang: 0000-0002-2305-3960

381

Haixia Wu: 0000-0003-4919-788X

382

Funding



383

This work was financially supported by the Singapore Ministry of Education (Grant No.

384

MOE2014-T2-1-134) and the Program of Higher-level Talents of Inner Mongolia University

385

(Grant No. 21300-5175151).

386

Notes

387 388 389 390 391 392 393 394 395 396 397 398

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501

TOC Graphic

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Scheme 1. Monitoring mechanism of HOCD-RB 43x20mm (600 x 600 DPI)

ACS Paragon Plus Environment


Figure 1. (A) Time courses of HOCD-RB (1 µM) in 100 mM PBS (pH 6.8) at r.t., the enhancement of fluorescence intensity at 585 nm was recorded (λex = 500 nm); (B) Selectivity of HOCD-RB towards biological ROS and reducing agents. The fluorescence responses were obtained upon addition of various biological species to HOCD-RB (0.1 µM) in 10 mM PBS (pH 7.4) for 10 min at r.t. (λex = 480 nm, λem = 576 nm). 83x121mm (300 x 300 DPI)


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Figure 2. Structures of flavonoids measured for 1O2 quenching assay 83x44mm (300 x 300 DPI)



Figure 3. Synergistic effect values (SE) of mixtures formed catechins with flavonols. 83x42mm (300 x 300 DPI)


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Figure 4. (A) Imaging of 1O2 in RAW 264.7 cells. (B) Quantitative increase in fluorescent intensity of HOCDRB (5 µM) following treatments mentioned in Figure S6. 83x129mm (600 x 600 DPI)



Scheme 2. Proposed structures of reaction products of EGCG with 1O2. 83x55mm (300 x 300 DPI)


Page 34 of 34

Development of a Fluorescent Probe for Measurement of

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