The chemical structures of polyphenols critically influence the toxicity

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The chemical structures of polyphenols critically influence the toxicity of ZnO nanoparticles Cao Zhang, Yining Li, Liangliang Liu, Yu Gong, Yixi Xie, and Yi Cao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00368 • Publication Date (Web): 31 Jan 2018 Downloaded from http://pubs.acs.org on February 2, 2018

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Journal of Agricultural and Food Chemistry

Title page

Title: The chemical structures of polyphenols critically influence the toxicity of ZnO nanoparticles

Authors: Cao Zhang1#, Yining Li1#, Liangliang Liu2, Yu Gong1, Yixi Xie1*, Yi Cao1,2*

Affiliations: Affiliations: 1. Key Laboratory of Environment-Friendly Chemistry and Applications of Ministry Education, Laboratory of Biochemistry, College of Chemistry, Xiangtan University, Xiangtan 411105, P.R. China

2. Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences, Changsha 410205, P.R. China

#: These two authors contributed equally to this work.

* Send correspondence to: Dr. Yixi Xie ([email protected]) and Dr. Yi Cao ([email protected])

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ACS Paragon Plus Environment


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Abstract: Recent studies suggested that phytochemicals as natural anti-oxidants in

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food could alleviate nanoparticle (NP) toxicity. This study investigated the combined

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toxicity of ZnO NPs and a panel of polyphenols. Surprisingly, both polyphenols with

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high and almost no radical scavenging activities could elicit cyto-protective effects

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against NP exposure in Caco-2 cells, which were primarily influenced by the

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positions of hydroxyl group. Polyphenols with different chemical structures variously

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influenced the hydrodynamic size, Zeta potential and solubility of ZnO NPs as well as

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NP induced intracellular superoxide and Zn ions, which could all contribute to the

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combined effects. Responses of human endothelial cells appeared to be different from

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the responses of Caco-2 cells, which may indicate cell type dependent responses to

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combined exposure of NPs and phytochemicals. In conclusion, the data from this

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study suggested a pivotal role of chemical structures of phytochemicals in

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determining their capacity to affect ZnO NP toxicity.

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Keywords: Phytochemicals; ZnO nanoparticles (NPs); Oxidative stress; Cytotoxicity;

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Caco-2 cells.

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Introduction

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Due to their small nano-size, nanoparticles (NPs) are used not only in cosmetics,1

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microelectronics2 and medicinal products,3,4 but also in food. In food and food related

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products, NPs could be used for, but not limited to, anti-microorganisms, color

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development and nutrient supplement.5,6 As such, oral exposure of human beings to

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NPs via food and food related products is likely increasing, and there is a need to

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assess the toxicity of NPs to cells lining gastrointestinal gut.7 Particularly, the

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assessment should consider the interactions between food components and NPs to

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better mimic the exposure of NPs in real life as we and others recently suggested.8,9

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Phytochemicals are secondary metabolites synthesized by plants as well as non-

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pathogenic microorganisms living within plants. They are healthy components widely

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present in food with well-documented beneficial effects such as anti-cancer,10

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prevention of cardiovascular diseases11 and regulation of inflammation.12 Interestingly,

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recent studies also showed that phytochemicals might influence the toxicity of NPs.13

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For example, Sarkar and Sil found that quercetin significantly protected murine

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hepatocytes from ion oxide NP induced cytotoxicity and apoptosis.14 Similarly,

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Shalini et al recently found that quercetin significantly reduced cytotoxicity induced

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by ZnO NPs to human lymphocytes by the inhibition of oxidative stress.15 Using

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Caco-2 cells, Martirosyan et al showed that phenolic compounds significantly reduced

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Ag NP induced cytotoxicity, oxidative stress and inflammatory responses.16,17

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Because phytochemicals are widely present in food which can interact with NPs

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added into food, we and others have recently suggested that the interactions between

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NPs and phytochemicals should be carefully evaluated to better predict the toxicity of

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NPs in food.8,9 However, most of the previous studies only investigated the 3



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interactions between NPs and phytochemicals with relatively high anti-oxidative

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properties, whereas relatively few studies evaluated the combined toxicity following

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co-exposure to NPs and phytochemicals with little to no anti-oxidative properties.13

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There are many different types of phytochemicals, and it has been shown before that

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the chemical structures of phytochemicals could critically influence the biological

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activity,18 interactions with biological molecules19 as well as the stability of

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phytochemicals.20 In this study, we investigated the interactions between ZnO NPs

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and a panel of polyphenols with different positions and numbers of hydroxyl groups,

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so the influence of chemical structures of phytochemicals could be evaluated. The

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radical scavenging activities of different types of polyphenols were evaluated by 1,1-

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diphenyl-2-picrylhydrazyl (DPPH) assay, and the influence of polyphenols with both

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high and almost no anti-oxidant properties on the biological effects of NPs was

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studied. Moreover, the production of intracellular superoxide following co-exposure

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to NPs and polyphenols was determined to further indicate the role of oxidative stress.

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ZnO NPs were selected because they are among the most popular NPs used in food

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for anti-bacterial and nutrition supplement, but their toxicity toward cells lining

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gastrointestinal gut still needs further investigation.21 Caco-2 cells were used as the in

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vitro model for human intestine, and the cytotoxicity, oxidative stress and intracellular

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Zn ions induced by ZnO NPs with or without the presence of different types of

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polyphenols were investigated. For comparison, human umbilical vein endothelial

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cells (HUVECs) were also used as a control because both NPs and polyphenols could

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be adsorbed to reach circulation, leading to combined exposure to endothelial cells.22-

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potential and solubility was also studied, because it has been suggested that NP

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colloidal stability could significantly affect the effects of NPs.9,25

The influence of polyphenols on NP UV-Vis spectra, hydrodynamic size, Zeta

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Materials and methods

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Cell culture

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The human colon epithelial Caco-2 cells (ATCC, HTB-37) were cultured in

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supplemented DMEM (Dulbecco's Modified Eagle Medium)/high glucose medium

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(Hyclone, GE Healthcare) as we described elsewhere.26 For the experiments, the cells

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were seeded at a density of 1.5╳104 per well on 96-well plates and grown for 2 days

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prior to exposure. For comparison, HUVECs were also used for cytotoxicity assay

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(see below), which were cultured in endothelial cell medium (ECM; purchased from

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ScienCell Research Laboratories, Carlsbad, CA) as we described elsewhere.27

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HUVECs were seeded at a density of 1╳104 per well on 96-well plates and grown for

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2 days prior to exposure.

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Particle characteristics and preparation

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ZnO NPs (code XFI06; 20 nm) were purchased from Nanjing XFNANO Materials

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Tech Co., Ltd (Nanjing, China) and have been thoroughly characterized as we

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reported earlier.28 X-ray diffractograms (XRD) indicated the hexagonal phase of NPs

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with an average size of 22.3 nm. The BET surface area was measured as 19.072 m2/g.

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The hydrodynamic size and Zeta potential of 16 µg/mL XFI06 suspended in water

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were measured as 234.4±3.5 nm and -15.5±0.3 mV, respectively. Herein, the

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morphology and topography of XFI06 were further investigated by using transmission

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electron microscopy (TEM) and atomic force microscope (AFM), respectively. For

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TEM, XFI06 was briefly sonicated in EtOH, and a drop of suspension was deposited 5



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and dried on a carbon-coated grid. The sample was then coated with a conductive

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layer of Au of 10–20 nm thickness and images were taken by the TEM accelerated at

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200 kV (FEI TECNAI G20, USA). The TEM size was measured based on the

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quantification of 50 randomly selected particles by using ImagJ (NIH). The AFM

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study was done on a Bruker MultiMode 8 by using Peakforce mode at room

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temperature. Briefly, the XFI06 suspension was prepared by sonication and a drop of

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the diluted aqueous dispersion was deposited on a new cleaved mica surface. A

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ScanAsyst-Fluid probe with a nominal spring constant of 0.7 N/m and a nominal tip

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radius R =20 nm was used for the sample (ScanAsyst Fluid+, Bruker AXS).

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Nanoscope analysis 1.8 software was employed to analyze the topographic images. A

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total of 50 randomly selected NPs was analyzed to calculate the average size.

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To make the suspension of XFI06, a stock solution of 1.28 mg/mL particles in MilliQ

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water was prepared by sonication continuously for twice of 8 min with continuously

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cooling on ice using an ultrasonic processor FS-250N (20% amplitude; Shanghai

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Shengxi, China) and then diluted in full cell culture medium for exposure. In this

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study, XFI06 was co-incubated with a panel of phytochemicals. These

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phytochemicals include: quercetin (S1; 95% purity), galangin (S2; ≥98% purity),

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fisetin (S3; ≥95% purity), 3,6-dihydroxyflavone (S4; 97% purity), 3’-hydroxyflavone

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(S5; 98% purity), 3’,4’-dihydroxyflavone (S6; 97% purity), 5-hydroxyflavone (S7; 97%

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purity), 3,4’-dihydroxyflavone (S8; >97% purity), 3-hydroxyflavone (S9; >98%

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purity), 6-hydroxyflavone (S10; >98% purity), 7-hydroxyflavone (S11; >97% purity),

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myricetin (S12; >98% purity), apigenin (S13; ≥98% purity), kaempferol (S14; >98%

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purity; S1 to S14 were purchased from Shanghai Yuanye Biotechnology Co., Ltd., 6


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Shanghai, China) and baicalein (S15; >98%; S15 was purchased from Tokyo

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Chemical Industry TCI, Tokyo, Japan).

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The hydrodynamic size distribution and Zeta potential of 32 µg/mL XFI06 suspended

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in MilliQ water with or without the presence of 50 µM polyphenols was measured by

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Zetasizer nano ZS90 (Malvern, UK).

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UV-Vis spectra of XFI06 in different suspensions

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The UV-Vis spectra of 32 µg/mL XFI06, 50 µM polyphenols and XFI06 plus

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polyphenols were recorded by the Agilent Cary 60 UV-Vis spectrophotometer

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(Agilent Technologies, Santa Clara, CA, USA), and MilliQ water was used as blank.

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DPPH assay

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The radical scavenging activity of different types of polyphenols was assessed by

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DPPH assay. Various concentrations of polyphenols were prepared from 50 µM to

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1.56 µM in methanol and then incubated with 0.3 mM DPPH (Sigma-Aldrich). After

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incubated for 30 min in the dark, the absorbance at 517 nm was read by an ELISA

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reader to indicate the radical scavenging activity.

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Atomic Absorption Spectrometer (AAS)

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AAS was used to determine the dissolution of XFI06 in different suspensions. The Zn

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standard was prepared in water as 0.2 µg/mL, 0.4 µg/mL, 0.6 µg/mL, 0.8 µg/mL, 1.2

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µg/mL and 1.6 µg/mL. 32 µg/mL XFI06 was suspended in water with or without the

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presence of 50 µM of different types of polyphenols and aged for 24 h at 37 °C in a

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CO2 incubator prior to centrifuge at 16000×G for 30 min. To induce the complete

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dissolution of particles, 32 µg/mL XFI06 was incubated with HCl for 24 h. All the 7



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samples and standard were measured by an AA7000 AAS (Shimadzu CO., LTD,

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Japan) equipped with a Zn Hollow Cathode Lamp. Experiment was done

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independently twice with n=2 for each (n=4 for total), and the concentrations of Zn

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ions were calculated according to the standard curve.

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Cell counting kit-8 (CCK-8) assay

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CCK-8 assay was used to indicate the cytotoxicity. The assay was done by using

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commercial kit following manufacturer’s instruction (Beyotime, Nantong, China). For

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Caco-2 cells, cells were grown on 96-well plates and grown for 2 days. After that,

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cells were exposed to 50 µM of different types of polyphenols with or without the

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presence of 32 µg/mL XFI06. Control cells were incubated with medium containing

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the same amount of vehicles. Since we observed a strong cyto-protective effects of

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S12 (myricetin) against XFI06 exposure, we further investigated the interactions

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between XFI06 and S12 with different concentrations. For this purpose, Caco-2 cells

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were exposed to various concentrations of XFI06 (from 64 µg/mL to 4 µg/mL) with

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or without the presence of 50 µM S12, or various concentrations of S12 (from 50 µM

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to 3.13 µM) with or without the presence of 32 µg/mL XFI06. After 24 h exposure,

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the cells were rinsed once with Hanks’ solution, and CCK-8 assay was done

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according to the manufacturer’s instruction. The yellow product was read at 450 nm

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with 690 nm as reference by an ELISA reader (Synergy HT, BioTek, USA). For

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comparison, HUVECs on 96-well plates were also exposed to 50 µM of different

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types of polyphenols with or without the presence of 32 µg/mL XFI06 as indicated

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above, followed by CCK-8 assay to indicate cytotoxcity.

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Intracellular superoxide

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The intracellular superoxide was estimated by using a probe dihydroethidium (DHE;

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Beyotime, China) as we previously described.28 Briefly, Caco-2 cells were grown on

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96-well black plates for 2 days before exposure. After that, the cells were incubated

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with 50 µM of different types of polyphenols with or without the presence of 32

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µg/mL XFI06. Control cells were incubated with medium containing the same

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amount of vehicles. After 3 h exposure, the cells were rinsed once with Hanks’

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solution, incubated with 10 µg/mL DHE (Beyotime, Nantong, China) for 30 min in

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the dark, and then rinsed with Hanks’ solution. The red fluorescence was read at Ex

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530±25 nm and Em 590±35 nm by an ELISA reader.

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Intracellular Zn ions

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The accumulation of intracellular Zn ions in Caco-2 cells after 3 h exposure to

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different types of polyphenols with or without the presence of XFI06 was measured

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by using a fluorescent probe Zinquin ethyl ester (Sigma-Aldrich, USA) as our

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previous described.29

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Statistics

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All the data were expressed as means±standard error (SE) of means of 3-5

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independent experiments. One-way ANOVA followed by Tukey HSD test using R

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3.2.2. The p value

The chemical structures of polyphenols critically influence the toxicity

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