Chemical Structures of Polyphenols That Critically Influence the

Jan 31, 2018 - Surprisingly, polyphenols with both high and almost no radical scavenging activities could elicit cytoprotective effects against NP exp...
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Cite This: J. Agric. Food Chem. 2018, 66, 1714−1722

Chemical Structures of Polyphenols That Critically Influence the Toxicity of ZnO Nanoparticles Cao Zhang,†,# Yining Li,†,# Liangliang Liu,‡ Yu Gong,† Yixi Xie,*,† and Yi Cao*,†,‡ †

Key Laboratory of Environment-Friendly Chemistry and Applications of Ministry Education, Laboratory of Biochemistry, College of Chemistry, Xiangtan University, Xiangtan 411105, P.R. China ‡ Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences, Changsha 410205, P.R. China S Supporting Information *

ABSTRACT: Recent studies suggested that phytochemicals as natural antioxidants in food could alleviate nanoparticle (NP) toxicity. This study investigated the combined toxicity of ZnO NPs and a panel of polyphenols. Surprisingly, polyphenols with both high and almost no radical scavenging activities could elicit cytoprotective effects against NP exposure in Caco-2 cells, which were primarily influenced by the positions of the hydroxyl group. Polyphenols with different chemical structures variously influenced the hydrodynamic size, zeta potential, and solubility of ZnO NPs as well as NP-induced intracellular superoxide and Zn ions, which could all contribute to the combined effects. Responses of human endothelial cells appeared to be different from the responses of Caco-2 cells, which may indicate cell-type dependent responses to combined exposure of NPs and phytochemicals. In conclusion, the data from this study suggested a pivotal role of chemical structures of phytochemicals in determining their capacity to affect ZnO NP toxicity. KEYWORDS: phytochemicals, ZnO nanoparticles (NPs), oxidative stress, cytotoxicity, Caco-2 cells

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INTRODUCTION Because of their small size, nanoparticles (NPs) are used not only in cosmetics,1 microelectronics2 and medicinal products3,4 but also in food. In food and food-related products, NPs could be used for, but not limited to, antimicroorganisms, color development, and nutrient supplement.5,6 As such, oral exposure of human beings to NPs via food and food-related products is likely increasing, and there is a need to assess the toxicity of NPs to cells lining the gastrointestinal tract.7 Particularly, the assessment should consider the interactions between food components and NPs to better mimic the exposure of NPs in real life as we and others recently suggested.8,9 Phytochemicals are secondary metabolites synthesized by plants as well as nonpathogenic microorganisms living within plants. They are healthy components widely present in food with well-documented beneficial effects such as anticancer,10 prevention of cardiovascular diseases,11 and regulation of inflammation.12 Interestingly, recent studies also showed that phytochemicals might influence the toxicity of NPs.13 For example, Sarkar and Sil found that quercetin significantly protected murine hepatocytes from ion oxide NP induced cytotoxicity and apoptosis.14 Similarly, Shalini et al. recently found that quercetin significantly reduced cytotoxicity induced by ZnO NPs to human lymphocytes by the inhibition of oxidative stress.15 Using Caco-2 cells, Martirosyan et al. showed that phenolic compounds significantly reduced Ag NP-induced cytotoxicity, oxidative stress, and inflammatory responses.16,17 Because phytochemicals are widely present in food that can interact with NPs added into food, we and others have recently suggested that the interactions between NPs and phytochemicals should be carefully evaluated to better predict the toxicity of NPs in food.8,9 However, most of the previous studies only © 2018 American Chemical Society

investigated the interactions between NPs and phytochemicals with relatively high antioxidative properties, whereas relatively few studies evaluated the combined toxicity following coexposure to NPs and phytochemicals with little to no antioxidative properties.13 There are many different types of phytochemicals, and it has been shown before that the chemical structures of phytochemicals could critically influence the biological activity,18 interactions with biological molecules,19 and the stability of phytochemicals.20 In this study, we investigated the interactions between ZnO NPs and a panel of polyphenols with different positions and numbers of hydroxyl groups, so the influence of chemical structures of phytochemicals could be evaluated. The radical scavenging activities of different types of polyphenols were evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay, and the influence of polyphenols with both high and almost no antioxidant properties on the biological effects of NPs was studied. Moreover, the production of intracellular superoxide following coexposure to NPs and polyphenols was determined to further indicate the role of oxidative stress. ZnO NPs were selected because they are among the most popular NPs used in food for antibacterial and nutrition supplement, but their toxicity toward cells lining the gastrointestinal tract still needs further investigation.21 Caco-2 cells were used as the in vitro model for human intestine, and the cytotoxicity, oxidative stress, and intracellular Zn ions induced by ZnO NPs with or without the presence of different types of polyphenols were investigated. For comparison, human umbilical vein endothelial Received: Revised: Accepted: Published: 1714

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2018 2018 2018 2018 DOI: 10.1021/acs.jafc.8b00368 J. Agric. Food Chem. 2018, 66, 1714−1722

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Journal of Agricultural and Food Chemistry

spectrophotometer (Agilent Technologies, Santa Clara, CA, U.S.A.), and Milli-Q water was used as blank. DPPH Assay. The radical scavenging activities of different types of polyphenols were assessed by DPPH assay. Various concentrations of polyphenols were prepared from 50 to 1.56 μM in methanol and then incubated with 0.3 mM DPPH (Sigma-Aldrich). After being incubated for 30 min in the dark, the absorbance at 517 nm was read by an enzyme-linked immunosorbent assay (ELISA) reader to indicate the radical scavenging activity. Atomic Absorption Spectrometer. An atomic absorption spectrometer (AAS) was used to determine the dissolution of XFI06 in different suspensions. The Zn standard was prepared in water as 0.2, 0.4, 0.6, 0.8, 1.2, and 1.6 μg/mL. Thirty-two μg/mL of XFI06 was suspended in water with or without the presence of 50 μM of different types of polyphenols and aged for 24 h at 37 °C in a CO2 incubator prior to centrifugation at 16 000 × G for 30 min. To induce the complete dissolution of particles, 32 μg/mL of XFI06 was incubated with HCl for 24 h. All the samples and standard were measured by an AA7000 AAS (Shimadzu Co., Ltd., Japan) equipped with a Zn hollow cathode lamp. The experiment was done independently twice with n = 2 for each (n = 4 for total), and the concentrations of Zn ions were calculated according to the standard curve. Cell Counting Kit-8 Assay. Cell counting kit-8 (CCK-8) assay was used to indicate the cytotoxicity. The assay was done by using the commercial kit and following the manufacturer’s instructions (Beyotime, Nantong, China). For Caco-2 cells, cells were grown on 96-well plates for 2 days. After that, cells were exposed to 50 μM of different types of polyphenols with or without the presence of 32 μg/ mL of XFI06. Control cells were incubated with medium containing the same amount of vehicles. Because we observed strong cytoprotective effects of S12 (myricetin) against XFI06 exposure, we further investigated the interactions between XFI06 and S12 with different concentrations. For this purpose, Caco-2 cells were exposed to various concentrations of XFI06 (from 64 to 4 μg/mL) with or without the presence of 50 μM S12, or various concentrations of S12 (from 50 to 3.13 μM) with or without the presence of 32 μg/mL of XFI06. After 24 h exposure, the cells were rinsed once with Hanks’ solution, and CCK-8 assay was done according to the manufacturer’s instruction. The yellow product was read at 450 nm with 690 nm as reference by an ELISA reader (Synergy HT, BioTek, U.S.A.). For comparison, HUVECs on 96-well plates were also exposed to 50 μM of different types of polyphenols with or without the presence of 32 μg/mL of XFI06 as indicated above, followed by CCK-8 assay to indicate cytotoxicity. Intracellular Superoxide. The intracellular superoxide was estimated by using a probe dihydroethidium (DHE; Beyotime, China) as we previously described.28 Briefly, Caco-2 cells were grown on 96-well black plates for 2 days before exposure. After that, the cells were incubated with 50 μM of different types of polyphenols with or without the presence of 32 μg/mL of XFI06. Control cells were incubated with medium containing the same amount of vehicles. After 3 h exposure, the cells were rinsed once with Hanks’ solution, incubated with 10 μg/mL of DHE (Beyotime, Nantong, China) for 30 min in the dark, and then rinsed with Hanks’ solution. The red fluorescence was read at Ex 530 ± 25 nm and Em 590 ± 35 nm by an ELISA reader. Intracellular Zn Ions. The accumulation of intracellular Zn ions in Caco-2 cells after 3 h exposure to different types of polyphenols with or without the presence of XFI06 was measured by using a fluorescent probe Zinquin ethyl ester (Sigma-Aldrich, U.S.A.) as we previous described.29 Statistics. All the data were expressed as means ± standard error (SE) of means of 3−5 independent experiments. One-way ANOVA was followed by Tukey HSD test using R 3.2.2. The p value 97% purity), 3hydroxyflavone (S9; >98% purity), 6-hydroxyflavone (S10; >98% purity), 7-hydroxyflavone (S11; >97% purity), myricetin (S12; >98% purity), apigenin (S13; ≥98% purity), kaempferol (S14; >98% purity; S1−S14 were purchased from Shanghai Yuanye Biotechnology Co., Ltd., Shanghai, China), and baicalein (S15; >98%; S15 was purchased from Tokyo Chemical Industry TCI, Tokyo, Japan). The hydrodynamic size distribution and zeta potential of 32 μg/mL of XFI06 suspended in Milli-Q water with or without the presence of 50 μM polyphenols was measured by Zetasizer nano ZS90 (Malvern, U.K.). UV−Vis Spectra of XFI06 in Different Suspensions. The UV− vis spectra of 32 μg/mL of XFI06, 50 μM polyphenols, and XFI06 plus polyphenols were recorded by the Agilent Cary 60 UV−vis

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RESULTS Morphology of XFI06. The TEM morphology is shown in Figure 1A. On the basis of the measurement of 50 randomly 1715

DOI: 10.1021/acs.jafc.8b00368 J. Agric. Food Chem. 2018, 66, 1714−1722

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Changes of Hydrodynamic Size and Zeta Potential. The hydrodynamic size and zeta potential distribution of XFI06 with or without the presence of polyphenols are shown in Figures S3 and S4, respectively. For hydrodynamic size distribution (Figure S3), XFI06 showed monodispersity with a peak at ∼800 nm, which indicated the agglomerates and/or aggregates in suspension. With the presence of polyphenols, there was a shift of the peaks to smaller sizes. For zeta potential distribution (Figure S4), XFI06 was almost neutral, and the presence of polyphenols changed the zeta potential of XFI06 to be negative. The average zeta potential of XFI06 with or without the presence of polyphenols is shown in Figure 2. All of

Figure 1. TEM morphology (A) and AFM topography (B) of XFI06 (ZnO NPs).

selected particles, the average particle size was calculated as 35.9 ± 12.8 nm (size range 15−70 nm). The AFM topography is shown in Figure 1B, which indicates uniform surfaces of NPs. The average particle size was calculated as 39.7 ± 12.2 nm (size range 20−75 nm). UV−Vis Spectra. A total of 15 polyphenols were used in this study, and their chemical structures are shown in Supporting Information, Figure S1. They are classified as flavones and flavonols with different numbers and positions of hydroxyl groups (Table 1). The UV−vis spectra of the Table 1. Substitution Positions of the Different Types of Polyphenols Used in This Study

Figure 2. Average zeta potential of XFI06 (ZnO NPs) with or without the presence of different types of polyphenols. Data represent means ± SD of three measurements.

the polyphenols decreased the zeta potential of XFI06 to different extents, with S1 (quercetin) being the most effective, followed by S3 (fisetin), S6 (3′,4′-dihydroxyflavone), S12 (myricetin), S13 (apigenin), and S15 (baicalein). Dissolution of XFI06. The Zn ion concentrations from the dissolution of XFI06 were determined by AAS, and the result is shown in Figure 3. Without the presence of polyphenols, XFI06 was partially soluble. The presence of polyphenols influenced the dissolution of XFI06 differently. Some of the polyphenols, such as S1 (quercetin; p < 0.01), S3 (fisetin; p < 0.01), S12 (myricetin; p < 0.05), and S13 (apigenin; p < 0.01), significantly promoted the concentrations of Zn ions released from the dissolution of XFI06. In contrast, S15 (baicalein; p < 0.01), and less effectively S10 (6-hydroxyflavone; p < 0.05) and S11 (7-hydroxyflavone; p < 0.05), significantly decreased the Zn ion concentrations released from the dissolution of XFI06. Some of the polyphenols, such as S4 (3,6-dihydroxyflavone), S5 (3′-hydroxyflavone), and S6 (3′,4′,-dihydroxyflavone), showed almost no effect on the dissolution of XFI06. Radical Scavenging Activity. The radical scavenging activity of all the polyphenols was assessed by DPPH assay, and the result is shown in Figure 4. On the basis of DPPH assay, the polyphenols were classified either as high radical scavenging polyphenols (radical scavenging activities >50% at the highest concentration) or as low radical scavenging polyphenols (radical scavenging activities 0.05; Figure 5B). Similarly, S1 (quercetin) also slightly increased the toxicity of XFI06 but acted even more slightly. The cytotoxicity of Caco-2 cells after coexposure to XFI06 and S12 with different concentrations is shown in Figure S5. Without the presence of 50 μM S12, exposure to 32 and 64 μg/ mL of XFI06 was associated with significantly reduced cellular viability (p < 0.01). However, with the presence of 50 μM S12, XFI06 only significantly reduced cellular viability at 64 μg/mL (p < 0.01; Figure S5A). Exposure to various concentrations of S12 did not significantly affect Caco-2 cellular viability (p > 0.05), whereas coexposure to 32 μg/mL XFI06 and 3.13 μM (p < 0.01), 6.25 μM (p < 0.05), 12.5 μM (p < 0.05), or 25 μM (p < 0.05) S12 significantly reduced cellular viability. Moreover, coexposure to 25 or 50 μM S12 and 32 μg/mL of XFI06 resulted in significantly higher Caco-2 viability compared with that after exposure to XFI06 alone (p < 0.01; Figure S5B). For comparison, HUVECs were also used, and the results are shown in Figure 6. Exposure to S1 (quercetin), S2 (galangin), S3 (fisetin), and S14 (kaempferol) was associated with significantly reduced cellular viability (p < 0.05), whereas exposure to S5 (3′-hydroxyflavone; p < 0.01) and S7 (5hydroxyflavone; p < 0.05) significantly increased cellular viability (Figure 6A). Exposure to XFI06 reduced the cellular viability of HUVECs to ∼12%, and only S12 (myricetin) significantly increased the cellular viability of HUVECs after XFI06 exposure (p < 0.01; Figure 6B). Intracellular Superoxide. As shown in Figure 7A, exposure to different types of polyphenols did not significantly affect intracellular superoxide. Exposure to XFI06 also did not significantly induce intracellular superoxide (p > 0.05), but coexposure of Caco-2 cells to XFI06 and S9 (3-hydroxyflavone) significantly enhanced intracellular superoxide (p < 0.01). In contrast, the presence of other types of polyphenols did not significantly influence intracellular superoxide in Caco-2 cells after XFI06 exposure (p > 0.05; Figure 7B). Intracellular Zn Ions. Exposure to different types of polyphenols did not significantly affect intracellular Zn ions (p

Figure 3. Zn ion concentrations released from the dissolution of XFI06 (ZnO NPs) with or without the presence of different types of polyphenols. Thirty-two μg/mL of XFI06 was aged for 24 h with or without the presence of different types of polyphenols. To induce the complete dissolution of XFI06, 32 μg/mL of XFI06 was also incubated with HCl for 24 h. Zn ions released from the dissolution of XFI06 were measured by AAS. Data represent means ± SD of two independent experiments with n = 2 (total n = 4). *p < 0.05, compared with XFI06.

polyphenols that almost completely scavenged DPPH at 50 μM (Figure 4A). In contrast, S10 (6-hydroxyflavone), S11 (7hydroxyflavone), and S13 (apigenin) are low radical scavenging polyphenols as they almost showed no effect to scavenge DPPH as high as 50 μM (Figure 4B). Cytotoxicity. Without exposure to XFI06 (Figure 5A), only S3 (fisetin) significantly reduced cellular viability of Caco-2 cells (p < 0.05). However, the cellular viability of Caco-2 cells after exposure to different types of polyphenols was >80%, which indicated relatively low cytotoxic potential of these polyphenols to Caco-2 cells. Exposure to XFI06 reduced the cellular viability to ∼20%. Some types of polyphenols, such as S8 (3,4′-dihydroxyflavone; p < 0.05), S10 (6-hydroxyflavone; p < 0.01), S11 (7-hydroxyflavone; p < 0.01), and S12 (myricetin;

Figure 4. Radical scavenging activities of different types of polyphenols as assessed by DPPH assay. The polyphenols were classified as high radical scavenging polyphenols (radical scavenging activities >50% at the highest concentration) and low radical scavenging polyphenols (radical scavenging activities 0.05; Figure 8A). Exposure to XFI06 induced >2-fold increase of intracellular Zn ions, and the presence of S9 (3hydroxyflavone) further enhanced XFI06 induced intracellular Zn ions. Some of the polyphenols, such as S4 (3,6dihydroxyflavone), S11 (7-hydroxyflavone), S12 (myricetin), and S15 (baicalein), slightly lowered XFI06 induced intracellular Zn ions, but this effect was not statistically significantly different (p > 0.05; Figure 8B).

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DISCUSSION The present study investigated the interactions between ZnO NPs and a panel of polyphenols with different numbers and positions of hydroxyl groups, so the influence of chemical structures of polyphenols on NP−polyphenol interactions could be studied. The radical scavenging activities of these polyphenols were indicated by DPPH assay, and results clearly showed that these polyphenols exhibited different antioxidative 1718

DOI: 10.1021/acs.jafc.8b00368 J. Agric. Food Chem. 2018, 66, 1714−1722

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Figure 7. Intracellular superoxide of Caco-2 cells. Caco-2 cells were exposed to different types of polyphenols (A) and XFI06 and XFI06 plus polyphenols (B) for 3 h, and intracellular superoxide was determined by using a fluorescence probe. Data represent means ± SE of three independent experiments (n = 3). #p < 0.01, compared with XFI06 exposed cells.

Figure 8. Intracellular Zn ions of Caco-2 cells. Caco-2 cells were exposed to different types of polyphenols (A) and XFI06 and XFI06 plus polyphenols (B) for 3 h, and intracellular Zn ions were determined by using a fluorescence probe. Data represent means ± SE of three independent experiments (n = 3). #p < 0.05, compared with XFI06 exposed cells.

further indicated that, at least in the case of polyphenols, the influence on NP colloidal stability is dependent on the chemical structures of phytochemicals. We then investigated the cytotoxicity of ZnO NPs to Caco-2 cells with or without the presence of different types of phytochemicals. Previous studies suggested that the cytoprotective effects of phytochemicals were dependent on the antioxidant properties.8,9 In this study, S12 (myricetin), which exhibited a strong radical scavenging activity (radical scavenging activities ∼80% at 50 μM), also significantly protected Caco-2 cells from ZnO NP exposure (p < 0.01). In addition, by using various concentrations of S12, the results from this study further showed that S12 could elicit a cytoprotective effect in a manner dependent on the doses of S12 (p < 0.01). However, not all the protective effects could be explained by the antioxidant properties of polyphenols. For example, S1 (quercetin), S3 (fisetin), and S6 (3′,4′-dihydroxyflavone) also

on the types of polyphenols. While some types of polyphenols, such as S1 (quercetin) and S2 (galangin), markedly increased the UV−vis absorbance of XFI06 at 370 nm, other types of polyphenols, such as S5 (3′-hydroxyflavone) and S7 (5hydroxyflavone), only slightly increased the UV−vis absorbance of XFI06 at 370 nm. Similarly, the hydrodynamic size, zeta potential, and dissolution of XFI06 could be variously altered by different types of polyphenols. In our recent study, we found that baicalein was more effective than baicalin to alter the UV− vis absorbance, hydrodynamic size, and zeta potential of XFI06.26 In other studies, we showed that palmitate or oleate reduced the solubility and increased hydrodynamic size of ZnO NPs in water.30,31 Although these techniques could not provide quantitative information about NP−polyphenol interactions, the results from this study in combination with previous studies indicated that the colloidal aspects of NPs could be influenced by food components. In addition, our data from this study 1719

DOI: 10.1021/acs.jafc.8b00368 J. Agric. Food Chem. 2018, 66, 1714−1722

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there is one exception: S8 (3,4′-dihydroxyflavone), which has one hydroxyl group at C-3 on ring C and one at C-4′ on ring B, significantly protected Caco-2 cells from ZnO NP exposure (p < 0.05). It should be noticed that we only used polyphenols with different positions and numbers of hydroxyl groups; it could be possible that other structures might also influence the combined effects of polyphenols and NPs, but thi may need further studies. It is worthy of noticing that the responses of HUVECs to combined exposure of ZnO NPs and polyphenols appeared to be quite different from the responses of Caco-2 cells. While S8 (3,4′-dihydroxyflavone; p < 0.05), S10 (6-hydroxyflavone; p < 0.01), S11 (7-hydroxyflavone; p < 0.01), and S12 (myricetin; p < 0.01) significantly protected Caco-2 cells from ZnO NP exposure, only S12 showed this effect in HUVECs (p < 0.01). To the best of our knowledge, relatively few studies compared the different responses of cell lines to combined exposure of NPs and phytochemicals. Our recent study showed that baicalein only slightly protected Caco-2 but not HepG2 cells from ZnO NP exposure.26 In another study, it was shown that isoorientin enhanced ZnO NP induced toxicity to cancerous liver cells but not the normal cells.33 If this is the case, it probably indicated that the combined effects of NPs and phytochemicals are dependent on the types of cells used for assessment and should be evaluated case to case. It has been suggested that reactive oxygen species overproduction and/or accumulation of intracellular Zn ions is involved in ZnO NP induced cytotoxicity.34 The results from this study showed that exposure to ZnO NPs was associated with increased intracellular Zn ions (>2-fold increase of intracellular Zn ions) but not superoxide (p > 0.05), which is in agreement with our previous studies using human macrophages,29,35 hepotocytes,28 and intestinal cells.26 This is also consistent with previous reports showing that the accumulation of intracellular Zn ions plays a pivotal role in ZnO NP induced toxicity to human cells.36−38 There was one exception: S9 (3hydroxyflavone) significantly promoted ZnO NP induced intracellular superoxide and Zn ions (p < 0.01) and showed a trend in the direction of higher cytotoxicity after combined exposure to S9 and ZnO NPs. A previous study showed that exposure to isoorientin and ZnO NPs, but not ZnO NPs alone, induced intracellular reactive oxygen species in liver cells. As a consequence, coexposure to isoorientin and ZnO NPs induced higher cytotoxicity compared with that induced by ZnO NPs alone.33 Therefore, it is possible that coexposure to S9 and XFI06 induced a relatively higher cytotoxicity due to augmented superoxide production and accumulation of intracellular Zn ions. In this study, we found that the solubility of XFI06 was altered by the presence of different types of polyphenols. This is consistent with previous reports showing that the amount of Zn ions released from the dissolution of ZnO NPs could be changed when NPs were suspended in different media.39−41 However, there appeared to be no obvious correlation between solubility of XFI06 and concentrations of intracellular Zn ions. For example, S1 (quercetin; p < 0.01), S3 (fisetin; p < 0.01), S12 (myricetin; p < 0.05), and S13 (apigenin; p < 0.01) significantly promoted the solubility of XFI06, but they did not significantly increase or even slightly decreased intracellular Zn ions (p > 0.05). In our recent studies, we found that oleate30 or palmitate31 could decrease the solubility of XFI06, whereas dipalmitoylphosphatidylcholine increased the solubility.42 However, only oleate significantly reduced intracellular Zn ions in cells after exposure to aged

exhibited strong radical scavenging activities (radical scavenging activities ∼95% at 50 μM) but showed no or almost no protective effects against ZnO NP induced cytotoxicity such that cellular viability of Caco-2 cells following coexposure to these polyphenols and XFI06 was comparable to that after exposure to XFI06 alone (p > 0.05). In contrast, S8 (3,4′dihydroxyflavone), S10 (6-hydroxyflavone), and S11 (7hydroxyflavone), which exhibited little to no radical scavenging activities (radical scavenging activities 0.05). The data from this study further suggested that the positions of hydroxyl groups are more important to influence the cytoprotective effects. By using S10 (6-hydroxyflavone) and S11 (7-hydroxyflavone), it was shown that hydroxyl groups at C-6 and C-7 on ring A could significantly reduce the toxicity of NPs (p < 0.01). However, hydroxyl groups at other positions, such as C-5 on ring A and C-3 on ring C, might weaken the protective effects of hydroxyl groups at C-6 and C-7 on ring A. This could be evidenced by the fact that S12 (galangin) and S4 (3,6-dihydroxyflavone) did not significantly protect Caco-2 cells from NP exposure (p > 0.05), although they have hydroxyl groups at C-6 and C-7 on ring A. Moreover, S7 (5-hydroxyflavone), which has a hydroxyl group at C-5 on ring A, and S9 (3-hydroxyflavone), which has a hydroxyl group at C-3 on ring C, did not significantly protect Caco-2 cells from NP exposure (p > 0.05). One possible explanation is that the hydroxyl group at C-3 and C-5 could provide hydrogen bonding to the oxo-group, which can consequently influence the stability and activity of polyphenols.18,32 Polyphenols with one or two hydroxyl groups on ring B, such as S5 (3′-hydroxyflavone) and S6 (3′,4′-dihydroxyflavone), did not significantly protect Caco-2 cells (p > 0.05), whereas S12 (myricetin), which has three hydroxyl groups on ring B, showed significantly cytoprotective effects (p < 0.01). This might indicate that one or two hydroxyl groups on ring B could also weaken the cytoprotective effects of polyphenols against ZnO NP exposure. Generally, polyphenols with one hydroxyl group at C-5 on ring A or C-3 on ring C or one or two hydroxyl groups on ring B showed little to no protective effects for Caco-2 cells against ZnO NP exposure. However, 1720

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XFI06. Thus, it is possible that intracellular Zn ion concentrations are not directly corrected with extracellular dissolution of ZnO NPs. It should be noticed that the accumulation of intracellular Zn ions could not completely explain the combined effects. For example, S12 (myricetin) significantly reduced ZnO NP induced cytotoxicity but only slightly decreased intracellular Zn ions. In our recent studies, we found that the presence of palmitate enhanced ZnO NP induced cytotoxicity without an influence on the accumulation of intracellular Zn ions.29,43 Previously, it has been shown before that the main food components enhanced Ag NP uptake without an effect on NP induced cytotoxicity.44 Thus, it is possible that the NP uptake is not completely connected with the combined toxicity of NPs and food components. Other factors, such as the changes of colloidal aspects by food components, may also critically contribute to the alterations of toxicity.9,25 In summary, the results from this study indicated that the chemical structures of polyphenols, beside or even rather than their antioxidant properties, contributed to their capacity to influence the toxicity of ZnO NPs to Caco-2 cells. The results suggested that the positions, but not the numbers of hydroxyl groups, are not crucial for the cytoprotective effects. More specifically, the hydroxyl groups at C-6 and C-7 on ring A could significantly reduce the toxicity of ZnO NPs, whereas hydroxyl groups at C-5 on ring A and C-3 on ring C might weaken the cytoprotective effects of polyphenols. The hydroxyl groups on ring B might also weaken the cytoprotective effects of polyphenols, unless there are three hydroxyl groups at ring B. The presence of polyphenols influenced ZnO NP UV−vis spectra, hydrodynamic size, zeta potential, and solubility as well as Zn ions, which could all contribute to the changes of cytotoxicity of ZnO NPs to Caco-2 cells. ZnO NP themselves and combined with polyphenols practically did not induce intracellular superoxide (with one exception, S9 (3-hydroxyflavone)), a further hint that generation of reactive oxidative species alone may not be responsible for toxicity. The results from this study also indicated that, when assessing the combined toxicity of NPs and phytochemicals, it may be necessary to evaluate the influence of phytochemicals on NP colloidal aspects.

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C.Z. and Y.L. contributed equally to this work.

Funding

This work was financially supported by Natural Science Foundation of Hunan Province (2017JJ3303) and National Natural Science Foundation of China (31701613). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We appreciate Prof. Yanhuai Ding for his scientific assistance in AFM study.

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ABBREVIATIONS USED AAS, atomic absorption spectrometer; AFM, atomic force microscope; CCK, cell counting kit-8; DHE, dihydroethidium; DPPH, 1,1-diphenyl-2-picrylhydrazyl; ECM, endothelial cell medium; HUVEC, human umbilical vein endothelial cell; NP, nanoparticle; SE, standard error; TEM, transmission electron microscopy; XRD, X-ray diffractograms

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(1) Antunes, A. F.; Pereira, P.; Reis, C.; Rijo, P.; Reis, C. Nanosystems for Skin Delivery: From Drugs to Cosmetics. Curr. Drug Metab. 2017, 18 (5), 412−425. (2) McIntyre, R. A. Common nano-materials and their use in real world applications. Sci. Prog. 2012, 95 (1), 1−22. (3) Liao, W.; Zhang, T. T.; Gao, L.; Lee, S. S.; Xu, J.; Zhang, H.; Yang, Z.; Liu, Z.; Li, W. Integration of Novel Materials and Advanced Genomic Technologies into New Vaccine Design. Curr. Top. Med. Chem. 2017, 17 (20), 2286−2301. (4) Zhao, P.; Xu, Q.; Tao, J.; Jin, Z.; Pan, Y.; Yu, C.; Yu, Z. Near infrared quantum dots in biomedical applications: current status and future perspective. Wiley. Interdiscip. Rev. Nanomed. Nanobiotechnol. 2017, e1483. (5) Hoseinnejad, M.; Jafari, S. M.; Katouzian, I. Inorganic and metal nanoparticles and their antimicrobial activity in food packaging applications. Crit. Rev. Microbiol. 2018, 44 (2), 161−181. (6) Pathakoti, K.; Manubolu, M.; Hwang, H. M. Nanostructures: Current uses and future applications in food science. J. Food Drug Anal. 2017, 25 (2), 245−253. (7) McCracken, C.; Dutta, P. K.; Waldman, W. J. Critical assessment of toxicological effects of ingested nanoparticles. Environ. Sci.: Nano 2016, 3 (2), 256−282. (8) Cao, Y.; Li, J.; Liu, F.; Li, X.; Jiang, Q.; Cheng, S.; Gu, Y. Consideration of interaction between nanoparticles and food components for the safety assessment of nanoparticles following oral exposure: A review. Environ. Toxicol. Pharmacol. 2016, 46, 206−210. (9) McClements, D. J.; Xiao, H.; Demokritou, P. Physicochemical and colloidal aspects of food matrix effects on gastrointestinal fate of ingested inorganic nanoparticles. Adv. Colloid Interface Sci. 2017, 246, 165−180. (10) Rothwell, J. A.; Knaze, V.; Zamora-Ros, R. Polyphenols: dietary assessment and role in the prevention of cancers. Curr. Opin. Clin. Nutr. Metab. Care 2017, 20 (6), 512−521. (11) Bahramsoltani, R.; Ebrahimi, F.; Farzaei, M. H.; Baratpourmoghaddam, A.; Ahmadi, P.; Rostamiasrabadi, P.; Rasouli Amirabadi, A. H.; Rahimi, R. Dietary polyphenols for atherosclerosis: A comprehensive review and future perspectives. Crit. Rev. Food Sci. Nutr. 2017, 1−19. (12) Shimizu, M. Multifunctions of dietary polyphenols in the regulation of intestinal inflammation. J. Food Drug Anal. 2017, 25 (1), 93−99. (13) Cao, Y.; Xie, Y.; Liu, L.; Xiao, A.; Li, Y.; Zhang, C.; Fang, X.; Zhou, Y. Influence of phytochemicals on the biocompatibility of

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.8b00368. Structures of the polyphenols used in this study; UV−vis spectra of polyphenols, XFI06 (ZnO NPs), and polyphenols + XFI06; representative hydrodynamic size distribution of XFI06 (ZnO NPs) with or without the presence of polyphenols; representative zeta potential distribution of XFI06 (ZnO NPs) with or without the presence of polyphenols; cytotoxicity to Caco-2 cells as assessed by CCK-8 assay (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Yi Cao: 0000-0002-9282-0051 1721

DOI: 10.1021/acs.jafc.8b00368 J. Agric. Food Chem. 2018, 66, 1714−1722

Article

Journal of Agricultural and Food Chemistry inorganic nanoparticles: a state-of-the-art review. Phytochem. Rev. 2017, 16, 555−563. (14) Sarkar, A.; Sil, P. C. Iron oxide nanoparticles mediated cytotoxicity via PI3K/AKT pathway: role of quercetin. Food Chem. Toxicol. 2014, 71, 106−115. (15) Shalini, D.; Senthilkumar, S.; Rajaguru, P. Effect of size and shape on toxicity of zinc oxide (ZnO) nanomaterials in human peripheral blood lymphocytes. Toxicol. Mech. Methods 2018, 28 (2), 87−94. (16) Martirosyan, A.; Bazes, A.; Schneider, Y. J. In vitro toxicity assessment of silver nanoparticles in the presence of phenolic compounds−preventive agents against the harmful effect? Nanotoxicology 2014, 8 (5), 573−582. (17) Martirosyan, A.; Grintzalis, K.; Polet, M.; Laloux, L.; Schneider, Y. J. Tuning the inflammatory response to silver nanoparticles via quercetin in Caco-2 (co-)cultures as model of the human intestinal mucosa. Toxicol. Lett. 2016, 253, 36−45. (18) Chen, L.; Teng, H.; Xie, Z.; Cao, H.; Cheang, W. S.; SkalickaWoniak, K.; Georgiev, M. I.; Xiao, J. Modifications of dietary flavonoids towards improved bioactivity: An update on structureactivity relationship. Crit. Rev. Food Sci. Nutr. 2016, 1−15. (19) Tang, F.; Xie, Y.; Cao, H.; Yang, H.; Chen, X.; Xiao, J. Fetal bovine serum influences the stability and bioactivity of resveratrol analogues: A polyphenol-protein interaction approach. Food Chem. 2017, 219, 321−328. (20) Xiao, J.; Hogger, P. Stability of dietary polyphenols under the cell culture conditions: avoiding erroneous conclusions. J. Agric. Food Chem. 2015, 63 (5), 1547−1557. (21) Krol, A.; Pomastowski, P.; Rafinska, K.; Railean-Plugaru, V.; Buszewski, B. Zinc oxide nanoparticles: Synthesis, antiseptic activity and toxicity mechanism. Adv. Colloid Interface Sci. 2017, 249, 37−52. (22) Cao, Y.; Gong, Y.; Liu, L.; Zhou, Y.; Fang, X.; Zhang, C.; Li, Y.; Li, J. The use of human umbilical vein endothelial cells (HUVECs) as an in vitro model to assess the toxicity of nanoparticles to endothelium: a review. J. Appl. Toxicol. 2017, 37 (12), 1359−1369. (23) Bittner, K.; Kemme, T.; Peters, K.; Kersten, S.; Danicke, S.; Humpf, H. U. Systemic absorption and metabolism of dietary procyanidin B4 in pigs. Mol. Nutr. Food Res. 2014, 58 (12), 2261− 2273. (24) Croft, K. D.; Yamashita, Y.; O’Donoghue, H.; Shirasaya, D.; Ward, N. C.; Ashida, H. Screening plant derived dietary phenolic compounds for bioactivity related to cardiovascular disease. Fitoterapia 2017, DOI:10.1016/j.fitote.2017.12.002. (25) Moore, T. L.; Rodriguez-Lorenzo, L.; Hirsch, V.; Balog, S.; Urban, D.; Jud, C.; Rothen-Rutishauser, B.; Lattuada, M.; Petri-Fink, A. Nanoparticle colloidal stability in cell culture media and impact on cellular interactions. Chem. Soc. Rev. 2015, 44 (17), 6287−6305. (26) Li, Y.; Zhang, C.; Liu, L.; Gong, Y.; Xie, Y.; Cao, Y. The effects of baicalein or baicalin on the colloidal stability of ZnO nanoparticles (NPs) and toxicity of NPs to Caco-2 cells. Toxicol. Mech. Methods 2018, 28 (3), 167−176. (27) Ji, Y.; Zhu, M.; Gong, Y.; Tang, H.; Li, J.; Cao, Y. Thermoresponsive Polymers with Lower Critical Solution Temperature- or Upper Critical Solution Temperature-Type Phase Behaviour Do Not Induce Toxicity to Human Endothelial Cells. Basic Clin. Pharmacol. Toxicol. 2017, 120 (1), 79−85. (28) Zhou, Y.; Fang, X.; Gong, Y.; Xiao, A.; Xie, Y.; Liu, L.; Cao, Y. The Interactions between ZnO Nanoparticles (NPs) and alphaLinolenic Acid (LNA) Complexed to BSA Did Not Influence the Toxicity of ZnO NPs on HepG2 Cells. Nanomaterials 2017, 7 (4), 91. (29) Jiang, Q.; Li, X.; Cheng, S.; Gu, Y.; Chen, G.; Shen, Y.; Xie, Y.; Cao, Y. Combined effects of low levels of palmitate on toxicity of ZnO nanoparticles to THP-1 macrophages. Environ. Toxicol. Pharmacol. 2016, 48, 103−109. (30) Fang, X.; Jiang, L.; Gong, Y.; Li, J.; Liu, L.; Cao, Y. The presence of oleate stabilized ZnO nanoparticles (NPs) and reduced the toxicity of aged NPs to Caco-2 and HepG2 cells. Chem.-Biol. Interact. 2017, 278, 40−47.

(31) Gong, Y.; Liu, L.; Li, J.; Cao, Y. The presence of palmitate affected the colloidal stability of ZnO NPs but not the toxicity to Caco-2 cells. J. Nanopart. Res. 2017, 19 (10), 335. (32) Ravishankar, D.; Rajora, A. K.; Greco, F.; Osborn, H. M. Flavonoids as prospective compounds for anti-cancer therapy. Int. J. Biochem. Cell Biol. 2013, 45 (12), 2821−2831. (33) Yuan, L.; Wang, Y.; Wang, J.; Xiao, H.; Liu, X. Additive effect of zinc oxide nanoparticles and isoorientin on apoptosis in human hepatoma cell line. Toxicol. Lett. 2014, 225 (2), 294−304. (34) Saptarshi, S. R.; Duschl, A.; Lopata, A. L. Biological reactivity of zinc oxide nanoparticles with mammalian test systems: an overview. Nanomedicine (London, U. K.) 2015, 10 (13), 2075−2092. (35) Chen, G.; Shen, Y.; Li, X.; Jiang, Q.; Cheng, S.; Gu, Y.; Liu, L.; Cao, Y. The endoplasmic reticulum stress inducer thapsigargin enhances the toxicity of ZnO nanoparticles to macrophages and macrophage-endothelial co-culture. Environ. Toxicol. Pharmacol. 2017, 50, 103−110. (36) Mu, Q.; David, C. A.; Galceran, J.; Rey-Castro, C.; Krzeminski, L.; Wallace, R.; Bamiduro, F.; Milne, S. J.; Hondow, N. S.; Brydson, R.; Vizcay-Barrena, G.; Routledge, M. N.; Jeuken, L. J.; Brown, A. P. Systematic investigation of the physicochemical factors that contribute to the toxicity of ZnO nanoparticles. Chem. Res. Toxicol. 2014, 27 (4), 558−567. (37) Gilbert, B.; Fakra, S. C.; Xia, T.; Pokhrel, S.; Madler, L.; Nel, A. E. The fate of ZnO nanoparticles administered to human bronchial epithelial cells. ACS Nano 2012, 6 (6), 4921−4930. (38) Shen, C.; James, S. A.; de Jonge, M. D.; Turney, T. W.; Wright, P. F.; Feltis, B. N. Relating cytotoxicity, zinc ions, and reactive oxygen in ZnO nanoparticle-exposed human immune cells. Toxicol. Sci. 2013, 136 (1), 120−130. (39) Reed, R. B.; Ladner, D. A.; Higgins, C. P.; Westerhoff, P.; Ranville, J. F. Solubility of nano-zinc oxide in environmentally and biologically important matrices. Environ. Toxicol. Chem. 2012, 31 (1), 93−99. (40) Wang, Y.; Yuan, L.; Yao, C.; Ding, L.; Li, C.; Fang, J.; Sui, K.; Liu, Y.; Wu, M. A combined toxicity study of zinc oxide nanoparticles and vitamin C in food additives. Nanoscale 2014, 6 (24), 15333− 15342. (41) Mudunkotuwa, I. A.; Rupasinghe, T.; Wu, C. M.; Grassian, V. H. Dissolution of ZnO nanoparticles at circumneutral pH: a study of size effects in the presence and absence of citric acid. Langmuir 2012, 28 (1), 396−403. (42) He, T.; Long, J.; Li, J.; Liu, L.; Cao, Y. Toxicity of ZnO nanoparticles (NPs) to A549 cells and A549 epithelium in vitro: Interactions with dipalmitoyl phosphatidylcholine (DPPC). Environ. Toxicol. Pharmacol. 2017, 56, 233−240. (43) Gong, Y.; Ji, Y.; Liu, F.; Li, J.; Cao, Y. Cytotoxicity, oxidative stress and inflammation induced by ZnO nanoparticles in endothelial cells: interaction with palmitate or lipopolysaccharide. J. Appl. Toxicol. 2017, 37 (8), 895−901. (44) Lichtenstein, D.; Ebmeyer, J.; Knappe, P.; Juling, S.; Bohmert, L.; Selve, S.; Niemann, B.; Braeuning, A.; Thunemann, A. F.; Lampen, A. Impact of food components during in vitro digestion of silver nanoparticles on cellular uptake and cytotoxicity in intestinal cells. Biol. Chem. 2015, 396 (11), 1255−1264.

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DOI: 10.1021/acs.jafc.8b00368 J. Agric. Food Chem. 2018, 66, 1714−1722