Protective effect of Caffeic Acid Phenethyl Ester (CAPE) against oxidative stress

Protective effect of Caffeic Acid Phenethyl Ester (CAPE) against oxidative stress

Journal of Functional Foods 29 (2017) 178–184 Contents lists available at ScienceDirect Journal of Functional Foods journal homepage: www.elsevier.c...

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Journal of Functional Foods 29 (2017) 178–184

Contents lists available at ScienceDirect

Journal of Functional Foods journal homepage: www.elsevier.com/locate/jff

Short communications

Protective effect of Caffeic Acid Phenethyl Ester (CAPE) against oxidative stress Ana Laura Carreño a,1, Efrain Alday a,1, Jael Quintero b, Lucía Pérez c, Dora Valencia d, Ramón Robles-Zepeda a, Judith Valdez-Ortega a, Javier Hernandez e, Carlos Velazquez a,⇑ a

Department of Chemistry-Biology, University of Sonora, Blvd. Luis Encinas y Rosales s/n, Hermosillo, Sonora C.P. 83000, Mexico Departamento de Ciencias de la Salud, Universidad de Sonora, Blvd. Bordo Nuevo s/n, Ejido Providencia, Cd Obregón, Sonora C.P. 85039, Mexico Departamento de Investigación en Ciencia y Tecnología, DICTUS, University of Sonora, Blvd. Luis Encinas y Rosales s/n, Hermosillo, Sonora C.P. 83000, Mexico d Department of Chemical Biological and Agropecuary Sciences, University of Sonora, Av. Universidad and Irigoyen, Caborca, Sonora C.P. 83600, Mexico e Unidad de Servicios de Apoyo en Resolución Analítica, Universidad Veracruzana, Xalapa, Ver. C.P. 575, Mexico b c

a r t i c l e

i n f o

Article history: Received 12 July 2016 Received in revised form 3 December 2016 Accepted 6 December 2016

Keywords: CAPE Cellular antioxidant activity Murine B-cell lymphoma cells Murine macrophage cells Sonoran propolis constituents

a b s t r a c t The antioxidant properties of several polyphenolics of propolis have been reported, however their protective effect against oxidative stress considering cell integrity is scarce. In this study, we evaluated the cellular antioxidant activity (CAA) of caffeic acid phenetyl ester (CAPE), rutin and galangin (Sonoran propolis constituents) using two murine cell lines derived from different immunological lineages (B-cell lymphoma and macrophages), based on the fluorescence of intracellularly oxidised 20 -70 -dichlorofluorescein (DCF) probe, together with cell morphology analysis and membrane integrity assessment by flow cytometry. CAPE (5 lM) showed the highest CAA (97.9%) on B-cell lymphoma cells against 1 mM H2O2, followed by rutin (25 lM; 30.9%), meanwhile galangin (25 lM) did not show CAA. CAPE exhibited a higher CAA than the antioxidant controls [quercetin (12.5 lM), ascorbic acid (50 lM) and trolox (10 lM)], and additionally it helped to preserve cellular morphology. Similar effects were observed on macrophage cells, indicating that CAPE has a cellular protective effect against ROS. Further studies are needed to investigate the potential health benefits of CAPE. Ó 2016 Elsevier Ltd. All rights reserved.

1. Introduction Reactive oxygen species (ROS) are highly unstable molecules continuously produced by cellular metabolism under normal conditions. ROS comprise free radicals such as superoxide anion (O 2 ), singlet oxygen (1O2), hydroxyl radicals (OH), peroxides (ROOR0 or ROOH), as well as non-radical oxidizing agents such as hydrogen peroxide (H2O2) and hypochlorous acid (HOCl), in addition to nitrogen containing oxidants, such as nitric oxide (Carocho & Ferreira, 2013; Eruslanov & Kusmartsev, 2010; Prasad, Gupta, & Tyagi, 2016). In order to modulate and limit the high reactivity and levels of ROS, the cellular environment is endowed with appropriate defence mechanisms, including the enzymatic systems [xanthine oxidase (XO), cytochrome P-450, NADPH oxidase, superoxide dismutase (SOD), catalase and glutathione peroxidase] and the nonenzymatic systems [reduced glutathione, zinc and uric acid] ⇑ Corresponding author. 1

E-mail address: [email protected] (C. Velazquez). Both authors contributed equally to this work.

http://dx.doi.org/10.1016/j.jff.2016.12.008 1756-4646/Ó 2016 Elsevier Ltd. All rights reserved.

(Carocho & Ferreira, 2013; Eruslanov & Kusmartsev, 2010; Prasad et al., 2016). Fluctuations at basal levels of ROS are essential to activate and regulate several cellular events, including development, proliferation and differentiation, as well as the activation of innate immune response (Mittler, 2016; Prasad et al., 2016). However, ROS produced by effector cells (activated lymphocytes and phagocytes), through innate and adaptive immune responses, induce some oxidative damage into parallel tissues (Tabas & Glass, 2013). Generally, an overproduction of ROS would shift the redox homeostasis towards the oxidative stress process, which is implicated in the pathogenesis of several chronic and inflammatory diseases (Mittler, 2016; Prasad et al., 2016; Tabas & Glass, 2013; Wang & Joseph, 1999). Interestingly, bioactive compounds present in food and functionalised food products provide further cellular protection against ROS. These compounds, generally termed nutraceuticals, comprise structurally diverse molecules, including water and lipid soluble vitamins, such as ascorbic acid (AA), retinoic acid, tocopherol, riboflavin, as well as plant secondary metabolites such as carotenoids and polyphenolic compounds, among others (Babich, Schuck, Weisburg, & Zuckerbraun, 2011; Cencic & Chingwaru, 2010;

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Lokesh, Channarayappa, & Venkatarangana, 2015; Prasad et al., 2016). It is broadly reported that polyphenolic compounds exert several health benefits, including antioxidant potential, mostly by their capacity to act as ROS scavengers (Babich et al., 2011; Medi c´-Šaric´ et al., 2009). Propolis is one of the richest sources of polyphenols, representing a potential matrix of nutraceuticals (Bankova, 2005; Medic´-Ša ric´ et al., 2009). In particular, Caffeic Acid Phenethyl Ester (CAPE) is a propolis constituent that has gained attention due to its broad pharmacological activities (Bankova, 2005; Zhang, Tang, Li, Zhu, & Duan, 2014), including antibacterial, antiproliferative, antiparasitic and antioxidant effects, among others (Alday-Provencio et al., 2015; Hernandez et al., 2007; Velazquez et al., 2007; Wu et al., 2007; Zhang et al., 2014). CAPE is more biologically effective than other natural hydroxycinnamic acid derivatives because of its structural properties, possessing better bioavailability in lipophilic systems due to its partition coefficient (Serafim, Milhazes, Borges, & Oliveira, 2015; Zhang et al., 2014). At present, some studies have evaluated the antioxidant potential of CAPE considering in vitro and in vivo experimental models under different oxidative stress conditions (Havermann, Chovolou, Humpf, & Wätjen, 2014; Song et al., 2012; Wang, Stavchansky, Kerwin, & Bowman, 2010; Wu et al., 2007). There are several methods to assess potential antioxidant compounds. The cellular antioxidant activity assay (CAA), designed by Wang and Joseph (1999), combines both biological model systems and chemical assays (Shahidi & Zhong, 2015; Wang & Joseph, 1999), and provides a quantitative measure of cellular redox state by using the cell-permeating DCFH-DA probe precursor, which is intracellularly de-esterified to DCFH2 and oxidised to the fluorescent DCF probe by cellular ROS (Eruslanov & Kusmartsev, 2010; Shahidi & Zhong, 2015; Wang & Joseph, 1999; Wolfe & Liu, 2007). In a previous study from our lab, CAPE, rutin and galangin, chemical constituents of Sonoran propolis, exhibited a moderate to high free-radical scavenging activity by using 2,2-diphenyl-1picrylhydrazyl radical (DPPH) (Velazquez et al., 2007). Here, we decided to evaluate the CAA of those Sonoran propolis constituents on two murine cell lines derived from different immunological effector cells (B-lymphocytes and macrophages). We report that CAPE shows a higher CAA than rutin and galangin on B-cell lymphoma cells, obtaining similar results on the macrophage cell line. In addition, CAPE helped to preserve cellular morphology after oxidative stress induction. Our results indicate that CAPE has a strong protective effect against oxidative stress.

2. Material and methods 2.1. Chemicals and Sonoran propolis Dimethyl sulphoxide (DMSO), 20 ,70 -dichlorofluorescein diacetate (DCFH-DA; P97.0%), 2,20 -Azobis(2-methylpropionamidine) dihydrochloride (AAPH; P97%), Dulbecco’s Modified Eagle’s Medium (DMEM), sodium bicarbonate (P99.5%), L-asparagine (98%), L-arginine

monohydrochloride (P98%), L-glutamine solution (200 mM), sodium pyruvate solution (100 mM), penicillinstreptomycin solution (1000 U/1 U per mL), ascorbic acid (AA) (P98.0%), caffeic acid (P98.0%), galangin (P95.0%), rutin hydrate (P94.0%), trolox (P98.0%), sodium chloride, sodium phosphate monobasic, sodium phosphate dibasic, methanol, phenethyl alcohol, toluene, p-toluenesulphonic acid, deuterated acetone-d6, propidium iodide (PI), hexane and ethyl acetate were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Quercetin dihydrate (P98.0%) was purchased from Fluka-BioChemika (Sigma-Aldrich Co.; Steinheim, Germany). Hydrogen peroxide (30%) was purchased from J. T. Baker Chemicals. Fetal bovine serum (FBS) was

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purchased from Gibco (Carlsbad, CA, USA). Ultrapure water (18X) was obtained by using a Milli-Q advantage A10 system (Millipore, Bedford, USA). Sonoran propolis samples were collected in Ures, Sonora, Mexico (N 29°270 18100 , W 110°230 39800 ). Propolis extractions were performed in methanol according to Hernandez et al. (2007). 2.2. Chemical synthesis of CAPE CAPE was synthesised from the esterification of caffeic acid (5.0 g; 27.7 mM) and phenetyl alcohol (100 mL; 834.9 mM), using dry toluene (200 mL), and p-toluenesulphonic acid as catalyst (Grunberger et al., 1988; Ojeda-Contreras et al., 2008). The reaction was stirred and carried out under reflux in an oil bath for 36 h in an inert atmosphere of nitrogen within a flask fitted with a reflux condenser, Dean-Stark trap, stirring bar and rubber septa. All the solvents were transferred by using syringe-septum and cannula technique. Solvent was removed by distillation under reduced pressure, then CAPE was chromatographed on silica gel using hexane/ethyl acetate (9:1 v/v) to obtain 3.15 g of a light yellow solid. This product was purified by flash column chromatography on silica gel 230–400-mesh using ethyl acetate/hexane (9:1) as eluent, followed by recrystallization with hexane/ethyl acetate (8:2) that yield a white powder. The chemical structure of CAPE was spectroscopically determined by 1H and 13C spectra on a Agilent Technologies 400/54 Premium Shielded spectrometer operating at 400 MHz with a probe temperature of 25 °C, using deuterated acetone-d6 as solvent, as reported by Ojeda-Contreras et al. (2008). 2.3. Cell lines Cell lines M12.C3.F6 (murine B-cell lymphoma) and RAW 264.7 (macrophage; Abelson murine leukaemia virus transformed) were kindly provided by Emil R. Unanue (Department of Pathology and Immunology, Washington University in St. Louis, MO, USA). M12. C3.F6 cells were cultured in DMEM supplemented with 5% heat inactivated FBS, whereas RAW 264.7 cells were cultured in DMEM at 10% heat inactivated FBS. 2.4. Cellular antioxidant activity (CAA) assays In order to evaluate the intracellular ROS level in M12.C3.F6 and RAW 264.7 cell lines after treatment with CAPE, rutin, galangin and Sonoran propolis, we used the Cellular Antioxidant Activity assay (CAA) as reported by Wolfe and Liu (2007), based on the quantitative method of cellular oxidative stress developed by Wang and Joseph (1999), and finally adapted to flow cytometry as described by Eruslanov and Kusmartsev (2010) with some modifications. Bcell lymphoma M12.C3.F6 cell line was used as a cellular model because of its oxidative stress sensibility, as well as its culture properties (Hernandez et al., 2007; Wade et al., 1989). Briefly, M12.C3.F6 were seeded in a 6-well plate (Costar, Corning Inc.; Kennebunk, ME, USA) at 2  105 cells/mL, and after 24 h incubation at 37 °C in a 5% CO2 atmosphere, CAPE (0, 1 and 5 lM), rutin (0, 12.5 and 25 lM) and galangin (0, 12.5, 25 and 50 lM) were added. Additionally, we evaluated the CAA of Sonoran propolis (0, 6.3, 12.5 and 25 lg/mL). Quercetin dihydrate (0, 6.3, 12.5 and 25 lM), trolox (0, 5 and 10 lM) and AA (0, 5, 50, 75 and 100 lM) were used as positive antioxidant controls in the CAA assays according to their in vitro antioxidant activity (Velazquez et al., 2007; Vergauwen et al., 2015). The most appropriate conditions for the tested compounds without achieving cytotoxicity in M12. C3.F6 cells were observed after 1 h of treatment exposure at the following concentrations: CAPE at 5 lM, rutin 25 lM and galangin 25 lM, while the most efficient concentrations for controls were: quercetin at 12.5 lM, AA at 50 lM and trolox at 10 lM. RAW

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264.7 cells were used since they produce high amounts of ROS molecules under specific conditions (respiratory burst), in addition to their sensitivity to propolis compounds (Hernandez et al., 2007). RAW 264.7 cells were seeded in a 6-well plate (Costar, Corning Inc.; Kennebunk, ME, USA) at 2  106 cells/mL and CAPE (0, 1, 5 and 10 lM) was added to cellular suspension. Afterwards, cells were incubated for 1 h at 37 °C and 5% CO2. After treatment, M12.C3.F6 and RAW 264.7 cells were harvested and washed two times in cold PBS (pH 7.2) (575g, 7 min, 4 °C). Then, cells were resuspended in DCFH-DA (1 lM) in cold PBS (pH 7.2) and were incubated in the dark for 30 min at 37 °C in a 5% CO2. Subsequently, cells were washed two times with cold PBS, and cellular oxidative stress was induced by AAPH (10 mM) (Wolfe & Liu, 2007) or H2O2 at several concentrations (0, 0.5, 1, 2.5, 5 y 10 mM) during 10 min. AAPH and H2O2 (10 mM) did not affect cell viability. H2O2 was more efficient than AAPH (data not shown) under the tested conditions, therefore in the followed experiments we used only H2O2 as oxidative stress inductor. After 10 min with H2O2, the reaction was diluted out with cold PBS, and cells were washed one time with cold PBS (575g, 7 min, 4 °C), and incubated with PI (1 lg/mL) for 10 min at room temperature in the dark. Finally, cells were washed and resuspended in cold PBS and immediately analysed by flow cytometry (FACS Canto II, Becton Dickinson Biosciences, San Jose, CA, USA).

2.5. Statistical analysis Data were analysed using nonparametric analysis with MannWhitney U test, IBM SPSS Statistics 20, 2011. The results were obtained by at least three independent experiments carried out in triplicate. Mean and standard deviation (SD) of data were graphed using Prism 5 (2007) for Windows, GraphPad Software, Inc. (La Jolla, CA, USA). Differences with a value of p less than 0.05 were considered significant. 3. Results We used the CAA assay in order to evaluate the protective capacity of CAPE, rutin, and galangin against ROS in the cellular environment of two different murine cell lines, B-cell lymphoma M12.C3.F6 and macrophages RAW 264.7 cells. After 1 h treatment, CAPE (5 lM) exhibited the highest CAA by reducing intracellular ROS in M12.C3.F6 cells (97.9, 58.4 and 23.8% at 1, 5 and 10 mM H2O2, respectively), followed by rutin (25 lM; 30.9, 45.4 and 27.9% at 1, 5 and 10 mM H2O2, respectively). In contrast, galangin (25 lM) did not induce any antioxidant effect (Fig. 1). CAPE (5 lM) showed higher CAA than the antioxidant controls: AA (50 lM), quercetin (12.5 lM) and trolox (10 lM) at 1 mM H2O2 (37.3%, 74.6% and 82.0%, respectively) on M12.C3.F6 cells, these

Fig. 1. Cellular antioxidant activity (CAA) of Sonoran propolis constituents in M12.C3.F6 cells determined by flow cytometry. A: CAPE (5 lM), B: Rutin (25 lM), and C: Galangin (25 mM) treatment, followed by ROS induction with H2O2(1, 5 and 10 mM). D: Ascorbic acid (50 mM), E: Trolox (10 mM), and F: Quercetin (12.5 mM), were used as antioxidant control compounds. Black bars correspond to the compound treatments, and open bars correspond to the dissolvent control (DMSO) treatments. Data on graphics represent mean fluorescence intensity (MFI) expressed as fluorescence intensity arbitrary units (FIAU) based on the fluorescence of DCF probe detected by flow cytometry. The results shown are representative of at least three independent experiments, all values represent mean of triplicate determinations ± SD. Significant differences (*, p < 0.05) were found from control dissolvent in all treatments for all tested conditions.

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results are summarised in Fig. 1. In addition, we did not observe a significant CAA with Sonoran propolis treatment (0, 6.3, 12.5 and 25 lg/mL) on M12.C3.F6 cells (data not shown). Sonoran propolis and its three constituents (CAPE, rutin and galangin), the control compounds (AA, quercetin and trolox) and DMSO (used as solvent) did not affect M12.C3.F6 cell viability at the tested concentrations (viability P90%; Fig. 2). The high CAA exhibited by CAPE (5 lM) followed by H2O2 (1 mM) on B-cell lymphoma cell line was additionally evidenced via analysis of morphology and fluorescence distribution on treated cells. The sole CAPE and DMSO treatment did not induce any effect on cell morphology and did not increase intracellular ROS concentration, as detected by DCF fluorescence (Supplementary material, Fig. 1S), whereas incubation with H2O2 (1, 5 and 10 mM) increased ROS concentration and alterations on cell morphology after H2O2 treatment were observed. Cells increased their

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size as H2O2 concentration was higher, which suggested a dosedependent effect on cell morphology (Fig. 3A and B). Cells pre-incubated with CAPE (5 lM) followed by H2O2 (1 mM) showed an evident negative displacement of fluorescence (MFI: 449 FIAU; Fig. 3A), in comparison with cells pre-incubated with DMSO (solvent) and the stress inductor H2O2 (1 mM) (MFI: 968 FIAU). Furthermore, CAPE protected M12.C3.F6 cells from the cell shape alterations induced by H2O2, resulting in a similar morphology to that shown by normal cells (Fig. 3B). Interestingly, the effect of CAPE (MFI: 449 FIAU), resulted in a reduction of the basal fluorescence of M12.C3.F6 cells (MFI: 656 FIAU; Fig. 3A). These observations support the protective effect of CAPE against oxidative stress induced by H2O2 on M12.C3.F6 cells. In order to investigate whether CAPE has CAA on phagocytic cells, we used the macrophage cell line RAW 264.7. CAPE prevented the H₂O₂-induced oxidative stress in RAW 264.7 macrophages (Fig. 4A), and the most evident CAA of CAPE (5 lM) on RAW 264.7 cells was observed at 0.5 mM H2O2 treatment (MFI: 3745 FIAU), in comparison with DMSO control treatment (MFI: 6468 FIAU) (Fig. 4B). In addition, none of the tested conditions achieved cytotoxicity on RAW 264.7 cells (viability P90%).

4. Discussion

Fig. 2. Effect of Sonoran propolis constituents (CAPE, rutin and galangin) and antioxidant controls (quercetin, ascorbic acid and trolox) on M12.C3.F6 cell viability. Propidium iodide staining was used to assess cell viability (%) after compound treatment, followed by oxidative stress induction with H2O2(1 mM). Dot plot graphics show the cellular distribution in size (FSC-A) vs. cell exclusion of propidium iodide dye (PE-A).

In this study, we determined the CAA of CAPE, rutin and galangin (Sonoran propolis constituents), in two different murine cell models (B-cell lymphoma and macrophages). Here, we found that CAPE treatment induced a protective effect against the H2O2-induced oxidative stress on both cell lines. CAPE showed the highest CAA, by diminishing ROS amount and preserving cellular integrity, viability and morphology in both cell models (Figs. 3 and 4). The CAA of CAPE was evidenced on B-cell lymphoma and macrophage cells, although it is important to notice that basal ROS amounts in those cells was different due to their biochemical characteristics and different immune response functions due to their cellular lineage, which is reflected in their MFI basal values. The antioxidant activity exerted by numerous substances with alimentary and pharmaceutical potential applications is often determined by spectrophotometric methods based on metal reducing power, oxidation products, or on the radical scavenging capacity (Shahidi & Zhong, 2015; Wolfe & Liu, 2007). However, the obtained data do not necessarily reflect the ability to reduce ROS in an intracellular environment, because a potential antioxidant compound could not be bioactive under physiological conditions such as temperature, pH, permeability, cellular internalization and metabolism that determined its bioavailability and biological function (Bender & Graziano, 2015; Eruslanov & Kusmartsev, 2010; Shahidi & Zhong, 2015; Wang & Joseph, 1999; Wolfe & Liu, 2007). In that sense, the results obtained in this study reflect the bioavailability and effect of an internalised amount of Sonoran propolis constituents prior cellular stress induction, demonstrating the antioxidant protective effect of CAPE under cellular physiological conditions, since the reaction is not taking place simultaneously as in chemistry methods. CAPE is a phenolic compound that represents a potential nutraceutical agent to supply antioxidant effects and health benefits (Bankova, 2005; Hernandez et al., 2007; Zhang et al., 2014). CAPE has been reported to induce a protective role against ROS by in vitro (including CAA assays) and in vivo studies. By in vitro studies, Burgazli et al. (2013) found that low doses of CAPE might modulate the nitric oxide levels in human umbilical vein endothelial cells in a dose-response manner, while higher doses would inflict some cellular damage (Burgazli et al., 2013). Moreover, Song et al. (2012) reported that CAPE induced anti-inflammatory

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Fig. 3. CAPE induces cellular antioxidant activity (CAA) and cell morphology protection against ROS in M12.C3.F6 cells. A: Overlaid histograms of CAPE treatment (5 mM) and dissolvent control treatment (DMSO) in M12.C3.F6 cells followed by ROS induction with H2O2 (1, 5 and 10 mM). The data represent mean fluorescence intensity (MFI) values expressed in fluorescence intensity arbitrary units (FIAU) based on the fluorescence of DCF probe detected by flow cytometry. A significant fluorescence displacement to the left (MFI value) after CAPE treatment in comparison with DMSO control can be observed in all the H2O2 concentrations tested. B: Cellular morphology distribution is shown in dot plots according to size (FSC-A) vs. cell complexity (SSC-A) after CAPE and DMSO treatments, followed to oxidative stress induction with H2O2(1, 5 and 10 mM).

and anti-oxidative effects in human middle ear epithelial cells by significantly inhibiting the H2O2-induced expression of TNF-a and COX-2 in a dose and time dependent manner, as well as by decreasing ROS levels and SOD expression upregulated by H2O2 (Song et al., 2012). By in vivo experiments, Havermann et al. (2014) have demonstrated that CAPE (100 lM) enhances the stress resistance associated to an improvement of lifespan in Caenorhabditis elegans by diminishing the ROS production induced by thermal stress (Havermann et al., 2014). Additionally, Gurel et al. (2004) reported that intraperitoneal administration of CAPE (10 lmol/kg) in Wistar rats, after local skin damage by thermal trauma, led to amelioration of endogenous enzymatic systems (SOD and XO) (Gurel et al., 2004). On the basis of those studies performed in different in vitro and in vivo models, CAPE has been demonstrated to protect against cellular oxidative stress by diminishing ROS levels and improving the efficiency of enzymatic mechanisms. However, further in vivo studies focused on the integrative evaluation of enzymatic systems efficiency, intracellular ROS amounts and cell integrity are needed. The CAA assay has been suggested as an appropriate model to correlate molecular effectiveness at cellular level to its in vivo performance (Shahidi & Zhong, 2015). The strong CAA of CAPE under

different conditions has been reported by other authors (Havermann et al., 2014; Song et al., 2012; Wang et al., 2010). Wang et al. (2010) treated human umbilical vein endothelial cells with CAPE (20 lM) for 2 h without including a stress inductor, measuring fluorescence intensity by a microplate reader. Song et al. (2012) simultaneously stimulated human middle ear epithelial cells with CAPE (100 lM) and H2O2 (100 lM) for 30 min, observing an evident antioxidant activity by flow cytometry, however nor the protective effect prior oxidative stress induction and nor the cellular viability were approached. Otherwise, Havermann et al. (2014) determined the CAA of CAPE (25 lM) on human colon carcinoma cells by flow cytometry, although they did not assess cell morphology and viability after a long-time exposure to CAPE (4 h), followed by H2O2 (1 h at 500 lM). In this study, CAA by flow cytometry in conjunction with cell morphology and cell integrity analysis, allowed us to ensure the evaluation of metabolically active cells considering membrane integrity (cell viability >90%), morphology, and complexity at the end of treatment period. Detection by microplate reader (fluorometry) does not assess these cellular characteristics. In that sense, here we were able to observe the protective effect of CAPE (5 lM) against ROS, preserving cell morphology and integrity in both cellular models at short treatment period (1 h) that would

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Fig. 4. CAPE induces Cellular antioxidant activity (CAA) and cell morphology protection against ROS in RAW 264.7 cells. A: CAPE treatment (1, 5 and 10 mM), followed by ROS induction with H2O2(0.5, 1, 2.5 and 5 mM). Black bars correspond to the CAPE treatment, and open bars correspond to the dissolvent control (DMSO) treatment. The results shown are representative of at least three independent experiments, all values represent mean of triplicate determinations ± SD. Significant differences (*,p < 0.05) were found from control dissolvent in all the experiments. Data represent mean fluorescence intensity (MFI) expressed as fluorescence intensity arbitrary units (FIAU) based on the fluorescence of DCF probe detected by flow cytometry. B: Overlaid histograms of CAPE treatment (5 mM) and dissolvent control treatment (DMSO) in RAW 264.7 cells, followed by ROS induction with H2O2 (0.5, 1, 2.5 and 5 mM). A significant fluorescence displacement to the left in MFI values after CAPE treatment in comparison with DMSO control can be observed in all the tested concentrations of H2O2.

have not been efficiently detected by flow cytometry without the PI staining. We consider that these modifications improve the stringency of CAA assay and should be evaluated by other authors. Rutin is a naturally occurring quercetin glycosylated derivative, previously reported with potent free-radical scavenging activity by DPPH assay (Velazquez et al., 2007). By CAA on M12.C3.F6 cells, we observed a higher effect of quercetin (74.6%, MFI: 119 FIAU at 12.5 lM) in comparison with rutin (30.9%, MFI: 386 FIAU at 25 lM), at a lesser concentration. These data would be associated to the structural differences between rutin and quercetin, comprised by the presence of hydroxyl groups in the disaccharide moiety (-O-a-L-rhamnopyranosyl-(1?6)-b-D-glucopyranoside) that could be able to react with DPPH radical in a more evident manner than with ROS in a cellular environment. Moreover, differences in bioavailability of both compounds could also explain these results (Wolfe & Liu, 2008). In contrast to CAPE and rutin, galangin did not exhibit a CAA, despite galangin showed a moderate antioxidant activity by DPPH assay (Velazquez et al., 2007). Moreover, the treatment with galangin (25 lM) seems to induce an increase in cellular ROS together with H2O2, suggesting a pro-oxidant effect, such as other flavonoids, as apigenin that has been reported to be an oxidative stress inducer that triggers apoptosis (Andueza et al., 2015). Subsequent structure-

activity relationship studies must be carried out in order to understand those differences in CAA of flavonoids. The positive controls AA, trolox and quercetin (50, 10 and 12.5 lM, respectively), were included according to their broadly reported antioxidant activity and their use in the food industry (Velazquez et al., 2007; Vergauwen et al., 2015). Trolox exhibited the highest CAA, followed by quercetin, meanwhile AA presented a slight CAA. This low efficiency of AA (50 lM) against oxidative stress could be explained by a low cellular uptake by its putative transporter proteins (Rivas et al., 2008). However, other studies reported CAA by AA (1 mM) on the porcine small intestinal epithelial cell line, suggesting then its uptake and defence against oxidative stress (Vergauwen et al., 2015). Although, in that study, AA was twenty-fold more concentrated and as well its treatment period. In our experiments, CAPE was slightly more effective than trolox and more active than AA and quercetin, suggesting a remarkable protective role against ROS at the intracellular environment.

5. Conclusions Our results suggest that CAPE induces CAA in addition to cellular protection against oxidative stress induced by ROS. To our

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