Green coffee hydroxycinnamic acids but not caffeine protect human HepG2 cells against oxidative stress

    Green coffee hydroxycinnamic acids but not caffeine protect human HepG2 cells against oxidative stress Gema Baeza, Miryam Amigo-Benavent, Beatriz Sarri´a, Luis Goya, Raquel Mateos, Laura Bravo PII: DOI: Reference:

S0963-9969(14)00350-0 doi: 10.1016/j.foodres.2014.05.035 FRIN 5277

To appear in:

Food Research International

Received date: Revised date: Accepted date:

31 January 2014 16 April 2014 28 May 2014

Please cite this article as: Baeza, G., Amigo-Benavent, M., Sarri´a, B., Goya, L., Mateos, R. & Bravo, L., Green coffee hydroxycinnamic acids but not caffeine protect human HepG2 cells against oxidative stress, Food Research International (2014), doi: 10.1016/j.foodres.2014.05.035

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ACCEPTED MANUSCRIPT Green coffee hydroxycinnamic acids but not caffeine protect human

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HepG2 cells against oxidative stress

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Gema Baeza, Miryam Amigo-Benavent, Beatriz Sarriá, Luis Goya, Raquel

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Mateos, Laura Bravo*

Department of Metabolism and Nutrition, Institute of Food Science, Technology

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and Nutrition (ICTAN - CSIC), C/ José Antonio Novais 10, 28040 Madrid, Spain

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* Corresponding author:

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Prof. Laura Bravo

Department of Metabolism and Nutrition

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Institute of Food Science, Technology and Nutrition (ICTAN - CSIC) C/ José Antonio Novais 10, 28040 Madrid, Spain. Tel: +34 91 5492300; Fax: +34 91 5493627.

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E-mail: [email protected]

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ACCEPTED MANUSCRIPT Abstract The intake of green coffee has been associated with a lower risk of diseases of oxidative etiology probably due to its high phenolic content. The present study

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investigated the effect of treating human HepG2 cells with different

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concentrations of a green coffee bean extract (GCBE) and its main

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hydroxycinnamic acids, 5-caffeoylquinic acid (5-CQA) and 3,5-dicaffeoylquinic acid (3,5-DCQA), and the methylxanthine caffeine (CAF), directly or prior to inducing an oxidative stress by incubating cells with 400

M tert-

butylhydroperoxide (t-BOOH). Direct treatment with GCBE (1-50 g/mL), 5-CQA

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and 3,5-DCQA (1-40 M) significantly decreased reactive oxygen species (ROS) production on HepG2 cells. Pre-treatment with GCBE, 5-CQA and 3,5-

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DCQA for 20 h prevented the cellular and macromolecular damage induced by t-BOOH, returning glutathione levels and the activity of antioxidant enzymes to

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values similar to control cells. Moreover, the increased ROS generation induced by t-BOOH was dose-dependently prevented when cells were pre-treated with

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GCBE, 5-CQA and 3,5-DCQA. CAF showed no protective effect. It can be concluded that GCBE and its main polyphenols, 5-CQA and 3,5-DCQA, but not

Keywords:

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caffeine, confer a significant protection against oxidative stress in vitro.

Green

coffee;

polyphenolic

compounds;

caffeine,

dietary

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antioxidants; oxidative stress biomarkers.

Abbreviations: 5-CQA, 5-caffeoylquinic acid; 3,5-DCQA, 3,5-dicaffeoylquinic acid; CAF, caffeine; FQA, feruloylquinic acids; CT, control; DCFH-DA, dichlorofluorescein 2`7`-diacetate; DMSO, dimethyl sulfoxide; DNPH, 2,4dinitrophenylhydrazine; FBS, fetal bovine serum; GCBE, green coffee bean extract; GPx, glutathione peroxidase; GR, glutathione reductase; GSH, reduced glutathione; LDH, lactate dehydrogenase; MDA, malondialdehyde; NADH, nicotine adenine dinucleotide reduced; NADPH, nicotine adenine dinucleotide phosphate reduced; OPT, o-phthalaldehyde; PBS, phosphate-buffered saline; RNS, reactive nitrogen species; ROS, reactive oxygen species; SD, standard deviation; SDS, sodium dodecyl sulfate; t-BOOH, tert-butylhydroperoxide. 2

ACCEPTED MANUSCRIPT 1. Introduction The balance between reactive oxygen/nitrogen species (ROS/RNS) and antioxidant defenses is essential to maintain correct cellular functions (Valko et

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al., 2007). When excessive reactive species are produced the endogenous

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protective system cannot completely prevent the deleterious effects of

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ROS/RNS causing oxidative/nitrosative stress leading to molecular injury that can contribute to the etiopathogenic mechanisms involved in the development of important diseases such as cancer, cardiovascular diseases or diabetes.

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Diet plays an important role as a source of exogenous antioxidants, such as certain vitamins, carotenoids, minerals like selenium, and specially polyphenols.

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Polyphenols are secondary plant metabolites widely distributed in vegetables, fruits and beverages, and have different health-beneficial effects as antioxidant, anti-inflammatory or antibacterial (Bravo, 1998). In particular, polyphenols act

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chelating metals, scavenging free radicals or modulating endogenous

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antioxidant (Alia et al., 2006a; Tanigawa, Fujii, & Hou, 2007; Granado-Serrano et al., 2010; Rodríguez-Ramiro, Ramos, Bravo, Goya, & Martin, 2011).

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Coffee is one of the most widely consumed beverages worldwide, with an annual consumption of approximately 7 million tons according to FAO (http://www.fao.org/docrep/007/y5143s/y5143s00.htm). Green coffee beans are

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rich in phenolic compounds, mainly hydroxycinnamic acids that may amount up to 4.1-11.3 % (w/w) of the coffee bean (Perrone, Donangelo, Donangelo, & Farah, 2010). Major hydroxycinnamic acids in green coffee are 3-, 4-, and 5caffeoylquinic acids (3-, 4-, and 5-CQA), 3,4-, 3,5-, and 4,5-dicaffeoylquinic acids (3,4-, 3,5-, and 4,5-DCQA), and 3-, 4-, and 5-feruloylquinic acids (3-, 4-, and 5-FQA), among others (Alonso-Salces, Serra, Reneiro, & Héberger, 2009). Other bioactive components found in green coffee are the methylxanthines caffeine (CAF), theobromine, and theophylline (Huck, Guggenbichler, & Bonn, 2005), being caffeine the major alkaloid in coffee. Roasting drastically affects the phenolic composition of green coffee beans resulting in the degradation and/or transformation of polyphenols, affecting the physical and chemical properties of roasted coffee beans (Schenker et al., 2002; Somporn, Kamtuo, Theerakulpisut, & Siriamornpun, 2011; Perrone, 3

ACCEPTED MANUSCRIPT Farah, & Donangelo, 2012). Conversely, new compounds formed during roasting of coffee, such as melanoidins, also have antioxidant, and hepato- and genoprotective effects (Goya, Delgado-Andrade, Rufian-Henares, Bravo, &

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Morales, 2007; Del Pino-García, González-San José, Rivero-Pérez, & Muñiz,

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2012), although not compensating the high loss of hydroxycinnamic acids and antioxidant capacity of green coffee beans (Daglia, Papetti, Gregotti, Bertè, &

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Gazzani, 2000; Del Castillo, Ames, & Gordon, 2002; Somporn, Kamtuo, Therrakulpisut, & Siriamornpun, 2011; Del Pino-García, González-San José, Rivero-Pérez, & Muñiz, 2012; Perrone, Farah, & Donangelo, 2012). This loss of

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biological activity has also been shown in animal experiments, where a green coffee aqueous extract was more effective in the inhibition of carrageenan-

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induced oedema and lipopolysaccharide-induced peritonitis than a roasted coffee aqueous extract (De Castro et al., 2013). Moreover, administration of green coffee improved lipid profile and redox status, and decreased glucose

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and insulin plasma levels in diabetic rats (Ahmed, El-Ghamery, & Samy, 2013),

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probably due to the effects of hydroxycinnamic acids. Thus, the intake of green coffee products has increased in recent years as a healthier alternative than

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roasted coffee, as its consumption has been associated with a lower risk of suffering diseases of oxidative etiology such as cancer, cardiovascular diseases or diabetes (Kozuma, Tsuchiya, Kohori, Hase, & Tokimitsu, 2005).

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The liver is one of the major metabolizing organs, receiving antioxidants and other bioactive compounds absorbed from the digestive tract together with xenobiotics. Therefore, it is more susceptible to the potential damaging effects of xenobiotics and molecules involved in their detoxification, including ROS and free radicals, which may result in inflammatory and fibrotic processes (Jaeschke et al., 2002). This substantiates the interest of studying oxidative stress and the effect of dietary antioxidants in this organ. Human hepatoma HepG2 cells have been used in biochemical and nutritional studies as hepatic human model to gain insight into the specific mechanisms involved in the biological activity of dietary compounds (Brandon et al., 2006; Goya, Martin, Ramos, Mateos, & Bravo, 2009). Bearing this in mind, the aim of this study was to investigate if a green coffee bean extract (GCBE) might directly affect HepG2 cell integrity and steady-state values of redox status, also evaluating the GCBE capacity to 4

ACCEPTED MANUSCRIPT protect HepG2 cells against an oxidative cell damage induced by tertbutylhydroperoxide (t-BOOH). In addition, the main phenolic constituents (5CQA and 3,5-DCQA) and the methylxanthine CAF were individually studied in

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HepG2 cells in order to further investigate their contribution to the preventive

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effect against oxidative stress.

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2. Material and methods

2.1. Reagents

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Green coffee (Coffea arabica L. from Colombia) was purchased in a local supermarket in Madrid (Spain). DMEM-F12 culture media and fetal bovine serum (FBS) were from Biowhitaker Europe (Lonza, Madrid, Spain). 5acid,

gentamicin,

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caffeoylquinic

caffeine,

penicillin,

streptomycin,

tert-

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butylhydroperoxide (t-BOOH), glutathione reductase (GR), reduced (GSH) and oxidized glutathione, nicotine adenine dinucleotide reduced (NADH), nicotine

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adenine dinucleotide phosphate reduced (NADPH), dimethyl sulfoxide (DMSO), o-phthalaldehyde (OPT), sodium dodecyl sulfate (SDS), dichlorofluorescein2’7’-diacetate

(DCFH-DA),

2,4-dinitrophenylhydrazine

(DNPH),

EDTA,

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mercaptoethanol and crystal violet were acquired from Sigma-Aldrich (Madrid, Spain). 3,5-dicaffeoylquinic acid was purchased from Biopurify (Chengdu, Sichuan, China). Bradford reagent was acquired from Bio-Rad (Madrid, Spain). All other chemicals were of analytical grade.

2.2. Characterization of phenolic and methylxanthine content of green coffee Green coffee beans were grounded to 0.5 µm particle size. Phenolic and methylxanthine compounds were extracted (Bravo, & Saura-Calixto, 1998) in triplicate from 1 g of ground green coffee with 2 N hydrochloric acid in aqueous methanol (50:50, v/v, 1 h at room temperature, constant shaking), centrifuged (10 min, 3000 g), the supernatants collected and the pellets extracted with acetone:water (70:30, v/v, 1 h at room temperature, constant shaking). After the second extraction, samples were centrifuged (10 min, 3000 g) and both 5

ACCEPTED MANUSCRIPT supernatants combined, evaporated under reduced pressure until dryness, resuspended in 1% (v/v) formic acid and filtered (0.45 µm) prior to injection of 20 µL into the HPLC.

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Analysis of polyphenols and methylxanthines was performed in a HPLC-DAD

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1200 series system (Agilent Technologies, Waldrom, Germany). Separation

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was performed on a Superspher 100 RP18 column (250 mm x 4.6 mm i.d., 4 m, Agilent Technologies) preceded by an ODS RP18 guard column and at flow rate of 1 mL/min and 30ºC. Two different gradient programs were used to characterize both phytochemical groups, using separate columns for each

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analysis. Polyphenols were separated by a binary gradient of 1% formic acid in deionized water (solvent A) and acetonitrile (solvent B) as follows: from 10% to

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20% solvent B over 5 min, 20% to 25% solvent B over 20 min, 25% to 35% solvent B over 10 min, isocratically for 25 min, returning to the initial conditions over 10 min. Methylxanthines were separated using the following gradient: from

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6% to 10% solvent B over 20 min, 10% to 13% solvent B over 5 min, 13% to

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15% solvent B over 5 min, 15% to 10% solvent B over 8 min, 10% to 6% solvent B over 4 min, and then isocratically for 3 min. Chromatograms were

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acquired at 280, 320, and 360 nm to register phenolic composition, and at 272 nm for methylxanthines. Quantification was done by using the calibration curves of 5-CQA and 3,5-DCQA to calculate the concentration of mono- and

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dicaffeoylquinic acids, respectively, and CAF to determine methylxanthine content.

2.3. Preparation of green coffee bean extract (GCBE) and pure compounds to test in cell culture The green coffee bean extract obtained as described above (section 2.2) was evaporated, lyophilized, dissolved in 1% DMSO in deionized water, and diluted with FBS-free DMEM-F12 medium to prepare 1, 10 and 50 µg/mL solutions (0.1% DMSO final concentration in cell culture). Pure standard 5-CQA, 3,5-DCQA, and CAF were dissolved in 1% DMSO in deionized water and diluted with serum free medium to prepare different test solutions (1, 10, 20 and 40 µM; 0.1% DMSO final concentration in cell culture). 6

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2.4. Cell culture and treatment Human hepatoma HepG2 cells were maintained in a humidified atmosphere of

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5% CO2 and 95% air at 37 ºC and grown in DMEM-F12 medium supplemented

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with 2.5% FBS and 50 mg/L of antibiotics (gentamicin, penicillin and

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streptomycin). The day before the experiments, cells were changed to FBS-free medium to avoid potential FBS interference with the assays affecting the final results.

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Two sets of experiments were designed for this study: i) cells were incubated with different concentration of GCBE (1, 10 and 50 µg/mL) and standards 5-

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CQA, 3,5-DCQA, and CAF (1, 10, 20 and 40 µM in the assay of ROS generation; 1, 10 and 20 µM in the rest of assays) to test the direct antioxidant effect; and ii) HepG2 cells were pre-treated with the same concentrations of

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GCBE and pure compounds for 20 h before inducing an oxidative stress by

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treating cells with 400 µM t-BOOH for 3 h, to test the protective effect of these phytochemicals. Control cells were treated with 0.1% of DMSO in comparison

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with treated cells.

2.5. Cell viability and cytotoxicity

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Cell viability was determined by the crystal violet assay (Granado-Serrano et al., 2007). Cells were seeded (104 cells/well) in 96-well plates, treated with the different compounds for 24 h, washed with 200 L PBS and then incubated with 50 L of 0.2% crystal violet solution (in 2% ethanol) for 20 min. After rinsing the plates twice with 200 L water, 100 L of 1% SDS were added to break down the cells and release the dye to estimate cell viability. The absorbance at 560 nm was measured using a microplate reader (Bio-Tek, Winooski, VT, USA). Results are expressed as percentage of cell viability referred to the absorbance measured in control cells. Cell toxicity was estimated by the lactate dehydrogenase (LDH) assay (Welder, & Acosta, 1994). Cells were plated at a concentration of 8x10 5 cells/ plate in 60 mm diameter plates. After the different treatments, the culture medium was 7

ACCEPTED MANUSCRIPT collected (3 mL), and the cells scraped in PBS (3 mL) and sonicated to break down the cell membrane. The absorbance at 340 nm in the culture medium and cell content was determined in a microplate reader after adding a mixture of

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1.35 M Tris, 0.08 M pyruvate and 2 mg/mL NADH. Percentage of LDH was

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estimated from the ratio between LDH activity in the culture medium and that of

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the whole cell content.

2.6. ROS generation

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The DCFH-DA assay was used to quantify cellular ROS production (Alia, Ramos, Mateos, Bravo, & Goya, 2005). Cells were seeded in 24-well plates

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(1.2x105 cells/well). To evaluate the direct effect, HepG2 cells were incubated with 10 µL DFCH-DA (10 µM in DMSO) for 30 min at 37ºC. Then, cells were washed with PBS twice and treated with test samples (GCBE and pure

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standards). Fluorescence (excitation and emission wavelengths 485 nm and

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530 nm, respectively) was measured at 0, 60, 120 and 180 min. To test the protective effect against oxidative stress, cells were pre-treated with compounds

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for 20 h, incubated with 10 μL of DCFH-DA (10 µM in DMSO) for 30 min at 37ºC and washed with PBS. Then, cells were exposed to 500 µL of 400 µM t-BOOH and fluorescence was measured. Control cells without t-BOOH treatment were

to time.

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used as negative control. Results are expressed as fluorescence units referred

2.7. Reduced glutathione evaluation GSH content was evaluated by a fluorometric assay which takes advantage of the reaction of GSH with OPT at pH 8 (Hissin, & Hilf, 1976). HepG2 cells were seeded in 60-mm diameter plates (2.4x105 cells/plate) and incubated with the different treatments. Cells were scraped and homogenized by ultrasound with 110 µL of 5% trichloroacetic acid containing 2 mM EDTA for 10 min. Following centrifugation (30 min, 7500 rpm), 50 µL of clear supernatant were mixed with 15 µL of 6 M NaOH, 175 µL of 0.1 M phosphate-buffer sodium containing 5 mM EDTA (pH 8.0) and 10 μL of 10 mg/mL OPT in methanol. After incubation in the dark at room temperature for 15 min, fluorescence was measured (excitation 8

ACCEPTED MANUSCRIPT wavelength 340 nm, emission wavelength 460 nm). A standard calibration curve of GSH (5-1000 ng) was used to determine GSH content.

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2.8. Glutathione peroxidase (GPx) and glutathione reductase (GR) activity

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HepG2 cells were seeded in 100-mm diameter plates (2x106 cells/plate) and,

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after treatment, were scraped in PBS and centrifuged for 7 min at 2000 rpm. Cell pellets were resuspended in 50 mM Tris, containing 5 mM EDTA and 0.5 mM mercaptoethanol, sonicated and then centrifuged again. Enzyme activities were evaluated in the supernatants. The determination of GPx activity is based

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on the oxidation of GSH by GPx using t-BOOH as a substrate coupled to the disappearance of NADPH by GR, which was monitored by following

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absorbance at 340 nm (UV-Visible Spectrophotometer, Beckman, DU-640, USA) (Günzler, Kremers, & Flohé, 1974). GR activity was determined by following the decrease in absorbance due to the oxidation of NADPH utilized in

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the reduction of oxidized glutathione (Goldberg, & Spooner, 1987). Results are

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expressed as mU/mg of protein. Protein concentration was measured using the

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Bio-Rad Protein assay following manual’s instructions.

2.9. Macromolecular damage

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Malondialdehyde (MDA) analysis was developed to determine the cellular lipid oxidation by HPLC after its transformation into 2,4-dinitrophenylhydrazone (Mateos, Goya, & Bravo, 2004). Cells were seeded (2.5x106 cells/plate) in 100mm diameter plates and submitted to the different treatments. Values are expressed as nmol of MDA/mg of protein. Protein concentration was measured using the Bio-Rad Protein assay reagent. Protein oxidation was determined by quantifying carbonyl groups following the protocol described by Levine et al. (1990). Cells were seeded in 100-mm diameter plates (2x106 cells/plate) and subjected to the different treatments. Culture medium was collected and cells were scrapped in PBS. Then, cells were centrifuged (2000 rpm, 10 min), resuspended in homogeneity buffer (0.25 M Tris pH 7.4, 0.2 M sucrose and 5 mM DTT) and sonicated. The cytoplasmic content was incubated with 10 mM DNPH in 2 N HCl for 1 h at room 9

ACCEPTED MANUSCRIPT temperature and protein content was precipitated with 10% trichloroacetic acid. After centrifuging and washing with ethyl acetate:ethanol (1:1, v/v), the pellet was resuspended in 6 M guanidine and the absorbance was measured at 360

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nm. Results are expressed as nmol carbonyl per mg protein.

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2.10. Statistics

SPSS 19.0 (SPSS Inc., IL, USA) was employed for the statistical analysis of data. Homogeneity of variances was checked by the Levene test. Multiple comparisons were carried out using One-way ANOVA followed by Bonferroni or

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Games-Howell tests depending on variances being homogeneous or not. The level of significance was fixed at p < 0.05. Results are expressed as mean±

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standard deviation (SD).

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3. Results

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3.1. Characterization and quantification of phenolic and methylxanthine content of green coffee

The chromatographic analysis of green coffee showed that the mono- and

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diesters of hydroxycinnamic acids and quinic acid are the major constituents of the phenolic fraction. Caffeoylquinic and dicaffeoylquinic acid isomers were the major components of the phenolic fraction of green coffee beans accounting for over 84% of the total phenolic content. A minor proportion of quinic acid esterified to ferulic acid (11%) or sinapic acid (1.85%) was quantified. Regarding methylxanthine content, caffeine and theobromine were present in the GCBE, caffeine accounting for 99.78% of total methylxanthines.

3.2. Antioxidant effects of GCBE and main component, 5-CQA, 3,5-DCQA and CAF, on HepG2 cells in basal conditions The treatment of HepG2 cells with GCBE (10 μg/mL) and 5-CQA, 3,5-DCQA and CAF (10 μM) for 20 h did not provoke changes on cell morphology which was very similar to that showed by control cells (Figure 1). Cell viability and 10

ACCEPTED MANUSCRIPT cytotoxicity determined by the crystal violet and LDH tests, respectively, remained unaltered after 20 h treatment with GCBE and standards at doses as high as 50 μg/mL and 20 μM, respectively (Table 1). Thus, it can be assumed

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that the range of concentration finally selected can be safely used to study the

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potential protective effect in vitro of GCBE and standards against a condition of oxidative stress. Likewise, there were no significant changes in antioxidant

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defenses (GSH content, and GPx and GR activity) nor lipid and protein oxidation after 20 h of treatment with these samples (Table 1). Treatment of cells with 1, 10, or 50 μg/mL of GCBE and 1, 10, 20, and 40 μM of

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hydroxycinnamic acids (5-CQA and 3,5-DQA) significantly decreased ROS production compared to control cells, including the lowest concentrations

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evaluated. On the contrary, ROS generation in cells exposed to CAF (1-40 µM)

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was similar to ROS levels in control cells (Figure 2).

3.3. Antioxidant effects of GCBE, 5-CQA, 3,5-DCQA and CAF on HepG2 cells in

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a condition of oxidative stress

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Cell cytotoxicity: Treatment of HepG2 cells with 400 µM t-BOOH for 3 h caused significant morphological changes, being rounder, smaller and widely separated. This altered morphology was also observed in cells pre-treated with 10 μM CAF before inducing oxidative stress with t-BOOH. However, when cells

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were pre-treated with GCBE (10 μg/mL), 5-CQA and 3,5-DCQA (10 μM) before incubation with t-BOOH, no significant morphological changes were observed in HepG2 cells (Figure 3). Accordingly, the percentage of LDH leaked to the culture medium enhanced significantly in cells treated with t-BOOH (≈ 60%), indicating an important cytotoxic effect of this oxidizing agent in HepG2 cells. Pre-treatment with GCBE, 5-CQA and 3,5-DCQA for 20 h significantly reduced the t-BOOH-induced increase of LDH resulting in LDH activity similar to controls when cells were pre-treated with the highest concentrations, although 3,5DCQA prevented t-BOOH oxidative effect at all tested concentrations (1-20 μM). On the contrary, CAF did not prevent cell damage caused by t-BOOH (Figure 4).

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ACCEPTED MANUSCRIPT ROS generation: The intracellular ROS production was estimated to evaluate the degree of cellular oxidative stress. The pro-oxidant t-BOOH induced a significant increase in ROS production over time as compared to non-stressed

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control cells, which was significantly prevented when cells were pre-treated with

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GCBE or pure test compounds, observing a dose-dependent response with

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concentration (Figure 5).

Antioxidant defenses: A significant and remarkable decrease (60-70%) of GSH content was observed in cells treated with 400 µM t-BOOH for 3 h, highlighting the oxidative condition induced by t-BOOH treatment. Pre-treatment with GCBE

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and hydroxycinnamic acids dose-dependently recovered normal GSH levels, whereas pre-treatment with CAF for 20 h did not (Figure 6).

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Treatment with t-BOOH for 3 h induced a significant increase in the activity of GPx and GR (Figure 7) as a defense response against the oxidative insult.

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Pretreatment with all doses of GCBE and standards (5-CQA and 3,5-DCQA) partially reversed the chemically induced increase in GPx and GR, with CAF

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showing no effect (Figure 7).

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Macromolecular damage: The cellular concentration of MDA was significantly increased when HepG2 cells were treated with t-BOOH for 3 h, indicating an important lipid oxidation. Likewise, cells incubated with 1 µg/mL GCBE and 10 µM CAF provided MDA values similar to stressed control cells. On the contrary,

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pre-treatment with the rest of GCBE concentrations and 10 µM 5-CQA and 3,5DCQA for 20 h completely prevented lipid oxidation caused by t-BOOH (Figure 8).

Finally, incubation of HepG2 cells with 400 µM t-BOOH caused a significant increase in the cellular concentration of carbonyl groups (≈ 70%), showing its capacity to induce protein oxidation. Pre-treatment with GCBE for 20 h completely prevented the increase of carbonyl groups induced by the prooxidant agent, whereas 10 µM of pure hydroxycinnamic acids partly reduced carbonyls levels, with CAF showing no protective effect (Figure 9).

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ACCEPTED MANUSCRIPT 4. Discussion

Human hepatoma HepG2 cells is a validated model of the human liver that has

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been well characterized and is widely used in biochemical and nutritional

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studies (Alía et al., 2006a; Brandon et al., 2006; Martin et al., 2008). Similarly, tBOOH has been commonly used in cell culture experiments as an inductor of

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oxidative stress (Alia, Ramos, Mateos, Bravo, & Goya, 2005; Lima et al., 2007). t-BOOH is an organic peroxide that can increase ROS production from other radicals and causes macromolecular damage increasing oxidized glutathione

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levels and ultimately leading to cell death (Sies, & Summer, 1975; Guirdelli, Cattabeni, & Cantoni, 1997). This study demonstrates for the first time that the

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main hydroxycinnamic acids from green coffee, 5-CQA and 3,5-DCQA, protect human liver cells against oxidative stress at physiological concentrations. Therefore, these results confirm that GCBE constitutes an important defense

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against oxidative damage by modulating cell cytotoxicity, ROS generation,

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antioxidant defenses and macromolecular damage, probably through interaction with free radicals and modulation of endogenous antioxidant defenses

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expression and activities, cell signaling and/or gene expression via NF-κB or Nrf2 (Alia et al., 2006a; Tanigawa, Fujii, & Hou, 2007; Granado-Serrano et al., 2010; Rodríguez-Ramiro, Ramos, Bravo, Goya, & Martín, 2011). On the

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contrary, caffeine showed no protective effect. Hydroxycinnamic acids are the major phenolic components of green coffee, amounting up to 4-12% in mass (Perrone, Donangelo, Donangelo, & Farah, 2010). Different studies have shown the high variety of hydroxycinnamic acids in green coffee beans (Alonso-Salces, Guillou, & Berrueta, 2009; Jaiswal, Patras, Eravuchira, & Kuhnert, 2010). In the present work, 5-CQA was the main phenolic component in green coffee beans from Colombia (54% of total polyphenols), which is in accordance with previous reports (Alonso-Salces, Serra, Reneiro, & Hèberger, 2009). Among dicaffeoylquinic acids, 3,5-DCQA predominated, whereas the major methylxanthine was CAF, also in agreement with previous data (Alonso-Salces, Serra, Reneiro, & Hèberger, 2009). Hydroxycinnamic acids have been associated with anti-cancer and antiinflammatory effects. In particular, 5-CQA can inhibit the proliferation of 13

ACCEPTED MANUSCRIPT carcinogenic cells in vitro and the neoplastic transformation of epidermal cells (Feng et al., 2005). Similarly, 3,5-DCQA inhibits the proliferation of colon cancer cells,

also

reducing

the

lipopolysaccharide-induced

inflammation

in

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macrophages by preventing NF-κB translocation and consequently the

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expression of cyclo-oxygenase 2 and pro-inflammatory mediators (Han, Kim-J, Kim-H, Chun, & Jeong, 2010; Puangprahant, Berhow, Vermillion, Potts, &

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Gonzalez de Mejia, 2011). However, the role of hydroxycinnamic acids in the cell cycle is not clear. Some studies have considered 5-CQA as stimulant of cell death (Yip, Chan, Pang, Tam, & Wong, 2006), whereas other researchers have

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shown inhibition of cellular apoptosis by 5-CQA effects through blockage of caspase-3 activity and modulation of Bcl-2 and Bax expression (Huang,

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Chuang, Wu, & Yen, 2008; Cho et al., 2009; Wu, Lin, & Zhang, 2012). Also, a lack of effect on cell viability, caspase-3 activity and Bcl-XL or Bax levels by 5CQA has also been reported in HepG2 (Ramos, Alía, Bravo, & Goya, 2005;

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Granado-Serrano et al., 2007), as well as on cell proliferation and cell cycle

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arrest elicited by a mulberry leaves water extract rich in caffeoylquinic acids (Naowaratwattana, De-Eknamkul, & De Mejia, 2010), showing the need for

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further studies on the effect of hydroxycinnamic acids. In the present study, incubation for 24 h with GCBE (1, 10 and 50 µg/mL) or 5CQA, 3,5-DCQA and CAF (1, 10 and 20 µM) did not affect cell viability not

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eliciting cytotoxic effects in agreement with previous reports (Ramos, Alía, Bravo, & Goya, 2005; Granado-Serrano et al., 2007; Cho et al., 2009; Wu, Lin, & Zhang, 2012). However, pretreatment with GCBE, 5-CQA and 3,5-DCQA partly or totally protected from the cell damage induced by t-BOOH, preventing ROS increase, macromolecular oxidation, depletion of GSH, and recovering antioxidant enzyme activities. To our knowledge, this is the first report on the antioxidant and hepatoprotective effect of a green coffee bean extract against tBOOH-induced damage in HepG2 cells and the contribution of the main monoand dicaffeoylquinic acids in coffee, also showing the lack of effect of methylxanthines. Similar results preventing ROS generation induced by t-BOOH or tumor necrosis factor  (TNF-) were seen when treating HepG2 cells with Hemerocallis fulva flower extracts (Lin, Lu, Huang, & Chen, 2011) and Gymnaster koraiensis extracts (Jho et al., 2013) rich in mono- and 14

ACCEPTED MANUSCRIPT dicaffeoylquinic acids, respectively. The results here reported are also in agreement with previous studies on HepG2 and other cell lines with cocoa phenolic extract (Martin et al., 2008), hydroxycinnamic acids (Granado-Serrano

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et al 2007; Zha, Xu, Wang, Dong, & Wang, 2007; Cho et al., 2009; León-

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González et al., 2012), hydroxytyrosol (Pereira-Caro et al., 2012), lutein (Lima et al., 2007), quercetin (Alia et al., 2006b), epicatechin (Martin et al., 2010) or

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procyanidin B2 (Rodriguez-Ramiro, Ramos, Bravo, Goya, & Martin, 2011). Choi et al. (2005) also showed a hepatic-protective role for 3,4-dicaffeoylquinic acid (3,5-DCQA isomer) against carbon tetrachloride induced hepatic

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cytotoxicity, improving aminotransferases and bilirubin levels. Moreover, both isomers have been related with neuroprotective effects against hydrogen

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peroxide, decreasing cellular death by inhibition of caspase-3 (Kim, Park, Jeon, Kwon, & Chun, 2005). Recently, the isomers 3,5-DCQA and 4,5-DCQA have shown a neuroprotective effect in SH-SY5Y neuroblastoma cells, attenuating

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the neurotoxic effects elicited by -amyloid peptide (Deng et al., 2013). 3,5-

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DCQA has also shown neuroprotective actions against retinal damage both in vitro in RGC-5 cells and in vivo, showing antioxidative and antiapoptotic effects

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(Ahn et al., 2014). The protective effect elicited by the GCBE can thus be ascribed to its phenolic fraction, since caffeine did not show protective effects in agreement with results reported for the main cocoa methylxanthine,

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theobromine (Martin et al., 2008). Physiologic effects of dietary compounds are limited by their absorption and metabolization rate. In this way, caffeine is quickly and completely absorbed in the small intestine and transported to the liver, where it is metabolized and distributed to the organism (Heckman, Weil, & Gonzalez de Mejia, 2010) prior to rapid urinary elimination mostly as methylated xanthines and uric acids (Martínez-López, Sarriá, Baeza, Mateos, & Bravo-Clemente, submitted). Plasma concentrations up to 10 µM have been reported after intake of a normal coffee serving (Martínez-López, Sarriá, Baeza, Mateos, & Bravo-Clemente, submitted), in the range of the doses used in the present study. On the contrary, the absorption rate of hydroxycinnamic acids is lower. The ester bond between caffeic acid and quinic acid in 5-CQA and 3,5-DCQA may affect their bioavailability (Zhao, & Moghadasian, 2010), although previous studies have 15

ACCEPTED MANUSCRIPT shown that 30% of consumed 5-CQA can be absorbed in the stomach and small intestine (Lafay, Morand, Manach, Besson, & Scalbert, 2006; Farah, Monteiro, Donangelo, & Lafay, 2008; Stalmach et al., 2009;). Plasma levels of

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hydroxycinnamic acids after roasted coffee consumption are in the nM range,

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although concentrations of the major colonic metabolites (dihydrocaffeic and dihydroferulic acids) approximate the µM levels (Stalmach et al., 2009; Renouf

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et al., 2010). Despite their low bioavailability, hydroxycinnamic acids may have an important role on health even at low concentrations (1 µM), suggesting that continued exposition to physiological levels of hydroxycinnamates through

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may have beneficial health effects.

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moderate consumption of coffee and other dietary sources of chlorogenic acids

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5. Conclusions

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GCBE and its main polyphenols, 5-CQA and 3,5-DCQA, but not caffeine, confer a significant protection against oxidative stress in vitro. In this way, the main

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polyphenols from green coffee could be responsible for the beneficial health effects associated with consumption of green coffee bean derived products such as extracts commercialized as nutraceuticals or soluble green coffee

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either alone or blended with roasted coffee.

Acknowledgements This work was funded by the Spanish Ministry of Science and Innovation (projects AGL2010-18269 and Consolider-Ingenio CSD2007-00063). G. B. is a FPI fellow funded by the Spanish Ministry of Science and Innovation. M. A.-B. is a postdoctoral researcher of the JAE Program funded by CSIC and the European Social Fund. L.B. conceived and designed the study. All authors revised and approved the final version of the manuscript. The authors declare no conflicts of interest.

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ACCEPTED MANUSCRIPT Zhao, Z., & Moghadasian, M.H. (2010). Bioavailability of hydroxycinnamates: a brief review of in vivo and in vitro studies. Phytochemistry Reviews, 9, 133-

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(10 µM) on HepG2 cell morphology after 20 h treatment (x100 magnification).

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Figure 2. Direct effect on intracellular ROS generation after treatment with the noted concentrations of GCBE (a), 5-CQA (b), 3,5-DCQA (c) and CAF (d) at 0,

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60, 120 and 180 min. Result are expressed as fluorescence units against time (n = 4 – 8). SD values are not included due to intense bar overlapping. Different letters denote significant differences (p < 0.05).

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Figure 3. HepG2 cell morphology after pre-treatment with GCBE (10 µg/mL) and 5-CQA, 3,5-DCQA and CAF (10 µM) for 20 h (upper photos) and after 400

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µM t-BOOH exposure for 3 h (lower photos) (x100 magnification). Figure 4. Protective effect of pre-treatment for 20 h with GCBE (1, 10 and 50 µg/mL) and 5-CQA, 3,5-DCQA and CAF (1, 10 and 20 µM) against oxidative

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stress induced by 400 µM t-BOOH for 3 h on HepG2 cytotoxicity (% LDH in

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medium). Results are means ± SD (n = 5 – 10). Different letters indicate statistically differences (p < 0.05).

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Figure 5. Protective effect on intracellular ROS production after pre-treatment with the noted concentrations of GCBE (a), 5-CQA (b), 3,5-DCQA (c) and CAF (d) for 20 h and after 400 µM t-BOOH exposure. Result are expressed as

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fluorescence units against time (n = 4 – 5). SD values are not included due to intense bar overlapping. Different letters denote significant differences (p < 0.05).

Figure 6. Protective effect of pre-treatment for 20 h with GCBE (1, 10 and 50 µg/mL) and 5-CQA, 3,5-DCQA and CAF (1, 10 and 20 µM) against oxidative stress induced by 400 µM t-BOOH for 3 h on GSH content. Results are means ± SD (n = 6 – 8). Different letters indicate statistically differences (p < 0.05). Figure 7. Protective effect of pre-treatment for 20 h with GCBE (1, 10 and 50 µg/mL) and 5-CQA, 3,5-DCQA and CAF (1, 10 and 20 µM) against oxidative stress induced by 400 µM t-BOOH for 3 h on GPx (a) and GR (b) activity. Results are means ± SD (n = 5 – 9). Different letters indicate statistically differences (p < 0.05). 25

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Figure 9. Protective effect of pre-treatment for 20 h with GCBE (1, 10 and 50 µg/mL) and 5-CQA, 3,5-DCQA and CAF (10µM) against oxidative stress induced by 400 µM t-BOOH for 3 h on protein-oxidation. Results are means ±

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Table 1. Direct effect of GCBE (1, 10 and 50 µg/mL) and 5-CQA, 3,5-DCQA and CAF (1, 10 and 20 µM) on cell viability and cytotoxicity (% LDH in medium), antioxidant defenses (GSH content, and GPx and GR activity) and macromolecular damage (MDA and carbonyl concentrations). HepG2 cells were treated with the noted concentrations for 20 h (24 h cell viability). Values are means ± SD (n = 10-16 cell viability; n = 4-8 rest of assays). Different letters within a column indicate statistically differences (p < 0.05).

100.00 ± 5.93

a

13.96 ± 1.85

a

ng GSH 212.53 ± 33.64

mU GPx/mg protein a

GCBE 10 µg/mL

98.68 ± 5.35

50 µg/mL

96.29 ± 8.99

Control

a

95.09 ± 5.32

a a

ab

100.00 ± 6.99

13.88 ± 1.62 13.65 ± 1.84 13.70 ± 1.55

11.19 ± 1.20

a a a

a

220.75 ± 19.95 202.99 ± 16.77 205.89 ± 18.46

212.53 ± 26.87

5-CQA 10 µM

94.90 ± 7.27

20 µM 3,5-DCQA

94.17 ± 8.79

1 µM

96.03 ± 5.03

10 µM

95.72 ± 6.76

20 µM

95.95 ± 7.26

1 µM

102.67 ± 5.70

a a

ac a ac

11.17 ± 1.39 10.34 ± 0.78 11.31 ± 0.94 10.34 ± 1.89 10.59 ± 1.24 10.14 ± 1.59

a a a

a a a

214.54 ± 27.45

CE P

a

95.45 ± 4.83

209.19 ± 21.03 208.28 ± 18.32

AC

1 µM

a a a

28.16 ± 2.83

TE D

1 µg/mL

US

Control

% LDH in medium

MA N

% Cell viability

Antioxidant defenses

219.17 ± 22.70 213.21 ± 31.39 210.47 ± 27.43

Macromolecular damage

CR

Cell viability and cytotoxicty

a

a a a

a a a

27.91 ± 2.39 26.43 ± 2.26 25.40 ± 2.76

40.72 ± 3.33

43.36 ± 5.18 42.60 ± 7.50 39.63 ± 3.40 43.59 ± 2.87 40.84 ± 2.22 40.34 ± 0.49

a

a a a

a

a a a

a a a

mU GR/mg protein 7.22 ± 0.76

7.04 ± 0.99 6.96 ± 0.91 6.87 ± 0.88

9.08 ± 1.53

9.94 ± 1.10 9.47 ± 1.49 9.74 ± 1.35

a

a a a

a

9.02 ± 0.36

0.48 ± 0.07

0.46 ± 0.06 0.49 ± 0.67 0.45 ± 0.06

0.48 ± 0.02

a

a a a

a

nmol carbonyl/mg protein 1.02 ± 0,18

1.09 ± 0.16 1.05 ± 0.17 1.04 ± 0.16

1.59 ± 0.32

a

a a a

a

a a

0.39 ± 0.03

a

1.39 ± 016

a

a

10.02 ± 1.71 9.31 ± 1.23

nmol MDA/mg protein

a

a

0.39 ± 0.05

a

1.51 ± 0.28

a

a

CAF 10 µM

102.70 ± 7.97

20 µM

105.46 ± 7.60

ab ab b

9.97 ± 1.22 10.02 ± 1.31 10.56 ± 1.38

a a a

222.89 ± 25.67 211.94 ± 28.06 213.20 ± 29.74

a a a

41.63 ± 5.07 42.93 ± 5.27 40.21 ± 1.64

36

a a a

9.45 ± 1.31 8.91 ± 1.05 9.30 ± 1.28

a a a

0.48 ± 0.12

a

1.81 ± 0.15

a

ACCEPTED MANUSCRIPT

IP

T

Highlights - GCBE, 5-CQA, 3,5-DCQA and CAF are not cytotoxic on HepG2 cells.

CR

- Treatment for 20h with GCBE, 5-CQA and 3,5-DCQA decreases ROS generation in HepG2 cells. - GCBE and hydroxycinnamic acids have a remarkable protective effect against oxidative stress.

US

- Pre-treatment with CAF for 20 h does not protect against oxidative damage in vitro.

AC

CE P

TE D

MA N

- Hydroxycinnamic acids could be responsible for green coffee beneficial effects.

37