Accepted Manuscript Short communication Assessment of white grape pomace from winemaking as source of bioactive compounds, and its antiproliferative activity M. José Jara-Palacios, Dolores Hernanz, M. Luisa Escudero-Gilete, Francisco J. Heredia, Jeremy P.E. Spencer PII: DOI: Reference:
S0308-8146(15)00373-8 http://dx.doi.org/10.1016/j.foodchem.2015.03.022 FOCH 17269
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
11 December 2014 19 February 2015 9 March 2015
Please cite this article as: José Jara-Palacios, M., Hernanz, D., Luisa Escudero-Gilete, M., Heredia, F.J., Spencer, J.P.E., Assessment of white grape pomace from winemaking as source of bioactive compounds, and its antiproliferative activity, Food Chemistry (2015), doi: http://dx.doi.org/10.1016/j.foodchem.2015.03.022
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Assessment of white grape pomace from winemaking as source of bioactive compounds,
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and its antiproliferative activity
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M. José Jara-Palacios1, Dolores Hernanz2, Tania Cifuentes-Gomez3, M. Luisa Escudero-Gilete1,
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Francisco J. Heredia1*, Jeremy P. E. Spencer3
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de Farmacia, Universidad de Sevilla, 41012 Sevilla, Spain.
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Sevilla, Spain.
Food Colour and Quality Laboratory, Department of Nutrition and Food Science, Facultad
Department of Analytical Chemistry, Facultad de Farmacia, Universidad de Sevilla, 41012
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Chemistry, Food and Pharmacy, University of Reading, Reading RG6 6AP, United Kingdom.
Molecular Nutrition Group, Department of Food and Nutritional Sciences, School of
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* Corresponding author:
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Francisco J. Heredia
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Food Colour & Quality Laboratory, Dept. Nutrition & Food Science, Universidad de Sevilla.
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Facultad de Farmacia, 41012 Sevilla, Spain.
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Tel.: +34 954556495
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e-mail: [email protected]
Fax: +34 954556110
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ABSTRACT
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The anti-proliferative effects of a purified white grape pomace extract (PWGPE), as well as
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of some phenolic standards on colon cancer cells were examined. The phenolic composition
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of the PWGPE was determined by rapid resolution liquid chromatography/mass
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spectrometry (RRLC/MS). The PWGPE had 92.6, 43.3 and 6.01 mg/g of flavanols, flavonols
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and phenolic acids, respectively and, along with pure catechin, epicatechin, quercetin and
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gallic acid, they were all found capable of inhibiting cellular proliferation. PWGPE (100
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µg/ml) inhibited the proliferation of cells by 52.1% at 48 h, whilst catechin, epicatechin,
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quercetin and gallic acid (60 µg/ml) inhibited growth by 65.2, 62.2, 81.0 and 71.0%,
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respectively, at 72 h. The PWGPE is an interesting source of phenolic compounds with
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antiproliferative properties, that could be of interest in the food and pharmaceutical
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industries.
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Keywords: Caco-2, colorectal cancer, grape pomace, phenolic compounds
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1. Introduction
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Wine seems to be beneficial to health and, therefore, a moderate and regular consumption
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of wine is recommended. During winemaking, the polyphenols of the grape are transferred
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to the wine, but a high proportion still remains in the solid winemaking by-products. Grape
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pomace, consisting of the waste seeds, skins and stems resulting from the winemaking
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process, has been postulated to be a relatively good source of dietary bioactive compounds,
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such as polyphenols (Anastasiadi, Pratsini, Kletsas, Skaltsounis, & Haroutounian, 2010;
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Rodríguez Montealegre, Romero Peces, Chacón Vozmediano, Martínez Gascueña, & García
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Romero, 2006), with recent data reporting its antioxidant, antiradical, antimicrobial, anti-
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inflammation and anticancer activities and its cardioprotective action (Xia, Deng, Guo, & Li,
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2010; Sagdic, Ozturk, Ozkan, Yetim, Ekici, & Yilmaz, 2011; Rodriguez-Rodriguez et al., 2012).
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Therefore, the consumption of polyphenols has been associated with reduced risk of chronic
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diseases, including cardiovascular and neurodegenerative diseases, and cancer, e.g. colon
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cancer (Vauzour, Rodríguez-Mateos, Corona, Oruna-Concha, & Spencer, 2010).
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The importance of polyphenols makes the study of byproducts of interest for the food,
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nutraceutical, pharmaceutical and chemical industries. In recent years, there had been a
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growing interest in the use of byproducts rich in polyphenols emanating, from the
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winemaking industry, to produce extracts and novel products for the maintenance of human
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health (Perumalla & Hettiarachchy, 2011; Kulkarni, De Santos, Kattamuri, Rossi, & Brewer,
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2011). As such, the extraction of polyphenols from waste material deriving from traditional
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industrial practices represents an attractive, sustainable and cost effective source of these
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high-value biological bioactives, which could be incorporated into foods. Due to the
3
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increasing demand for nutraceutical and antioxidant compounds, the study of grape
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pomace may be useful for industrial purposes.
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Colorectal cancer is the third most prevalent cancer worldwide (Chan & Giovannucci, 2010)
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and there is intense interest in the development of novel chemopreventive products and/or
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the production of anti-cancer agents from novel, industrial waste-stream sources. Several
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studies have indicated the anti-cancer potential of polyphenolic compounds, and in
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particular their ability to inhibit the proliferation of cancer cells via their effects on the cell-
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cycle and/or apoptosis (Lee et al., 2006; Ramos, Rodríguez-Ramiro, Martín, Goya, & Bravo,
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2011; Vu et al., 2012), and have suggested their use as novel dietary chemopreventive
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agents (Araújo, Gonçalves, & Martel, 2011). For example, the anti-proliferative effects of
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polyphenolic extracts from olive oil (Corona, Deiana, Incani, Vauzour, Dessì, & Spencer,
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2007), red wine (Gómez-Alonso, Collins, Vauzour, Rodríguez-Mateos, Corona, & Spencer,
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2012), tomato (Saunders, 2009), araçá (Medina et al., 2011), raspberry (Coates et al., 2007),
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and cranberry (Vu et al., 2012) have been reported. However, to date there are no previous
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data on the actions of grape pomace extracts, although their potential to protect against
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oxidative stress has been shown in Caenorhabditis elegans (Jara-Palacios et al., 2013). These
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phenolic extracts are a relatively rich source of bioactive compounds, which could be
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exploited in the food and pharmaceutical industries.
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The current study we aimed to address this by investigating the potential anti-cancer effects
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of a purified white grape pomace extract (PWGPE), rich in phenolic compounds, on colon
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adenocarcinoma cell growth, in relation to the individual effects of pure polyphenols
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relevant to PWGPE, notably epicatechin, catechin, quercetin and gallic acid. 4
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2. Materials and methods
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2.1. Reagents and standards
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Methanol, n-hexane and hydrochloric acid were purchased from Labscan (Dublin, Ireland).
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Formic acid, HPLC-grade acetonitrile, sodium carbonate, and Folin-Ciocalteau reagent were
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obtained from Panreac (Barcelona, Spain). Gallic acid, protocatechuic acid, (+)-catechin, (−)-
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epicatechin, quercetin, kaempferol, quercetin-3-O-rutinoside (rutin), ferulic acid, caffeic
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acid, p-coumaric acid, and phosphate buffered saline (PBS) were purchased from Sigma-
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Aldrich (Madrid, Spain). Quercetin-3-O-glucoside and kaempferol-3-O-glucoside were
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obtained from Extrasynthese (Genay, France). Alamar Blue was from Abd Serotec
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(Kidlington, UK). Fehling’s reagents, dimethyl sulfoxide (DMSO), sulforhodamine B-based
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assay kit and all other reagents used for assay with cells were obtained from Sigma Aldrich
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(UK).
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The stock solutions of phenolic standards were prepared in water at concentration of 100
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mg/l. The corresponding dilutions were made up from the stock solutions to assess the
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antioxidant activity and the anti-proliferative effects.
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2.2. Extraction of phenolic compounds from grape pomace
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Grape pomace of the variety Zalema, D.O. “Condado de Huelva” (Spain), collected after
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winemaking, was supplied by “Vinícola del Condado” winery (Bollullos Par del Condado,
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Spain). Grape pomace was immediately frozen at -20 °C, freeze-dried for 48 hours and
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finally ground to a fine powder. The freeze-dried grape pomace (50 g) was extracted with
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75% methanol as extraction solvent. The mixture was sonicated for 1 h followed by further
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constant shaking for 12 h (VWR Incubating mini-shaker), centrifuged at 4190 x g for 15 min 5
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and finally the supernatant was collected. The remaining residue was subjected to a further
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two extractions and the supernatants were combined to obtain the phenolic extract. This
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methanolic phenolic extract was dried under vacuum and the aqueous extract was washed
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with n-hexane. The resulting extract was passed through a C18 column (10 × 5 cm) to
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remove sugars, which was achieved by their elution, following addition of 100 ml of acidified
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water (pH 2; HCl), followed by 300 ml of Milli-Q® water at a constant rate (0.5 ml/min),
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using a diaphragm-metering pump (STEPDOS, Scientific Laboratory Suppliers). The Fehling
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test was carried out to ascertain the presence of sugars in the water extracts, as described
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previously (Eid, Al-Awadi, Vauzour, Oruna-Concha, & Spencer, 2013). Following the removal
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of sugars, the elution of phenolic compounds was achieved by addition of 400 ml of
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methanol. This methanolic extract was concentrated to dryness, using a rotatory vacuum
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evaporator at 40 °C, and freeze-dried, yielding the PWGPE.
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2.3. Determination of individual phenolic compounds: RRLC/MS analysis.
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Chromatographic analysis was carried out as previously described (Jara-Palacios, Hernanz,
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González-Manzano, Santos-Buelga, Escudero-Gilete, & Heredia, 2014). An Agilent 1260
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chromatograph (Agilent Technologies, Palo Alto, CA, USA), equipped with a diode-array
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detector, and an API 3200 Qtrap (Applied Biosystems, Darmstadt, Germany), equipped with
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an ESI source, and a triple-quadrupole ion trap mass analyser were used. Phenolic
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compounds were identified by comparing their retention time, UV-vis spectra, and mass
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spectral data with authentic standards where available. The compounds were quantified
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using peak area data of resolved peaks at 280, 320 and 370 nm, for flavanols, phenolic acids
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and flavonols, respectively. Procyanidins were quantified, using catechin as the standard,
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whilst caftaric, fertaric and coutaric acids were quantified using caffeic, ferulic and p6
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coumaric acids, respectively. Quercetin and isorhamnetin derivatives were quantified as
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quercetin, and kaempferol derivates as kaempferol. Detailed retention times, UV-vis data
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and mass spectrometric data were shown previously in another paper (Jara-Palacios et al.,
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2014). The samples were analyzed in triplicate, and the results were expressed as mg of
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phenolic compound per g of PWGPE.
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2.4. Determination of total phenolic content
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The total phenolic content (TPC) was determined by the Folin-Ciocalteu spectrophotometric
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method, as described previously (Jara-Palacios et al., 2014). Analyses were conducted in
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triplicate and gallic acid was employed as a calibration standard. Results were expressed as
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mg of gallic acid per g of PWGPE.
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2.5. Cell culture
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2.5.1. General
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Human colon adenocarcinoma cells (Caco-2, ECACC Salisbury, Wiltshire, UK) were cultured
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in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 20% heat-inactivated
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bovine serum, 2 mM L-glutamine, 1% non-essential amino acids, 100 U/ml of penicillin, and
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100 µg/ml of streptomycin. Cells were seeded in 24-well plates at low confluence (2.5 x 104
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per well) and then exposed to the PWGPE (10, 25, 50 and 100 µg/ml), catechin (30 and 60
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µg/ml), epicatechin (30 and 60 µg/ml), quercetin (30 and 60 µg/ml), gallic acid (30 and 60
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µg/ml), or vehicle (DMSO 1%), 8 h after seeding.
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2.5.2. Cellular proliferation and viability
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The ability of the PWGPE to inhibit cellular proliferation was tested, using the
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sulforhodamine B (SRB) assay. Cells were harvested following 24, 48 and 72 h in culture and
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treated by the addition of 125 µl of ice-cold TCA (4 °C; 1 h). After fixing, media were
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removed, cells were washed and total biomass was determined using SRB (250 µl of 0.4%
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SRB; 30 min). Unincorporated dye was removed by washing with 1% acetic acid, whilst cell
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incorporated dye was solubilised using Tris-base (10 mM). Dye incorporation, reflecting cell
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biomass, was measured at 492 nm, using a GENios microplate reader (TECAN, Reading, UK).
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Viability was assessed after 24 h of treatment with PWGPE at different concentrations by
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the Alamar blue assay, as described previously (Vauzour, Vafeiadou, Rice-Evans, Williams, &
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Spencer, 2007). Briefly, Alamar Blue solution (10% v/v) was added to each well, and plates
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were incubated for 3 h, prior to fluorescence being measured at 540 nm excitation and 612
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nm emission on the GENios microplate reader.
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2.6. Statistical analysis
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For the statistical treatment of the data, the Statistica v.8.0 software was used. One-way
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analysis of variance (ANOVA) was employed to establish if antioxidant and anti-proliferative
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effects differed significantly between (a) the PWGPE and the phenolic standards and (b)
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those with the vehicle (DMSO 1%).
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3. Results and discussion
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3.1. Phenolic composition and antioxidant activity
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The total phenolic content of the PWGPE was 365 mg/g (Table 1). 11 flavanols, 9 flavonols
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and 5 phenolic acids were identified and quantified by RRLC/MS, with flavanols being the
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major compounds detected accounting for 65% of total phenolic compounds (92.6 mg 8
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flavanols/g PWGPE), followed by flavonols (43.3 mg/g PWGPE, 31%) and phenolic acids
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(6.01 mg/g PWGPE, 4%) (Table 1). The most abundant flavanol was procyanidin B1 (15.5
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mg/g PWGPE), followed by catechin, procyanidin B2-3-O-gallate, and procyanidin tetramer 1
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(12.2, 11.8 and 10.8 mg/g PWGPE, respectively). The main flavonols were quercetin-3-O-
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glucoside and quercetin-3-O-glucuronide (16.9 and 15.8 mg/g PWGPE, respectively). The
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most abundant phenolic acid was gallic acid (3.03 mg/g PWGPE). These phenolic compounds
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were found in other extracts from Zalema grape pomace, but in different amounts (Jara-
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Palacios et al., 2013; Jara-Palacios et al., 2014), which may be due to the extraction process.
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3.2. Effect on cell proliferation
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PWGPE, at different concentrations (10, 25, 50 and 100 µg/ml), induced dose-dependent
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inhibition of cancer cell proliferation following 24, 48 and 72 h of exposure (Figure 1). A
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significant increase (p < 0.05) in growth inhibition treated cells was observed after all
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treatments, relative to vehicle, with the exception of PWGPE at 10 µg/ml and 72 h of
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exposure (p > 0.05), whilst the 100 µg/ml dose for 48 h induced the greatest degree of
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inhibition of proliferation (52% growth inhibition). Our data agree with previous cell data
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indicating that polyphenols are capable of inhibiting the proliferation of colon
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adenocarcinoma cells in a dose-dependent manner. Notably, the polyphenol rich foods, e.g.
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olive oil (100 µg/ml), red wine (100 µg/ml) and a tomato extract (2.5 mg/ml) inhibited
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cancer cell growth over a similar time frame (Corona et al., 2007; Saunders, 2009; Gómez-
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Alonso et al., 2012).
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Previous studies have indicated that such activity may be mediated via the potential of
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polyphenols and their metabolites to induce cell cycle blockage in the G2/M phase in 9
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adenocarcinoma cells, perhaps through their effects on COX-2 activity (Shan, Wang., & Li,
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2009; Yasui, Kim, Oyama, & Tanaka, 2009). On the other hand, these anti-proliferative
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effects may be mediated, in part, by their direct cytotoxic action (Gómez-Alonso et al.,
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2012). With respect to the latter, our data suggest that cell viability at 24 h was significantly
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(p < 0.05) reduced at higher exposure concentrations (Figure 2). Our data agree with
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previous observations that a polyphenol-rich red wine and araçá fruit extracts also reduce
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cell viability (Gómez-Alonso et al., 2012; Medina et al., 2011).
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The assayed concentrations of polyphenols (10-100 µg/ml) in the culture medium could be
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associated with those present in some food sources, such as white wine, cocoa, black
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currant, or tea (Recamales, Sayago, González-Miret, & Hernanz, 2006; Lamuela-Raventós,
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Romero-Pérez, Andrés-Lacueva, &, Tornero , 2005; Williamson & Clifford, 2010; Wang, Lu,
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Miao, Xie, & Yang, 2008). These amounts of polyphenols have shown antiproliferative
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effects in the cells; however, human health effects of these compounds depend on their
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bioavailability. A previous study suggests that the phenolic composition of a food source
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could affect the microbiota and its catabolic activity, inducing changes that could in turn
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affect the bioavailability and potential bioactivity of polyphenols producing metabolites
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which might be responsible for observed health effects in vivo (Cueva et al., 2013).
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Therefore, although in vitro assays provide information on bioactivity of polyphenols, it is
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essential to know their bioavailability by in vivo studies, which would be interesting to look
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at in future.
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In order to elaborate the mediating bioactive components of the extract used, individual
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phenolic compounds, gallic acid, catechin, quercetin and epicatechin were also tested for 10
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their effects on cancer cell growth (Figure 3). A significant inhibition (p < 0.05) of
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proliferation was observed for all compounds, relative to vehicle-treated cells, ranging from
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17-81% for epicatechin (30 µg/ml) and quercetin (60 µg/ml), respectively (Figure 3). Indeed,
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the anti-proliferative effects of the PWGPE were comparable to effects of catechin,
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epicatechin and gallic acid at similar concentrations to that found in the extract (p > 0.05).
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Although the precise concentrations of catechin, epicatechin, gallic acid and quercetin
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varied in the extract (12.2, 6.3, 3.0 and 2.9 mg/g PWGPE, respectively), as reported
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previously (Araújo et al., 2011), a combination of polyphenols may target overlapping and
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complementary phases of the carcinogenic process, thus increasing the efficacy and potency
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of its chemopreventive effects. Previous data support this, with a variety of phenolic
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compounds previously described as anticancer compounds with respect to colorectal cancer
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(Salucci, Stivala, Maiani, Bugianesi, & Vannini, 2002; Araújo et al., 2011; Ramos et al., 2011).
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4. Conclusions
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In summary, a PWGPE obtained from the winemaking process (Zalema white grape variety)
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is capable of inhibiting adenocarcinoma cell proliferation by a combination of anti-
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proliferative activity and via a direct initiation of cell death. Further studies are required to
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elucidate the specific components present in PWGPE which are responsible for these effects
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and the mechanisms involved, although our data support the conclusion that major
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polyphenols present in the extract, such as catechin and quercetin, may be the mediating
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components. This study has described the inhibitory nature of dietary polyphenolic
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compounds on colon cancer cell growth, but there is no clear evidence that consumption of
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polyphenols prevents cancer; therefore future work is necessary to establish this.
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Acknowledgements
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This work was partially supported by “V Plan Propio de Investigación” of Universidad de
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Sevilla. The authors acknowledge the collaboration of “Consejo Regulador D.O. Condado de
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Huelva” and assistance of the technical staff of Biology Service (SGI, Universidad de Sevilla).
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M. J. Jara-Palacios holds a predoctoral Research Grant (FPU) from the Spanish Ministry of
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Education.
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References
262 263
Anastasiadi, M., Pratsinis, H., Kletsas, D., Skaltsounis, A. L., & Haroutounian, S. A. (2010).
264
Bioactive non-coloured polyphenols content of grapes, wines and vinification by-products.
265
Evaluation of the antioxidant activities of their extracts. Food Research International, 43,
266
805-813.
267 268
Araújo, J. R., Gonçalves, P., & Martel, F. (2011). Chemopreventive effect of dietary
269
polyphenols in colorectal cancer cell lines. Nutrition Research, 31, 77-87.
270 271
Chan, A. T., & Giovannucci, E. L. (2010). Primary prevention of colorectal cancer.
272
Gastroenterology, 138, 2029-2043.
273 274
Coates, E. M., Popa, G., Gill, C. I., McCann, M. J., McDougall, G. J., Stewart, D., & Rowland, I.
275
(2007). Colon-available raspberry polyphenols exhibit anti-cancer effects on in vitro models
276
of colon cancer. Journal of Carcinogenesis, 6, 1-10.
277 278
Corona, G., Deiana, M., Incani, A., Vauzour, D., Dessì, M. A., & Spencer, J. P. (2007).
279
Inhibition of p38/CREB phosphorylation and COX-2 expression by olive oil polyphenols
280
underlies
281
Communications, 362, 606-611.
their
anti-proliferative
effects.
Biochemical
and
Biophysical
Research
282 283
Cueva, C., Sánchez-Patán, F., Monagas, M., Walton, G. E., Gibson, G. R., Martín-Álvarez, P. J.,
284
Bartolomé, B., & Moreno-Arribas, M. V. (2013). In vitro fermentation of grape seed flavan-313
285
ol fractions by human faecal microbiota: changes in microbial groups and phenolic
286
metabolites. FEMS Microbiology Ecology, 83, 792-805.
287 288
Eid, N. M. S., Al-Awadi, B., Vauzour, D., Oruna-Concha, M. J., & Spencer, J. P. E. (2013). The
289
effect of cultivar type and ripening on the polyphenol content of date palm fruit. Journal of
290
Agricultural and Food Chemistry, 61, 2453-2460.
291 292
Gómez-Alonso, S., Collins, V. J., Vauzour, D., Rodríguez-Mateos, A., Corona, G., & Spencer, J.
293
P.E. (2012). Inhibition of colon adenocarcinoma cell proliferation by flavonols is linked to a
294
G2/M cell cycle block and reduction in cyclin D1 expression. Food Chemistry, 130, 493-500.
295 296
Jara-Palacios, M. J., González-Manzano, S., Escudero-Gilete, M. L., Hernanz, D., Dueñas, M.,
297
González-Paramás, A. M., Heredia, F. J., & Santos-Buelga, C. (2013). Study of Zalema grape
298
pomace: phenolic composition and biological effects in Caenorhabditis elegans. Journal of
299
Agricultural and Food Chemistry, 61, 5114-5121.
300 301
Jara-Palacios, M. J., Hernanz, D., González-Manzano, S., Santos-Buelga, C., Escudero-Gilete,
302
M. L., & Heredia, F. J. (2014). Detailed phenolic composition of white grape by-products by
303
RRLC/MS and measurement of the antioxidant activity. Talanta, 125, 51-57.
304 305
Kulkarni, S., De Santos, F. A., Kattamuri, S., Rossi, S. J., & Brewer, M. S. (2011). Effect of
306
grape seed extract on oxidative, color and sensory stability of a pre-cooked, frozen, re-
307
heated beef sausage model system. Meat Science, 88, 139-144.
308 14
309
Lamuela-Raventós, R. M., Romero-Pérez, A. I., Andrés-Lacueva, C., Tornero, A. (2005).
310
Review: health effects of cocoa flavonoids. Food Science and Technology International, 11,
311
159-176.
312 313
Lee, S. Y. H., Munerol, B., Pollard, S., Youdim, K. A., Pannala, A. S., Kuhnle, G. G., Debnam,
314
E.S., Rice-Evans, C., & Spencer, J.P. (2006). The reaction of flavanols with nitrous acid
315
protects against N-nitrosamine formation and leads to the formation of nitroso derivatives
316
which inhibit cancer cell growth. Free Radical Biology and Medicine, 40, 323-334.
317 318
Medina, A. L., Haas, L. I. R., Chaves, F. C., Salvador, M., Zambiazi, R. C., Silva, W. P., Nora, L.,
319
& Rombaldi, C. V. (2011). Araçá (Psidium cattleianum Sabine) fruit extracts with antioxidant
320
and antimicrobial activities and antiproliferative effect on human cancer cells. Food
321
Chemistry, 128, 916-922.
322 323
Perumalla, A. V. S., & Hettiarachchy, N. S. (2011). Green tea and grape seed extracts-
324
potential applications in food safety and quality. Food Research International, 44, 827-839.
325 326
Ramos, S., Rodríguez-Ramiro, I., Martín, M. A., Goya, L., & Bravo, L. (2011). Dietary flavanols
327
exert different effects on antioxidant defenses and apoptosis/proliferation in Caco-2 and
328
SW480 colon cancer cells. Toxicology in Vitro, 25, 1771-1781.
329 330
Recamales, A. F., Sayago, A., González-Miret, M. L., & Hernanz, D. (2006). The effect of time
331
and storage conditions on the phenolic composition and colour of white wine. Food
332
Research International, 39, 220-229. 15
333 334
Rodríguez Montealegre, R., Romero Peces, R., Chacón Vozmediano, J. L., Marơnez Gascueña,
335
J., & García Romero, E. (2006). Phenolic compounds in skins and seeds of ten grape Vitis
336
vinifera varieties grown in a warm climate. Journal of Food Composition and Analysis, 2006,
337
19, 687-693.
338 339
Rodríguez-Rodríguez, R., Justo, M. L., Claro, C. M., Vila, E., Parrado, J., Herrera, M. D., &
340
Alvarez de Sotomayor, M. (2012). Endothelium dependent vasodilator and antioxidant
341
properties of a novel enzymatic extract of grape pomace from wine industrial waste. Food
342
Chemistry, 135, 1044-1051.
343 344
Sagdic, O., Ozturk, I., Ozkan, G., Yetim, H., Ekici, L., & Yilmaz, M. T. (2011). RP-HPLC-DAD
345
analysis of phenolic compounds in pomace extracts from five grape cultivars: Evaluation of
346
their antioxidant, antiradical and antifungal activities in orange and apple juices. Food
347
Chemistry, 126, 1749-1758.
348 349
Salucci, M., Stivala L. A., Maiani, G., Bugianesi, R., & Vannini, V. (2002). Flavonoids uptake
350
and their effect on cell cycle of human colon adenocarcinoma cells (Caco2). British Journal of
351
Cancer, 86, 1645-1651.
352 353
Saunders, C. (2009). The anti-proliferative effect of different tomato varieties on the human
354
colon adenocarcinoma cells. Bioscience Horizons, 2, 172-179.
355
16
356
Shan, B. E., Wang, M. X., & Li, R. G. (2009). Quercetin inhibit human sw480 colon cancer
357
growth in association with inhibition of cyclin d1 and surviving expression through Wnt/β-
358
catenin signaling pathway. Cancer Investigation, 27, 604-612.
359 360
Vauzour, D., Vafeiadou, K., Rice-Evans, C., Williams, R. J., & Spencer, J. P. E. (2007).
361
Activation of pro-survival Akt and ERK1/2 signalling pathways underlie the anti-apoptotic
362
effects of flavanones in cortical neurons. Journal of Neurochemistry, 103, 1355-1367.
363 364
Vauzour, D., Rodriguez-Mateos, A., Corona, G., Oruna-Concha, M. J., & Spencer, J. P. E.
365
(2010). Polyphenols and human health: prevention of disease and mechanisms of action.
366
Nutrients, 2, 1106-1131.
367 368
Vu, K. D., Carlettini, H., Bouvet, J., Côté, J., Doyon, G., Sylvain, J. F., & Lacroix M. (2012).
369
Effect of different cranberry extracts and juices during cranberry juice processing on the
370
antiproliferative activity against two colon cancer cell lines. Food Chemistry, 132, 959-967.
371 372
Wang, D., Lu, J., Miao, A., Xie, Z., & Yang, D. (2008). HPLC-DAD-ESIMS/MS analysis of
373
polyphenols and purine alkaloids in leaves of 22 tea cultivars in China. Journal of Food and
374
Composition and Analysis, 21, 361-369.
375 376
Williamson, G., & Clifford, M. N. (2010). Colonic metabolites of berry polyphenols: the
377
missing link to biological activity? British Journal of Nutrition, 2010, 104 (Suppl. 3), 48-66.
378
17
379
Xia, E. Q., Deng, G. F., Guo, Y. J., & Li, H. B. (2010). Biological activities of polyphenols from
380
grapes. International. Journal of Molecular Sciences, 11, 622-646.
381 382
Yasui, Y., Kim, M., Oyama, T., & Tanaka, T. (2009). Colorectal carcinogensis and suppression
383
of tumor development by inhibition of enzymes and molecular targets. Current Enzyme
384
Inhibition, 5, 1-26.
18
385
Figure Captions
386 387
Figure 1. Growth inhibition induced by treatment with PWGPE at different concentrations.
388
Caco-2 cells were exposed to the treatment for 24, 48 and 72 h before SRB assay was
389
conducted. Each value represents mean (n=12) ± SD. a= p < 0.001; b= p < 0.01; c= p < 0.05.
390 391
Figure 2. Viable cells determined after treatment for 24 h with different concentrations of
392
PWGPE. Caco-2 cells were exposed to the treatment for 24 h before Alamar blue assay was
393
conducted. Each value represents mean (n=12) ± SD. a= p < 0.001; b= p < 0.01; c= p < 0.05.
394 395
Figure 3. Growth inhibition induced by treatment with catechin, epicatechin, quercetin and
396
gallic acid at different concentrations. Caco-2 cells were exposed to the treatment for 24, 48
397
and 72h before SRB assay was conducted. Each value represents mean (n=9) ± SD.
398
19
399
Table 1. Concentrations of phenolic compounds identified by RRLC/MS and TPC of PWGPE Phenolic compound
mg/g PWGPE
Flavanols Catechin Epicatechin Procyanidin B1 Procyanidin B2 Procyanidin B3 Galloyled procyanidin Procyanidin B2-3-O-gallate Procyanidin trimer 1 Procyanidin trimer 2 Procyanidin tetramer 1 Procyanidin tetramer 2
12.2 ± 0.07 6.34 ± 0.02 15.5 ± 0.00 4.94 ± 0.80 4.35 ± 0.10 7.97 ± 0.00 11.8 ± 0.29 5.65 ± 0.35 7.27 ± 0.27 10.8 ± 0.10 5.74 ± 0.02
Flavonols Quercetin (Q) Kaempferol (K) Q-3-O-rutinoside Q-3-O-glucuronide Q-3-O-galactoside Q-3-O-glucoside K-3-O-glucuronide K-3-O-glucoside I-3-O-glucoside
2.87 ± 0.00 0.15 ± 0.00 0.69 ± 0.00 15.8 ± 0.01 2.12 ± 0.02 16.9 ± 0.07 0.31 ± 0.00 3.67 ± 1.73 0.70 ± 0.01
Phenolic acids Gallic acid Protocatechuic acid Caftaric acid Caffeic acid p-Coumaric acid
3.03 ± 0.13 0.94 ± 0.13 0.88 ± 0.00 0.63 ± 0.01 0.51 ± 0.01
Total phenolic compounds1
142± 1.41
2
400
Total phenolic content 365± 3.35 I,isorhamnetin. Each value represents mean (n=9) ± SD
401
1
sum of individual phenolic compounds
402
2
TPC determined by Folin Ciocalteu assay
403 404
20
405 406 407 408 409 410 411
21
412 413 414
22
24 h
90
48 h
72 h
Growth Inhibition (%)
80 70 60 50 40 30 20 10 0 30
60
30
60
30
60
30
60
Catechin (µg/mL) Epicatechin (µg/mL) Quercetin (µg/mL) Gallic acid (µg/mL) 415 416 417
23
418
Highlights
419
A white grape pomace extract, rich in bioactive compounds, was obtained
420
The extract and its individual phenolics inhibited colon adenocarcinoma cells proliferation
421
The extract could be used as a good source of functional ingredients for foodstuffs
422 423 424 425 426 427 428
24
S0308-8146(15)00373-8 http://dx.doi.org/10.1016/j.foodchem.2015.03.022 FOCH 17269
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
11 December 2014 19 February 2015 9 March 2015
Please cite this article as: José Jara-Palacios, M., Hernanz, D., Luisa Escudero-Gilete, M., Heredia, F.J., Spencer, J.P.E., Assessment of white grape pomace from winemaking as source of bioactive compounds, and its antiproliferative activity, Food Chemistry (2015), doi: http://dx.doi.org/10.1016/j.foodchem.2015.03.022
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Assessment of white grape pomace from winemaking as source of bioactive compounds,
2
and its antiproliferative activity
3
M. José Jara-Palacios1, Dolores Hernanz2, Tania Cifuentes-Gomez3, M. Luisa Escudero-Gilete1,
4
Francisco J. Heredia1*, Jeremy P. E. Spencer3
5 6
1
7
de Farmacia, Universidad de Sevilla, 41012 Sevilla, Spain.
8
2
9
Sevilla, Spain.
Food Colour and Quality Laboratory, Department of Nutrition and Food Science, Facultad
Department of Analytical Chemistry, Facultad de Farmacia, Universidad de Sevilla, 41012
10
3
11
Chemistry, Food and Pharmacy, University of Reading, Reading RG6 6AP, United Kingdom.
Molecular Nutrition Group, Department of Food and Nutritional Sciences, School of
12 13
* Corresponding author:
14
Francisco J. Heredia
15
Food Colour & Quality Laboratory, Dept. Nutrition & Food Science, Universidad de Sevilla.
16
Facultad de Farmacia, 41012 Sevilla, Spain.
17
Tel.: +34 954556495
18
e-mail: [email protected]
Fax: +34 954556110
19
1
20
ABSTRACT
21
The anti-proliferative effects of a purified white grape pomace extract (PWGPE), as well as
22
of some phenolic standards on colon cancer cells were examined. The phenolic composition
23
of the PWGPE was determined by rapid resolution liquid chromatography/mass
24
spectrometry (RRLC/MS). The PWGPE had 92.6, 43.3 and 6.01 mg/g of flavanols, flavonols
25
and phenolic acids, respectively and, along with pure catechin, epicatechin, quercetin and
26
gallic acid, they were all found capable of inhibiting cellular proliferation. PWGPE (100
27
µg/ml) inhibited the proliferation of cells by 52.1% at 48 h, whilst catechin, epicatechin,
28
quercetin and gallic acid (60 µg/ml) inhibited growth by 65.2, 62.2, 81.0 and 71.0%,
29
respectively, at 72 h. The PWGPE is an interesting source of phenolic compounds with
30
antiproliferative properties, that could be of interest in the food and pharmaceutical
31
industries.
32 33 34 35
Keywords: Caco-2, colorectal cancer, grape pomace, phenolic compounds
36 37
2
38
1. Introduction
39
Wine seems to be beneficial to health and, therefore, a moderate and regular consumption
40
of wine is recommended. During winemaking, the polyphenols of the grape are transferred
41
to the wine, but a high proportion still remains in the solid winemaking by-products. Grape
42
pomace, consisting of the waste seeds, skins and stems resulting from the winemaking
43
process, has been postulated to be a relatively good source of dietary bioactive compounds,
44
such as polyphenols (Anastasiadi, Pratsini, Kletsas, Skaltsounis, & Haroutounian, 2010;
45
Rodríguez Montealegre, Romero Peces, Chacón Vozmediano, Martínez Gascueña, & García
46
Romero, 2006), with recent data reporting its antioxidant, antiradical, antimicrobial, anti-
47
inflammation and anticancer activities and its cardioprotective action (Xia, Deng, Guo, & Li,
48
2010; Sagdic, Ozturk, Ozkan, Yetim, Ekici, & Yilmaz, 2011; Rodriguez-Rodriguez et al., 2012).
49
Therefore, the consumption of polyphenols has been associated with reduced risk of chronic
50
diseases, including cardiovascular and neurodegenerative diseases, and cancer, e.g. colon
51
cancer (Vauzour, Rodríguez-Mateos, Corona, Oruna-Concha, & Spencer, 2010).
52 53
The importance of polyphenols makes the study of byproducts of interest for the food,
54
nutraceutical, pharmaceutical and chemical industries. In recent years, there had been a
55
growing interest in the use of byproducts rich in polyphenols emanating, from the
56
winemaking industry, to produce extracts and novel products for the maintenance of human
57
health (Perumalla & Hettiarachchy, 2011; Kulkarni, De Santos, Kattamuri, Rossi, & Brewer,
58
2011). As such, the extraction of polyphenols from waste material deriving from traditional
59
industrial practices represents an attractive, sustainable and cost effective source of these
60
high-value biological bioactives, which could be incorporated into foods. Due to the
3
61
increasing demand for nutraceutical and antioxidant compounds, the study of grape
62
pomace may be useful for industrial purposes.
63 64
Colorectal cancer is the third most prevalent cancer worldwide (Chan & Giovannucci, 2010)
65
and there is intense interest in the development of novel chemopreventive products and/or
66
the production of anti-cancer agents from novel, industrial waste-stream sources. Several
67
studies have indicated the anti-cancer potential of polyphenolic compounds, and in
68
particular their ability to inhibit the proliferation of cancer cells via their effects on the cell-
69
cycle and/or apoptosis (Lee et al., 2006; Ramos, Rodríguez-Ramiro, Martín, Goya, & Bravo,
70
2011; Vu et al., 2012), and have suggested their use as novel dietary chemopreventive
71
agents (Araújo, Gonçalves, & Martel, 2011). For example, the anti-proliferative effects of
72
polyphenolic extracts from olive oil (Corona, Deiana, Incani, Vauzour, Dessì, & Spencer,
73
2007), red wine (Gómez-Alonso, Collins, Vauzour, Rodríguez-Mateos, Corona, & Spencer,
74
2012), tomato (Saunders, 2009), araçá (Medina et al., 2011), raspberry (Coates et al., 2007),
75
and cranberry (Vu et al., 2012) have been reported. However, to date there are no previous
76
data on the actions of grape pomace extracts, although their potential to protect against
77
oxidative stress has been shown in Caenorhabditis elegans (Jara-Palacios et al., 2013). These
78
phenolic extracts are a relatively rich source of bioactive compounds, which could be
79
exploited in the food and pharmaceutical industries.
80 81
The current study we aimed to address this by investigating the potential anti-cancer effects
82
of a purified white grape pomace extract (PWGPE), rich in phenolic compounds, on colon
83
adenocarcinoma cell growth, in relation to the individual effects of pure polyphenols
84
relevant to PWGPE, notably epicatechin, catechin, quercetin and gallic acid. 4
85
2. Materials and methods
86 87
2.1. Reagents and standards
88
Methanol, n-hexane and hydrochloric acid were purchased from Labscan (Dublin, Ireland).
89
Formic acid, HPLC-grade acetonitrile, sodium carbonate, and Folin-Ciocalteau reagent were
90
obtained from Panreac (Barcelona, Spain). Gallic acid, protocatechuic acid, (+)-catechin, (−)-
91
epicatechin, quercetin, kaempferol, quercetin-3-O-rutinoside (rutin), ferulic acid, caffeic
92
acid, p-coumaric acid, and phosphate buffered saline (PBS) were purchased from Sigma-
93
Aldrich (Madrid, Spain). Quercetin-3-O-glucoside and kaempferol-3-O-glucoside were
94
obtained from Extrasynthese (Genay, France). Alamar Blue was from Abd Serotec
95
(Kidlington, UK). Fehling’s reagents, dimethyl sulfoxide (DMSO), sulforhodamine B-based
96
assay kit and all other reagents used for assay with cells were obtained from Sigma Aldrich
97
(UK).
98
The stock solutions of phenolic standards were prepared in water at concentration of 100
99
mg/l. The corresponding dilutions were made up from the stock solutions to assess the
100
antioxidant activity and the anti-proliferative effects.
101 102
2.2. Extraction of phenolic compounds from grape pomace
103
Grape pomace of the variety Zalema, D.O. “Condado de Huelva” (Spain), collected after
104
winemaking, was supplied by “Vinícola del Condado” winery (Bollullos Par del Condado,
105
Spain). Grape pomace was immediately frozen at -20 °C, freeze-dried for 48 hours and
106
finally ground to a fine powder. The freeze-dried grape pomace (50 g) was extracted with
107
75% methanol as extraction solvent. The mixture was sonicated for 1 h followed by further
108
constant shaking for 12 h (VWR Incubating mini-shaker), centrifuged at 4190 x g for 15 min 5
109
and finally the supernatant was collected. The remaining residue was subjected to a further
110
two extractions and the supernatants were combined to obtain the phenolic extract. This
111
methanolic phenolic extract was dried under vacuum and the aqueous extract was washed
112
with n-hexane. The resulting extract was passed through a C18 column (10 × 5 cm) to
113
remove sugars, which was achieved by their elution, following addition of 100 ml of acidified
114
water (pH 2; HCl), followed by 300 ml of Milli-Q® water at a constant rate (0.5 ml/min),
115
using a diaphragm-metering pump (STEPDOS, Scientific Laboratory Suppliers). The Fehling
116
test was carried out to ascertain the presence of sugars in the water extracts, as described
117
previously (Eid, Al-Awadi, Vauzour, Oruna-Concha, & Spencer, 2013). Following the removal
118
of sugars, the elution of phenolic compounds was achieved by addition of 400 ml of
119
methanol. This methanolic extract was concentrated to dryness, using a rotatory vacuum
120
evaporator at 40 °C, and freeze-dried, yielding the PWGPE.
121 122
2.3. Determination of individual phenolic compounds: RRLC/MS analysis.
123
Chromatographic analysis was carried out as previously described (Jara-Palacios, Hernanz,
124
González-Manzano, Santos-Buelga, Escudero-Gilete, & Heredia, 2014). An Agilent 1260
125
chromatograph (Agilent Technologies, Palo Alto, CA, USA), equipped with a diode-array
126
detector, and an API 3200 Qtrap (Applied Biosystems, Darmstadt, Germany), equipped with
127
an ESI source, and a triple-quadrupole ion trap mass analyser were used. Phenolic
128
compounds were identified by comparing their retention time, UV-vis spectra, and mass
129
spectral data with authentic standards where available. The compounds were quantified
130
using peak area data of resolved peaks at 280, 320 and 370 nm, for flavanols, phenolic acids
131
and flavonols, respectively. Procyanidins were quantified, using catechin as the standard,
132
whilst caftaric, fertaric and coutaric acids were quantified using caffeic, ferulic and p6
133
coumaric acids, respectively. Quercetin and isorhamnetin derivatives were quantified as
134
quercetin, and kaempferol derivates as kaempferol. Detailed retention times, UV-vis data
135
and mass spectrometric data were shown previously in another paper (Jara-Palacios et al.,
136
2014). The samples were analyzed in triplicate, and the results were expressed as mg of
137
phenolic compound per g of PWGPE.
138 139
2.4. Determination of total phenolic content
140
The total phenolic content (TPC) was determined by the Folin-Ciocalteu spectrophotometric
141
method, as described previously (Jara-Palacios et al., 2014). Analyses were conducted in
142
triplicate and gallic acid was employed as a calibration standard. Results were expressed as
143
mg of gallic acid per g of PWGPE.
144 145
2.5. Cell culture
146
2.5.1. General
147
Human colon adenocarcinoma cells (Caco-2, ECACC Salisbury, Wiltshire, UK) were cultured
148
in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 20% heat-inactivated
149
bovine serum, 2 mM L-glutamine, 1% non-essential amino acids, 100 U/ml of penicillin, and
150
100 µg/ml of streptomycin. Cells were seeded in 24-well plates at low confluence (2.5 x 104
151
per well) and then exposed to the PWGPE (10, 25, 50 and 100 µg/ml), catechin (30 and 60
152
µg/ml), epicatechin (30 and 60 µg/ml), quercetin (30 and 60 µg/ml), gallic acid (30 and 60
153
µg/ml), or vehicle (DMSO 1%), 8 h after seeding.
154 155
2.5.2. Cellular proliferation and viability
7
156
The ability of the PWGPE to inhibit cellular proliferation was tested, using the
157
sulforhodamine B (SRB) assay. Cells were harvested following 24, 48 and 72 h in culture and
158
treated by the addition of 125 µl of ice-cold TCA (4 °C; 1 h). After fixing, media were
159
removed, cells were washed and total biomass was determined using SRB (250 µl of 0.4%
160
SRB; 30 min). Unincorporated dye was removed by washing with 1% acetic acid, whilst cell
161
incorporated dye was solubilised using Tris-base (10 mM). Dye incorporation, reflecting cell
162
biomass, was measured at 492 nm, using a GENios microplate reader (TECAN, Reading, UK).
163
Viability was assessed after 24 h of treatment with PWGPE at different concentrations by
164
the Alamar blue assay, as described previously (Vauzour, Vafeiadou, Rice-Evans, Williams, &
165
Spencer, 2007). Briefly, Alamar Blue solution (10% v/v) was added to each well, and plates
166
were incubated for 3 h, prior to fluorescence being measured at 540 nm excitation and 612
167
nm emission on the GENios microplate reader.
168 169
2.6. Statistical analysis
170
For the statistical treatment of the data, the Statistica v.8.0 software was used. One-way
171
analysis of variance (ANOVA) was employed to establish if antioxidant and anti-proliferative
172
effects differed significantly between (a) the PWGPE and the phenolic standards and (b)
173
those with the vehicle (DMSO 1%).
174 175
3. Results and discussion
176
3.1. Phenolic composition and antioxidant activity
177
The total phenolic content of the PWGPE was 365 mg/g (Table 1). 11 flavanols, 9 flavonols
178
and 5 phenolic acids were identified and quantified by RRLC/MS, with flavanols being the
179
major compounds detected accounting for 65% of total phenolic compounds (92.6 mg 8
180
flavanols/g PWGPE), followed by flavonols (43.3 mg/g PWGPE, 31%) and phenolic acids
181
(6.01 mg/g PWGPE, 4%) (Table 1). The most abundant flavanol was procyanidin B1 (15.5
182
mg/g PWGPE), followed by catechin, procyanidin B2-3-O-gallate, and procyanidin tetramer 1
183
(12.2, 11.8 and 10.8 mg/g PWGPE, respectively). The main flavonols were quercetin-3-O-
184
glucoside and quercetin-3-O-glucuronide (16.9 and 15.8 mg/g PWGPE, respectively). The
185
most abundant phenolic acid was gallic acid (3.03 mg/g PWGPE). These phenolic compounds
186
were found in other extracts from Zalema grape pomace, but in different amounts (Jara-
187
Palacios et al., 2013; Jara-Palacios et al., 2014), which may be due to the extraction process.
188 189
3.2. Effect on cell proliferation
190
PWGPE, at different concentrations (10, 25, 50 and 100 µg/ml), induced dose-dependent
191
inhibition of cancer cell proliferation following 24, 48 and 72 h of exposure (Figure 1). A
192
significant increase (p < 0.05) in growth inhibition treated cells was observed after all
193
treatments, relative to vehicle, with the exception of PWGPE at 10 µg/ml and 72 h of
194
exposure (p > 0.05), whilst the 100 µg/ml dose for 48 h induced the greatest degree of
195
inhibition of proliferation (52% growth inhibition). Our data agree with previous cell data
196
indicating that polyphenols are capable of inhibiting the proliferation of colon
197
adenocarcinoma cells in a dose-dependent manner. Notably, the polyphenol rich foods, e.g.
198
olive oil (100 µg/ml), red wine (100 µg/ml) and a tomato extract (2.5 mg/ml) inhibited
199
cancer cell growth over a similar time frame (Corona et al., 2007; Saunders, 2009; Gómez-
200
Alonso et al., 2012).
201 202
Previous studies have indicated that such activity may be mediated via the potential of
203
polyphenols and their metabolites to induce cell cycle blockage in the G2/M phase in 9
204
adenocarcinoma cells, perhaps through their effects on COX-2 activity (Shan, Wang., & Li,
205
2009; Yasui, Kim, Oyama, & Tanaka, 2009). On the other hand, these anti-proliferative
206
effects may be mediated, in part, by their direct cytotoxic action (Gómez-Alonso et al.,
207
2012). With respect to the latter, our data suggest that cell viability at 24 h was significantly
208
(p < 0.05) reduced at higher exposure concentrations (Figure 2). Our data agree with
209
previous observations that a polyphenol-rich red wine and araçá fruit extracts also reduce
210
cell viability (Gómez-Alonso et al., 2012; Medina et al., 2011).
211 212
The assayed concentrations of polyphenols (10-100 µg/ml) in the culture medium could be
213
associated with those present in some food sources, such as white wine, cocoa, black
214
currant, or tea (Recamales, Sayago, González-Miret, & Hernanz, 2006; Lamuela-Raventós,
215
Romero-Pérez, Andrés-Lacueva, &, Tornero , 2005; Williamson & Clifford, 2010; Wang, Lu,
216
Miao, Xie, & Yang, 2008). These amounts of polyphenols have shown antiproliferative
217
effects in the cells; however, human health effects of these compounds depend on their
218
bioavailability. A previous study suggests that the phenolic composition of a food source
219
could affect the microbiota and its catabolic activity, inducing changes that could in turn
220
affect the bioavailability and potential bioactivity of polyphenols producing metabolites
221
which might be responsible for observed health effects in vivo (Cueva et al., 2013).
222
Therefore, although in vitro assays provide information on bioactivity of polyphenols, it is
223
essential to know their bioavailability by in vivo studies, which would be interesting to look
224
at in future.
225 226
In order to elaborate the mediating bioactive components of the extract used, individual
227
phenolic compounds, gallic acid, catechin, quercetin and epicatechin were also tested for 10
228
their effects on cancer cell growth (Figure 3). A significant inhibition (p < 0.05) of
229
proliferation was observed for all compounds, relative to vehicle-treated cells, ranging from
230
17-81% for epicatechin (30 µg/ml) and quercetin (60 µg/ml), respectively (Figure 3). Indeed,
231
the anti-proliferative effects of the PWGPE were comparable to effects of catechin,
232
epicatechin and gallic acid at similar concentrations to that found in the extract (p > 0.05).
233
Although the precise concentrations of catechin, epicatechin, gallic acid and quercetin
234
varied in the extract (12.2, 6.3, 3.0 and 2.9 mg/g PWGPE, respectively), as reported
235
previously (Araújo et al., 2011), a combination of polyphenols may target overlapping and
236
complementary phases of the carcinogenic process, thus increasing the efficacy and potency
237
of its chemopreventive effects. Previous data support this, with a variety of phenolic
238
compounds previously described as anticancer compounds with respect to colorectal cancer
239
(Salucci, Stivala, Maiani, Bugianesi, & Vannini, 2002; Araújo et al., 2011; Ramos et al., 2011).
240 241
4. Conclusions
242
In summary, a PWGPE obtained from the winemaking process (Zalema white grape variety)
243
is capable of inhibiting adenocarcinoma cell proliferation by a combination of anti-
244
proliferative activity and via a direct initiation of cell death. Further studies are required to
245
elucidate the specific components present in PWGPE which are responsible for these effects
246
and the mechanisms involved, although our data support the conclusion that major
247
polyphenols present in the extract, such as catechin and quercetin, may be the mediating
248
components. This study has described the inhibitory nature of dietary polyphenolic
249
compounds on colon cancer cell growth, but there is no clear evidence that consumption of
250
polyphenols prevents cancer; therefore future work is necessary to establish this.
251 11
252 253 254
Acknowledgements
255
This work was partially supported by “V Plan Propio de Investigación” of Universidad de
256
Sevilla. The authors acknowledge the collaboration of “Consejo Regulador D.O. Condado de
257
Huelva” and assistance of the technical staff of Biology Service (SGI, Universidad de Sevilla).
258
M. J. Jara-Palacios holds a predoctoral Research Grant (FPU) from the Spanish Ministry of
259
Education.
260
12
261
References
262 263
Anastasiadi, M., Pratsinis, H., Kletsas, D., Skaltsounis, A. L., & Haroutounian, S. A. (2010).
264
Bioactive non-coloured polyphenols content of grapes, wines and vinification by-products.
265
Evaluation of the antioxidant activities of their extracts. Food Research International, 43,
266
805-813.
267 268
Araújo, J. R., Gonçalves, P., & Martel, F. (2011). Chemopreventive effect of dietary
269
polyphenols in colorectal cancer cell lines. Nutrition Research, 31, 77-87.
270 271
Chan, A. T., & Giovannucci, E. L. (2010). Primary prevention of colorectal cancer.
272
Gastroenterology, 138, 2029-2043.
273 274
Coates, E. M., Popa, G., Gill, C. I., McCann, M. J., McDougall, G. J., Stewart, D., & Rowland, I.
275
(2007). Colon-available raspberry polyphenols exhibit anti-cancer effects on in vitro models
276
of colon cancer. Journal of Carcinogenesis, 6, 1-10.
277 278
Corona, G., Deiana, M., Incani, A., Vauzour, D., Dessì, M. A., & Spencer, J. P. (2007).
279
Inhibition of p38/CREB phosphorylation and COX-2 expression by olive oil polyphenols
280
underlies
281
Communications, 362, 606-611.
their
anti-proliferative
effects.
Biochemical
and
Biophysical
Research
282 283
Cueva, C., Sánchez-Patán, F., Monagas, M., Walton, G. E., Gibson, G. R., Martín-Álvarez, P. J.,
284
Bartolomé, B., & Moreno-Arribas, M. V. (2013). In vitro fermentation of grape seed flavan-313
285
ol fractions by human faecal microbiota: changes in microbial groups and phenolic
286
metabolites. FEMS Microbiology Ecology, 83, 792-805.
287 288
Eid, N. M. S., Al-Awadi, B., Vauzour, D., Oruna-Concha, M. J., & Spencer, J. P. E. (2013). The
289
effect of cultivar type and ripening on the polyphenol content of date palm fruit. Journal of
290
Agricultural and Food Chemistry, 61, 2453-2460.
291 292
Gómez-Alonso, S., Collins, V. J., Vauzour, D., Rodríguez-Mateos, A., Corona, G., & Spencer, J.
293
P.E. (2012). Inhibition of colon adenocarcinoma cell proliferation by flavonols is linked to a
294
G2/M cell cycle block and reduction in cyclin D1 expression. Food Chemistry, 130, 493-500.
295 296
Jara-Palacios, M. J., González-Manzano, S., Escudero-Gilete, M. L., Hernanz, D., Dueñas, M.,
297
González-Paramás, A. M., Heredia, F. J., & Santos-Buelga, C. (2013). Study of Zalema grape
298
pomace: phenolic composition and biological effects in Caenorhabditis elegans. Journal of
299
Agricultural and Food Chemistry, 61, 5114-5121.
300 301
Jara-Palacios, M. J., Hernanz, D., González-Manzano, S., Santos-Buelga, C., Escudero-Gilete,
302
M. L., & Heredia, F. J. (2014). Detailed phenolic composition of white grape by-products by
303
RRLC/MS and measurement of the antioxidant activity. Talanta, 125, 51-57.
304 305
Kulkarni, S., De Santos, F. A., Kattamuri, S., Rossi, S. J., & Brewer, M. S. (2011). Effect of
306
grape seed extract on oxidative, color and sensory stability of a pre-cooked, frozen, re-
307
heated beef sausage model system. Meat Science, 88, 139-144.
308 14
309
Lamuela-Raventós, R. M., Romero-Pérez, A. I., Andrés-Lacueva, C., Tornero, A. (2005).
310
Review: health effects of cocoa flavonoids. Food Science and Technology International, 11,
311
159-176.
312 313
Lee, S. Y. H., Munerol, B., Pollard, S., Youdim, K. A., Pannala, A. S., Kuhnle, G. G., Debnam,
314
E.S., Rice-Evans, C., & Spencer, J.P. (2006). The reaction of flavanols with nitrous acid
315
protects against N-nitrosamine formation and leads to the formation of nitroso derivatives
316
which inhibit cancer cell growth. Free Radical Biology and Medicine, 40, 323-334.
317 318
Medina, A. L., Haas, L. I. R., Chaves, F. C., Salvador, M., Zambiazi, R. C., Silva, W. P., Nora, L.,
319
& Rombaldi, C. V. (2011). Araçá (Psidium cattleianum Sabine) fruit extracts with antioxidant
320
and antimicrobial activities and antiproliferative effect on human cancer cells. Food
321
Chemistry, 128, 916-922.
322 323
Perumalla, A. V. S., & Hettiarachchy, N. S. (2011). Green tea and grape seed extracts-
324
potential applications in food safety and quality. Food Research International, 44, 827-839.
325 326
Ramos, S., Rodríguez-Ramiro, I., Martín, M. A., Goya, L., & Bravo, L. (2011). Dietary flavanols
327
exert different effects on antioxidant defenses and apoptosis/proliferation in Caco-2 and
328
SW480 colon cancer cells. Toxicology in Vitro, 25, 1771-1781.
329 330
Recamales, A. F., Sayago, A., González-Miret, M. L., & Hernanz, D. (2006). The effect of time
331
and storage conditions on the phenolic composition and colour of white wine. Food
332
Research International, 39, 220-229. 15
333 334
Rodríguez Montealegre, R., Romero Peces, R., Chacón Vozmediano, J. L., Marơnez Gascueña,
335
J., & García Romero, E. (2006). Phenolic compounds in skins and seeds of ten grape Vitis
336
vinifera varieties grown in a warm climate. Journal of Food Composition and Analysis, 2006,
337
19, 687-693.
338 339
Rodríguez-Rodríguez, R., Justo, M. L., Claro, C. M., Vila, E., Parrado, J., Herrera, M. D., &
340
Alvarez de Sotomayor, M. (2012). Endothelium dependent vasodilator and antioxidant
341
properties of a novel enzymatic extract of grape pomace from wine industrial waste. Food
342
Chemistry, 135, 1044-1051.
343 344
Sagdic, O., Ozturk, I., Ozkan, G., Yetim, H., Ekici, L., & Yilmaz, M. T. (2011). RP-HPLC-DAD
345
analysis of phenolic compounds in pomace extracts from five grape cultivars: Evaluation of
346
their antioxidant, antiradical and antifungal activities in orange and apple juices. Food
347
Chemistry, 126, 1749-1758.
348 349
Salucci, M., Stivala L. A., Maiani, G., Bugianesi, R., & Vannini, V. (2002). Flavonoids uptake
350
and their effect on cell cycle of human colon adenocarcinoma cells (Caco2). British Journal of
351
Cancer, 86, 1645-1651.
352 353
Saunders, C. (2009). The anti-proliferative effect of different tomato varieties on the human
354
colon adenocarcinoma cells. Bioscience Horizons, 2, 172-179.
355
16
356
Shan, B. E., Wang, M. X., & Li, R. G. (2009). Quercetin inhibit human sw480 colon cancer
357
growth in association with inhibition of cyclin d1 and surviving expression through Wnt/β-
358
catenin signaling pathway. Cancer Investigation, 27, 604-612.
359 360
Vauzour, D., Vafeiadou, K., Rice-Evans, C., Williams, R. J., & Spencer, J. P. E. (2007).
361
Activation of pro-survival Akt and ERK1/2 signalling pathways underlie the anti-apoptotic
362
effects of flavanones in cortical neurons. Journal of Neurochemistry, 103, 1355-1367.
363 364
Vauzour, D., Rodriguez-Mateos, A., Corona, G., Oruna-Concha, M. J., & Spencer, J. P. E.
365
(2010). Polyphenols and human health: prevention of disease and mechanisms of action.
366
Nutrients, 2, 1106-1131.
367 368
Vu, K. D., Carlettini, H., Bouvet, J., Côté, J., Doyon, G., Sylvain, J. F., & Lacroix M. (2012).
369
Effect of different cranberry extracts and juices during cranberry juice processing on the
370
antiproliferative activity against two colon cancer cell lines. Food Chemistry, 132, 959-967.
371 372
Wang, D., Lu, J., Miao, A., Xie, Z., & Yang, D. (2008). HPLC-DAD-ESIMS/MS analysis of
373
polyphenols and purine alkaloids in leaves of 22 tea cultivars in China. Journal of Food and
374
Composition and Analysis, 21, 361-369.
375 376
Williamson, G., & Clifford, M. N. (2010). Colonic metabolites of berry polyphenols: the
377
missing link to biological activity? British Journal of Nutrition, 2010, 104 (Suppl. 3), 48-66.
378
17
379
Xia, E. Q., Deng, G. F., Guo, Y. J., & Li, H. B. (2010). Biological activities of polyphenols from
380
grapes. International. Journal of Molecular Sciences, 11, 622-646.
381 382
Yasui, Y., Kim, M., Oyama, T., & Tanaka, T. (2009). Colorectal carcinogensis and suppression
383
of tumor development by inhibition of enzymes and molecular targets. Current Enzyme
384
Inhibition, 5, 1-26.
18
385
Figure Captions
386 387
Figure 1. Growth inhibition induced by treatment with PWGPE at different concentrations.
388
Caco-2 cells were exposed to the treatment for 24, 48 and 72 h before SRB assay was
389
conducted. Each value represents mean (n=12) ± SD. a= p < 0.001; b= p < 0.01; c= p < 0.05.
390 391
Figure 2. Viable cells determined after treatment for 24 h with different concentrations of
392
PWGPE. Caco-2 cells were exposed to the treatment for 24 h before Alamar blue assay was
393
conducted. Each value represents mean (n=12) ± SD. a= p < 0.001; b= p < 0.01; c= p < 0.05.
394 395
Figure 3. Growth inhibition induced by treatment with catechin, epicatechin, quercetin and
396
gallic acid at different concentrations. Caco-2 cells were exposed to the treatment for 24, 48
397
and 72h before SRB assay was conducted. Each value represents mean (n=9) ± SD.
398
19
399
Table 1. Concentrations of phenolic compounds identified by RRLC/MS and TPC of PWGPE Phenolic compound
mg/g PWGPE
Flavanols Catechin Epicatechin Procyanidin B1 Procyanidin B2 Procyanidin B3 Galloyled procyanidin Procyanidin B2-3-O-gallate Procyanidin trimer 1 Procyanidin trimer 2 Procyanidin tetramer 1 Procyanidin tetramer 2
12.2 ± 0.07 6.34 ± 0.02 15.5 ± 0.00 4.94 ± 0.80 4.35 ± 0.10 7.97 ± 0.00 11.8 ± 0.29 5.65 ± 0.35 7.27 ± 0.27 10.8 ± 0.10 5.74 ± 0.02
Flavonols Quercetin (Q) Kaempferol (K) Q-3-O-rutinoside Q-3-O-glucuronide Q-3-O-galactoside Q-3-O-glucoside K-3-O-glucuronide K-3-O-glucoside I-3-O-glucoside
2.87 ± 0.00 0.15 ± 0.00 0.69 ± 0.00 15.8 ± 0.01 2.12 ± 0.02 16.9 ± 0.07 0.31 ± 0.00 3.67 ± 1.73 0.70 ± 0.01
Phenolic acids Gallic acid Protocatechuic acid Caftaric acid Caffeic acid p-Coumaric acid
3.03 ± 0.13 0.94 ± 0.13 0.88 ± 0.00 0.63 ± 0.01 0.51 ± 0.01
Total phenolic compounds1
142± 1.41
2
400
Total phenolic content 365± 3.35 I,isorhamnetin. Each value represents mean (n=9) ± SD
401
1
sum of individual phenolic compounds
402
2
TPC determined by Folin Ciocalteu assay
403 404
20
405 406 407 408 409 410 411
21
412 413 414
22
24 h
90
48 h
72 h
Growth Inhibition (%)
80 70 60 50 40 30 20 10 0 30
60
30
60
30
60
30
60
Catechin (µg/mL) Epicatechin (µg/mL) Quercetin (µg/mL) Gallic acid (µg/mL) 415 416 417
23
418
Highlights
419
A white grape pomace extract, rich in bioactive compounds, was obtained
420
The extract and its individual phenolics inhibited colon adenocarcinoma cells proliferation
421
The extract could be used as a good source of functional ingredients for foodstuffs
422 423 424 425 426 427 428
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