Grape stem extracts with potential anticancer and antioxidant properties

antioxidants Article Grape Stem Extracts with Potential Anticancer and Antioxidant Properties Javier Quero 1, Nerea Jiménez-Moreno 2, Irene Esparza 2, Jesús Osada 3,4 , Elena Cerrada 5, Carmen Ancín-Azpilicueta 2,* and María Jesús Rodríguez-Yoldi 1,4,*   Citation: Quero, J.; Jiménez-Moreno, N.; Esparza, I.; Osada, J.; Cerrada, E.; Ancín-Azpilicueta, C.; Rodríguez-Yoldi, M.J. Grape Stem Extracts with Potential Anticancer and Antioxidant Properties. Antioxidants 2021,10, 243. https:// doi.org/10.3390/antiox10020243 Academic Editor: Isabel Seiquer Received: 27 December 2020 Accepted: 29 January 2021 Published: 5 February 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1Department of Pharmacology and Physiology, Veterinary Faculty, University of Zaragoza, 50013 Zaragoza, Spain; javierquer[email protected] 2Department of Science, Public University of Navarra, Institute for Advanced Materials and Mathematics (INAMAT2), 31006 Pamplona, Navarra, Spain; [email protected] (N.J.-M.); [email protected] (I.E.) 3Department of Biochemistry and Molecular Cell Biology, Veterinary Faculty, University of Zaragoza, 50013 Zaragoza, Spain; [email protected] 4CIBERobn, ISCIII, IIS Aragón, IA2, 50009 Zaragoza, Spain 5Department of Inorganic Chemistry, Sciences Faculty, University of Zaragoza, 50009 Zaragoza, Spain; [email protected] * Correspondence: [email protected] (C.A.-A.); [email protected] (M.J.R.-Y.); Tel.: +34-948-169596 (C.A.-A.); +34-976-761649 (M.J.R.-Y.) Abstract: The application of plant extracts for therapeutic purposes has been used in traditional medicine because plants contain bioactive compounds with beneficial properties for health. Currently, the use of these compounds that are rich in polyphenols for the treatment and prevention of diseases such as cancer, diabetes, and cardiovascular diseases, many of them related to oxidative stress, is gaining certain relevance. Polyphenols have been shown to have antimutagenic, antioxidant, and anti-inflammatory properties. Therefore, the objective of the present work was to study the potential effect of grape stem extracts (GSE), rich in phenolic compounds, in the treatment of cancer, as well as their role in the prevention of this disease associated with its antioxidant power. For that purpose, three cancer lines (Caco-2, MCF-7, and MDA-MB-231) were used, and the results showed that grape stem extracts were capable of showing an antiproliferative effect in these cells through apoptosis cell death associated with a modification of the mitochondrial potential and reactive oxygen species (ROS) levels. Additionally, grape stem extracts showed an antioxidant effect on differentiated intestinal cells that could protect the intestine from diseases related to oxidative stress. Therefore, grape extracts contain bioactive principles with important biological properties and could be used as bio-functional food ingredients to prevent diseases or even to improve certain aspects of human health. Keywords: cancer cells; polyphenols; grape stem; proteasome; ROS; TrxR1 1. Introduction Grape stems are by-products generated in great quantity in the winemaking process, and their elimination causes environmental problems. Therefore, it is important to find strategies that allow the reuse of these products. This residue is a rich source of phenolic compounds, celluloses, hemicelluloses, and lignins [ 1 – 6 ]. Among them, phenolic compounds confer antioxidant properties to the extracts obtained from grape stems. For this reason, different studies have been conducted in order to determine the polyphenolic composition of grape stems, and several proanthocyanidins, anthocyanidins, flavonols, hydroxycinnamic acids, and stilbenes have been found. Among them, the most characteristic polyphenolic substances referred to in most of the studies are trans-resveratrol, ε -viniferin, caftaric acid, gallic acid, catechin (one of the most abundant polyphenols), epicatechin, malvidin derivatives, quercetin, and glycosylated derivatives of quercetin in position 3 [2,3,5,7]. Antioxidants 2021,10, 243. https://doi.org/10.3390/antiox10020243 https://www.mdpi.com/journal/antioxidants Antioxidants 2021,10, 243 2 of 17 According to current research results, grape stem extracts possess important biological activities with multiple benefits for human health due to antioxidant and anti-inflammatory properties [ 5 , 7 – 11 ]. Veskoukis et al. [ 10 ] found that this by-product is particularly rich in flavonoids and stilbenes, such as trans-resveratrol and viniferin, which are found in considerably high concentrations. These authors also found that such extracts exhibited significant antioxidant properties, and, even at low concentrations, they showed a strong ability to prevent the oxidation of low-density lipoprotein (LDL) and to reduce intracellular levels of reactive oxygen species (ROS). In this way, Gonzalez-Centeno et al. [ 12 ] and Veskoukis et al. [10] evaluated the total phenolic and total proanthocyanidin composition of different grape stem varieties, as well as their antioxidant potentials. Grape stem extracts also prevent ROS-induced DNA damage and have inhibitory activity against liver and cervical cancer cell growth, suggesting their potential as chemopreventive agents [ 13 ]. Using human epidermal keratinocytes, Domínguez-Perles et al. [ 14 ] observed a protective effect of grape stem extracts against oxidative stress. These researchers found a close correlation between the concentration of phenolic compounds in the extracts and the potential to regulate the redox balance in vitro , as well as the capacity of these extracts to efficiently modulate apoptosis in HaCaT keratinocytes. Cho et al. [ 15 ] studied the effect of the topical administration of grape stem extracts to mice skin before subjecting them to UVB radiation for three minutes, thrice a week for one month. These authors demonstrated that these extracts significantly inhibited oxidative damage induced by UVB radiation and observed decreased epidermal hyperplasia, melanin pigmentation, and collagen degradation in the skin of mice. In addition, grape pomace—consisting of peel, seed, stem, and pulps—is discarded during grape processing, including juice extraction and winemaking, despite its substantial phenolic content [ 8 ]. In this way, Del Pino-Garcia et al. [ 16 ] studied the chemopreventive potential of powdered red wine seasonings against colorectal cancer in HT-29 cells. Grape seed extracts have also been applied as photochemopreventive agents against UVB-induced skin cancer [ 17 ]. Likewise, Vitis vinifera extracts have shown an antidiabetic effect by inhibiting the enzyme glycogen phosphorylase [18]. On the other hand, grape stem extracts have significant antimicrobial activities that seem to be influenced by the structure and function of phenolic compounds, as well as by their interspecific relation with different bacterial strains [ 19 , 20 ]. For example, these types of extracts inhibited the growth of both Gram-positive (Listeria monocytogenes, Staphylococcus aureus and Enterococcus faecalis) and Gram-negative (Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumoniae) digestive pathogens under in vitro conditions [ 21 ], and their bioactive compounds are used in oral care [22]. In addition, the extracts could be used to control the presence of human pathogenic bacteria in fresh leafy vegetables [ 6 ]. Leal et al. [ 23 ] studied the potential of grape stem extracts from different white grape varieties as antimicrobial agents to reduce the use of antibiotics. These authors found that the bactericidal activity of the extracts was higher, in general, against Gram-positive than Gram-negative bacteria, although they used a different methodology from that of Dias et al. [21] to evaluate the antimicrobial activity (minimum inhibitory concentration vs. disc diffusion). Other studies have shown that grape stem extracts are highly effective against foot wound ulcers produced by Gram-positive bacteria, and they also have anti-inflammatory action, inhibiting the production of nitric oxide lipopolysaccharide-stimulated macrophages by up to 35.25% [24]. During the last few years, our research group has investigated the chemopreventive properties of extracts obtained from different plant matrices such as rosehips, fenugreek, pine bark, and artichoke waste on human colon cancer [3,25–27]. Though there have only been few publications on this subject, the anticarcinogenic potential of grape stem extracts has also been studied in different cell lines [ 13 , 28 , 29 ]. Additionally, grape stem extracts possess important bioactivities such as antiangiogenic properties [30]. However, the phenolic composition—and therefore the biological activity and efficacy—of a specific grape stem extract depends on the procedure used to obtain the extract. In a previous study, we selected an optimized extraction method for grape stems [ 3 ], and the Antioxidants 2021,10, 243 3 of 17 extracts obtained by this method presented high antioxidant potential and were demonstrated to be good candidates for SO 2 substitution in wines [ 4 ]. With this background, the aim of the present research was to complete the characterization of those grape stem extracts by studying their potential for the treatment of human colorectal adenocarcinoma (Caco-2) and human breast adenocarcinoma (MCF-7 and MDA-MB-231) cell lines. Thus, we measured the possible antiproliferative effects of these extracts on cancer cells and their mechanisms of action. Furthermore, the protective effects of these extracts, in a model of intestinal barrier (differentiated Caco-2 cells), were also tested through the measurement of the intracellular levels of ROS. 2. Materials and Methods 2.1. Extracts The grape stem extract was obtained through an extraction method using GRAS solvents from Mazuelo-variety stems harvested in the 2016 vintage [ 3 ]. Briefly, grape stems were oven-dried at 25 ◦ C, ground, and sieved ( φ < 0.3 mm). The extract was obtained after macerating the ground and sieved stems in 50% ethanol/water, with a 1:100 (w/v) ratio and at 40 ◦ C for 24 h. Then, the extract was centrifuged (8000 rpm for 15 min), filtered through filter paper, and lyophilized (Telstar Cryodos freeze drier, Madrid, Spain). 2.2. Chemicals All the used HPLC solvents were from Scharlab (Barcelona, Spain). All the used phenolic standards were from Sigma-Aldrich (Madrid, Spain), with the exception of malvidin-3-glucoside (enyn-chloride, Extrasynthese, Genay, France). Among the chemicals for spectrophotometric analysis, the Folin–Ciocalteu reagent, Trolox (6-hydroxy-2,5,7,8tetramethylchroman-2-carboxylic acid), gallic acid, and quercetin were supplied by SigmaAldrich (Madrid, Spain); glacial acetic acid, anhydrous sodium carbonate, and aluminum chloride 6-hydrate were supplied by PanReac AppliChem (Barcelona, Spain). 2.3. Identification and Quantification of Phenolic Composition of Grape Stem Extracts by HPLC-DAD The identification and quantification of the phenolic compounds present in the grape stem extracts were performed using high-performance liquid chromatography. The chromatograph was equipped with two 510 pumps, a 717 Plus autosampler, and a 996 photodiode array detector (Waters Div., Milford, MA, USA). A Zorbax Eclipse Plus C18 reversed phase column (250 × 4.6 mm; particle size of 5 µ m) (Agilent, Santa Clara, CA, USA) was used. For the analyses of the extract, between 45.0 ± 0.1 and 70.0 ± 0.1 mg of each sample were weighted and dissolved in 10 mL of methanol with the aid of an ultrasonic bath (JP Selecta, Barcelona, Spain). Samples were prepared in triplicate and analyzed once. The chromatographic analyses were carried out according to a modified method of Barros et al. [ 31 ]. Two mobile phases, A (water: 85% formic acid, 99:1 v/v) and B (acetonitrile: 85% formic acid, 99:1 v/v) were used. The flow rate was 1 mL/min using the following linear gradient scheme (t in min; % A): 0, 95%; 15, 85%; 22, 80%; 25, 80%; 35, 70%; 45, 50%; 50, 5%; 55, 95%; and 60, 95%. The column temperature was 30 ◦ C, and the injection volume was 40 µ L. The identification of the different compounds was performed by the double coincidence of the retention time of its corresponding standard and the UV–Vis spectrum of each compound. Quantification was carried out using calibration curves for each analyzed compound. The calibration curves used for resveratrol, gallic acid, quercetin, malvidin-3-glucoside, and caftaric acid presented linear correlation coefficients higher than 0.999. The calibration curves obtained for the rest of compounds (viniferin, catechin, and the derivative of quercetin) showed linear correlation coefficients higher than 0.998. In the case of the unidentified anthocyanin, it was not possible to identify its structure with the method used in the laboratory. However, most of the anthocyanins described in the literature that are present in grape stems correspond to derivatives of malvidin. For this reason, and given the fact that all anthocyanins have similar general structures, we Antioxidants 2021,10, 243 4 of 17 used the calibration curve of malvidin-3-glucoside to estimate the concentration of the unknown anthocyanin. 2.4. Determination of Antioxidant Capacity of the Grape Stem Extracts by DPPH The DPPH (2,2-diphenyl-1-pycrilhydracyl) assay was based on the method proposed by Brand-Williams et al. [ 32 ]. A standard solution of 24 mg of DPPH in 100 mL of methanol was prepared, and then it was diluted in methanol until we obtained an absorbance of 0.9 ± 0.1 at 517 nm in a UV–Vis spectrophotometer (Jenway, Staffordshire, UK). For the calibration curve, seven different Trolox standards were prepared in methanol in concentrations from 0.05 to 0.73 mM. For sample preparation, between 50.0 ± 0.1 and 72.0 ±0.1 mg of extract were dissolved in 10 mL of methanol, and the resulting mixture was diluted 10 times with methanol. For analysis, 150 µ L of the Trolox standard solution or processed sample were mixed with 2.85 mL of the DPPH solution. After 30 min in darkness, the antioxidant capacities of all the standards and samples were determined by measuring the absorbance at 517 nm. For each batch of extract, three different processed samples were prepared, and each of them was analyzed once. The linear correlation coefficient obtained for the calibration curve was R 2 > 0.998. The results of antioxidant capacity were expressed as mmol Trolox/g of extract. 2.5. Spectrophotometric Determination of Total Phenolic and Flavonoid Content of the Grape Stem Extracts Total phenolic content was analyzed using the Folin–Ciocalteu method, as described by Singleton et al. [ 33 ]. For the calibration curve, different gallic acid standards were prepared in methanol in concentrations from 0.2 to 4.6 mM. For sample preparation, between 50.0 ±0.1 and 72.0 ± 0.1 mg of extract were dissolved in 10 mL of methanol. For analysis, 100 µ L of the gallic acid standard solution or processed sample were mixed with 0.5 L of the Folin–Ciocalteu reagent, 7.9 mL of deionized water, and 1.5 mL of Na 2 CO 3 (20% w/w), and the resulting solutions were left for 2 h in darkness. The absorbance was measured at 765 nm in a UV–Vis spectrophotometer (Jenway, Staffordshire, UK). The standard used for the calibration curve was gallic acid, ranging between 0.2 and 5.08 mM. The linear correlation coefficient obtained for the calibration curve was R 2 > 0.999. For each batch of extract, three different processed samples were prepared, and each of them was analyzed once. The results of total phenolic content were expressed as mg gallic acid/g extracts. The total flavonoid content was determined by the colorimetric method of aluminum chloride using a solution of 2% AlCl 3 in 5% acetic acid [ 34 ]. For the calibration curve, different quercetin standard solutions were prepared in methanol in concentrations from 3 to 30 µ g/mL. For sample preparation, between 50.0 ± 0.1 and 72.0 ± 0.1 mg of extract were dissolved in 10 mL of methanol. For analysis, 1.5 mL of the quercetin standard solution or sample were mixed with 1.5 mL of the AlCl 3 solution, and the resulting solutions were left for 30 min in darkness. Then, absorbance was measured on a Jenway UV–Vis spectrophotometer at 420 nm. The linear correlation coefficient obtained for the calibration curve was R 2 > 0.999. For each batch of extract, three different processed samples were prepared, and each of them was analyzed once. The results were expressed as mg of quercetin/g extracts. In all cases, the samples were analyzed in triplicate. 2.6. Cell Culture Human Caco-2 cell line (TC7 clone) was kindly provided by Dr. Edith Brot-Laroche (UniversitéPierre et Marie Curie-Paris 6, UMR S 872, Les Cordeliers, France). Human breast adenocarcinoma MDA-MB-231 cells were kindly provided by Dr. Carlos J. Ciudad and Dr. Verònica Noé(Departamento de Bioquímica y Fisiología, Facultad de Farmacia, Universidad de Barcelona, Spain). Human breast adenocarcinoma MCF-7 cells were kindly provided by Cristina Sanchez-de-Diego (Departamento de Fisiología II, Universidad de Barcelona, Spain). Human fibroblast cells were kindly provided by Dr. Julio Montoya (Departamento de Bioquimica, Universidad de Zaragoza, Spain). All cell lines Antioxidants 2021,10, 243 5 of 17 were maintained in a humidified atmosphere of 5% CO 2 at 37 ◦ C. Cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 20% fetal bovine serum (FBS), 1% non-essential amino acids, 1% penicillin (1000 U/mL), 1% streptomycin (1000 µ g/mL), and 1% amphotericin (250 U/mL). The cells were enzymatically passaged with 0.25% trypsin–1 mM EDTA and sub-cultured on 25 cm 2 plastic flasks at a density of 5×105cells/cm2 . The culture medium was replaced every 2 days. Extract treatments were added 24 h post-seeding for assays on undifferentiated Caco-2 cells [ 35 ] and 10–15-days post-seeding on differentiated Caco-2, MCF-7, and MDA cells. Cell confluence (80%) was confirmed by optical microscopy observance. 2.7. Cell Treatment and Antiproliferative Property Analysis Extracts from grape stems were diluted in a cell culture medium to a final concentration 1.5 mg/mL. For cytotoxicity screening assays, the cells were seeded in 96-well plates at a density of 4 × 10 3 cells/well. The culture medium was replaced with a medium containing plant extracts, and cells were incubated for 48 or 72 h. The antiproliferative effect was measured with the sulforhodamine B (SRB) assay, as previously described [ 36 ]. Absorbance at 540/620 nm was measured with the SPECTROstar Nano (BMG Labtech, Ortenberg, Germany). The effect on cell growth was expressed as a percentage of the control. Finally, the IC 50 value was calculated under all conditions tested. IC 50 represents the concentration of compound that halves cell proliferation or viability. This value was selected for further analysis to elucidate the extracts’ mechanism of action on cancer cells. 2.8. Measurements of Apoptosis The cells were seeded in 25 cm 2 flasks (5 × 10 5 cells/cm 2 ), exposed to plant extracts for 48 h at the IC 50 concentration, and then collected and stained with annexin V-FITC and propidium iodide, as previously described [ 37 ]. A negative control was prepared by untreated cells, and it was used to define the basal level of apoptotic and necrotic or dead cells. After incubation, cells were transferred to flow cytometry tubes and washed twice with phosphate-buffered saline (PBS), followed by a resuspension in 100 µ L of the annexing V binding buffer (100 mM HEPES/NaOH pH 7.4, 140 mM NaCl, and 2.5 mM CaCl 2 ). To each tube, 5 µ L of annexin V-FITC and 5 µ L of propidium iodide were added. After 15 min of incubation at room temperature in the dark, 400 µ L of the annexin binding buffer were added and analyzed by flow cytometry within 1 h. The signal intensity was measured using a BD FACSAria TM cell sorter (BD Biosciences, San Jose, CA, USA) and analyzed using the BD FASCDivaTM software (BD Biosciences, San Jose, CA, USA). 2.9. Flow Cytometry Mitochondrial Membrane Potential Assay Cells were seeded in 25 cm 2 flasks and then exposed to plant extracts for 48 h. The control cells were incubated with a new medium without treatment. Then, cells were washed twice with PBS. The pellet was resuspended in PBS at concentration of 10 6 cell/mL, and 5 µ L of 10 µ M 1,1 0 ,3,3,3 0 -hexamethylindodicarbo-cyanine iodide (DiIC1) were added to each sample. Tubes were incubated at 37 ◦ C for 15 min, and 400 µ L of PBS were added prior to analyze fluorescence with BD FACSarray TM (BD Biosciences, San Jose, CA, USA) equipped with an argon ion laser. The excitation and emission settings were 633 and 658 nm, respectively [37]. 2.10. Determination of Intracellular Levels of Reactive Oxygen Species (ROS) The cells were seeded in 96-wells plate at a density of 4 × 10 3 cells/well. The intracellular level of ROS was assessed using the dichlorofluorescein assay, as previously described [ 37 ]. Cells were cultured 24 h before being incubated with stem extracts and then underwent oxidative stress induction by adding H 2 O 2 (80 mM) for 20 min. After that, the medium was removed, cells were washed twice with PBS, and cells were incubated for 1 h with 20 µ M 2 0 ,7 0 -dichlorofluorescein diacetate (DCFH-DA) in PBS at 37 ◦ C. The formation of the fluorescence oxidized derivative of DCF was monitored at an emission wavelength Antioxidants 2021,10, 243 6 of 17 of 535 nm and an excitation of 485 nm in a FLUOstar Omega (BMG Labtech, Ortenberg, Germany) multiplate reader. A measure at time “zero” was performed, cells were incubated at 37 ◦ C in the multiplate reader, and the generation of fluorescence was measured after 20 min. ROS levels were expressed as a percentage of fluorescence compared to the control. The obtained values of fluorescence intensity are considered as a reflection of total intracellular ROS content. 2.11. Determination of Proteasome Activity The cells (5 × 10 5 cells/cm 2 ) were seeded in a cell culture flask (25 cm 2 ). The determination of the proteasome activity was carried out with a fluorometric assay using a proteasome 20S activity assay kit (MAK172, Sigma-Aldrich, Madrid, Spain) based on SucLLVy-AMC, a fluorogenic substrate of the proteasome β 5 submit. Caco-2 cells were treated with grape stem extracts for 24 h post-seeding and then processed following instructions in the kit protocol. The fluorescence levels correspond to the proteasomal chymotrypsin-like activity (CT-L activity). The activity was measured in lysed cells with FLUOstar Omega (BMG Labtech, Ortenberg, Germany), and the value was obtained per mg of protein. The data are expressed in % CT-L activity. 2.12. Thioredoxin Reductase 1 (TrxR1) Activity The cells were seeded in a 96-well plate with grape stem extracts for 24 h. The cells were then lysed and incubated with a shaking motion for 20 min before adding 25 µ L/well of the reaction buffer (500 µ L of PBS pH 7.4, 80 µ L of 100 mM EDTA pH 7.5, 20 µ L of 0.05% BSA, 100 µ L of 20 mM NADPH, and 300 µ L of distilled H 2 O), and the reaction was started with DTNB (20 mM in pure ethanol), as previously described by Allaoui et al. [ 26 ]. The absorbance increase was followed at 405 nm with SPECTROstar Nano (BMG Labtech, Ortenberg, Germany) every minute for 6 min. The value was obtained per mg of protein and expressed in % thioredoxin reductase (TrxR) activity. 2.13. Statistical Analysis All assays were performed at least three times. Data are presented as mean ± SD. Means were compared using ANOVA. Significant differences at p< 0.05 were compared using a Bonferroni’s multiple comparison test. The statistical analyses were performed and the graphics were obtained using the GraphPad Prism Version 5.02 software for Windows (GraphPad Software San Diego, CA, USA). 3. Results and Discussion Plant polyphenols represent a variety of bioactive compounds that are capable of preventing and controlling cancer and diabetes, as well as neurodegenerative, autoimmune, cardiovascular, and ophthalmic diseases [ 38 ]. The presence of polyphenol compounds in grape stems gives them exceptional biological value [ 39 ]. It has been shown that extracts derived from grape stems possess potent antioxidant activity in vitro [ 40 ] and in cell lines [7], whereas their anticarcinogenic role has not been widely reported. The present study investigated the biological properties of grape stem extracts on different cancer cells, as well as their mechanisms of action. Furthermore, the extracts’ effects on the prevention of oxidative stress in a model of differentiated intestinal cells was also studied. 3.1. Phenolic Composition and Antioxidant Activity in Mazuelo Stem Extracts The phenolic composition, as well as the total polyphenol and flavonoid contents, of Mazuelo stem extracts are presented in Table 1. In this extract, nine phenolic compounds were found, of which the most abundant were (+)-catechin and the quercetin-3-derivative. Likewise, Leal et al. [ 24 ] found that (+)-catechin was the most abundant phenolic compound in Portuguese grape stem extracts from different varieties (Tinta Roriz, Touriga Nacional, Castelão, Syrah, Arinto, and Fernão Pires). Anastasiadi et al. [ 11 ] also reported the presence Antioxidants 2021,10, 243 7 of 17 of several phenolic compounds in grape stem extracts from six red and white varieties from Greece. In comparison to their results with our Mazuelo stem extract, trans-resveratrol, ε -viniferin, (+)-catechin, and caftaric acid coincide, (+)-catechin was found to be the most abundant in both studies. Regarding the concentrations of resveratrol and viniferin, these authors observed differences among varieties and vintages. Lambert et al. [ 41 ] analyzed the stilbene content of pruning canes of the Carignan variety, which is the name given in France to the Mazuelo variety. These authors found a higher amount of resveratrol and viniferin in their extracts (0.88 mg resveratrol/g extract and 0.97 mg viniferin/g extract), although it must be considered that grapevine canes are probably richer in stilbenes than grape stems [ 42 , 43 ]. In addition to the phenolic compounds found in the Mazuelo stems analyzed in this work, other compounds have been identified in grape stem extracts of different varieties. For instance, in stem extracts from Portuguese grapes, kaempferol and isorhamnetin were identified [ 2 ], and in grape stems from Greek varieties, ferulic, coumaric, caffeic, and syringic acids were identified [28]. Table 1. Phenolic composition (mg/g extract) and antioxidant capacity of the Mazuelo stem extract. Phenolic Composition & Antioxidant Capacity Grape Stem Extract Gallic acid 0.21 ±0.03 Caftaric acid 0.14 ±0.03 (+)-Catechin 0.98 ±0.20 Quercetin 0.05 ±0.01 Quercetin-derivative 10.91 ±0.08 Malvidin-3-glucoside 0.10 ±0.02 Unknown anthocyanin 20.15 ±0.02 Trans-resveratrol 0.26 ±0.04 Trans-ε-viniferin 0.59 ±0.09 Total phenolic content 383 ±2 Total flavonoid content 42.6 ±0.1 Antioxidant capacity by DPPH 50.47 ±0.04 1 Expressed as quercetin-3-glucoside; 2 expressed as malvidin-3-glucoside; 3 expressed as mg gallic acid/g extract; 4expressed as mg quercetin/g extract; 5expressed as mmol Trolox/g extract. Regarding the antioxidant capacity measured by the DPPH assay (Table 1), the result of the Mazuelo stem extract was similar to that of the Syrah (0.44 ± 0.04 mmol Trolox/g) and Fernão Pires (0.55 ± 0.01 mmol Trolox/g) extracts and higher than that of the Castelão (0.31 ± 0.01 mmol Trolox/g) and Arinto (0.15 ± 0.01 mmol Trolox/g) varieties found by Leal et al. (2020). 3.2. Effect of Extracts From Grape Stem on Cancer Cells 3.2.1. Antiproliferative Activity The toxicity of extracts from grape stems was evaluated on undifferentiated Caco-2, MCF-7, and MDA-MB-231 cells by an SRB assay. Initially, a range of concentrations of grape extracts (62.5, 125, 250, 500, and 1000 µ g/mL) was tested. The concentrations chosen were in relation to previous work carried out by our research group with other plant extracts [ 25 , 36 ]. The IC 50 was calculated in the different cell lines at two time-points of 48 and 72 h. However, in the MDA-MB-231 and MCF-7 cells, when treated for 72 h, this range was lethal in most concentrations and the IC 50 could not be calculated, so the range was modified to decreased concentrations (range: 9, 18. 37.5, 75, and 200 µ g/mL). These results suggest that cytotoxic effect of grape stem extracts (GSE) is concentrationand timedependent and that Caco-2 cells are less sensitive to GSE at the highest incubation time. At 48 h, similar viability curves were obtained in the three different cell lines ( Figure 1 , Table 2). Antioxidants 2021,10, 243 8 of 17 Antioxidants 2021, 10, x FOR PEER REVIEW 8 of 17 3.2. Effect of Extracts From Grape Stem on Cancer Cells 3.2.1. Antiproliferative Activity The toxicity of extracts from grape stems was evaluated on undifferentiated Caco-2, MCF-7, and MDA-MB-231 cells by an SRB assay. Initially, a range of concentrations of grape extracts (62.5, 125, 250, 500, and 1000 µg/mL) was tested. The concentrations chosen were in relation to previous work carried out by our research group with other plant extracts [25,36]. The IC50 was calculated in the different cell lines at two time-points of 48 and 72 h. However, in the MDA-MB-231 and MCF-7 cells, when treated for 72 h, this range was lethal in most concentrations and the IC50 could not be calculated, so the range was modified to decreased concentrations (range: 9, 18. 37.5, 75, and 200 µg/mL). These results suggest that cytotoxic effect of grape stem extracts (GSE) is concentrationand timedependent and that Caco-2 cells are less sensitive to GSE at the highest incubation time. At 48 h, similar viability curves were obtained in the three different cell lines (Figure 1, Table 2). Figure 1. Measurement of Caco-2, MCF-7, MDA-MB-231, and fibroblast cell viability at 48 and 72 h after incubation with grape stem extracts (GSE). The GSE concentrations tested in the four types of cells were 62.5, 125, 250, 500, and 1000 µg/mL, but at 72 h in the MCF-7 and MDA-MB-232 cells, the chosen concentrations were 9, 18, 37.5, 75, and 200 µg/mL. Table 2. IC50 (the concentration of compound that halves cell proliferation or viability) values of grape stem extracts on Caco-2, MCF-7, MDA-MB-231, and fibroblast cells after 72 and 48 h of incubation. IC50 (µg/mL) 72 h IC50 (µg/mL) 48 h Selectivity Index Caco-2 759 ± 51 661 ± 48 2.9 MCF-7 203 ± 53 817 ± 52 * 7.2 MDA-MB-231 85 ± 9 911 ± 10 * 17.0 Fibroblast 1454 ± 6 - - * p < 0.05; incubation time 48 vs. 72 h. The results showed that the grape stem extracts were not selective for a single cancer line, but they produced a decrease in viability in the three tested cell lines (Figure 1). The effect was faster in Caco-2 cells, although their effectiveness was greater in breast cells (MCF-7 and MDA-MB-231) at longer times (72 h). In order to determine the action of these extracts on a noncancerous model, the IC50 was calculated on human fibroblast cells, after 72 h of incubation, where we observed a significantly lower effect. These data could be Figure 1. Measurement of Caco-2, MCF-7, MDA-MB-231, and fibroblast cell viability at 48 and 72 h after incubation with grape stem extracts (GSE). The GSE concentrations tested in the four types of cells were 62.5, 125, 250, 500, and 1000 µ g/mL, but at 72 h in the MCF-7 and MDA-MB-232 cells, the chosen concentrations were 9, 18, 37.5, 75, and 200 µg/mL. Table 2. IC 50 (the concentration of compound that halves cell proliferation or viability) values of grape stem extracts on Caco-2, MCF-7, MDA-MB-231, and fibroblast cells after 72 and 48 h of incubation. IC50 (µg/mL) 72 h IC50 (µg/mL) 48 h Selectivity Index Caco-2 759 ±51 661 ±48 2.9 MCF-7 203 ±53 817 ±52 * 7.2 MDA-MB-231 85 ±9 911 ±10 * 17.0 Fibroblast 1454 ±6 - - *p< 0.05; incubation time 48 vs. 72 h. The results showed that the grape stem extracts were not selective for a single cancer line, but they produced a decrease in viability in the three tested cell lines (Figure 1). The effect was faster in Caco-2 cells, although their effectiveness was greater in breast cells (MCF-7 and MDA-MB-231) at longer times (72 h). In order to determine the action of these extracts on a noncancerous model, the IC 50 was calculated on human fibroblast cells, after 72 h of incubation, where we observed a significantly lower effect. These data could be used to obtain a selectivity index (SI), as previously described by Badisa el al. [ 44 ]. The SI results are shown in Table 2, with the highest value being for the MDA-MB-231 cell line, according with the highest effective response of the extracts towards these cells after 72 h of incubation. The observed difference in the two breast cancer lines could have been due to the fact that the action of these extracts could be related to the receptors’ expression for estrogens, which are only present in MCF-7 cells [45]. 3.2.2. Cell Death Studies Since the grape stem extracts produce a reduction in cell viability, it was decided to determine what type of cell death occurred. Thus, flow cytometry analyses over 48 h were performed using biomarkers of cell death. The results showed that treatment for 48 h with the IC 50 concentration corresponding to each cell line mainly produced early apoptosis in Caco-2 cells, while late apoptosis was mainly detected in MDA cells. However, no significant apoptosis was found in MCF-7 cells. Treatment with longer time (72 h) induced Antioxidants 2021,10, 243 9 of 17 a significant death of these cells by late apoptosis (Figure 2). Therefore, the obtained results showed that grape stem extracts at their IC 50 produced apoptosis in all tested cancerous cells by activating apoptotic pathways, thereby reducing their ability to non-selectively react with biological targets to cause necrosis and its related side effects. Since previous studies on plant extracts suggested mitochondrial dysfunction and intrinsic apoptosis induction [ 25 , 26 ], the mitochondrial membrane potential change was analyzed. Mitochondria play a pivotal role in life and cell death inasmuch as they produce the majority of the energy required for survival and regulate the intrinsic apoptosis pathway. The involvement of mitochondria in cell death is generally measured by following mitochondrial membrane depolarization [ 46 ]. The results showed that grape stem extracts significantly altered the mitochondrial potential of the tested cancer cells compared to the untreated ones (Figure 3); therefore, the changes in mitochondrial potential could be related to the observed apoptosis (Figure 2). Antioxidants 2021, 10, x FOR PEER REVIEW 9 of 17 used to obtain a selectivity index (SI), as previously described by Badisa el al. [44]. The SI results are shown in Table 2, with the highest value being for the MDA-MB-231 cell line, according with the highest effective response of the extracts towards these cells after 72 h of incubation. The observed difference in the two breast cancer lines could have been due to the fact that the action of these extracts could be related to the receptors’ expression for estrogens, which are only present in MCF-7 cells [45]. 3.2.2. Cell Death Studies Since the grape stem extracts produce a reduction in cell viability, it was decided to determine what type of cell death occurred. Thus, flow cytometry analyses over 48 h were performed using biomarkers of cell death. The results showed that treatment for 48 h with the IC50 concentration corresponding to each cell line mainly produced early apoptosis in Caco-2 cells, while late apoptosis was mainly detected in MDA cells. However, no significant apoptosis was found in MCF-7 cells. Treatment with longer time (72 h) induced a significant death of these cells by late apoptosis (Figure 2). Therefore, the obtained results showed that grape stem extracts at their IC50 produced apoptosis in all tested cancerous cells by activating apoptotic pathways, thereby reducing their ability to nonselectively react with biological targets to cause necrosis and its related side effects. Figure 2. Cont. Antioxidants 2021,10, 243 16 of 17 30. Stagos, D.; Apostolou, A.; Poulios, E.; Kermeliotou, E.; Mpatzilioti, A.; Kreatsouli, K.; Koulocheri, S.D.; Haroutounian, S.A.; Kouretas, D. 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