Effects of in vitro gastrointestinal digestion on phenolic compounds and antioxidant activity of different white winemaking byproducts extracts

Effects of in vitro gastrointestinal digestion on phenolic compounds and antioxidant activity of different white winemaking byproducts extracts M. José Jara-Palacios1, Sandra Gonçalves2, Dolores Hernanz3, Francisco José Heredia1*, Anabela Romano2. 1Food Colour and Quality Laboratory, Facultad de Farmacia, Universidad de Sevilla, 41012, Sevilla, Spain. 2MeditBio, Faculty of Sciences and Technology. University of Algarve, Faro, Portugal. 3Department of Analytical Chemistry, Facultad de Farmacia, Universidad de Sevilla, 41012 Sevilla, Spain * Corresponding author: Francisco J. Heredia Food Colour and Quality Laboratory, Facultad de Farmacia, Universidad de Sevilla, 41012, Sevilla, Spain Tel.: +34 954556495 e-mail: her[email protected] Abstract The effect of in vitro gastrointestinal digestion on phenolic composition and antioxidant activity of different white winemaking byproducts extracts (grape pomace and its parts: seeds, skins and stems) was evaluated. Fourteen individual phenolic compounds were evaluated by UHPLC. The antioxidant activity was measured by DPPH and ORAC assays. Differences on phenolic profile and antioxidant activity were observed depending on the digestion phase, the type of byproduct, the phenolic group and the antioxidant activity method. In general, digestion had a reducing effect on TPC and antioxidant activity; however, ORAC values of seed and stem extracts increased after digestion and some recovery indexes of the phenolic groups were very high. Results indicate that extracts from white winemaking byproducts are a reliable source of bioaccessible antioxidant compounds, which could be used as functional food ingredients. Keywords Winemaking byproducts; phenolic compounds; in vitro gastrointestinal digestion; antioxidant activity Abbreviations AAPH: 2,20-azobis(2-methylpropionamidine) dihydrochloride; DPPH: 1,1-diphenyl-2- picrylhydrazyl; LOD: limit of detection; LOQ: limit of quantification; ORAC: oxygen radical absorbance capacity; PCA: Principal Component Analysis; RI: Recovery index SGF: Simulated gastric fluid; SIF: Simulated intestinal fluid TE: Trolox equivalents TPC: Total phenolic content. 1. Introduction Nowadays, consumers are very interested in leading a correct and balanced diet that includes healthy and natural foods. Therefore, food industry has become well aware of the importance of producing foods with health beneficial properties. In this sense, the incorporation of bioactive compounds extracted from natural products into foods as functional ingredients is rising (García-Lomillo & González-SanJosé, 2017; Kowalska, Czajkowska, Cichowska, & Lenart, 2017; Martins, Pinho, & Ferreira, 2017; Nunes et al., 2016). Grape pomace, a byproduct obtained from winemaking in large amounts, is an important source of bioactive compounds. This fact and the potential market interest of this product have led food researchers searching new alternatives for its exploration (García-Lomillo & González-SanJosé, 2017). The extraction of phenolic compounds from this byproduct is an attractive, sustainable and cost-effective source of high-value bioactives. Phenolic compounds are very abundant in grape pomace and they are interesting due to their antioxidant activity and other biological effects, which have been related to the prevention of common disorders (Del Pino-García et al., 2017; Falchi et al., 2016; Fraternale et al., 2016). The phenolic composition and antioxidant activity of grape pomace and its constituents, such as seeds, skins and stems, have been widely studied. Previous studies of our group indicated that phenolic rich extracts from grape pomace attenuate reactive oxygen species (ROS) accumulation and increase the resistance against oxidative stress in Caenorhabditis elegans (Jara-Palacios et al., 2013), exert antiproliferative activity in colon cancer cells (Jara-Palacios et al., 2015), and induce apoptosis in leukemia cells (León-González, Jara-Palacios, Abbas, Heredia, & Schini-Kerth, 2017). Moreover, the addition of seeds and skins from grape pomace during the fermentative step of the winemaking process increase the phenolic content (Gordillo et al., 2014) and antioxidant potential of wines (Jara-Palacios, Hernanz, Escudero-Gilete, & Heredia, 2016). Phenolic extracts from winemaking byproducts have also been added to diverse food products, e.g. cheese (Han et al., 2011), tomate puree (Lavelli, Sri Harsha, Torri, & Zeppa, 2014), yogurt (Marchiani et al., 2016) and milk (Dos Santos et al., 2017), to improve their bioactive properties. Grape pomace appears to be a promising source of functional ingredients to improve the phenolic profile and the antioxidant properties of food products. However, to evaluate the real bioactive potential of extracts from grape pomace is important to determine the effect of gastrointestinal digestion on phenolic compounds and to establish the bioaccessibility (Garbetta et al., 2018). As reported previously (Saura-Calixto, Serrano, & Goñi, 2007), bioaccessibility is defined as the amount of a food constituent that is released from a complex food matrix in the lumen of the gastrointestinal tract and could potentially be available for absorption into the body and promote biological actions (bioavailability). Therefore, the first step in determining the possible effects of a compound is to evaluate its stability during gastrointestinal digestion. In vitro methods simulating digestion process can be used to study the bioaccesibility from food sources. These methods have some advantages compared to in vivo methods: are more rapid, economic, safe and do not have ethical restrictions (Hur, Lim, Decker, & McClements, 2011). Despite the importance of bioaccesibility, studies to determine the effect of digestion on phenolic composition and biological activity of extracts from winemaking byproducts are scarce (Corrêa et al., 2017; Fernández & Labra, 2013; Gil-Sánchez et al., in press; Janisch, Ölschläger, Treutter, & Elstner, 2006). As far as we know no reports on stem grape extracts were found in the literature and no previous studies regarding grape pomace extracts from white grape have been published in this respect. In this sense, the aim of this study was to evaluate the effect of in vitro gastrointestinal digestion on phenolic composition of extracts from different white winemaking byproducts. Phenolic profile of extracts from grape pomace, seeds, skins and stems before and after digestion were analyzed by UHPLC. In addition, changes in the antioxidant activity of the extracts were investigated also during the digestion process. 2. Materials and Methods 2.1. Reagents and standards Formic acid, HPLC-grade acetonitrile, methanol, ethanol, sodium carbonate and Folin- Ciocalteau reagent were obtained from Panreac (Barcelona, Spain). Gallic acid, protocatechuic acid, catechin, epicatechin, quercetin, kaempferol and quercetin-3-O- rutinoside (rutin) were purchased from Sigma-Aldrich (Madrid, Spain). Quercetin-3-O- glucoside and kaempferol-3-O-glucoside were obtained from Extrasynthese (Genay, France). 2,2-diphenyl-1-picrylhydrazyl (DPPH), phosphate-buffered saline (PBS), pepsin from porcine gastric mucosa (CAS:9001-75-6) and pancreatin from porcine pancreas (CAS: 8049-47-6) and all other reagents used to prepare simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) were purchased from Sigma-Aldrich (Steinheim, Germany). 6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) and 2,20-azobis(2-methylpropionamidine) dihydrochloride (AAPH) and fluorescein were acquired from Acros Organics (Geel, Germany). 2.2. Samples and extraction procedure The white winemaking byproducts from Zalema grapes (Vitis vinifera sp.) used in this study were grape pomace (including a mixture of seeds, skins, stems and rests of pulp), and skins, seeds, and stems separated manually from the pomace. Grape pomace samples were supplied by a winery located in Condado de Huelva Designation of Origin (southwestern Spain). Samples were lyophilized and powdered in a blender to achieve a mean particle size less than 2 mm. Extraction of phenolic compounds was performed using a water and ethanol mixture (1:1). The plant material (5 g) was soaked overnight at room temperature in 200 ml of solvent and the resulting extract was filtered through a 5-13 µm membrane. Finally, seed, skin, stem and pomace extracts were concentrated to dryness in a rotary evaporator and stored at -20 C. 2.3. Gastrointestinal in vitro digestion The in vitro digestion consisted of an initial gastric phase followed by an intestinal phase using simulated gastric fluid (SGF) and simulated intestinal fluid (SIF), respectively, prepared with KCl, KH2PO4, NaHCO3, NaCl, MgCl2(H2O)6 and (NH4)2CO3, as previously described by Minekus et al. (2014). For gastric phase, 5 mL of each extract (50 mg/mL) were mixed with 3.75 mL of SGF, 0.8 mL porcine pepsin solution (25000 U/mL) and 2.5 L of 0.3 M CaCl2. The pH of the reaction mixture was adjusted to 3 by 1 M HCl and SGF was added to obtain a final volume of 10 mL. Mixture was incubated in a water bath at 37 °C for 2 h with stirring. Immediately, aliquots (gastric digests) were taken, stored at -20ºC and were analyzed within 2 weeks. For intestinal phase, 7 mL of gastric digest was mixed with 3.85 mL of SIF, 1.75 mL of a pancreatin solution (800 U/mL), 20 mg of bovine bile extract, 20 mg of porcine bile extract and 14 L of 0.3 M CaCl2. The pH of the reaction mixture was adjusted to 6 by 1 M NaOH and SIF was added to obtain a final volume of 14 mL. Mixture was also incubated in a water bath at 37 °C for 2 h with stirring. Immediately, aliquots (intestinal digests) were taken, stored at -20ºC and were analyzed within 2 weeks. Three independent experiments were performed for extracts of each type of byproduct and in each experiment three replicates were evaluated. 2.4. Total phenolic content Total phenolic content (TPC) was determined using the Folin-Ciocalteu assay with some modifications (Gonçalves, Grevenstuk, Martins, Romano et al., 2015). Samples (undigested extracts, gastric and intestinal digests) were appropriately defrosted and stirred; then, the samples were diluted to an adequate concentration in relation to the standard (gallic acid). Briefly, 100 µL of sample, 200 µL of 10% (v/v) Folin-Ciocalteu reagent, and 800 µL of a solution of sodium carbonate (700 nM) were mixed and the solution was left to stand for 120 min for the reaction to take place and stabilize. Absorbance was measured at 765 nm. Gallic acid was employed as a calibration standard and results were expressed as gallic acid equivalents (mg GAE/g extract). 2.5. Individual phenolic compounds Chromatographic analyses were carried out in an Agilent 1290 chromatograph (Agilent Technologies, Palo Alto, CA, USA) equipped with a diode-array detector, which was set to scan from 200 to 770 nm, and a C18 Eclipse Plus 120 column (1.8 µm, 50 x 2.1 mm). The solvents were 0.01% formic acid in water (solvent A), and acetonitrile (solvent B) at the following gradient: 0-5 min, 5% B linear; 5-20 min 50% B linear; 20- 25 min, 100% A linear, washing and re-equilibration of the column. The flow-rate was 0.8 mL/min, and the temperature of the column was set at 25 ºC. For analysis, samples (undigested extracts, gastric and intestinal digests) were defrosted, stirred and filtered filter, L of sample was injected in the column. Gallic acid, protocatechuic acid, catechin, epicatechin, procyanidins B1 and B2, quercetin-3-O-rutinoside, quercetin-3-Oglucoside, kaempferol-3-O-glucoside were identified by their retention time and UV-vis spectra and by comparison with our data library and standards. The identity of these compounds was confirmed by mass spectrometry (Jara-Palacios et al., 2014). B2-O-gallate, trimer, tetramer, kaempferol-3- O-galactoside and isorhamnetin-3-O-glucoside) were directly identified by mass spectrometry. The compounds were quantified with phenolic standards using peak area data of resolved peaks at 280 nm for flavanols and phenolic acids and 370 nm for flavonols. The corresponding calibration curves were made up for the following phenolic compounds: catechin (r2 = 0.999), gallic acid (r2 = 0.994), protocatechuic acid (r2 = 0.996) and quercetin (r2 = 0.996). The limits of detection (LOD) and quantification (LOQ) were calculated from calibrates curves. The LOD were calculated as three times the relative standard deviation of the analytical blank values calculated from the calibration curve. The LOQ were calculated as ten times the relative standard of the analytical blank values calculated from the calibration curve. The LOD and LOQ were: catechin (LOD: 1.15 ng and LOQ: 3.83 ng), gallic acid (LOD: 3.35 ng and LOQ: 11.18 ng), protocatechuic acid (LOD: 1.31 ng and LOQ: 4.37 ng) and quercetin (LOD: 0.91 ng and LOQ: 3.02 ng). Total content of three phenolic groups (phenolic acids, flavanols and flavonols) were also estimated by summing the content of each individual phenolic compound quantified by UHPLC. Samples (undigested extracts, gastric and intestinal digests) of each type of byproduct were analyzed in triplicate, and the results were expressed as mg of phenolic compounds per gram of extract (mg/g). 2.6. Assessment of recovery index of phenolic compounds To evaluate the effect of each digestion phase (gastric and intestinal) on the three phenolic groups (phenolic acids, flavanols and flavonols), the recovery index (RI) was calculated according to the equation below (Martínez-Las Heras, Pinazo, Heredia, & Andrés, 2017): RI (%) = (A/B) x 100 much lower than those for undigested samples (Figure 2). A previous study reported that the ability of phenolic significantly after intestinal phase (between 5-18%) compared to that observed in the gastric digests, and this activity was also higher in intestinal digests than in undigested samples (Fawole & Opara, 2016), which was not in accordance with our results. Alkaline pH, as in intestinal conditions, has been reported to significantly increase scavenging ability of grape polyphenols (Bouayed et al., 2011). In this study, despite the reduction of antioxidant activity after digestion process, the intestinal digests showed antioxidant activity. Regarding the results of ORAC assay, the antioxidant activity trend was different between seed and stem extracts, and skin and pomace extracts. As can be observed in Figure 3, the ORAC values increased significantly (p<0.05) during in vitro digestion for seed and stems extracts (from 2715.87 to 4048.96, and from 1052.50 to 2311.39 mol TE/g, respectively). Nevertheless, values for skin and pomace extracts decreased after digestion (from 3384.30 to 1783.64 mol TE/g, and from 3022.25 to 2279.51 mol TE/g, respectively). Skin and pomace extracts showed the greatest antioxidant activity before digestion and the lowest one after digestion, which could be related to contents of flavonols in these extracts (higher values than in seed and stem extracts), that also experimented a reduction with the digestion process. In addition, the reduction or increase observed for the antioxidant activity could be linked to the presence of other not analyzed substances in the extracts, such as peptides, that could be involved in this activity (Pavan et al., 2014). Also, the assay employed could also affect the antioxidant activity assessment since pH modifications may alter structure of phenolic compounds affecting the antioxidant activity (Arenas & Trinidad, 2017). According to some authors, assays carried out with buffer pH 7, such as ORAC and ABTS, are more suitable for the intestinal digests (Bouayed et al., 2011; Guldiken et al., 2016). A large number of papers have reported the strong correlation between phenolic compounds and antioxidant activity. Thus, different statistical analyses were carried out to study this correlation between our data. Multiple regressions were performed to check the most influential phenolic compounds (independent variables) total on the antioxidant activity (dependent variable) measured by DPPH and ORAC assay, depending on: undigested samples, gastric and intestinal digests. For these regressions all samples were considered, without differentiating by type of winemaking byproduct. On the one hand, regarding ORAC assay, catechin, epicatechin, procyanidins B1 and B2, tetramer and kaempferol-3-O-glucoside significantly influenced antioxidant activity for undigested samples; gallic acid for gastric digests; and procyanidins B1 and B2-3-O- gallate, gallic acid and quercetin-3-O-glucoside for intestinal digests. On the other hand, regarding DPPH assay, the most influential phenolic compounds were phenolic acids for undigested samples; gallic acid and flavonols for gastric digests; and gallic acid, catechin and procyanidin B1 for intestinal digests. Finally, a PCA was also performed in order to attribute which phenolic groups are more important for each type of white winemaking byproduct. After applying PCA to the data, it was seen that the first two principal components (PCs) explained 99.71% of the total variance. It can be seen from Figure 4, the first component (PC1) led to the separation of the seed from skin, stem and pomace samples, mainly due to the flavanol content. The second component (PC2) led to the separation of pomace from the rest of the byproduct samples, being mainly due to flavonol content. Our results show that the antioxidant activity is strongly influenced by the phenolic profile. Therefore, if phenolic stability is affected by the digestion process, the antioxidant activity could be also affected. In a previous study with white grape skin, authors indicated that the in vitro digestion process attenuated the biological effect of skin polyphenols on intestinal cell line (Garbetta et al., 2018). However, other studies indicated an increase of the antioxidant activity of fruit extracts after digestion, which was associated with the release of phenolic compounds after the digestion (Pavan et al., 2014). The phenolic composition (and therefore the antioxidant activity) of a food source could affect the microbiota and its catabolic activity. In this sense, a previous study suggests that simulated colonic fermentation seems to have a positive effect over bioactive potential. Therefore, although results from gastric and intestinal digestion provide valuable information on potential bioavailability of polyphenols, it is important to evaluate the effects of colonic fermentation, which would be interesting to look at in future. 4. Conclusions The present study reported the influence of in vitro gastrointestinal digestion on phenolic profile and antioxidant activity of different white winemaking byproducts extracts. The results varied depending on the type of byproduct (seeds, skins, stems and pomace), due to different phenolic profile and different recovery indexes of phenolic groups. Although, gastrointestinal digestion had a reducing effect on TPC and antioxidant activity, digested extracts continued to have high values. In addition, some recovery indexes of the phenolic groups were very high and some individual phenolic compounds showed high stability.In some cases, such as protocatechuic acid, catechin, epicatechin, and dimeric proanthocyanidins, levels of these compounds increased after the simulated digestion, whichcould indicate high bioaccessibility and potential bioavailability. The main novelty of this work is the study of effects of in vitro gastrointestinal digestion using white winemaking by products for the first time. The results obtained showed that white winemaking byproducts extracts studied are a promising source of functional ingredients for food and dietary supplements which can provide potentially bioavailable antioxidants to consumers. In addition, the present work provides information about components of agriculture byproducts with healthpromoting, which can be used in the development of new products by the food industry. Acknowledgements The authors acknowledge the assistance of the technical staff of Biology Service (SGI, Universidad de Sevilla). MJJP thanks Professor A. Romano for her acceptance for a research stay in Meditbio (Univertity of Algarve). S. Gonçalves acknowledges a grant from the Foundation for Science and Technology (FCT), Portugal (SFRH/BPD/84112/2012) financed by POPH-QREN and subsidized by the European Science Foundation. Funding: This work was supported by the Ministerio de Economia y Competitividad, Gobierno de España (Project AGL2014-58486-C2-2-R). Conflict of interest/Disclosure All the authors declare no conflict of interest. References Arenas, E. H., & Trinidad, T. P. (2017). Fate of polyphenols in pili (Canarium ovatum Engl.) pomace after in vitro simulated digestion. Asian Pacific Journal of Tropical Biomedicine, 7, 53-58. Bouayed, J., Hoffmann, L., & Bohn, T. (2011). 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