Assessment of polyphenolic profile stability and changes in the antioxidant potential of maqui berry (Aristotelia chilensis (Molina) Stuntz) during in vitro gastrointestinal digestion

Industrial Crops and Products 94 (2016) 774–782

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Assessment of polyphenolic profile stability and changes in the antioxidant potential of maqui berry (Aristotelia chilensis (Molina) Stuntz) during in vitro gastrointestinal digestion Raquel Lucas-Gonzalez a , Sergio Navarro-Coves a , José A. Pérez-Álvarez a , ˜ b , Manuel Viuda-Martos a,∗ Juana Fernández-López a , Loreto A. Munoz a IPOA Research Group (UMH-1 and REVIV-Generalitat Valenciana), Agro-Food Technology Department, Escuela Politécnica Superior de Orihuela, Miguel Hernández University, Orihuela, Alicante, Spain b Universidad Central de Chile, Facultad de Ingeniería, Chile

a r t i c l e

i n f o

Article history: Received 27 June 2016 Received in revised form 16 September 2016 Accepted 23 September 2016 Keywords: Maqui berry In vitro digestion Antioxidant Polyphenolic profile Bioaccessibility

a b s t r a c t Maqui berry (Aristotelia chilensis (Molina) Stuntz) has demonstrated a great potential as functional ingredient. However, determining the effect to which the maqui extract or its phytochemicals will benefit the consumers requires further knowledge. Thus, the aim of the present study was to determine the effect of in vitro gastrointestinal digestion (GID) on (i) the recovery and bioaccessibility indexes, (ii) the stability of polyphenolic compounds (phenolic acids, flavonoids and anthocyanins) and (iii) the changes in antioxidant activity of maqui berry grown in Chile. The extracts obtained in each phase (oral, gastric and intestinal) of GID were used to analyse the stability of polyphenolic compounds by means of HPLC whereas the antioxidant activity was determined using four different methodologies. All polyphenolic compounds decreased their concentration after GID and principally the anthocyanins content which was severely affected. The GID process decreased the scavenging properties in 89.9% and 84.2% with DPPH and ABTS assays, respectively, as well as the reducing power 74.1% with respect to non-digested sample. On the other hand, the chelating activity was increased (126.8%). At the end of GID process, the bioaccessibility of phenolic and flavonoid compounds was 78.19 and 14.20%, respectively. The results obtained suggest that although a great amount of maqui berry polyphenolic compounds are lost during digestion process they still have a great potential as antioxidants agents. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Due to the increasingly interest of population in consuming foods rich in healthy promoting compounds, the last years food science has focused its efforts on the search of new ingredients from fruits and vegetables. Today, these bioactive components are being intensively studied to evaluate their health properties. The evidence suggests that increasing the consume of plant-based foods can prevent some chronic diseases such as heart diseases, cancer, stroke, diabetes, etc. (Liu, 2013). The relation between plant-based food and health is the presence of bioactive compounds (Chen et al., 2007).

∗ Corresponding author. E-mail address: [email protected] (M. Viuda-Martos). http://dx.doi.org/10.1016/j.indcrop.2016.09.057 0926-6690/© 2016 Elsevier B.V. All rights reserved.

Aristotelia chilensis (Molina) Stuntz commonly known as maqui berry, Chilean blackberry or “maqui” is a wild edible berry from central and southern Chile. There are numerous health benefits related with maqui consumption such as anti-diabetic, cardio protective, inhibition of adipogenesis and inflammation, prevention of digestive disorders and prevention of LDL oxidation (Céspedes et al., 2008; Schreckinger et al., 2010; Rojo et al., 2012; Gironés-Vilaplana et al., 2014). The most significant effect has been attributed to phenolic compounds with high antioxidant capacity (Brauch et al., 2016). Regarding to its potential biological activity, maqui has the best performance in terms of ␣-glucosidase and lipase inhibitions compared with other fruits rich in phytochemicals (GironésVilaplana et al., 2014), therefore it also can help in the digestive process of carbohydrates, delaying the break-down of oligosaccharides and disaccharides into monosaccharides decreasing glucose absorption (Rubilar et al., 2011).

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Fig. 1. Graphic representation of the static in vitro gastrointestinal digestion procedure carried out with maqui berry extracts.

Recent studies have demonstrated the great potential of maqui as a potential functional ingredient to the development of functional food due to (i) its high content in polyphenolic compounds mainly anthocyanins and flavonoids and (ii) its antioxidant properties (Genskowsky et al., 2015, 2016; González et al., 2015). The role of anthocyanins as health promoter includes anti-inflammatory and anti-cancer activities and even protective effects against various metabolic, degenerative and cardiovascular diseases (Riaz et al., 2016). Its content in fruits and vegetables is directly proportional to antioxidant potential and has strong scavenging capacity against ˜ chemically generated reactive oxygen species (Castaneda-Ovando et al., 2009). Therefore, the interest in this berry as a functional food and its bioactive components has significantly increased. However, determining if the effect of maqui extract or its bioactive compounds will benefit the consumers requires further knowledge on its bioaccessibility and bioavailability. Consequently, the use of in vitro gastrointestinal digestion models has been useful to mimic the events occurring during the digestion and offer a good opportunity to investigate about the effect of physical and chemical parameters and its role in the bioaccessibility of these phytochemicals (Ting et al., 2015). Additionally, a in vitro gastrointestinal digestion models appear to provide an useful alternative to animal and human models for rapidly screening of food ingredients; furthermore in vitro techniques are ethically superior, faster and less expensive than in vivo techniques (Minekus et al., 2014). Thus, the purpose of this work was to determine the effect of in vitro gastrointestinal digestion on (i) the recovery and bioaccessibility indexes, (ii) the stability of polyphenolic compounds (phenolic acids, flavonoids and anthocyanins) and (iii) the changes in antioxidant activity of maqui berry grown in Chile.

2. Materials and methods 2.1. Plant material Maqui berry (Aristotelia chilensis (Molina) Stuntz) samples were provided by South-Am Freeze Dry S.A. The samples were collected ˜ from the Canete city in Bio-Bio Region (Chile) and lyophilized during 72 h. The product obtained was crushed in a mortar and finally sieved to remove seeds. 2.2. Simulated in vitro gastrointestinal digestion In vitro gastrointestinal digestion of maqui was performed according to the method described by Gullón et al. (2015a) (Fig. 1). The method included three different phases: oral, gastric and intestinal digestion. In the last phase of intestinal digestion, a segment (10 cm) of dialysis tubing (12–14 kDa molecular weight cut-off) filled with NaHCO3 (1 M) was placed inside of screw topped bottles filled with digested samples and incubated for 2 h in a shaking water bath at 37 ◦ C and 50 rpm. At the end of the incubation process, the solution left outside the dialysis tubing was taken as the OUT sample representing material that remained in the gastrointestinal tract (colon-available) and the solution that managed to diffuse into the dialysis tubing was taken as the IN sample (serumavailable). Instead of withdrawing aliquots from the reaction vessel at the end of the oral, gastric or intestinal digestion, individual digestions were carried out for each phase of digestion. Finally, all digestion mixtures were centrifuged for 12 min at 8000g at 4 ◦ C, yielding the chyme soluble fraction (CSF) and the pellet fraction (PF). Both fractions were lyophilized and stored until further use.

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2.3. Recovery index and bioaccessibility index To analyse the effect of the matrix composition on the digestion of the phenolic group (phenolic acids and flavonoids) two different indexes were studied following the indications of Ortega et al. (2011): The percentage of recovery and the percentage of bioaccessibility. The percentage of recovery allows the amount of phenolic group present in the complete digest (CSF and PF) after oral, gastric and intestinal digestion of test food to be measured according to: (i)Recoveryindex(%) = (PCDF /PCTF ) × 100 Where PCDF is the total phenol content (mg) in the digested (CSF + PF) and PCTF is the total phenol content (mg) quantified in test matrix. For each phenol group, the bioaccessibility is defined as the percentage of polyphenolic compounds that are solubilised in IN sample after intestinal digestion phase. Thus, this index defines the proportion of the polyphenolic compounds that could become available for absorption into the systematic circulation: (ii)Bioaccessibilityindex(%) = (PCS /PCDF ) × 100 Where: PCS is the total phenol content (mg) in the IN sample after the intestinal digestion phase and PCDF is the total phenol content (mg) in the digested sample (IN + OUT) after the intestinal digestion phase. 2.4. Total phenol and total flavonoid content The total phenol content (TPC) of lyophilized samples obtained during the different phases of in vitro gastrointestinal digestion was determined using the Folin-Ciocalteu’s reagent (Singleton and Rossi, 1965) while for the total flavonoid content (TFC), the method based on Blasa et al. (2005) was used. Methanolic solutions of lyophilized samples, with concentration comprise between 10 and 50 mg/mL, were used for both analysis. In TPC, gallic acid (GA) was the reference standard and the results were expressed as mg GA equivalents/g sample. In TFC, different concentrations of rutin (8.5–170 ␮g/mL) were used for calibration. The results were expressed in mg rutin equivalents (RE)/g of sample. 2.5. Determination of polyphenolic compounds Polyphenolic profiles of all samples obtained in each phase of in vitro gastrointestinal digestion were determined by High Performance Liquid Chromatography (HPLC) following the methodology described by Genskowsky et al. (2016). Samples were injected into a Hewlett-Packard HPLC series 1200 instrument equipped with C18 column (Mediterranea sea18 , 25 × 0.4 cm, 5 ␮m particle size) from Teknokroma, (Barcelona, Spain). Phenolic compounds were analyzed, in standard and sample solutions, using a gradient elution at 1 mL/min. The mobile phases were composed by formic acid in water (4.5:95.5, v/v) as solvent A and acetonitrile as solvent B. The chromatograms were recorded at 280, 360 and 520 nm. Polyphenolic compounds identification was carried out by comparing UV absorption spectra and retention times of each compound with those of pure standards injected in the same conditions. When standards were unavailable, the compounds were tentatively identified by comparing their UV/Vis spectra with previously published data (Fischer et al., 2011; Fredes et al., 2014; Brauch et al., 2016). Quantification of anthocyanins was executed based on linear curves of authentic standards. Delphinidin 3-glucoside calibration was used for the quantification of delphinidin derivatives, while the cyanidin 3-glucoside calibration was used for cyanidin-derivatives. The estimated concentrations were subse-

quently multiplied by a respective molecular-weight-correction factor according to Chandra et al. (2001). 2.6. Antioxidant activity 2.6.1. DPPH radical scavenging assay The free radical scavenging activity of lyophilized samples obtained during the different phases of in vitro gastrointestinal digestion was measured according to the methodology described by Brand-Williams et al. (1995) using the stable radical DPPH. Absorbance values were measured on a spectrophotometer at 517 nm. Results were expressed in mg Trolox equivalent/g sample. •

2.6.2. ABTS radical cation (ABTS + ) scavenging activity assay • The ABTS + scavenging activity assay of lyophilized samples obtained during the different phases of in vitro gastrointestinal digestion was determined as described by Leite et al. (2011). Absorbance values were measured on a spectrophotometer at 734 nm. The results were expressed as mg Trolox equivalent (TE)/g of sample. 2.6.3. Ferric reducing antioxidant power The ferric reducing antioxidant power (FRAP) of lyophilized samples obtained during the different phases of in vitro gastrointestinal digestion was determined using the methodology described by Oyaizu (1986). The FRAP values were estimated in mg Trolox equivalents (TE)/g of sample. 2.6.4. Ferrous ion-chelating ability assay Ferrous ions chelating activity (FIC) of lyophilized samples obtained during the different phases of in vitro gastrointestinal digestion was measured by inhibiting the formation of Fe2+ ferrozine complex, following the method of Carter (1971). Results were expressed in mg EDTA equivalent/g sample. 2.7. Statistical assay Statistical analysis and comparisons among means were carried out using the statistical package SPSS 19.0 (SPSS Inc., Chicago, IL). All experiments were carried out in triplicates and data were reported as mean ± standard deviation. The differences of mean values among concentration of bioactive compounds or antioxidant activity obtained in the different steps of the in vitro gastrointestinal digestion were analysed by one-way analysis of variance (ANOVA). The Tukey’s post hoc test was applied for comparisons of means, differences were considered significant at p < 0.05. Correlation analysis was performed between polyphenolic compounds contents and antioxidant activities of extracts using Pearson correlation analysis. 3. Results and discussion 3.1. Recovery index and bioaccessibility index Polyphenolic compounds (phenolic acids, flavonoids and anthocyanins) are considered the most important bioactive constituents of plants and fruits. The presence of polyphenolic compounds in foods and especially in fruits could be particularly important for consumers, due to their beneficial health properties. In addition, these bioactive compounds can be used as an important indicator of several functional properties like antioxidant capacities (Gullón et al., 2016). The total phenolic (TP) and total flavonoid (TF) recovery index of maqui berry fruits obtained after the different phases (oral, gastric and intestinal) of in vitro gastrointestinal digestion were shown in Fig. 2. The values for test matrix were obtained using a methanol extraction and these values were assumed as the 100%

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777

120

TPC

Recovery Index %

100

TFC

80

60

40

20

0

Test matrix

Oral

Gastric

Intestinal

Fig. 2. Recovery index of total phenolic content (TPC) and total flavonoids content (TFC) obtained after each step (oral, gastric and intestinal) of in vitro gastrointestinal digestion of maqui berry extracts.

of TPC or TFC of sample. After oral digestion, minimal modifications (p < 0.05) were obtained in the TF recovery since the percentage of flavonoids recuperated was 103.81%. Nevertheless, this phase had a moderate effect (p < 0.05) in TPC with a recovery index value of 79.79%. In this phase minimal modifications in TP or TF recovery index were expected due to the short exposure time and marginal effects of ␣-amylase as mentioned Mosele et al. (2016). Gastric digestion had a slight effect on TF recovery index which values decreased (102.63%) with reference to the values obtained after oral digestion (p > 0.05). On the other hand, a great increased, respect both test matrix and oral phase, was observed in TPC, with recovery index value of 112.92%. The phenolic compounds released from the test matrix after gastric digestion could be due to the break the bond of these compounds to proteins, fibre or sugar residues, as mentioned Gullón et al. (2015a). This fact could be attributed to the acidic pH and enzymatic activity which increases the extractability of polyphenolic compounds (phenolic acids and mainly flavonoids) from food matrix (Rodríguez-Roque et al., 2013). At the last phase of gastrointestinal digestion, intestinal phase, both TF and TP recovery indexes were deeply affected (p < 0.05) with recovery values of 17.14 and 21.76% respectively. This observation was in agreement with the work carried out by Mosele et al. (2015) in which an important decreased, in the TPC, was observed after intestinal phase digestion of pomegranate juice (42.2%) and pomegranate pulp (27,7%). Similarly, Gullón et al. (2015b) informed that the TP recovery index of apple bagasse flour and date pit flour was deeply affected with a decrease of 80 and 46%, respectively. This phenomenon could be explained since it is possible that when food matrix is exposed to gastrointestinal conditions, a proportion of polyphenolic compounds are transformed into different structural forms with different chemical properties and different bioavailability and biological activity (Bermúdez-Soto et al., 2007). It is well known that the bioactive polyphenols must be released from the food matrix to exert biological effects on human health. In this respect, the phenolic bioaccessibility is defined as the amount of the ingested polyphenols that are available for absorption in the gut after digestion (Ahmad-Qasem et al., 2014). The bioaccessibility of phenolic and flavonoid compounds present in maqui berry fruit, in the last phase of gastrointestinal digestion was 78.19 and 14.10% respectively. These values suggest, as mentioned Gullón et al. (2015a), that several changes in phenolic and flavonoid compounds such as (i) modification of chemical structure, (ii) increased

or reduced solubility or (iii) interaction with other compounds could have occurred during the development of the gastrointestinal digestion of maqui berry, which influenced their bioaccessibility. These results were in agreement with He et al. (2016) who informed that the bioaccessibility, after intestinal digestion, of phenolic compounds of grape juice and orange juice was 26.6 and 13.3%, respectively. In the same way Chitindingu et al. (2015) analysed the bioaccessibility of phenolic compounds in five wild and two domesticated cereal grains found in Zimbabwe. These authors reported that bioaccessibility of these cereals in the small intestine ranges between 16.76 and 33.06%. 3.2. Stability of polyphenolic compounds present in maqui berry during simulated in vitro gastrointestinal digestion The HPLC analysis of the maqui berry extracts (Table 1), showed a total of twenty polyphenolic compounds, identified as anthocyanins (eight compounds), flavonols (eleven compounds) and ellagic acid. With reference to anthocyanins content, only delphinidin derivatives and cyanidin derivatives were detected. These results were in agreement with several authors (Schreckinger et al., 2010; Brauch et al., 2016) who reported the occurrence of eight anthocyanins in maqui berry fruits identified as delphinidin or cyanidin-derivatives. Delphinidin-3-glucoside and delphinidin-3-sambuboside, with no statistical differences (p > 0.05) between them, were the predominant anthocyanins detected. In oral phase (Table 1), all detected anthocyanins decreases their concentration (p < 0.05) with respect to the initial one (Test matrix). Delphinidin-3-glucoside content decreased 54.40% and delphinidin-3-sambuboside content decreased 54.30% while cyanidin-3-sambuboside diminished 80.61%. This fact suggests that during oral phase anthocyanins were less effectively released than during the chemical extraction maybe due to the low contact time with the enzyme. In gastric phase (Table 1), all anthocyanins detected had an increment (p < 0.05) of their concentration with reference to oral phase values, although their content was still much lower than in test matrix. This small increased of anthocyanins in gastric fraction is probably due to the partial degradation of proanthocyanin oligomers into cyanidins as reported Stanisavljevic´ et al. (2015). In addition, the enzymatic activity and/or pH conditions might facilitate the breakage of high molecular weight phenols which initially may be bound to proteins or fibre.

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Table 1 Polyphenolic profile of the two fractions (pellet fraction (PF) and soluble chime fraction (SCF)) obtained after each step (oral, gastric and intestinal) of in vitro gastrointestinal digestion of maqui berry extracts. Compound

Flavonoids Myricetin-3-galactoside Myricetin-3-glucoside Quercetin-galloyl-hexoside Quercetin-galloyl-hexoside Rutin Ellagic acid Quercetin-3-galactoside Quercetin-3-glucoside Quercetin-3-xyloside Dimethoxy-quercetin Myricetin Quercetin

matrix

Mouth Step

Gastric step

Intestinal step

PF

SCF

Total

PF

SCF

Total

IN

OUT

Total

2.91 ± 0.02aE 4.13 ± 0.09aB 2.63 ± 0.11aF 3.86 ± 0.09aC 6.92 ± 0.10aA 7.15 ± 0.12aA 3.26 ± 0.04aD 1.85 ± 0.01aG

1.40 ± 0.02 1.83 ± 0.01 0.90 ± 0.01 1.52 ± 0.14 2.43 ± 0.55 2.71 ± 0.26 0.45 ± 0.12 0.45 ± 0.09

0.33 ± 0.00 0.41 ± 0.01 0.57 ± 0.01 0.53 ± 0.01 0.73 ± 0.01 0.55 ± 0.00 0.21 ± 0.01 0.16 ± 0.00

1.73 ± 0.01cD 2.24 ± 0.04cB 1.47 ± 0.06cE 2.05 ± 0.05cC 3.16 ± 0.15cA 3.26 ± 0.25cA 0.65 ± 0.11cF 0.61 ± 0.02cF

1.47 ± 0.01 2.26 ± 0.02 1.08 ± 0.02 1.44 ± 0.03 2.72 ± 0.06 3.30 ± 0.02 0.53 ± 0.02 0.66 ± 0.01

0.61 ± 0.02 0.92 ± 0.01 0.69 ± 0.00 0.82 ± 0.00 1.47 ± 0.02 1.56 ± 0.02 0.39 ± 0.01 0.35 ± 0.02

2.08 ± 0.01bE 3.18 ± 0.00bC 1.77 ± 0.02bF 2.26 ± 0.01bD 4.19 ± 0.04bB 4.86 ± 0.03bA 0.92 ± 0.01bH 1.01 ± 0.00bG

N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D.

N.D. N.D. N.D. N.D. N.D. N.D. 0.02 ± 0.0 0.01 ± 0.0

– – – – – – 0.02 ± 0.0dA 0.01 ± 0.0dB

50.62 ± 2.4aI 165.57 ± 3.01aD 161.40 ± 2.19aD 116.66 ± 2.06aE 450.67 ± 2.57aC 936.59 ± 1.40aA 43.74 ± 0.61aJ 99.42 ± 1.47aG 32.69 ± 0.26aK 502.17 ± 0.91aB 103.13 ± 0.30aF 85.41 ± 0.88aH

16.35 ± 0.30 54.19 ± 0.12 39.99 ± 0.09 49.08 ± 0.28 110.53 ± 0.8 258.0 ± 0.11 4.36 ± 0.02 33.37 ± 0.06 13.98 ± 0.13 188.3 ± 0.35 38.65 ± 0.20 51.37 ± 0.09

7.68 ± 0.33 31.15 ± 0.84 12.61 ± 0.30 4.75 ± 0.18 131.19 ± 0.48 68.15 ± 0.24 1.93 ± 0.25 12.61 ± 0.30 4.75 ± 0.18 45.67 ± 0.21 5.91 ± 0.17 6.10 ± 0.04

24.03 ± 0.21cH 85.34 ± 0.54cD 52.60 ± 0.15cF 53.83 ± 0.24cF 241.72 ± 0.61cB 326.15 ± 0.16cA 6.29 ± 0.09cJ 45.98 ± 0.18cG 18.73 ± 0.15cI 233.99 ± 0.29cC 44.56 ± 0.18cG 57.47 ± 0.06cE

19.81 ± 0.38 56.56 ± 0.31 47.05 ± 0.73 40.04 ± 0.34 111.67 ± 1.68 262.74 ± 2.12 4.51 + 0.14 30.95 ± 0.23 13.69 ± 0.30 170.13 ± 1.13 35.83 ± 0.31 49.31 ± 0.32

13.61 ± 0.02 44.00 ± 0.11 28.38 ± 0.03 32.74 ± 0.06 95.79 ± 0.06 146.12 ± 0.05 3.34 ± 0.07 19.27 ± 0.03 7.36 ± 0.04 83.03 ± 0.02 14.76 ± 0.75 16.43 ± 0.05

33.42 ± 0.25bI 100.56 ± 0.23bD 75.43 ± 0.32bE 72.78 ± 0.14bF 207.46 ± 0.78bC 408.86 ± 1.52bA 7.85 ± 0.09bK 50.22 ± 0.11bH 21.05 ± 0.13bJ 253.16 ± 0.43bB 50.59 ± 0.53bH 65.74 ± 0.14bG

N.D. N.D. N.D. N.D. 9.82 ± 0.28 3.22 ± 0.09 2.15 ± 0.04 N.D. N.D. 19.48 ± 0.02 N.D. N.D.

N.D. N.D. N.D. N.D. 74.75 ± 1.38 28.63 ± 0.48 14.25 ± 0.32 N.D. N.D. 118.52 ± 1.87 N.D. N.D.

– – – – 84.57 ± 0.68dB 31.85 ± 0.28dC 16.40 ± 0.15dD – – 138.00 ± 0.89dA – –

Anthocyanins (Values expressed as mg of each compound/g sample). Flavonoids (Values expressed as ␮g of each compound/g sample). For the same phenolic compound, values in the same row followed with same lower case letter (a–d) are not significantly different (p > 0.05) according to Tukey’s Multiple Range Test. For the same digestion step, values in the same column followed with same upper case letter are not significantly different (p > 0.05) according to Tukey’s Multiple Range Test.

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Anthocyanins Delphinidin-3-sambubioside-5-glucoside Delphinidin-3,5-diglucoside Cyanidin-3-sambubioside-5-glucoside Cyanidin-3,5-diglucoside Delphinidin-3-sambubioside Delphinidin-3-glucoside Cyanidin-3-sambubioside Cyanidin-3-glucoside

Test

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Regarding individual compounds, delphinidin-3-glucoside content increased 49.08% whilst delphinidin-3-sambuboside content increased 32.59% with respect to oral phase values. In the intestinal phase, a drastically reduction of anthocyanins was obtained (Table 1). Only, cyanidin-3-sambubioside and cyanidin-3-glucoside were detected. These results could be explained by the incomplete release of anthocyanins from the matrix due to the possible interactions with other compounds, such as fibre and lipids, by poor enzymatic hydrolysis, and especially by the instability of anthocyanins under alkaline conditions (Kosinska-Cagnazzo et al., 2015). In this way, Correa-Betanzo et al. (2014) reported that after intestinal digestion of blueberry extracts there were a clear disappearance of the absorbance peaks from anthocyanins, indicating ring cleavage of most anthocyanins as the pH of the medium changes from acidic (pH 2) to alkaline (pH 8) conditions. According to PérezVicente et al. (2002), it is important to consider that, after intestinal digestion, the anthocyanins flavylium form would not be the predominant one due to the slightly alkaline pH condition. Also, the possibility that part of the anthocyanins was metabolized to some other non-colored, oxidized forms or degraded into other chemicals should be considered as mentioned Soriano-Sancho et al. (2015). Likewise, Mosele et al. (2016) reported that after the intestinal digestion of Arbutus unedo fruit, important losses of anthocyanins (80%) were found. In the same way, these authors (Mosele et al., 2015) informed that the reduction of anthocyanins content in pomegranate juice, pomegranate pulp and pomegranate extracts after intestinal digestion was 70.0, 69.5 and 28.1% respectively. Nevertheless, despite the evidence of significant reductions in monomeric anthocyanins after digestion the fact that anthocyanins were not detected in intestinal digestion extracts does not imply the complete loss or no absorption of these compounds. There are several works which informed that gastric absorption of anthocyanins has been found and about 20% anthocyanins could be absorbed through this way (Fernandes et al., 2014). On the opposite side, Manach et al. (2005) reported that intestinal absorption of anthocyanins is just 1% of the orally ingested amount. Regarding to flavonoids and phenolic acids (Table 1) twelve compounds were identified, principally quercetin and quercetinderivatives (eight compounds) and myricetin and myricetinderivatives (three compounds); ellagic acid which was found in the highest (p < 0.05) concentration was also identified. In reference to quercetin and its derivatives, dimethoxy-quercetin was found in the highest concentration (p < 0.05) followed by rutin while for myricetin and its derivatives, myricetin-3-glucoside showed the highest (p < 0.05) concentration. The results obtained were in agreement with those reported by Brauch et al. (2016) who informed that quercetin derivatives and myricetin derivatives were the major non-anthocyanin constituents of maqui berry fruits. In order to assess the stability of each individual flavonoid and phenolic acid compounds, present in maqui berry, the concentration was measured after each phase of the in vitro gastrointestinal digestion. At the end of oral digestion, all flavonoids and phenolic acids identified decreased the concentration (p < 0.05) with respect to the test matrix. Thus, ellagic acid, the main component, decreased 65.17% while dimethoxy-quercetin decreased 53.40% in reference to test matrix (p < 0.05). This phenomenon could be explained due to these compounds might be bound to proteins or fibre in the test matrix, through different mechanisms such as hydrogen bonding, covalent bonding or hydrophobic interactions thus making them unavailable for released in this phase due to, as mentioned above, the marginal activity of ␣-amylase. In gastric phase, the same twelve compounds were identified (Table 1) and as occurs with anthocyanins, there was a slight increased (p < 0.05) in the concentration values with respect to the values obtained in the oral digestion. Thus, ellagic acid content increased 25.36% and dimethoxy-quercetin content increased 8.19% in ref-

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erence to oral phase. In any case, there was a decrease (p < 0.05), in concentration values, with respect to the values of test matrix. The results obtained suggested that the decreased in the concentration of flavonoids and phenolic acids of maqui berry with reference to test matrix could be explained by interaction with other food constituents, causing changes in their molecular weight, solubility and chemical structure (Scalbert and Williamson, 2000), among the others like the gastric conditions (mainly pH value and enzymatic activity) which affected the release and stability of these compounds. It is noteworthy that the flavonoids and phenolic acid compounds released in the gastric digestion may be absorbed and they may have some local antioxidant effect in the small intestine (Chandrasekara and Shahidi, 2012). At the end of intestinal digestion (Table 1), a drastic reduction of polyphenolic compounds was found. Only four flavonoid and phenolic acid compounds were identified (rutin, ellagic acid, quercetin-3-galactoside and dimethoxy-quercetin). The concentration values were higher (p < 0.05) in OUT fraction than IN fraction. In both, IN and OUT fractions dimethoxy-quercetin was the main (p < 0.05) component followed by rutin, ellagic acid and finally, quercetin-3-galactoside. The results obtained were in agreement with Gullón et al. (2015a) who reported that after intestinal digestion, the phenolic acids and flavonoids present in pomegranate peel flour were severely reduced compared to the non-digested samples. These important losses of flavonoids and phenolic acids after the intestinal digestion phase could be explained by the instability of these compounds under alkaline conditions. Thus, Tenore et al. (2013) suggested that some flavonoid glycosides seemed to have a good stability to the gastric acidic medium but they had a great instability under the near alkaline conditions of intestinal digestion. Chen et al. (2016) mentioned that the instability of these compounds, under alkaline conditions, could be attributed to the fact that these compounds undergo several changes, such as oxidation, polymerisation and transformation. In addition, Saura-Calixto et al. (2007) reported that the reduction, in the concentration of phenolic compounds, under alkaline conditions was related to the complexes formation between these compounds and metal ions, proteins and/or fibre. 3.3. Antioxidant properties In this work, the influence of gastrointestinal digestion on antioxidant activity was assessed by using an in vitro model that better simulates physiological conditions. The antioxidant activity of maqui berry, after each phase of in vitro gastrointestinal digestion, was unchanged compared with methanolic maqui berry extract. To determine the antioxidant properties of maqui berry fruits, four different methodologies were used in this work due to a single analytical assay is not able to fully quantify the total antioxidant properties of a sample, basically by the different mechanisms naturally involved. For this reason, a number of complementary antioxidant assays is typically employed in combination. Table 2 shows the antioxidant activity values obtained, after each phase of in vitro gastrointestinal digestion, of maqui berry fruits using DPPH, ABTS, FRAP and FIC assays. In DPPH assay, non-digested sample had values of 12.40 mg TE/g sample. After oral phase, DPPH values had a slight reduction 8.62% (p < 0.05) with respect to initial extract. The chyme soluble fraction (CSF) values were lower (p < 0.05) than pellet fraction (PF). Gastric and intestinal phases had a profound impact on antioxidant activity of maqui berry determined with DPPH assay. Thus, gastric digestion caused an increase of 12.50% in the antioxidant capacity (p < 0.05) with respect to initial values of maqui extract. However, the intestinal digestion produced a reduction of 75.40% in the antioxidant activity with reference to non-digested sample. In scientific literature, there is contradictory information about the effect of gastric and intestinal digestions on antioxidant activity

−75.4 −74.1 126.9 −84.2 3.05 ± 0.01 9.38 ± 0.10c 0.58 ± 0.01a 1.44 ± 0.02d 3.40 ± 0.00 13.24 ± 0.12B 0.00 ± 0.00 3.97 ± 0.05B −8.6 −3.4 −42.6 −10.7 12.40 ± 0.10 36.19 ± 1.60b 0.30 ± 0.01 9.08 ± 0.62b b

DPPH FRAP FIC ABTS

B

3.33 ± 0.00 10.71 ± 0.36B 0.11 ± 0.00A 2.65 ± 0.16B

A

8.01 ± 0.00 24.26 ± 0.96A 0.06 ± 0.00B 5.46 ± 0.10A

c

11.33 ± 0.00 34.97 ± 0.52b 0.17 ± 0.00c 8.11 ± 0.11c

SCF Total PF

Test Matrix Assay

PF: Pellet fraction; SCF: Soluble Chyme Fraction. % Var.: Percentage of variation between the initial values and the values obtained after digestion. For the same antioxidant assay, values in the same row followed with same lower case letter (a–d) are not significantly different (p > 0.05) according to Tukey’s Multiple Range Test. For the same antioxidant assay and the same phase of GID, values in the same row followed with same upper case letter (A–B) are not significantly different (p > 0.05) according to Tukey’s Multiple Range Test. FIC: expressed as mg EDTA equivalent/g.; FRAP: expressed as mg Trolox equivalent/g; ABTS: expressed as mg Trolox equivalent/g.; DPPH: expressed as mg Trolox equivalent/g.

2.38 ± 0.00 7.28 ± 0.18A 0.54 ± 0.00A 1.12 ± 0.16A 0.67 ± 0.01 2.11 ± 0.01B 0.14 ± 0.00B 0.32 ± 0.01B 12.5 24.8 −100.0 14.1 10.55 ± 0.01 31.92 ± 0.78A 0.00 ± 0.00 6.39 ± 0.76A

13.95 ± 0.57 45.16 ± 0.55a 0.00 ± 0.00 10.37 ± 0.42a

IN

Gastric

SCF

% var. Oral

B

PF

A

Total

a

% var.

Intestinal

B

OUT

A

Total

d

% var.

R. Lucas-Gonzalez et al. / Industrial Crops and Products 94 (2016) 774–782 Table 2 Antioxidant properties of the two fractions (pellet fraction (PF) and soluble chime fraction (SCF)) measured with DPPH, FRAP, FIC and ABTS assays after each step (oral, gastric and intestinal) of in vitro gastrointestinal digestion of maqui berry extracts.

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measure with DPPH assay. Thus, Celep et al. (2015) reported that antioxidant activity (determined with DPPH assay) of blueberry and cherry wines significant decreased with respect to non-digested sample. Similarly, Correa-Betanzo et al. (2014) reported that DPPH scavenging activity of blueberry extracts decreased over 50% after intestinal digestion. On the other hand, Gullón et al. (2015a) mentioned that the antioxidant effect of pomegranate peel after gastric and intestinal digestion increased with respect to non-digested sample. As regards to ABTS assay, as occurs with DPPH, a slight reduction (p < 0.05) in the antioxidant activity was achieved after oral phase with respect to non-digested sample which had values of 9.08 mg TE/g sample. Likewise, the CSF values were lower (p < 0.05) than PF. In gastric digestion the ABTS values increased 14.13% with respect to initial values. These values were in concordance with Chandrasekara and Shahidi (2012) who reported that gastric digestion of millet grains increased the ABTS values. However, Tagliazucchi et al. (2010) reported that ABTS radical scavenging activity of grape polyphenols decreased during gastric digestion. In the intestinal digestion, a deep reduction (p < 0.05) of antioxidant activity was obtained (84.16%) with respect to non-digested sample. This decrease in antioxidant activity could arise from the lower concentration of polyphenolic compounds, mainly anthocyanins, obtained in this step of the gastrointestinal digestion. Additionally, as mentioned Celep et al. (2015), interactions with other food components as well as variations in pH values could cause variations in the antioxidant activity. In any case, this reduction in the antioxidant activity obtained in this phase of the gastrointestinal digestion was in agreement with those reported by several authors (Correa-Betanzo et al., 2014; Gullón et al., 2015b). With reference to ferric reducing activity power (FRAP). The results obtained in this work (Table 2) showed that a slight reduction (p > 0.05) in the FRAP values were achieved at the end of oral digestion with respect to non-digested sample. After gastric phase, the FRAP values increased (p < 0.05) 24.77% with respect to initial values of maqui extracts. This fact was in concordance with Gullón et al. (2015a) who reported that FRAP values of digested pomegranate peel increased after gastric digestion. In the last phase of gastrointestinal digestion, the FRAP values lesser 74.08% with respect to initial values (p < 0.05). These results were in agreement with those Bouayed et al. (2011) who informed that reducing power of digested apple were 57% lower compared to total antioxidants in fresh apples. The FRAP values obtained in “IN” fraction were 2.11 mg TE/g sample which means that a minor reducing power is achieved. Table 2 showed the ferrous ion chelating activity (FIC) values of digested maqui extracts. At the end of oral digestion, FIC values were reduced 42.64% (p < 0.05) with reference to initial sample. These values suggested that bioactive compounds with chelating properties were strongly bonded to other matrix constituents such as fibre or proteins and its release did not occur, in an active way, at this stage of the digestive process. The gastric digestion significantly affects the chelating ability. At this stage, a reduction of 100% was obtained with respect to the non-digested sample which means that the pH conditions and/or enzymes exerted a considerable effect on bioactive compounds with chelating ability. However, at the end of intestinal digestion the chelating activity increased 126.85% with respect to non-digested sample (p < 0.05). The results obtained were in agreement with Stanisavljevic´ et al. (2015) who informed that chelating activity of chokeberry juice increased after intestinal digestion and Chandrasekara and Shahidi (2012) who reported that chelating ability of millet grain augmented significantly at the end of intestinal digestion. This increase in chelating capacity might be due to the liberation of several compounds like: phenolic acids, flavonoids or other constituents, such as ascorbates, reducing carbohydrates, tocopherols, carotenoids, or pigments which might contribute to the chelating activity of digested sample

R. Lucas-Gonzalez et al. / Industrial Crops and Products 94 (2016) 774–782 Table 3 Coefficient values of correlation between the Total phenolic content (TPC) or Total flavonoid content (TFC) and antioxidant activity measure with DPPH, FRAP, FIC and ABTS assays after each step (oral, gastric and intestinal) of in vitro gastrointestinal digestion. Antioxidant Assay

DPPH FRAP FIC ABTS

Oral

Gastric

Intestinal

TPC

TFC

TPC

TFC

TPC

TFC

0.998 0.991 −0.972 0.999

1.000 0.996 −0.983 0.999

0.999 0.987 0.000 0.950

−0.763 −0.844 0.000 −0.913

1.000 0.998 1.000 0.983

0.995 0.989 0.993 0.996

(Gullón et al., 2015a). In the same way Stanisavljevic´ et al. (2015) reported that the increase in chelating capacity is probably due to the release of small peptides originating from matrix proteins. The antioxidant activity of the extract solutions always depended on the composition and the concentration of its antioxidants, mainly polyphenolic compounds. Genskowsky et al. (2016) mentioned that, the antioxidant properties of polyphenolic compounds present in fruits, in general, and in maqui berry in particular are complicated to link to a specific compound or group compounds due to their complexity and variability. Therefore, the antioxidant activity could be caused by the major compounds presented in maqui berry or a synergistic effect between the major compounds and the minor ones. Correlation analysis could be used to explain this relationship. Therefore, Table 3 showed the correlations between polyphenolic profile (i.e. TPC and TFC) and antioxidant activity in maqui berry extracts measured with ABTS, DPPH, FIC and FRAP assays, after each phase (oral, gastric and intestinal) of in vitro gastrointestinal digestion. In oral digestion (Table 3) a strong and positive correlation was obtained between TPC and TFC and the antioxidant activity measured with DPPH, FRAP and ABTS assays. However, a negative correlation was achieved for FIC assay and TPC and FIC assay and TFC. In gastric digestion a high correlation (Table 3) was found between TPC and the antioxidant activity analysed with the different methodologies. On the other hand, a negative correlation was achieved between TFC and antioxidant activity which suggested that the antioxidant activity obtained in this phase is due to other compounds. Regarding to intestinal phase, again a high and positive correlation was found among TPC and TFC and the antioxidant activity obtained with DPPH, ABTS FIC and FRAP assays. These results were consistent with previous studies (Chandrasekara and Shahidi 2012; Huang et al., 2014; Gullón et al., 2015a,b) which reported a high correlation between polyphenolic compounds and antioxidant activity. 4. Conclusion This is the first time that the bioaccessibility and stability of polyphenolic compounds of maqui berry has been analysed throughout the different phases of gastrointestinal digestion. This work demonstrates that polyphenolic compounds presents in maqui are released, mainly, in the early phases of gastrointestinal digestion where they could exert bioactivity, as antioxidant compounds, after their absorption in gastric digestion. However, their stability, mainly of anthocyanin compounds, is profoundly affected in the last phase of digestion, probably modifying their physico-chemical properties which are reflected in their antioxidant properties and bioaccessibility. Several studies are necessary mainly for the stabilization of flavonoids and anthocyanin compounds through food matrix interactions which might help to provide sufficient levels for absorption during gastrointestinal digestion.

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