Effect of in Vitro Gastrointestinal Digestion on Encapsulated and

Article pubs.acs.org/JAFC

Effect of in Vitro Gastrointestinal Digestion on Encapsulated and Nonencapsulated Phenolic Compounds of Carob (Ceratonia siliqua L.) Pulp Extracts and Their Antioxidant Capacity Siham Ydjedd,† Sihem Bouriche,‡ Rubén López-Nicolás,§ Teresa Sánchez-Moya,§ Carmen Frontela-Saseta,*,§ Gaspar Ros-Berruezo,§ Farouk Rezgui,‡ Hayette Louaileche,† and Djamel-Edine Kati† †

Laboratoire de Biochimie Appliquée, Faculté des Sciences de la Nature et de la Vie, and ‡Laboratoire des Matériaux organiques, Département de Génie des Procédés, Faculté de Technologie, Université de Bejaia, Bejaia 06000, Algeria § Department of Food Science and Nutrition, Faculty of Veterinary Sciences, Regional Campus of International Excellence Campus Mare Nostrum, University of Murcia, Murcia 30071, Spain ABSTRACT: To determine the effect of in vitro gastrointestinal digestion on the release and antioxidant capacity of encapsulated and nonencapsulated phenolics carob pulp extracts, unripe and ripe carob pulp extracts were microencapsulated with polycaprolactone via double emulsion/solvent evaporation technique. Microcapsules’ characterization was performed using scanning electron microscopy and Fourier transform infrared spectrometry analysis. Total phenolics and flavonoids content and antioxidant activities (ORAC, DPPH, and FRAP) were evaluated after each digestion step. The release of phenolic acids and flavonoids was measured along the digestion process by HPLC−MS/MS analysis. The most important phenolics and flavonoids content as well as antioxidant activities were observed after gastric and intestinal phases for nonencapsulated and encapsulated extracts, respectively. The microencapsulation of carob polyphenols showed a protective effect against pH changes and enzymatic activities along digestion, thereby promoting a controlled release and targeted delivery of the encapsulated compound, which contributed to an increase in its bioaccessibility in the gut. KEYWORDS: carob pulp, microencapsulation, in vitro gastrointestinal digestion, phenolic compounds, antioxidant activity

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INTRODUCTION Carob (Ceratonia siliqua L.) is an evergreen tree belonging to the Leguminosae family, widely cultivated in the Mediterranean region usually in mild and dry places with poor soils.1 It is mainly produced in Spain, Italy, Morocco, Portugal, and Greece; however, Algeria was the sixth major world producer of carob, with a production of around 3.53 tons in 2014.2 Carob fruit is used as a raw material in industries such as food, pharmaceutical, and cosmetics. Because of its sweetness (about 40−50% of sugars, mainly sucrose) and flavor similar to chocolate, carob pods have long been used as cacao substitute for food production (sweets, biscuits, etc.).3 Earlier studies on carob fruit extracts demonstrated their various health-promoting effects mainly attributed to their high phenolic contents such as antioxidant properties in the in vitro and in vivo test systems,4,5 and antiproliferative activity on rat N1 × 10−115 neuroblastoma cells, and on human HeLa cervical and MCF-7 breast cancer cell lines.6 In past years, the encapsulation process has been usually performed to improve the stability of bioactive compounds and hence preserve their bioactivity during processing and storage, as well as to prevent undesirable interactions with food matrixes.7 Furthermore, encapsulation was used to mask any off-flavors and to ensure that the active compounds are maintained in a bioactive form within the gastrointestinal tract.8 Numerous techniques have been developed on bioactive substances encapsulation including spray drying, cocrystallization, emulsions, coacervation, and freeze-drying.7 Each of these © XXXX American Chemical Society

has its own specific advantages and drawbacks in encapsulation, protection, delivery, cost, regulatory status, and ease of use. Among these, water-in-oil-in-water (W1/O/W2) multiple emulsions have been widely considered as the major method of encapsulation and delivery systems for a wide range of lipophilic, hydrophilic, and amphiphilic bioactive molecules,9 due to their high-efficiency encapsulation, maintenance of chemical stability of encapsulated molecules, and controlled release.10 Polycaprolactone (PCL) is a biodegradable polymer with one relatively polar ester group and five nonpolar methylene groups. This polyester is globally hydrophobic and semicrystalline.11 The polymer has been used to microencapsulate antioxidants extracted from Helichrysum stoechas (L.).12 There are numerous reports on encapsulation of phenolic compounds derived from plant extracts by different methods using several biopolymers as coating material,13,14 but no study was found on the encapsulation of phenolic carob extracts. The biological effects of phenolic substances and their values in human health have been well demonstrated.15 However, the bioavailability and stability of these compounds in the digestion and absorption process affect greatly their health benefits. Many previous investigations on the stability and availability of dietary Received: November 14, 2016 Revised: January 4, 2017 Accepted: January 9, 2017

A

DOI: 10.1021/acs.jafc.6b05103 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

resulting microcapsules were collected by centrifugation at 3000 rpm for 15 min, washed with distilled water, dried in a desiccator, and then stored at 4 °C. The encapsulation efficiency (EE) was determined by measuring the total phenolic contents of sample before encapsulation (TP0) and the total phenolic contents of the supernatant after centrifugation (TPS). The EE (%) was calculated, using the following equation:

phenolics after gastrointestinal digestion showed that these factors varied widely from one polyphenol to another depending on their release from the dietary matrix.16−18 Moreover, the role of microbiota in the metabolism and bioavailability of dietary phenolics is also key.19,20 Nevertheless, to our knowledge, there is only one study regarding the effects of in vitro digestion on carob polyphenols, which is the investigation of Ortega et al.21 on the matrix composition effect on the digestibility of carob flour phenols. Thus, the objective of the current study was to determine how gastrointestinal digestion affects both encapsulated and nonencapsulated phenolic carob pulp extracts as well as their antioxidant activities. In addition, our work highlights the bioactive potential of these extracts at two ripening stages (unripe and ripe).

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EE% =

TP0 − TPS × 100 TP0

Microcapsules’ Characterization. Analysis of Microcapsules’ Morphology. The shape and surface morphology of carob pulp extract loaded PCL microparticles were observed using scanning electron microscopy (SEM) (Quanta 200 system, FEI, Hillsboro, OR). Encapsulated sample was spread on a stub, and the micrographs were recorded at 15 kV. FTIR Analysis of PCL, Lyophilized Extract, and Microcapsules. The physicochemical properties of PCL, phenolic carob pulp extracts, and prepared microparticles were analyzed with Fourier transform infrared spectrometry (FTIR) (IR Affinity-1 CE spectrometer, Shimadzu, Japan). Two milligrams of sample was finely grounded with 200 mg of purified potassium bromide. This powder mixture was then pressed in a mechanical die press to form a pellet. These pellets were scanned, and spectra were recorded from 400 to 4000 cm−1 at a resolution of 4 cm−1. In Vitro Gastrointestinal Digestion. The in vitro gastrointestinal digestion (IVGID) of samples consists of a three-step procedure, which simulates the digestion in mouth (oral digestion), stomach (gastric digestion), and small intestine (duodenal digestion). The stock solutions of digestion fluids were prepared with purified water as given in Table 1.

MATERIALS AND METHODS

Chemicals. Poly(ε-caprolactone) (PCL; Capa 6800, Mw = 123 g/ mol) was provided by Solvay (Lyon, France). Poly(vinyl alcohol) (PVA-124, 95% hydrolyzed), methyl cellulose, methylene chloride (CH2Cl2), Folin−Ciocalteu reagent, sodium carbonate anhydrous, and acetone were purchased from Biochem, Chemopharm (Quebec, Canada). Aluminum chloride hexahydrate (≥97% purity) was from Biochem, Chemopharma (GA); gallic acid was from Prolabo (Montreuil, France); 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,4,6-tri(2-pyridyl)-s-triazine (TPTZ) were from Sigma-Aldrich (Sternheim, Germany). 2,2′-Azobis (2-amidino-propane) dihydrochloride (AAPH) was purchased from Acros (Fair Lawn, NJ). 6Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) and Fluorescein (Na salt) (FL) were obtained from Sigma (St. Louis, MO). All of the enzymes (α-amylase, pepsin, pancreatin) used for in vitro digestion were purchased from Sigma−Aldrich Co. (St. Louis, MO). Acetic acid and methanol (grade HPLC−MS) were from Scharlau S.L. (Barcelona, Spain). Materials. Fresh carob (Ceratonia siliqua L.) pods of Lahlou variety, typically cultivated in the area of Bejaia (Algeria), were collected from different points of the tree and in various parts of the parcel. Fruits were harvested in good sanitary conditions at unripe and ripe stages. The sample was randomly selected and washed carefully; seeds were removed and pulps were lyophilized (Alpha1-4 LD Plus, Christ, Osterode am Harz, Germany), ground with a crusher (IKA, A 11 basic, Staufen, Germany), and passed through a 500 μm sieve before analyses. Extraction Procedure. Carob pulp powder (10 g) was homogenized in 100 mL (three times) of acetone/water (70:30, v/ v) and shaken using a water bath shaker at a temperature of 40 °C for 60 min (WB 22, Memmert, Germany). The mixture was centrifuged at 3000 rpm (Nüve NF 200, Ankara, Turkey) for 5 min and filtered using Whatman paper (no. 4). The solvent then was evaporated using a rotary evaporator under reduced pressure at a temperature of 40 °C. The remained aqueous phase was lyophilized (Alpha1-4 LD Plus, Christ, Osterode am Harz, Germany). The dry extracts were stored at 4 °C until use. The extraction process was carried out in triplicate. Microencapsulation of the Carob Extracts. Microcapsules containing lyophilized aqueous acetone carob pulp extracts were prepared by the double emulsion/solvent evaporation technique as reported by Barroso et al.12 with slight modifications. An aqueous solution of the extract (70 mg of dry extract in 4 mL of distilled water) with 4 mg of methyl cellulose (W1) was emulsified into an organic solution (800 mg of poly-ε-caprolactone (PCL)/20 mL of dichloromethane (DCM)) (O) by a high-speed homogenizer (IKA, yellow line DI 25 basic, Germany) at 13 500 rpm during 2 min. This primary emulsion (W1/O) was poured into 200 mL of solution of poly(vinyl alcohol) (PVA) (W2) as emulsifying agent. A stable double emulsion (W1/O/W2) was obtained under continuous stirring (400 rpm) for 60 min. Microparticles’ consolidation was achieved by DCM evaporation using a Heidolph rotary evaporator at a temperature of 30 °C and under reduced pressure (600 mbar) for 60 min. The

Table 1. Preparation of Stock Solutions of Simulated Digestion Fluids salt (stock solutions) KCl (0.5 M) KH2PO4 (0.5 M) NaHCO3 (1 M) NaCl (2 M) MgCl2(H2O)6 (0.15 M) NH4(CO3)2 (0.5 M) HCl (6 M)

stock solution added to prepare 250 mL of SSF (mL)

stock solution added to prepare 250 mL of SGF (mL)

stock solution added to prepare 250 mL of SIF (mL)

7.55 1.85

3.45 0.45

3.4 0.4

3.4 0.25

6.25 5.9 0.2

21.25 4.8 0.55

0.03

0.25

0.045

0.65

0.7

The digestion was started by introducing 5 mL of simulated salivary fluid (SSF) containing 7.5 mg of α-amylase and 25 μL of CaCl2(H2O)6 (0.3 M) to 1 g of sample (carob pulp extract, microcapsules). The mixture (pH 7) was gently stirred for 2 min at 37 °C. In the next step, 5 mL of simulated gastric fluid (SGF) (20 mg of pepsin and 2.5 μL of CaCl2 (H2O)6) was added to the oral phase, pH was adjusted to 3 with HCl (6 M), and the mixture was incubated at 37 °C in a shaking water bath for 2 h at a speed of 50 rpm. After oral and gastric steps, a volume of 1 mL of sample was taken for further analysis. To simulate the intestinal digestion, 10 mL of simulated intestinal fluid (SIF) with 37.5 mg of pancreatin and 40 mg of bile salts then was added to the gastric phase, the pH was adjusted to 7 with NaOH (1 M), and the mixture was incubated for 2 h at a speed of 50 rpm. Finally, the gastrointestinal mixtures were recovered and frozen at −24 °C until analysis. The samples were centrifuged and filtered before each analyses. Total Phenolics Content (TPC). Total phenolics content of the undigested and digested extracts was estimated according to the method described by Singleton and Rossi.22 One hundred microliters of sample was mixed with 1 mL of Folin−Ciocalteu reagent and 0.8 mL of 7.5% sodium carbonate solution. The test tubes were allowed to B


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Figure 1. Scanning electron microscopy (SEM) pictures of PCL-carob pulp extracts microparticles: (A) unripe carob pulp extract loaded PCL microcapsules; (B) ripe carob pulp extract loaded PCL microcapsules. (A1, B1) Microparticles morphology; (A2,3, B2,3) microparticles surface. stand in the dark at room temperature for 30 min. Absorbance was measured at 765 nm with a spectrophotometer (Thermo Scientific Evolution 300, Leicestershire, UK). Total phenolics content was expressed as milligrams gallic acid equivalents per 100 g of dry weight (mg GAE/100 g DW). Total Flavonoids Content (TFC). Total flavonoids content of undigested and digested samples was determined according to the method of Quettier-Deleu et al.23 based on the formation of the flavonoids−aluminum complex. Equal volumes of extract and aluminum chloride solution (2%) were mixed. The absorbance of the reaction mixture was measured at 430 nm after 15 min of incubation at room temperature. Total flavonoids content was expressed as milligrams quercetin equivalents per 100 g of dry weight (mg QE/100 g DW). Antioxidant Activities. Oxygen Radical Absorbance Capacity (ORAC) Assay. This method is based on the inhibition of the peroxylradical-induced oxidation initiated by thermal decomposition of azocompounds such as 2,2′-azobis (2-amidino-propane) dihydrochloride. The ORAC assay was carried out according to the methodology described by Prior et al.24 An aliquot (20 μL) of sample was added to a well in a 96-well bottom reading microplate (Costar, NY). Two hundred microliters of fluorescein solution (0.095 μM) was added to each well of the plate. The microplate was incubated at 37 °C for 15 min before an aliquot of 20 μL of AAPH solution (79.6 mM) was added. Fluorescence readings were done in a plate reader (Synergy HT, Biotek Instruments Inc., Winooski, VT) using excitation and emission wavelengths of 485 and 528 nm, respectively. Data are expressed as micromoles of Trolox equivalents per 100 g of dry weight (μmol TE/100 g DW). Ferric-Reducing Antioxidant Power (FRAP) Assay. The FRAP assay was carried out according to the procedure of Benzie and Strain.25 Briefly, the FRAP reagent was prepared from acetate buffer (pH 3.6), 10 mM TPTZ (2,4,6-tri(2-pyridyl)-s-triazine) solution in 40 mM HCl, and 20 mM iron(III) chloride solution in proportions of 10:1:1 (v/v/v), respectively. The FRAP reagent was freshly prepared prior to use. One hundred microliters of sample was added to 1 mL of the FRAP reagent. The absorbance of the reaction mixture was recorded at 593 nm after 4 min. The results were expressed as micromoles of Trolox equivalents per 100 g of dry weight (μmol TE/ 100 g DW). DPPH Radical Scavenging Assay. DPPH radical scavenging activity of different extracts was measured according to the procedure described by Brand-Williams et al.26 One milliliter of DPPH solution (60 μM) was mixed with 100 μL of sample. The decrease in absorbance was determined at 517 nm, after 30 min of incubation. The

results were expressed as micromoles of Trolox equivalents per 100 g of dry weight (μmol TE/100 g DW). HPLC−MS/MS Analysis. The most important phenolic compounds of undigested and digested carob pulp extracts were determined. The analysis were carried out on a HPLC−MS/MS system consisting of an Agilent 1100 Series HPLC (Agilent Technologies, Santa Clara, CA) equipped with a microwell plate autosampler and a capillary pump, and connected to an Agilent Ion Trap XCT Plus mass spectrometer (Agilent Technologies, Santa Clara, CA) using an electrospray interface (ESI). Samples were centrifuged at 14 000 rpm during 10 min, and the supernatants were filtered through (0.22 μm PVDF filter). Forty microliters of each filtered sample was injected into an Agilent Zorbax SB-Aq HPLC column (5 μm, 150 × 4.6 mm), thermostated at 40 °C, and eluted at a flow rate of 200 μL/min. Mobile phase A, consisting of water + 0.1% acetic acid, and mobile phase B, consisting of pure methanol, were used for the chromatographic separation. The elution program consisted of 0−5 min 5% B; 5−30 min 50% B; 40−45 min 100% B; and 50−60 min 5% B. UV chromatograms were recorded at 210, 275, and 340 nm. The mass spectrometer was operated in the negative mode with a capillary spray high voltage of 3500 V, and a scan speed of 22 000 (m/z)/s from 50 to 500 m/z, with the target mass located at 200 m/z. The Smart ICC target was set to 200.000 counts, whereas the maximum accumulating time was 20 ms, and three spectra were averaged in each scan. The nebulizer gas pressure was set to 30 psi, whereas the drying gas was set to a flow of 8 L/min at a temperature of 350 °C. The selected ions were extracted and analyzed both in scan (MS) and in MRM mode (MS/MS). The selected ions were sequentially fragmented using helium collision-induced dissociation (CID) with an isolation width of 1 m/z and a relative collision energy of 35%. Mass spectra were obtained using the Data Analysis program for LC/MSD Trap Version 3.2 (Bruker Daltonik, GmbH, Germany). Quantification of phenolic components (relative amounts) was expressed as a percentage. The percentage was calculated from the area of individual phenolic compound in the digested sample in each step of digestion relative to the area of this compound in the undigested sample (raw material), which was considered as 100%. Statistical Analysis. Statistical analysis and comparisons among means were carried out using STATISTICA 5.5 software (Tulsa, OK). All experiments were carried out in triplicate, and data were reported as mean ± standard deviation. The differences of mean values among the concentration of bioactive compounds or antioxidant activity and that obtained in the different steps of the IVGID were analyzed by one-way analysis of variance (ANOVA). The LSD post hoc test was applied for comparisons of means; differences were considered C


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Figure 2. FTIR spectra of PCL, carob pulp extract, unripe pulp extract loaded PCL microcapsules (PCL-US), and ripe pulp extract loaded PCL microcapsules (PCL). significant at p < 0.05. Correlation analysis was performed between total phenolics, total flavonoids, and antioxidant activities of extracts using Pearson correlation analysis.

the particles were not uniform and most of them were in spherical form with rough surface. However, at high magnification (Figure 1A2,3, B2,3), tiny pores were easily observed at the surface of microcapsules. These pores may be generated from the evaporation of imbibed water during the evaporation step where the water molecules were trapped in the microcapsule matrix during the dispersion process.29 A similar result was obtained on the morphology of the disodium norcantharidate-loaded PCL microcapsules.30 Fourier Transform Infrared (FTIR) Analysis. FTIR spectroscopy of PCL, carob pulp extracts (R-CP, U-CP), and polyphenols loaded PCL microcapsules was conducted to study structural information and PCL-carob extract interactions, as shown in Figure 2. Regarding pure PCL, a broad band at 3448 cm−1 is attributed to the O−H stretching vibration of the hydroxyl groups. Furthermore, PCL displays a signal for C−H stretching at 2943 and 2854 cm−1. A strong peak at 1730 cm−1 is assigned to the CO stretching vibrations of PCL. Peaks at 1453 and 1493 cm−1 correspond to the CH2 bending vibration, whereas C−O stretching vibrations were observed at 1161 and 1076 cm−1.11 The spectra of phenolic carob extracts present a similar profile with different bands intensities. The absorption band observed at 3446 cm−1 corresponded to the hydroxyl stretching vibrations of the different O−H groups (alcohols, carboxylic acids, and phenols). The strong band at 1638 cm−1 is assigned to the CO stretching of conjugated acids and CC

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RESULTS AND DISCUSSION Encapsulation Efficiency (EE) and Microcapsules’ Morphology. The encapsulation efficiencies of both unripe and ripe carob pulp extracts were determined. The EE value was higher in the unripe carob pulp extract (78.38 ± 0.37%) as compared to that in the ripe carob pulp extract (56.63 ± 0.94%). This can be explained by the presence of high amounts of phenolic compounds in the unripe carob pulp extract as compared to the ripe one, which had higher sugar and fiber amounts than the unripe carob pulp extract, which led to an overestimation of phenolics content at this stage. These sugars and fibers had a great affinity to the aqueous phase. Thus, the ripe carob pulp extract could lose, in addition to these compounds (sugars and fibers), some of the phenolics linked to them, in the external PVA solution during the second emulsion where they were solubilized. Kalogeropoulos et al.27 and Rosa et al.28 evaluated the encapsulation efficiency of crude phenolic extract of Hypericum perforatum and blackberry phenolic compounds, respectively, and reported that the efficiency depended on the type of encapsulated compound. Scanning electron microscopy (SEM) analysis was performed to obtain information about the morphology of microparticles as well as their appropriate surfaces. As shown in Figure 1, SEM pictures of PCL-carob pulp extract microparticles showed that D


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Table 2. Changes in the Total Phenolic Content (TPC), Total Flavonoid Content (TFC), and Antioxidant Activities of Carob Pulp Extracts before and after IVGIDa ripe carob pulp nonencapsulated raw material TPC (mg GAE/100 g DW) TFC (mg QE/100 g DW) ORAC (μmol TE/100 g DW) FRAP (μmol TE/100 g DW) DPPH (μmol TE/100 g DW)

oral

3448.5 ± 18.4 76.3 ± 0.9

11.3 ± 0.1 b 0.6 ± 0.01 c

29581.5 ± 187.3 15604.6 ± 37.5 30975.6 ± 25.4

109.6 ± 14.0 c 59.9 ± 0.1 b 90.8 ± 0.1 c

gastric

encapsulated intestinal

Antioxidant Compounds 16.0 ± 0.1 a 16.3 ± 0.2 a 1.35 ± 0.01 a 0.9 ± 0.02 b Antioxidant Activities 228.14 ± 5.8 b 310.91 ± 21.6 a 76.70 ± 0.2 a 64.5 ± 0.2 b 143.21 ± 1.3 a 119.3 ± 0.8 b unripe carob pulp

oral 9.0 ± 0.3 c 0.5 ± 0.1 c 90.3 ± 2.1 c 9.4 ± 0.3 c 248.6 ± 1.0 b

nonencapsulated raw material

a

TPC (mg GAE/100 g DW) TFC (mg QE/100 g DW)

14355.5 ± 40.3 126.1 ± 0.4

ORAC (μmol TE/100 g DW) FRAP (μmol TE/100 g DW) DPPH (μmol TE/100 g DW)

43630.7 ± 557.5 50554.1 ± 381.6 83996.1 ± 249.1

oral

gastric

gastric

intestinal

34.1 ± 0.5 b 0.5 ± 0.04 b

201.7 ± 0.3 a 3.0 ± 0.1 a

190.6 ± 6.8 b 17.1 ± 0.2 b 469.2 ± 3.3 a

2229.5 ± 188.8 a 42.2 ± 0.8 a 498.7 ± 30.3 a

encapsulated intestinal

Antioxidant Compounds 29.1 ± 0.1 b 124.7 ± 0.6 a 33.94 0.3 ± 0.01 b 0.8 ± 0.01 a 0.3 Antioxidant Activities 145.9 ± 2.1 c 352.6 ± 26.0 a 301.8 439.7 ± 0.7 a 328.9 ± 0.8 b 226.7 400.0 ± 1.6 b 800.7 ± 1.0 a 280.6

oral

gastric

intestinal

± 0.3 b ± 0.01 b

16.0 ± 0.3 c 0.8 ± 0.1 c

65.8 ± 0.6 b 1.5 ± 0.1 b

287.3 ± 0.8 a 17.9 ± 0.2 a

± 8.4 b ± 1.7 c ± 0.3 c

85.4 ± 5.7 c 34.3 ± 0.8 c 143.6 ± 1.3 c

505.1 ± 54.3 b 108.6 ± 0.9 b 311.4 ± 3.7 b

2835.6 ± 194.1 a 390.1 ± 0.7 a 599.0 ± 10.9 a

Letters within the same row indicate significant differences at p < 0.05.

vibration of aromatic. Aromatic C−H bending was observed in the range 860−680 cm−1.31 Regarding the phenolic carob extracts loaded PCL microspheres, the bands corresponding to CO and OH stretching showed a broader behavior as compared to pure PCL, and the bands are shifted to lower wavenumbers, indicating that polyphenols are associated with the PCL polymer by interactions between the carbonyl and the carboxyl groups of the polyphenols and the polymer. These results confirm that carob polyphenols were incorporated in the biopolymer (PCL) matrix by intermolecular interactions.12 Total Phenolic (TPC) and Flavonoid Contents (TFC). The digestive process is the first step, before absorption and metabolism, in modulating the bioavailability of polyphenols by affecting their stability and bioaccessibility.32 The impact of gastrointestinal digestion on TPC and TFC of carob pulp extracts is shown in Table 2. The results revealed that the TPC and TFC decreased strongly after the in vitro digestion for both unripe and ripe carob pulp extracts in comparison to the undigested samples. These results are in agreement with the findings of Granese et al.33 in their study on the variation of polyphenols, anthocyanins, and antioxidant power in the strawberry grape after simulated gastrointestinal transit. For the nonencapsulated ripe carob pulp extract, there are no significant differences (p < 0.05) between the amounts of total phenolic after the digestion process, whereas, for the encapsulated extract, the TPC increased during the digestion process, and the highest value was recorded in the intestinal phase. For nonencapsulated unripe carob pulp extract, the TPC after digestion was higher in the gastric phase, while,the contents recorded in the oral and intestinal phases did not exhibit significant differences. However, for the encapsulated extract, the phenolic amounts increased with the digestion phases (oral < gastric < intestinal). Concerning the TFC, after the gastrointestinal digestion, the highest amounts were recorded in the gastric medium for both

nonencapsulated extracts. However, for both encapsulated extracts, the release of flavonoids was higher in the intestinal medium. The reduction of both TPC and TFC, at the end of intestinal step, in nonencapsulated extracts is in agreement with the work performed by Ortega et al.21 in which recovered TP and TF of washed carob flour decreased after the intestinal digestion step. Gullon et al.18 also obtained that the TP and TF recovery index decreased after intestinal digestion of pomegranate peel flour. Low values of TPC and TFC in the oral phase (after 2 min of digestion) can be explained by the low solubility of these compounds in salivary fluid and to the short period of this step. As reported by Ortega et al.,21 the phenolic losses during IVGID could be explained by the physicochemical transformations, such as oxidation or the interactions with other components, like polysaccharides in the digestion mixture. Furthermore, the decrease of phenolic compounds could arise from precipitation of some phenolics like tannins with proteins ́ et al.15 (enzymes) in the digest. In this regard, González-Sarrias reported that ellagitannins and ellagic acid bioavailability were limited by the pH and protein environment. However, for the encapsulated extracts, the TPC and TFC are more released in the intestinal phase and less in the gastric phase as compared to the nonencapsulated extracts. Hence, this result means that the encapsulated phenolics are protected against the condition changes of digestion such as the type of enzyme and pH variations. Our results for the encapsulated extracts are in accordance with the results of Flores et al.34 where they studied the in vitro release properties of encapsulated blueberry extracts, finding that phenolic contents increased throughout gastric to intestinal digestion. However, Saikia et al.35 in their study on the microencapsulation of phenolic compounds of Averrhoa carambola pomace with maltodextrin by spray and freeze-drying showed in both techniques that the release of phenolic compounds was higher in gastric simulated medium (pH 1.2) than in the simulated intestinal medium (pH 6.8). According to Saura-Calixto et al.32 E


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Table 3. HPLC−MS/MS Phenolic Profiles of Carob Pulp Extracts after the in Vitro Simulated Gastrointestinal Digestiona ripe carob pulp nonencapsulated oral cinnamic acid p-coumaric acid gallic acid syringic acid ellagic acid apigenin naringenin kaempferol quercetin rhamnoside myricetin rhamnoside

encapsulated

gastric

71.6 45.9 145.3 5.6 98.3

± ± ± ± ±

1.0 1.8 5.1 0.03 0.3

73.4 43.6 149.6 6.1 88.6

± ± ± ± ±

1.3 0.4 3.9 0.1 1.6

37.4 97.8 58.0 65.1 67.9

± ± ± ± ±

0.9 2.1 1.3 0.9 1.31

63.9 135.8 105.7 111.3 107.5

± ± ± ± ±

0.9 5.4 1.0 1.0 3.0

intestinal

oral

Phenolic Acids 62.5 ± 0.3 9.4 26.8 ± 0.4 21.3 0.6 ± 0.01 5.9 4.7 ± 0.3 3.0 178.5 ± 5.3 92.2 Flavonoids 50.8 ± 2.0 1137.0 85.8 ± 1.2 800.8 38.5 ± 0.02 4.4 60.1 ± 1.6 1137.0 17.3 ± 1.0 800.8 unripe carob pulp

gastric

± ± ± ± ±

0.02 0.5 0.1 0.1 1.5

10.6 52.5 5.3 4.9 149.4

± ± ± ± ±

0.2 3.4 0.2 0.1 3.3

17.3 42.8 4.9 3.5 148.0

± ± ± ± ±

0.2 2.3 0.1 0.2 3.2

± ± ± ± ±

45.0 41.5 0.08 6.1 1.9

1110.1 810.5 4.6 1110.1 810.5

± ± ± ± ±

5.5 20.5 0.07 9.2 2.3

784.5 572.2 6.7 784.5 572.2

± ± ± ± ±

2.0 3.4 0.3 7.1 2.0

nonencapsulated oral

a

encapsulated

gastric

cinnamic acid p-coumaric acid gallic acid syringic acid ellagic acid

98.8 38.7 361.4 31.5 66.2

± ± ± ± ±

1.7 0.1 1.7 0.7 2.2

80.2 45.7 194.6 39.0 60.5

± ± ± ± ±

0.8 2.4 5.1 0.4 1.0

apigenin naringenin kaempferol quercetin rhamnoside myricetin rhamnoside

57.1 104.5 89.5 42.7 45.5

± ± ± ± ±

1.3 0.8 2.6 1.0 0.9

92.8 111.1 87.2 74.4 67.8

± ± ± ± ±

2.8 0.9 1.0 3.7 0.3

intestinal

intestinal Phenolic Acids 95.8 ± 4.2 16.3 ± 0.2 407.0 ± 5.5 22.6 ± 0.5 392.3 ± 15.2 Flavonoids 82.9 ± 0.5 109.2 ± 3.7 73.9 ± 0.9 47.9 ± 2.4 40.2 ± 1.6

oral

gastric

intestinal

16.7 11.1 2.9 20.8 277.1

± ± ± ± ±

0.3 0.2 0.03 1.4 8.7

15.9 22.3 22.3 8.9 111.7

± ± ± ± ±

0.2 0.4 0.1 1.1 1.7

22.2 8.22 1.4 9.5 321.1

± ± ± ± ±

0.2 0.2 0.01 0.4 2.0

140.0 81.3 230.9 407.7 289.7

± ± ± ± ±

1.6 3.0 1.7 15.0 10.4

577.5 205.4 181.8 2098.7 592.6

± ± ± ± ±

10.6 3.4 63.7 32.7 8.7

416.1 150.2 166.3 1427.4 133.2

± ± ± ± ±

20.4 3.0 4.4 13.3 4.3

Results are expressed as % of starting material.

and Saikia et al.,35 the behavior of the microcapsules in a simulated gastrointestinal medium is dependent on the type and property of the coating material used for encapsulation and their resistance or susceptibility to digestive enzymes as well as on the gastrointestinal conditions like pH range. Antioxidant Activities. The antioxidant activity of plant extracts is mainly linked to their phenolic compounds. Nonetheless, the antioxidant properties of these compounds might change due to the chemical transformations resulting from different mechanisms during the gastrointestinal digestion. Therefore, to evaluate the influence of IVGID on the antioxidant capacity of carob pulp extracts, several assays were performed (ORAC, FRAP, and DPPH). The antioxidant activity results of encapsulated and nonencapsulated phenolic carob pulp extracts were shown in Table 2. All activities tested decreased strongly after the digestion process in comparison to the raw material. This reduction in activity could be due to the decrease in phenolics after the digestive process. Regarding the oxygen radical absorbance capacity (ORAC) of extracts after digestion, the nonencapsulated extracts showed significant differences (p < 0.05) during IVGID. Ripe extract had the highest ORAC activity in the intestinal phase, whereas for the unripe extract this activity was higher in the gastric phase. However, for encapsulated extracts, the highest activity was noted in the intestinal medium. Gullon et al.18 in their study on the effect of IVGID on ORAC assay of pomegranate peel flour found that this activity was significantly (p < 0.05) higher in the gastric step.

Regarding the ferric-reducing antioxidant power (FRAP) of digested extracts, both nonencapsulated extracts presented significant differences (p < 0.05) during the digestion process. The highest FRAP value was recorded in the gastric and oral phases for ripe and unripe extracts, respectively. For encapsulated ones, this activity increased with the digestion steps where the most important FRAP activity was recorded in the intestinal phase for both ripe and unripe extracts. Flores et al.34 showed that the ferric reducing power increased with digestion steps and was more pronounced for the encapsulated blueberry extracts in the intestinal phase. The study of Chen et al.36 on nutraceutical potential and antioxidant benefits of 11 fruit seeds subjected to an in vitro digestion reported that FRAP values of studied fruit seeds decreased after the duodenal phase of digestion. Phenolic compounds are a large and diverse group of phytochemicals, which include many different classes of bioactive substances. In this regard, the difference in FRAP and ORAC activities, in oral and intestinal phases, respectively, may not be due to phenolics and flavonoids content, but rather to the possible diversity of the polyphenols present. However, the highest activities (FRAP and ORAC) after the gastric digestion could be attributed to the higher released phenolics and flavonoids content, with quenching and reducing properties, in acidic medium. Moreover, according to Moran et al.,37 the effect of the pH could be different for various polyphenols. At neutral pH some phenolics have displayed pro-oxidant F


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and cinamic acids) after oral and gastric steps. However, the ellagic acid was well released after the intestinal step of digestion. This result is in concordance with the findings of ́ et al.15 in their study on the identification of González-Sarrias the limits of ellagic acid bioavailability after oral administration. By contrast, the syringic acid amount decreased strongly during the digestion process and presented a loss of 95% and 78% in ripe and unripe pulp extracts, respectively, after the intestinal step. For ripe pulp extract, gallic acid was completely digested after the intestinal step, whereas for the unripe it was well released. A similar result was observed by Chen et al.36 where they found that gallic acid was completely digested for some fruit seeds, after the duodenal phase. According to Ortega et al.,21 the trend of the phenolic acids after digestion could be highly influenced by the food matrix composition. The free phenolic acids (gallic acid) resulting from the digestion of the soluble fiber present in ripe carob pulp extract could probably compensate the instability of the gallic acid in the intestinal conditions. The high values of gallic and ellagic acids after the digestion process could be due to the hydrolyze of gallotannins (monogalloyl-glucoside, digalloyl-glucoside, tetragalloyl-glucoside, and tetragalloylglucoside) and ellagitannins. By contrast, after the gastric step, increased values of flavonoids were noted for all of them mainly for naringenin, kaempferol, quercetin-rhamnoside, and myricetin-rhamnoside. Nevertheless, losses of flavonoids were observed after the intestinal phase, although they still maintained high release stability as compared to phenolic acids. The release of phenolic compounds from the microcapsules was performed during the IVGID process. The release of individual phenolics during digestion differs from one compound to another; some of them are well released after the gastric phase in acidic medium, such as gallic and pcoumaric acids, and some flavonoids (naringenin, quercetinrhamnoside, and myricetin-rhamnoside). Other phenolics were well released in neutral medium after oral and intestinal phases. That could be due to the pH changes through the IVGID phase: mouth (pH ≈ 7), stomach (pH ≈ 3), and small intestine (pH ≈ 7), which may cause appreciable alterations in the structure and physicochemical properties of the bioactive compounds.8 It is worth mentioning that the percentage of phenolic compounds is lower in encapsulated samples than in nonencapsulated ones, and remained fairly more constant along different gastrointestinal digestion steps. These results were also expected, because the starting amount of phenolic in encapsulated samples was lower (encapsulated efficiency and amount of polymer should be taken into account). The properties of encapsulating polymer play a major role in enhancing the encapsulation efficiency and controlled release of the core material. Some phenolics were slowly released from the microcapsules and need more time than 2 h in the intestinal phase (neutral medium) to be completely released, after a complete degradation of the polymer. Correlation between Total Phenolics, Total Flavonoids, and Antioxidant Activities. It is known that the Folin−Ciocalteu reagent interacts with numerous reducing nonphenolic substances such as sugars, amino acids, vitamin C, and other organic acids, which thus may lead to overestimation of TPC. Therefore, the higher antioxidant activity cannot always be attributed to the higher TPC in the samples. The correlation between antioxidant capacities (ORAC, FRAP, and DPPH) and the TPC and TFC of the samples was calculated,

activities, whereas at lower pH others have exhibited antioxidant activities. Otherwise, both nonencapsulated extracts revealed significant differences (p < 0.05) on DPPH radical scavenging activity. This last assay was more important in the gastric medium. For the encapsulated extracts, a strong activity was observed in intestinal medium. Many reports studying the effect of IVGID on DPPH scavenging activity informed that the gastric phase affected strongly the DPPH assay. Indeed, Chen et al.36 and Correa-Betanzo et al.17 mentioned that DPPH scavenging values increased significantly (P < 0.05) after the gastric phase of digestion for fruit seeds and blueberry extracts, respectively. According to Gullon et al.,18 the increment in antioxidant activity could be attributed to higher release of bioactive compounds, with scavenging properties, from the samples under the acidic conditions of gastric digestion. Furthermore, Rice-Evans et al.38 reported that the chemical structure of phenolics also plays a role in the free radical-scavenging activity, which is mainly dependent on the number and position of hydrogen-donating hydroxyl groups on the aromatic rings of the phenolic molecules. In this sense, it is well-known that the aglycone compounds possess higher antioxidant power than their glycosides. The pH of a substance is known to affect the racemization of molecules, which probably creates two chiral enantiomers with different biological reactivities. Thereby, during the digestive process, antioxidant compounds could be more reactive particularly at acidic pH (gastric medium) than at neutral pH (intestinal medium), as racemization could increase with pH in other compounds.39 Under the intestinal conditions, the decrease in antioxidant activity (ORAC, FRAP, and DPPH), for nonencapsulated extracts, might be attributed to the lower TPC and TFC and/or to the fact that some phenolics could be transformed into different structural forms with other chemical proprieties due to their sensitivity to the neutral pH.40 The highest antioxidant activities (ORAC, FRAP, and DPPH), for encapsulated extracts, in the intestinal phase, could be explained by the highest phenolics and flavonoids amount released from the microcapsules via the degradation of PCL polymer at neutral pH (7). The weak activities recorded in oral and gastric phases of digestion may be due to the low phenolic compounds content at these levels, which were released from the microcapsules surface and/or via the penetration of salivary and gastric fluids into the microcapsules through their surface pores. HPLC−MS/MS Analysis. Table 3 summarized the HPLC− MS/MS phenolic profiles of encapsulated and nonencapsulated carob pulp extracts along IVGID. Five free phenolic acids and five flavonoids were identified and semiquantified after each digestion phase in both unripe and ripe carob pulp extracts as well as in both encapsulated and nonencapsulated samples. These results are in agreement with those reported by Ortega et al.21 on the analysis of phenolic profiles of carob flour by UPLC−MS/MS, after the simulated gastrointestinal digestion. The results obtained showed also that the 10 phenolic compounds were released from the microcapsules after the digestion process, which means that these phenolics are well microencapsulated by the PCL coating. The phenolics presented different behaviors during simulated digestion. The analysis of phenolics release of nonencapsulated carob pulp extracts during gastrointestinal digestion showed high stability for the main phenolic acids (gallic, p-coumaric, G


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Table 4. Correlation Coefficients between Total Phenolic Content (TPC), Total Flavonoid Content (TFC), and Antioxidant Activities (ORAC, FRAP, DPPH)a ripe carob pulp

unripe carob pulp

nonencapsulated

TPC-ORAC TPC-FRAP TPC-DPPH TFC-ORAC TFC-FRAP TFC-DPPH a

encapsulated

raw material

oral

gastric

intestinal

oral

gastric

0.43 0.54 0.99* 0.48 0.94* 0.70

0.99* 0.89* 1.00* 0.58 0.94 0.64

0.91* 0.64 0.94* 1.00* 0.86* 1.00*

0.63 1.00* 0.96* 0.97* 0.43 0.41

0.70 0.41 0.97* 0.42 0.88* 0.61

0.53 0.79 0.71 0.96 0.69 0.63

nonencapsulated

intestinal

oral

gastric

intestinal

oral

gastric

intestinal

1.00* 0.85* 0.99* 0.96* 0.97* 0.89*

0.75 0.98* 0.82* 0.62 0.95* 0.49

0.77 0.80 0.96* 1.00* 0.30 0.61

0.76 1.00* 1.00* 1.00* 0.76 0.72

0.75 0.53 0.65 0.61 0.45 0.55

0.94* 0.64 0.54 0.73 1.00* 0.60

0.98* 0.72 0.60 0.51 1.00* 0.79

0.80* 0.96* 0.89* 0.82* 0.99* 0.84*

*The correlation coefficients are significant at p < 0.05.

■

ABBREVIATIONS USED SEM, scanning electron microscopy; FTIR, Fourier transform infrared; IVGID, in vitro gastrointestinal digestion; ORAC, oxygen radical absorbance capacity; FRAP, ferric-reducing antioxidant power; DPPH, 2,2-diphenyl-1-picrylhydrazyl radical; HPLC−MS, high performance liquid chromatography− mass spectrometry

along IVGID. As shown in Table 4, the TPC and TFC exhibited positive relationships with the antioxidant activities tested. This result was in concordance with the findings of Chen et al.36 Before IVGID, TPC and TFC were significantly (p < 0.05) correlated with DPPH and FRAP, respectively, for both unripe and ripe pulp extracts, whereas the correlation coefficients between ORAC and TPC and TFC were not significant (p < 0.05). On the other hand, after gastric digestion, all activities were significantly (p < 0.05) correlated with TFC and TPC for ripe and unripe pulp extracts, respectively. For encapsulated extracts, all activities were significantly (p < 0.05) correlated and exhibited a positive relationship with TPC and TFC, after the intestinal digestion. Thus, these results indicated that phenolic compounds widely contributed to the antioxidant activity of carob fruit. Otherwise, a weak correlation can be attributed to the presence of other nonphenolic compounds, which contributed to the antioxidant capacity of the samples. Furthermore, the antioxidant activity of phenolics is essentially dependent on their molecular structure. It was shown that the CHCH−COOH grouping of the hydroxycinnamic acids ensures a great capacity to transfer a proton and to stabilize later the radicals than the carboxyl (COOH) grouping of the hydroxybenzoic acids. Besides, the flavonoids alones, with some structures, can act as donors of protons or electrons, which lead to a good correlation with them.38 Moreover, the extracts are very complex mixtures of many different compounds with distinct activities. The difference in activity can be due to the synergistic or antagonist effects of these compounds.

■

encapsulated

raw material

■

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +34 868887985. Fax: +34 868887167. E-mail: [email protected]. ORCID

Carmen Frontela-Saseta: 0000-0002-6383-5713 Funding

The present research was performed under the financial support of the Algerian Ministry of Higher Education and Scientific Research in collaboration with the Human Nutrition and Food Science Research Group (NUTBRO) of the University of Murcia (Spain) (E098-02) and Ministerio de Economı ́a y Competitividad (Spain) through the Project AGL2016-78125-R. Notes

The authors declare no competing financial interest. H


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Effect of in Vitro Gastrointestinal Digestion on Encapsulated and

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