Characterization and Influence of Static In Vitro Digestion on Bioaccessibility of Bioactive Polyphenols from an Olive Leaf Extract

  Citation: Duque-Soto, C.; Quirantes-Piné, R.; Borrás-Linares, I.; Segura-Carretero, A.; Lozano-Sánchez, J. Characterization and Influence of Static In Vitro Digestion on Bioaccessibility of Bioactive Polyphenols from an Olive Leaf Extract. Foods 2022,11, 743. https://doi.org/10.3390/ foods11050743 Academic Editor: Angela Conte Received: 9 February 2022 Accepted: 1 March 2022 Published: 3 March 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). foods Article Characterization and Influence of Static In Vitro Digestion on Bioaccessibility of Bioactive Polyphenols from an Olive Leaf Extract Carmen Duque-Soto 1,2, Rosa Quirantes-Piné1, Isabel Borrás-Linares 1,3,* , Antonio Segura-Carretero 3,† and Jesús Lozano-Sánchez 1,2,† 1 Research and Development Functional Food Centre (CIDAF), Health Science Technological Park, Avenida del Conocimiento 37, Edificio BioRegión, 18016 Granada, Spain; [email protected].es (C.D.-S.); [email protected] (R.Q.-P.); [email protected] (J.L.-S.) 2Department of Food Science and Nutrition, University of Granada, Campus Universitario s/n, 18071 Granada, Spain 3Department of Analytical Chemistry, Faculty of Sciences, Avda Fuentenueva s/n, University of Granada, 18071 Granada, Spain; [email protected] *Correspondence: [email protected]; Tel.: +34-958-637-206 † These authors contributed equally to this work. Abstract: Olive leaves, one of the most abundant olive production by-products, have shown incredible potential for their characteristic bioactive compound composition, with unique compounds such as the polyphenol oleuropein. In order to evaluate the bioaccessibility of bioactive compounds present in an olive leaf extract, samples were submitted to an in vitro digestion process following INFOGEST protocol, and qualitative and quantitative characterization of the original extract and digestive samples at different times were carried out using HPLC-ESI-TOF-MS. The analyzed extract presented an abundance of phenolic compounds, such as secoiridoids, with oleuropein being the main identified compound. The in vitro digestion process showed an effect on the phenolic profile of the extract, with a lower recovery in the gastric phase and an increase at the beginning of the intestinal phase. Most of the studied compounds showed high bioaccessibility at the end of the digestion, with oleuropein, ligstroside, and quercetin-3-O-galactoside being among the ones with higher value. These findings show the potential for future use of olive leaf polyphenols. However, further research is needed in order to evaluate the absorption, delivery, and interaction of these compounds with the colon. Keywords: olive leaf extract; polyphenols; HPLC; in vitro digestion; bioaccessibility 1. Introduction The olive tree (Olea europaea L.) has been cultivated for centuries in the Mediterranean for the production of some of its most renowned products: oil and table olives. This area holds 98% of the crop area and 97% of the global production, with Spain being the first producing country [ 1 , 2 ]. As a result, abundant quantities of associated residues are produced. Only in Spain, 1–5 t/ha of the pruning residue in the form of leaves and branches are generated [ 1 , 3 ]. Its elimination has traditionally consisted of the grinding and burning of these tree-by-products. However, the environmental implications of these processes have led to an interest in the search for new innovative processing alternatives. In this respect, their reutilization has been proposed, as a way of propelling the circular economy and the development of products with high added-value. As some of the most abundant by-products, the use of olive leaves for industrial purposes has been considered an innovative alternative, as this matrix poses as an interesting source of chemical compounds of great industrial potential. These structures Foods 2022,11, 743. https://doi.org/10.3390/foods11050743 https://www.mdpi.com/journal/foods Foods 2022,11, 743 2 of 20 present a wide variety of compounds ranging from mannitol, widely used as a sweetener for sugar-free products, to lignocellulosic compounds. In particular, there is a rising interest in their bioactive composition, from which phenolic compounds are the most important constituents. Phenolic compounds are an extensive and heterogeneous group of molecules deriving from plants’ secondary metabolism, which have gained great interest in the scientific community in recent years. These molecules have demonstrated varied biological activities such as the antioxidant, anti-inflammatory, and antihypertensive effects related to some of the health benefits associated with olive leaves [ 4 ]. In this sense, olive leaf extracts rich in phenolic compounds are being studied for the treatment of diverse infections and as astringent and antiseptic agents [ 4 – 6 ]. Even though they have been considered for their therapeutic applications, the mentioned extracts could also be of great relevance for the food industry. Some of their pondered uses are in the production of functional foods or even as additives for the improvement of products’ shelf life, due to their natural action as antioxidants and preservatives. In this sense, they are already being evaluated for improving the oxidative stability of dietary oils [7]. Phenolic content in the olive tree may vary between different tree structures. Previous studies have shown a significantly higher concentration of these compounds in leaves than in other parts of the tree, which makes their extraction from this matrix of great interest to industry [ 7 , 8 ]. Due to their bioactive composition and its potential use, obtaining phenolic rich extracts from olive leaves and its conversion into high added-value ingredients for the food industry would suppose the promotion of a sustainable processing method of these olive by-products, generating natural nutritional products with minimal residue production, thus improving, and potentiating the circular economy. However, in order to efficiently implement these extracts for their potential functional use, a wider knowledge of their chemical composition, biological activity, and behavior under gastrointestinal conditions is needed. The most popular technique for studying the phenolic profile of olive leaves in literature has been High Pressure Liquid Chromatography (HPLC) mainly coupled to mass spectrometry (MS). The bioactive compound profile of olive leaves is quite diverse, with oleuropein, a secoiridoid exclusive from the Oleaceae family, which represents from 60 to 90 mg/g of dry weight of leaves, being one of the most abundant [ 5 , 8 ]. Even though the isolation of single compounds could be considered, evidence on the synergistic effect of the interaction between polyphenols on its bioactivity shows a greater interest on the use of their corresponding extracts [6]. However, there still are challenges to its application in the food industry. As it is usually orally administrated, one of the most prominent challenges is the stability of the molecules of interest through the digestive tract, which influences its potential absorption and, therefore, hinders the accomplishment of their biological activity. In order to properly address this issue, the bioaccessibility and bioavailability of the compounds of interest must be considered, as those molecules need to reach their target areas before exerting their beneficial activity. Polyphenols are labile compounds, sensitive to both light and high temperatures, as well as rapidly metabolized and eliminated from the body. Thus, digestive conditions may contribute to a reduction in stability and directly affect their effectiveness, lowering their bioaccessibility and diminishing their absorption [ 7 , 9 , 10 ]. Additionally, absorption can also depend on their chemical structure, as some molecules might require previous metabolization in order to be accessible [ 4 , 7 , 11 – 13 ]. All these factors may affect and produce a different phenolic profile available for absorption than that observed in the olive leaf extract and should be studied in detail. In order to study the bioaccessibility and bioavailability of phenolic compounds as influenced by the digestive process, in vivo approaches have been considered. Even though it seems to be a closer representation of the digestive process, there are disadvantages to their use, as a wide variety of parameters can alter the results, such as gender, age, or diverse disorders and physiological alterations. These models are also complex, expensive, and require extended periods of time [ 13 , 14 ]. These facts can also suppose an issue for Foods 2022,11, 743 3 of 20 the accurate and adequate interpretation of the bioaccessibility results, hindering the comparison between studies. On the other hand, in vitro models have also been developed in an effort to reproduce the digestive process as close as possible. However, one of the main challenges is the variety of models developed to date. The variety and range of models and conditions considered hinder the accurate comparison between different studies. In order to solve these difficulties, the INFOGEST protocol was developed as the result of more than 2 years of discussion between multidisciplinary researchers to get a standardized and harmonized protocol of static in vitro digestion which allows comparison between studies. It applies constant ratios between food and digestive fluids at a constant pH for each digestive phase. Food samples are submitted to a sequential digestion consisting of oral, gastric, and intestinal phases, maintaining constants parameters such as electrolytes, concentration and enzymatic activity, bile, dilution, pH, and digestion time, based on physiological data and evidence [15]. Recently, a revision on this method has been published, called INFOGEST 2.0, which considers the possible problems associated with the original method, including an oral phase or the use of gastric lipase. This method also gathers some modifications for the study of the liberation of micronutrients during digestion. As for the evaluation of bioaccessibility of phytochemicals such as polyphenols, this model permits the realistic liberation of the compound to an aqueous phase [ 16 ]. However, in vitro digestion studies of polyphenolrich olive leaf extracts seem to be scarce, and those available only monitor very few of these compounds. Additionally, as far as we are concerned, this study is one of the first to apply this method to olive-leaf extract. Therefore, the aim of this study is the characterization of an olive leaf extract and the evaluation of the effect that the INFOGEST in vitro digestive protocol had on its polyphenolic profile, with the intention of identifying the resulting metabolites and the compounds available after the gastrointestinal process in order to determine their bioaccessibility. 2. Materials and Methods 2.1. Chemicals All chemicals were of analytical reagent grade and used as received. Bovine bile salts (Sigma B-8631) and enzymes for in vitro digestion (pepsin 3412 U/mg protein and pancreatin 4xUSP) were obtained from Sigma Aldrich (Saint Louis, MO, USA). Sodium hydroxide (NaOH), hydrochloric acid (HCl), potassium chloride (KCl), potassium dihydrogen phosphate (KH 2 PO 4 ), sodium hydrogen carbonate (NaHCO 3 ), sodium chloride (NaCl), and ammonium carbonate ([(NH 4 ) 2 CO 3 ]) that were used to prepare the simulated digestive fluids were obtained from Fisher Chemicals (Waltham, MA, USA). HPLC–MS grade acetonitrile and formic acid were purchased from Fisher (Thermo Fisher Scientific, Leicestershire, UK ). Standards of luteolin-7-O-glucoside (purity ≥ 98%), verbascoside ( purity ≥99% ), and loganin (purity ≥ 97.0%) were purchased from Sigma Aldrich, oleuropein (purity ≥ 98%) was purchased from Extrasynthese (Lyon, France), and hydroxytyrosol (purity ≥ 98%) was purchased from Cayman Chemical (Ann Arbor, MI, USA). Distilled water with a resistance of 18.2 MΩwas deionized in a Milli-Q system (Bedford, MA, USA). 2.2. Plant Material Commercial olive leaf extract was provided by NATAC S.L., and it was obtained from the solid-liquid extraction of grinded leaves, with 80% ethanol as the extraction solvent with a solvent to sample ratio of 20:1, a temperature of 45 ◦ C, and an extraction time of 2 h. 2.3. Static In Vitro Digestion INFOGEST Static in vitro gastrointestinal digestion was performed following the INFOGEST 2.0 protocol described by Minekus et al. [ 15 ], taking into consideration the modifications provided by Brodkorb et al. [ 16 ] for the study of phenolic compounds, as well as the nature of the samples of study. The following process was carried out as a triplicate for each Foods 2022,11, 743 4 of 20 sample. In order to replicate oral digestion, 5 g of commercial olive-leaf dry extract were resuspended in 5 mL (1:1, w/v) of Simulated Salivary Fluid (SSF) in a 50 mL Falcon tube. This was stirred for 5 min, protecting the resultant mixture from light. For the gastric phase simulation, the bolus was mixed with 7.5 mL of Simulated Gastric Fluid (SGF), 2000 U/mL of pepsin, and 5 µ L of CaCl 2 0.3 M. The pH was adjusted to 3.0, adding the necessary volumes of 1 M HCl. The final volume for this step was adjusted to 18 mL by addition of MilliQ H 2 O. The mixture was homogenized and inertized with N 2 . The gastric phase was carried out for 2 h at 37 ◦ C under constant agitation at 150 rpm using a refrigerated incubator (MaxQTM 6000 SHKE6000-8CE, Thermo Scientific, Waltham, MA, USA). An aliquot of 1 mL was recovered and stored in an Eppendorf tube at the end of the present phase, labelled as GP. For preparing the intestinal phase, 9.8 mL of Simulated Intestinal Fluid (SIF), 100 U/mL of pancreatin, 2.5 mL of bile, and 40 µ L of CaCl 2 0.3 M were added to the existent simulated chyme. Then, the pH was fixed to 7.0, adding the required volumes of 1 M NaOH, and MilliQ H 2 O was added to achieve a final volume of 40 mL. This was homogenized and inertized with N 2 . The intestinal phase was carried out for 2 h at 37 ◦ C under conditions of constant agitation at 150 rpm using a refrigerated incubator (MaxQTM 6000 SHKE60008CE, Thermo Scientific, Waltham, MA, USA). Aliquots of 1 mL were recovered at 30 min intervals and stored in an Eppendorf tube, labelled as IP1, IP2, IP3, and IP4. For pH controlling purposes, through both the gastric and intestinal phases, pH measurements were at 30 min intervals, adjusting the value to 3.0 and 7.0, respectively, when necessary. Sample tubes were stored at − 80 ◦ C until further use. This process was carried out as a triplicate. 2.4. Bioaccessibility Bioaccessibility, corresponding to the fraction of phenolic compounds freed from its food matrix into the gastrointestinal tract and, therefore, accessible for intestinal absorption, has been calculated using Equation (1) [ 17 ]. For each digestive phase, the accumulative presence of these compounds in the bioaccessible fraction was expressed as a percentage of the initial phenolic content, that is, according to the initial composition of the extract, using Equation (2) [ 18 ]. In this case, recovery at 240 min constitutes the final bioaccessibillity of each considered compound. In order to determine the initial phenolic content of the original extract to calculate the Bioaccessibility (Equation (1)), 5 g of extract was resuspended in the final volume of the intestinal phase (18 mL) and subjected to the sample treatment described for the bioaccessible fractions (Section 2.3): Bioaccessibility (%)=PC content in IP4 (mg) Initial PC content (mg)×100% (1) Recovery (%)=PC content in DS (mg) Initial PC content (mg)×100% (2) where PC is the phenolic compounds; IP4 is the final aliquot of the intestinal phase; DS is the digested samples; and Initial PC content refers to the presence of phenolic compounds in the olive leaf extract. 2.5. Bioactive Compound Extraction Digested samples stored at − 80 ◦ C were processed before their characterization. For this purpose, samples stored in the Eppendorf tubes were defrosted in ice for 2 h and those in the Falcon tubes were stored overnight in the refrigerator. Samples GP, IP1, IP2, IP3, and IP4 (Gastric Phase and Intestinal Phase 1–4 taken at 150, 180, 210, and 240 min, respectively) were then homogenized and centrifuged at 14,800 rpm, 10 min, and 4 ◦ C, conserving both bioaccessible (supernatants) and residual fractions (pellets). For the extraction of phenolic compounds from the bioaccessible fraction, 200 µ L were added to 100 µ L of MeOH:EtOh 50:50 (v/v), agitated in vortex, and maintained at Foods 2022,11, 743 5 of 20 −20 ◦C for 30 min in order to precipitate the proteins. Then, the samples were centrifuged at 14,800 rpm, 10 min, and 4 ◦ C, and the supernatants were evaporated in a vacuum concentrator for 4–5 h, later stored at − 20 ◦ C. Before characterization, 100 µ L MeOH was added, and the resultant was homogenized in a refrigerated ultrasound bath for 4 h. After that, the processed digested samples were centrifuged under the previous conditions and the supernatants were introduced in HPLC vials for their later analysis. As for the residual fraction, 1 mL of MeOH was added to 100 mg of residue, then homogenized and introduced in a refrigerated ultrasound bath for 15 min. Then, it was introduced into an incubator with agitation at 4 ◦ C and centrifuged at 14,800 rpm, 10 min, and 4 ◦ C. The supernatants were then evaporated in a vacuum concentrator for 2–3 h and stored at − 20 ◦ C. Before analysis, the samples were resuspended in a MeOH volume in order to achieve a concentration of 500 µ g/mL, with the aid of a refrigerated ultrasound bath. Then, the processed residual fractions were centrifuged under the aforementioned conditions and the supernatants, diluted when necessary, were introduced in HPLC vials for their later analysis. 2.6. Characterization of Phenolic Compounds For the characterization of the samples, stock solutions of 1 mg/mL in methanol were prepared from the following commercial compounds: hydroxytyrosol, oleuropein, verbascoside, luteolin-7-O-glucoside, and loganin. These solutions were filtrated using regenerated cellulose filters of 0.45 µ m pore diameter and stored at − 20 ◦ C in amber screw cap bottles. Calibration curves with concentrations 1, 10, 25, 50, 100, and 150 µ g/mL (hydroxytyrosol and oleuropein) and 1, 5, 10, 20, 30, and 40 µ g/mL (verbascoside, luteolin7-O-glucoside, and loganin) were prepared and analyzed as a triplicate. Analyses were made using an Agilent 1200 Liquid Chromatography system (Agilent Technologies, Palo Alto, CA, USA) equipped with a micro vacuum degasser, binary pump, autosampler, thermostated column compartment, and diode array detector. The HPLC column used was an Agilent Zorbax Eclipse Plus C18 (1.8 µ m, 4.6 × 150 mm). The mobile phases consisted of water plus 0.5% acetic acid (A) and acetonitrile (B). The multistep linear gradient applied was the following: 0 min, 5% B; 2 min, 30% B; 25 min, 95% B; 30 min, 95% B; and 42 min, 5% B. Then, the initial conditions were maintained for 3 min. The flow was 0.5 mL/min, temperature was maintained fixed at 30 ◦ C, and injection volume in the HPLC system was 5 µL. The HPLC system was coupled to a microTOF-Q II mass spectrometer (Bruker Daltoniks, Bremen, Germany) equipped with an ESI interface (Agilent Technologies, Palo Alto, CA, USA) operating in negative ion mode, in a mass range of 50–1000 m/z. Nitrogen was used as nebulizing/ionizing and drying gas at conditions of 2 bar and 10 L/min, respectively. Drying temperature was set at 190 ◦ C, capillary voltage of +4 kV, and End Plate Offset at − 500 V. Other optimum values for parameters were output voltage, 120 V; Skimmer 1, 40 V; Hexapole 1, 23 V; Hexapole RF, 100 Vpp; skimmer 2, 22.5 V; Lens 1 transfer, 50 µs; and Lens 1 Pre-Pulse Storage, 3 µs. In order to recalibrate the mass spectra obtained during analysis to achieve a mass precision of 5 ppm, 5 mM sodium formate was used as a calibration agent at the beginning of each analysis, with an m/zrange of 50–1200 Da. 2.7. Data Processing For the phenolic compounds’ characterization, ion mass data were processed in the software DataAnalysis 4.0 (Bruker Daltoniks, Bremen, Germany), creating a molecular formulae list of the analyzed substances with a tolerance error of 2 ppm. Identification was carried out by comparison with literature and personal databases of phenolic compounds present in olive leaf, allowing for the identification of most of the compounds. As for the quantification of both the extract and the different digested samples, chromatograms were also processed in DataAnalysis 4.0, where areas under each peak were calculated. Analyses were carried out in triplicate for each sample. Later, an adequate Foods 2022,11, 743 6 of 20 standard was selected for each compound according to their structural similarity, and its concentration was calculated by the interpolation of peak area detected in the replicate analysis of the samples in the calibration curve of the selected surrogate standard. Phenolic content in mg was calculated for each sample replicate and the mean concentration as well as statistic deviation was obtained. The quantitative content as well as the selected commercial standard for each identified compound are summarized in Supplementary Table S2 for bioaccesssible fractions and in Supplementary Table S3 for residual fractions. 3. Results 3.1. Olive Leaf Extract Characterization Previous to the evaluation of the influence that the gastrointestinal conditions had on its chemical profile, it is necessary to evaluate the characterization of the extract of study, identifying all previous phenolic compounds of interest. Characterization of the olive leaf extract was carried out by HPLC-ESI-TOF-MS. As can be observed in Figure 1, the base peak chromatogram of this extract presented a high complexity. Therefore, major peaks were selected for their tentative identification, with a total of 74 compounds considered. Numbers were given according to retention time where, due to the nature of the chromatographic column, polar analytes were eluted at low retention times. Identification was carried out by the comparison of retention times, m/zvalues, and molecular formulae proposed, and MS spectra provided by TOF-MS, with data provided by previous literature and personal databases of the phenolic compounds in olive leaf. Table 1summarizes the MS data of the identified compounds, including the retention time, experimental and calculated m/zfor the molecular formulae provided for each [M − H] − , and error, sigma value, and name of the proposed compound for each peak. MiliSigma value (mSigma) is a numeric value which indicates the level of similitude between theorical and experimental isotopic distributions, where a low value indicates statistical similitude. Its tolerance is normally established at 50, although factors such as coeluting analytes or compounds from the matrix could lead to higher values [ 19 ]. In this case, due to the complexity of the sample, some compounds achieved mSigma values higher than 50. Foods 2022, 11, x FOR PEER REVIEW 6 of 21 2.7. Data Processing For the phenolic compounds’ characterization, ion mass data were processed in the software DataAnalysis 4.0 (Bruker Daltoniks, Bremen, Germany), creating a molecular formulae list of the analyzed substances with a tolerance error of 2 ppm. Identification was carried out by comparison with literature and personal databases of phenolic compounds present in olive leaf, allowing for the identification of most of the compounds. As for the quantification of both the extract and the different digested samples, chromatograms were also processed in DataAnalysis 4.0, where areas under each peak were calculated. Analyses were carried out in triplicate for each sample. Later, an adequate standard was selected for each compound according to their structural similarity, and its concentration was calculated by the interpolation of peak area detected in the replicate analysis of the samples in the calibration curve of the selected surrogate standard. Phenolic content in mg was calculated for each sample replicate and the mean concentration as well as statistic deviation was obtained. The quantitative content as well as the selected commercial standard for each identified compound are summarized in Supplementary Table S2 for bioaccesssible fractions and in Supplementary Table S3 for residual fractions. 3. Results 3.1. Olive Leaf Extract Characterization Previous to the evaluation of the influence that the gastrointestinal conditions had on its chemical profile, it is necessary to evaluate the characterization of the extract of study, identifying all previous phenolic compounds of interest. Characterization of the olive leaf extract was carried out by HPLC-ESI-TOF-MS. As can be observed in Figure 1, the base peak chromatogram of this extract presented a high complexity. Therefore, major peaks were selected for their tentative identification, with a total of 74 compounds considered. Numbers were given according to retention time where, due to the nature of the chromatographic column, polar analytes were eluted at low retention times. 1 2 3 4 5 6 7 8910 29 30 31 32 33 34 3536 46 47 48 49 56 57 58 59 606162 63 64 6665 67 68 69 70 7172 73 74 a. b. c. Figure 1. Cont. Foods 2022,11, 743 7 of 20 Foods 2022, 11, x FOR PEER REVIEW 7 of 21 Figure 1. Base peak chromatogram (BPC, 50–1000 m/z) of the olive leaf extract, indicating zoomed zones (a–c). Identification was carried out by the comparison of retention times, m/z values, and molecular formulae proposed, and MS spectra provided by TOF-MS, with data provided by previous literature and personal databases of the phenolic compounds in olive leaf. Table 1 summarizes the MS data of the identified compounds, including the retention time, experimental and calculated m/z for the molecular formulae provided for each [M−H]-, and error, sigma value, and name of the proposed compound for each peak. MiliSigma value (mSigma) is a numeric value which indicates the level of similitude between theorical and experimental isotopic distributions, where a low value indicates statistical similitude. Its tolerance is normally established at 50, although factors such as coeluting analytes or compounds from the matrix could lead to higher values [19]. In this case, due to the complexity of the sample, some compounds achieved mSigma values higher than 50. Table 1. Identified compounds in olive leaf extract. Peak RT (min) Theoretical m/z Formula Measured m/z Error (ppm) mSigma Proposed Compound 1 3.01 181.0732 C6H13O6 181.0718 −7.7 1.39 Sorbitol isomer 1 2 3.06 195.0528 C6H11O7 195.0510 −8.8 2.08 Glucuronic acid 3 3.30 191.0601 C7H11O6 191.0561 −20.7 25.67 Quinic acid 4 4.03 191.0224 C6H7O7 191.0197 −13.9 4.05 Citric acid isomer 1 5 4.40 421.1346 C17H25O12 421.1351 1.2 5.82 Unknown 6 4.63 191.0206 C6H7O7 191.0197 −4.8 2.02 Citric acid isomer 2 7 5.17 151.0400 C8H7O3 151.0401 0.3 18.49 Hydroxyphenylacetic acid/Vainillin 8 5.34 295.1029 C11H19O9 295.1035 1.9 24.55 Rhamnosylarabinose isomer 1 9 5.92 181.0718 C6H13O6 181.0718 −0.4 5.83 Sorbitol isomer 2 10 6.22 375.1289 C16H23O10 375.1297 2.0 14.10 Loganic/epiloganic acid isomer 1 11 6.57 295.1036 C11H19O9 295.1035 −0.4 4.76 Rhamnosylarabinose isomer 2 12 6.64 375.1294 C16H23O10 375.1297 0.6 23.03 Loganic/epiloganic acid isomer 2 13 6.84 315.1086 C14H19O8 315.1085 −0.1 8.24 Hydroxytyrosol glucoside 14 7.15 389.1114 C16H21O11 389.1089 −6.3 10.00 Oleoside/Secologanoside 15 7.25 153.0581 C8H9O3 153.0557 −15.8 15.20 Hydroxytyrosol 7 8 9 10 11 12 13 14 Time [min] 0.0 0.2 0.4 0.6 0.8 6 x10 Intens. Extracto_concFG_59_01_18574.d: BPC 49.0004-1001.0182 - 11 12 13 15 14 17 16 18 1920 21 22 23 24 2526 27 28 37 38 39 4041 42 43 44 45 50 51 52 53 54 55 a. b. c. Figure 1. Base peak chromatogram (BPC, 50–1000 m/z) of the olive leaf extract, indicating zoomed zones (a–c). Table 1. Identified compounds in olive leaf extract. Peak RT (min) Theoretical m/z Formula Measured m/z Error (ppm) mSigma Proposed Compound 1 3.01 181.0732 C6H13O6181.0718 −7.7 1.39 Sorbitol isomer 1 2 3.06 195.0528 C6H11O7195.0510 −8.8 2.08 Glucuronic acid 3 3.30 191.0601 C7H11O6191.0561 −20.7 25.67 Quinic acid 4 4.03 191.0224 C6H7O7191.0197 −13.9 4.05 Citric acid isomer 1 5 4.40 421.1346 C17H25O12 421.1351 1.2 5.82 Unknown 6 4.63 191.0206 C6H7O7191.0197 −4.8 2.02 Citric acid isomer 2 7 5.17 151.0400 C8H7O3151.0401 0.3 18.49 Hydroxyphenylacetic acid/Vainillin 8 5.34 295.1029 C11H19O9295.1035 1.9 24.55 Rhamnosylarabinose isomer 1 9 5.92 181.0718 C6H13O6181.0718 −0.4 5.83 Sorbitol isomer 2 10 6.22 375.1289 C16H23O10 375.1297 2.0 14.10 Loganic/epiloganic acid isomer 1 11 6.57 295.1036 C11H19O9295.1035 −0.4 4.76 Rhamnosylarabinose isomer 2 12 6.64 375.1294 C16H23O10 375.1297 0.6 23.03 Loganic/epiloganic acid isomer 2 13 6.84 315.1086 C14H19O8315.1085 −0.1 8.24 Hydroxytyrosol glucoside 14 7.15 389.1114 C16H21O11 389.1089 −6.3 10.00 Oleoside/Secologanoside 15 7.25 153.0581 C8H9O3153.0557 −15.8 15.20 Hydroxytyrosol 16 7.40 377.1451 C16H25O10 377.1453 0.5 15.65 Lamiol 17 7.67 461.1671 C20H29O12 461.1664 −1.3 18.50 Diosmetin−7-glucoside 18 7.82 623.2006 C29H35O15 623.1981 −3.9 1.27 Verbascoside/Isoverbascoside 19 7.87 701.2309 C31H41O18 701.2298 −1.6 8.61 Oleuropein diglucoside 20 7.97 447.0939 C21H19O11 447.0933 −1.4 15.50 Luteolin-glucoside isomer 1 21 8.05 447.0930 C21H19O11 447.0933 0.5 6.84 Luteolin-glucoside isomer 2 22 8.15 555.1730 C18H35O14 555.1778 −1.4 15.96 Hydroxyoleuropein 23 8.27 333.1552 C15H25O8333.1555 0.7 12.65 Unknown 24 8.35 463.1475 C19H27O13 463.1457 −3.8 8.18 Quercetin-3-O-galactoside 25 8.55 447.0936 C21H19O11 447.0933 −0.8 11.33 Luteolin-glucoside isomer 3 26 8.67 541.1949 C25H33O13 541.1927 −4.2 8.90 Hydro-oleuropein 27 8.74 539.1784 C25H31O13 539.1770 −2.7 71.11 Oleuropein isomer 1 28 8.85 569.1879 C26H33O14 569.1876 −0.6 8.90 Metoxyoleuropein 29 9.07 539.1785 C25H31O13 539.1770 −1.6 24.76 Oleuropein Foods 2022,11, 743 8 of 20 Table 1. Cont. Peak RT (min) Theoretical m/z Formula Measured m/z Error (ppm) mSigma Proposed Compound 30 9.26 539.1779 C25H31O13 539.1770 1.3 4.37 Oleuropein isomer 2 31 9.56 601.2150 C27H37O15 601.2138 −2.1 16.07 Unknown 32 9.81 301.1293 C14H21O7301.1293 −0.2 5.40 Unknown 33 9.99 523.1840 C25H31O12 523.1821 −3.6 13.64 Ligstroside 34 10.18 377.1233 C19H21O8377.1242 2.2 24.41 Oleuropein aglycone isomer 1 35 10.41 241.0717 C11H13O6241.0718 0.5 1.50 Elenolic acid 36 10.68 377.1252 C19H21O8377.1242 −2.6 15.60 Oleuropein aglycone isomer 2 37 11.31 285.0395 C15H9O6285.0405 3.2 42.95 Luteolin 38 11.55 301.0355 C15H9O7301.0354 −0.6 2.56 Quercetin 39 11.66 377.1249 C19H21O8377.1242 −1.9 14.63 Oleuropein aglycone isomer 3 40 11.85 393.1188 C19H21O9393.1191 0.8 10.88 10-Hydroxyoleuropein aglycone 41 11.93 327.2167 C18H31O5327.2177 3.0 28.79 Unknown 42 12.08 327.2175 C18H31O5327.2177 0.5 4.41 Unknown 43 12.25 377.1241 C19H21O8377.1242 0.2 18.41 Oleuropein aglycone isomer 4 44 12.48 331.2487 C18H35O5331.2490 0.9 1.86 Trihydroxystearic acid 45 12.78 377.1237 C19H21O8377.1242 1.4 18.55 Oleuropein aglycone isomer 5 46 13.04 377.1249 C19H21O8377.1242 −1.9 14.33 Oleuropein aglycone isomer 6 47 14.11 377.1310 C19H21O8377.1336 6.9 7.27 Oleuropein aglycone isomer 7 48 14.59 377.1261 C19H21O8377.1242 −4.9 24.57 Oleuropein aglycone isomer 8 49 14.81 377.1248 C19H21O8377.1242 −3.5 21.14 Oleuropein aglycone isomer 9 50 14.97 309.2045 C18H29O4309.2071 8.4 22.65 13-hydroperoxide linolenic acid isomer 1 51 15.14 391.1380 C20H23O8391.1398 4.8 8.53 Methyl oleuropein aglycone isomer 1 52 15.31 457.2785 C31H37O3457.2748 −8.1 9.16 Unknown 53 15.46 345.1314 C19H21O6345.1344 8.6 28.93 Gibberellic acid 54 15.63 307.1893 C18H27O4307.1915 7.0 5.91 Phytoprostane 55 15.81 513.1740 C27H29O10 513.1766 5.1 7.91 Unknown 56 16.06 359.1123 C26H15O2359.1078 −12.5 40.60 Unknown 57 16.55 391.1390 C20H23O8391.1398 2.1 16.47 Methyl oleuropein aglycone isomer 2 58 16.95 361.1278 C19H21O7361.1293 4.0 8.96 Ligstroside aglycone 59 17.38 305.1739 C18H25O4305.1758 6.3 13.45 Unknown 60 17.95 721.3616 C34H57O16 721.3652 5.0 33.73 Unknown 61 18.15 405.1536 C21H25O8405.1555 4.7 7.18 Dimethyl oleuropein aglycone 62 18.52 309.2057 C18H29O4309.2071 4.8 7.78 13-Hydroperoxide linolenic acid isomer 2 63 19.14 485.3247 C30H45O5485.3272 7.6 85.38 Dihydroxy-oxooleanolic/hydroxyoleanenedoic 64 19.86 647.3260 C31H51O14 647.3284 3.8 2.18 Unknown 65 21.53 293.2145 C18H29O3293.2122 −7.9 47.09 Hydroxylindenic acid isomer 1 66 21.88 293.2149 C18H29O3293.2122 −9.1 43.73 Hydroxylindenic acid isomer 2 67 22.28 487.2910 C25H43O9487.2913 0.5 5.63 Unknown 68 22.55 425.3407 C29H45O2425.3425 4.2 15.02 7-Ketostigmasterol 69 23.02 291.1954 C18H27O3291.1966 4.0 35.63 Unknown 70 23.54 295.2281 C18H31O3295.2279 −0.8 5.42 Oxo-octadecanoic acid 71 24.22 469.3307 C30H45O4469.3323 3.4 57.77 11-Oxo-oleanolic acid 72 24.42 293.2115 C18H29O3293.2122 2.6 15.79 Hydroxylindenic acid isomer 3 73 24.84 293.2108 C18H29O3293.2122 4.8 1.13 Hydroxylindenic acid isomer 4 74 25.38 471.3456 C30H47O4471.3480 5.0 16.94 Masilinic acid Foods 2022,11, 743 9 of 20 As can be observed in Table 1, most of the identified compounds belonged to the category of phenolic compounds, such as secoirioids, flavonoids, and simple phenols. Secoiridoids. Olea europaea L. has presented an abundance of secoiridoids, such as oleosides, which are specific to this specie. In the extract of study, the most abundant compounds identified belonged to this class, as is the case for oleuropein ( compound 29 ), the most abundant and characteristic polyphenol found in this tree. This compound has been previously reported in literature as one of the main components of different tree structures, such as leaves, which is supported by this study, where it presented the most prominent peak by both intensity and area, which was later translated in a higher concentration [ 19 – 27 ]. Other isomers of oleuropein were also found at close retention times (compounds 27 and 30 ). The extracted ion chromatogram (EIC) for m/z701 presented a peak at retention time 7.85 ( 19 ), which has been identified as oleuropein diglucoside. Coinciding with the loss of two hexose residues (162 Da) in relation to the previous compound, 9 peaks were identified at m/z377 and proposed as oleuropein aglycone in different isomeric forms ( 34 , 36 , 39 , 43 , 45 – 49 ). Additionally, two other oleuropein derivates were proposed such as hydroxyoleuropein ( 22 ), hydro-oleuropein ( 26 ), metoxyoleuropein ( 28 ), 10-hydroxyoleuropein aglycone ( 40 ), methyl oleuropein aglycone ( 51 , 57 ), and dimethyl oleuropein aglycone ( 61 ). Identification of these compounds has been confirmed with previous literature for the olive leaf [ 27 , 28 ], where 7 isomers of oleuropein aglycone were found in olive extracts. Secoiridoids derivates from the structure of tyrosol have also been identified in this extract. A peak ( 33 ) with m/z523 in EIC was found whose molecular formula corresponds with ligstroside, as well as an ion in EIC m/z361 with ligstroside aglycone ( 58 ) [ 14 , 19 , 26 – 30 ]. Additionally, an [M-H] − ion with m/z389 ( 14 ) was assigned as oleoside or secologanoside, both of which have been previously identified in olive. Simple phenols. Different simple phenols have been found in this extract, which have also been previously observed in different parts of the olive tree. Compound 15 has been identified as hydroxytyrosol, with a peak corresponding to an [M-H] − ion with m/z153. It has also been found in its glucoside form, corresponding with compound 13 , with an [M-H]−ion with m/z315 [20–22,25,27]. Other simple phenols were identified in the present extract as verbascoside/ isoverbascoside (compound 18 ,m/z623) and hydroxyphenilacetic acid/vainillin (compound 7 ,m/z151) [ 14 , 19 , 29 , 31 – 33 ]. Peak 16 with m/z377 (retention time 7.4 min) has been proposed as lamiol. Flavonoids. Flavonoids are a characteristic group of compounds found in olivetree related structures. Compounds from this class have been proposed as diosmetin-7glucoside ( 17 ), three isomers of luteolin-glucoside ( 20 , 21 and 25 ), luteolin ( 37 ), quercetin-3O-galactoside (24), and quercetin (38) [34]. Other compounds. There have been other compounds tentatively identified in the extract. Among them, we can find maslinic acid ( 74 ), widely described in olive leaf and related extracts, as well as compounds 10 and 12 , proposed as isomers of loganic/epiloganic acid, and compound 35, identified as elenolic acid [35,36]. The [M-H] − ion with an m/z307 has been assigned as a phytoprostane, considered oxidative stress markers in superior plants [ 37 ]. Additionally, the peak found at EIC m/z 345 has been proposed as giberelic acid (53), a hormonal diterpene [38]. There have also been found oleanolic acid derivatives such as dihydroxy-oxo-oleanolic acid ( 63 ) and 11-oxooleanolic acid ( 71 ). The [M-H] − ion with a 425 ( 68 ) has been proposed as 7-ketostigmasterol, a phytosterol, m/z295 ( 70 ) as oxo-octadecenoic acid, and m/z293 as diverse isomers of hydroxylindenic acid (65,66,72,73). Carbohydrates and organic acids, such as sorbitol ( 1 , 9 ), glucuronic acid ( 2 ), citric acid ( 4 , 6 ), quinic acid ( 3 ), and rhamnosylarabinose ( 8 , 11 ), were proposed as compounds found at lower retention times. These are compounds found extensively in vegetal organisms, functioning as carbohydrate sources as well as metabolic intermediates, as citric acid is Foods 2022,11, 743 16 of 20 activity was observed in the extract after the gastric phase, related to a higher presence of polyphenols, reaching even higher values than before the digestion process. Oleuropein, verbascoside, and luteolin-7-O-glucoside standards submitted to the digestive process showed a decrease through digestion time. This can prove the importance that the interaction between polyphenols and compounds derived from their natural matrix has and their role in bioaccessibility. Degradation of these complex structures during the intestinal phase can be explained through the high sensibility of dietary polyphenols to these conditions, with a higher pH than the gastric phase [ 48 ]. This would mean the degradation of complex molecules, probably associated with polysaccharides or proteins such as enzymes, allowing the increase of their free forms, in this case detectable and, therefore, justifying that high concentration. The sustaining of those conditions could also lead to a periodical degradation of free forms, as reflected in the evolution shown in the graphics. As has been observed, some compounds present a different behavior. Oleuropein aglycone decrease and 10-hydroxyoleuropein aglycone increase could be related as a result of intestinal chemical reactions that benefit the hydroxylation of the first generating the second, which establishes the instability of oleuropein aglycone under intestinal conditions. The low recovery percentage of quercetin and luteolin could be related to the low solubility of flavonoids in aqueous media and their high stability. Oleuropein being an ester of both elenolic acid and hydroxytyrosol is interesting in the evaluation of these degradation metabolites. In the digested samples, both hydroxytyrosol and its glycosylated form are found, showing a similar behavior as described. On the other hand, elenolic acid appears to decrease significantly at the beginning of the intestinal phase, being relatively stable during the rest of the digestion process. Oleuropein and derivatives reduction could be translated into an increase in both hydroxytyrosol and elenolic acid. However, the decrease of the latter could be indicative of instability of these compounds, which under intestinal conditions appears to suffer an intense degradation into other unknown or undetected molecules, modulating its bioaccessibility. As for their bioaccessibility, due to the lack in literature regarding in vitro digestion of olive leaf extracts, these results have also been compared with those on other matrixes. Results from the previous study contrast with those presented by González et al. (2019) for an olive leaf extract, where bioaccessibility of oleuropein was lower than 50%, even though secoiridoids reached more than 300%. In López de Lacey et al. (2012), where bioaccessibility of quercetin-3-O-galactoside was evaluated in green tea extracts, the values were lower than ours, at 60%. For the rest of the phenolic compounds of study, this value is relatively high, i.e., above 50%, showing a great amount of them that can be absorbed, or at least be in the zone of absorption. Therefore, most of the phenolic content present in the extract can reach this area, being able to be absorbed into systemic circulation to later accomplish their biological activity. However, it is also known that most polyphenols can reach the colon, hence the mentioned compounds may be able to reach the mentioned area without being absorbed and interacting with colonic microbiota. For this reason, later studies focusing on the controlled delivery and release of polyphenols specifically to the colon, through the application of encapsulation techniques, would guarantee an increase in the active potential of these compounds. On the other hand, the importance that the digestive process has on olive oil phenolic content has also been observed [ 49 ]. The observed bioaccessibility was rather low for secoiridoids, where hydrolysis of oleuropein derivatives led to values lower than 5%. In olive pomace, however, this parameter was closer to 100% for hydroxytyrosol, tyrosol, and oleuropein. The nature of differences between these results could be due to variability in the plant source as well as extraction conditions. As some results consider polyphenols in oils, the difference in polarity could also alter the phenolic profile observed. In addition, the interaction with molecules such as fibers has been shown to alter bioaccessibility [ 50 , 51 ]. The diversity of in vitro digestion conditions applied for different studies must also be Foods 2022,11, 743 17 of 20 considered. This entails the introduction of multiple variability factors between studies that could influence stability and, therefore, the obtained results. Furthermore, the diversity in nature of the studied samples, as well as the differences in data expression, can also involve a higher effort into the comparison process. The obtained data indicate the presence of significative concentrations of phenolic compounds in relation to those present in the extract that reach the intestinal phase and that, therefore, can be available for absorption. This constitutes a promising approach to the study of the impact of olive leaf polyphenols on human health. The influence of the intestinal absorption on its bioavailability, bioaccessibility, and bioactivity, also needs to be taken into consideration, as not all polyphenol structures are absorbed at the same rate. There are two main hypotheses on the intestinal absorption mechanisms: introduction through a Na+ dependent glucose transporters with a posterior deglycosylation and the absorption of aglycone forms by passive diffusion. Therefore, from the identified forms, only aglycones and glycosylated structures can be absorbed [52]. Although the in vitro static process gathers the most important conditions and phases of the digestion, the simplicity of itself makes it an adequate method of study for foods with simple compositions and purified components. Therefore, these data can constitute a preliminary study and sustain the continuation of research on bioaccessibility, taking into consideration a higher number of factors, such as the interaction with digestive microbiota. The presence and action of microbial enzymes could also present an impact on the final bioaccessibility of these molecules [13]. Part of the great importance of polyphenols on human health is derived from their action on microbiota, especially on its colonic phase. Non-absorbed phenolic compounds go into the large intestine, where they are degraded to phenolic acids by the action of the colonic microbiota [ 53 ]. This has raised an interest for its anti-inflammatory and immunomodulating activities in neoplasia, where the pro-inflammatory environment could promote carcinogenesis [ 54 ]. In this way, the non-absorbed fraction under intestinal conditions could still present an interest in the study of their potential health effects. Therefore, it can be considered as a future aspect for analysis of olive leaf extract polyphenols. 5. Conclusions The phenolic profile of the present olive leaf extract was affected by the static in vitro digestion process. A total of 24 compounds have been monitored and their stability under gastrointestinal conditions has been studied. Degradation kinetics of major phenolic compounds in the bioaccessible fraction of samples showed similarity, with reduced concentration in the gastric phase, which increased in the first moments of the intestinal phase. Oleuropein and most derived compounds seem to present best recovery at the beginning of the intestinal phase, which was reduced at later times. This tendency has also been observed for the other monitored compounds. However, other compounds differ from this tendency, with quercetin and luteolin showing a low and stable recovery, as well as elenolic acid, which did not present a higher recovery at the beginning of the intestinal phase. During most of the intestinal phase, concentrations were stabilized, leading to high bioaccessibility values for most compounds such as oleuropein, the main compound identified, or ligstroside and quercetin-3-O-galactoside. Most of the studied compounds may be able to reach the colon, which may allow for future studies evaluating their stabilities under different colon segments, as well as their interaction with their correspondent microbiota. Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/foods11050743/s1, Table S1: Main analytical parameters of calibration curves. Table S2: Identified compounds in the bioaccessible fraction expressed as total mg and standard deviation (SD) for each digestive phase. The used surrogate standard is indicated for each compound: Oleuropein (OLE), Luteolin-7-O-glucoside (LUT-GLU), Loganine (LOG), Hydroxytyrosol (HYTY), and Verbascoside (VERB). Table S3: Identified compounds in the residual fraction expressed as total mg and standard deviation (SD) for each digestive phase. The used surrogate standard is indicated for each Foods 2022,11, 743 18 of 20 compound: Oleuropein (OLE), Luteolin-7-O-glucoside (LUT-GLU), Loganine (LOG), Hydroxytyrosol (HYTY), and Verbascoside (VERB). Author Contributions: Conceptualization, A.S.-C. and J.L.-S.; methodology, I.B.-L. and R.Q.-P.; software, C.D.-S.; validation, I.B.-L. and R.Q.-P.; formal analysis, R.Q.-P.; investigation, C.D.-S., R.Q.-P. and I.B.-L.; resources, A.S.-C., J.L.-S. and I.B.-L.; data curation, C.D.-S. and R.Q.-P.; writing—original draft preparation, C.D.-S., R.Q.-P.; writing—review and editing, J.L.-S. and I.B.-L.; visualization, J.L.-S.; supervision, A.S.-C.; project administration, A.S.-C., J.L.-S. and I.B.-L.; funding acquisition, A.S.-C., J.L.-S. and I.B.-L. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by Ministerio de Ciencia, Innovación y Universidades under the “Proyectos de I+D+i «RETOS INVESTIGACIÓN»” (grant number RTI2018-096724-B-C22); and by Junta de Andalucía, under the “Proyectos I+D+i del Programa Operativo FEDER 2018” (grant number B-AGR-466-UGR18), the “Ayudas a proyectos de I+D+I de entidades privadas calificadas como agentes del sistema andaluz del conocimiento” (grant number PY18-RE-0033), the “Ayudas al funcionamiento de los Grupos operativos de la Asociación Europea para la Innovación (AEI) en materia de productividad y sostenibilidad agrícolas en el sector del olivar, 2020” (grant number GOPOGR-20-0001), and, finally, the “Ayudas a los agentes públicos del Sistema Andaluz del Conocimiento, para la realización de proyectos de I+D+i” (grant number A-AGR-226-AGR20). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: All the data generated by this research have been included in the article. For any assistance, it is possible to contact with the corresponding authors. Acknowledgments: The authors thank NATAC S.L. for providing the olive extract. CDS is grateful for the Doctoral Program in Nutrition and Food Sciences, University of Granada (grant from the Regional Government of Andalucía PREDOC_00110). Conflicts of Interest: The authors declare no conflict of interest. References 1. Niaounakis, M.; Halvadakis, C.P. Olive Processing Waste Management: Literature Review and Patent Survey, 2nd ed.; Elsevier: Amsterdam, The Netherlands, 2006. [CrossRef] 2. FAOSTAT. 2019. Available online: http://www.fao.org/faostat/en/#data/QC (accessed on 17 March 2021). 3. Spinelli, R.; Picchi, G. Industrial harvesting of olive tree pruning residue for energy biomass. Bioresour. Technol. 2010 ,101, 730–735. [CrossRef] [PubMed] 4. Marín, L.; Miguélez, E.M.; Villar, C.J.; Lombó, F. 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