Influence of in vitro gastric digestion of olive leaf extracts on their bioactive properties against H. pylori

Citation: Villalva, M.; Silvan, J.M.; Guerrero-Hurtado, E.; Gutierrez-Docio, A.; Navarro del Hierro, J.; Alarcón-Cavero, T.; Prodanov, M.; Martin, D.; Martinez-Rodriguez, A.J. Influence of In Vitro Gastric Digestion of Olive Leaf Extracts on Their Bioactive Properties against H. pylori.Foods 2022,11, 1832. https://doi.org/ 10.3390/foods11131832 Academic Editor: Maria D. Guillen Received: 13 May 2022 Accepted: 20 June 2022 Published: 22 June 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 Influence of In Vitro Gastric Digestion of Olive Leaf Extracts on Their Bioactive Properties against H. pylori Marisol Villalva 1, Jose Manuel Silvan 1, Esperanza Guerrero-Hurtado 2, Alba Gutierrez-Docio 2, Joaquín Navarro del Hierro 2, Teresa Alarcón-Cavero 3,4 , Marin Prodanov 2, Diana Martin 2 and Adolfo J. Martinez-Rodriguez 1,* 1Microbiology and Food Biocatalysis Group (MICROBIO), Department of Biotechnology and Food Microbiology, Institute of Food Science Research (CIAL, CSIC-UAM), C/Nicolás Cabrera, 9. Cantoblanco Campus, Autonomous University of Madrid, 28049 Madrid, Spain; [email protected] (M.V.); [email protected] (J.M.S.) 2Department of Production and Characterization of Novel Foods, Institute of Food Science Research (CIAL, CSIC-UAM), C/Nicolas Cabrera, 9. Cantoblanco Campus, Autonomous University of Madrid, 28049 Madrid, Spain; esperanza.guerrer[email protected] (E.G.-H.); [email protected] (A.G.-D.); [email protected] (J.N.d.H.); marin.pr[email protected] (M.P.); [email protected] (D.M.) 3Microbiology Department, Hospital Universitario de La Princesa, Sanitaria Princesa Research Institute, 28006 Madrid, Spain; [email protected] 4Department of Preventive Medicine, Public Health and Microbiology, School of Medicine, Autonomous University of Madrid, 28029 Madrid, Spain *Correspondence: [email protected]; Tel.: +34-91-001-7964 Abstract: The aim of this work was to evaluate the influence of in vitro gastric digestion of two olive leaf extracts (E1 and E2) on their chemical composition and bioactive properties against Helicobacter pylori (H. pylori), one of the most successful and prevalent human pathogens. HPLC-PAD/MS analysis and anti-inflammatory, antioxidant, and antibacterial activities of both olive leaf extracts were carried out before and after their in vitro gastric digestion. The results showed that gastric digestion produced modifications of the chemical composition and bioactive properties of both olive leaf extracts. The main compounds in the extract E1 were hydroxytyrosol and its glucoside derivatives (14,556 mg/100 g), presenting all the identified compounds a more polar character than those found in the E2 extract. E2 showed a higher concentration of less polar compounds than E1 extract, with oleuropein (21,419 mg/100 g) being the major component. Gastric digestion during the fasted state (pH 2) induced an overall decrease of the most identified compounds. In the extract E1, while the anti-inflammatory capacity showed only a slight decrease (9% of IL-8 production), the antioxidant properties suffered a drastic drop (23% of ROS inhibition), as well as the antibacterial capacity. However, in the extract E2, these changes caused an increase in the anti-inflammatory (19% of IL-8 production) and antioxidant activity (9% of ROS inhibition), which could be due to the hydrolysis of oleuropein and ligustroside into their main degradation products, hydroxytyrosol and tyrosol, but the antibacterial activity was reduced. Gastric digestion during fed state (pH 5) had less influence on the composition of the extracts, affecting in a lesser degree their anti-inflammatory and antioxidant activity, although there was a decrease in the antibacterial activity in both extracts similar to that observed at pH 2. Keywords: gastric digestion; olive leaf extract; Helicobacter pylori; anti-inflammatory activity; antioxidant activity; antibacterial activity; HPLC-PAD-MS characterization 1. Introduction Helicobacter pylori (H. pylori) is a Gram-negative bacterium found on the luminal surface of the gastric epithelium. It is one of the most successful and prevalent human pathogens that infect more than 50% of the world’s population [ 1 , 2 ]. H. pylori infection is Foods 2022,11, 1832. https://doi.org/10.3390/foods11131832 https://www.mdpi.com/journal/foods Foods 2022,11, 1832 2 of 15 recognized as a worldwide concern because it can cause several gastrointestinal disorders, including chronic active gastritis without clinical symptoms, as well as peptic ulceration, gastric adenocarcinoma, gastric mucosa-associated lymphoid tissue lymphoma, and other extra-gastric pathologies [ 2 , 3 ]. H. pylori has been classified as a type I carcinogen by the International Agency for Research on Cancer (IARC) [ 4 ] and it is considered the main infectious agent associated with cancer. In fact, the IARC estimated that 6.2% of all registered cancers were attributable to H. pylori [5]. Due to this high correlation between H. pylori infection and development of gastric cancer, most therapeutic international guidelines aim to eradicate this pathogen using a combination of antibiotics with a proton pump inhibitor in a triple or quadruple therapy [6,7] . However, the eradication rate of H. pylori treatment is markedly decreasing in recent years, mainly because the prevalence of antibiotic resistance strains appears to increase over time [ 8 , 9 ]. Failures in H. pylori eradication are alarming at present, but they are likely to be critical in the near future, since global rates of antibiotic resistance are rising, and H. pylori therapy is increasingly prescribed. This fact and the relationship that has been observed between the eradication of H. pylori and the onset or worsening of other pathologies, such as esophageal reflux, has led many researchers to question whether it is always essential to carry out an eradicative therapy, even in the absence of symptoms, or whether it is possible to consider milder and more sustainable treatments to fight against H. pylori although they do not have the eradicating power of antibiotics [ 10 ]. Therefore, the use of alternative therapies that could enhance antibiotic activity or modulate inflammatory response to support H. pylori eradicative therapy appears to be reasonable. In this regard, extracts with bioactive properties obtained from food ingredients or food by-products are showed as potentially useful candidates in the complementary therapy against H. pylori [11–15]. Olive industry by-products (including olive mill wastewaters, olive pomace, and olive leaves) are considered a relevant source of bioactive compounds and olive leaves represent 10% of the total weight of the olives arriving at mills, containing considerable amounts of bioactive secondary metabolites which have particular importance in their health properties [ 16 ]. Recently, we have reported that olive leaf extracts with different composition showed anti-inflammatory, antioxidant, and antibacterial activity against H. pylori. These properties were significantly related with the phenolic and secoiridoid composition of these extracts [ 13 ], which can vary depending on factors, such as olive variety and growing, climatic, and storage conditions. Oleuropein and hydroxytyrosol are mayor constituents of olive leaf extracts, followed by verbascoside, apigenin-7-glucoside, luteolin-7-glucoside, and tyrosol. These compounds have been related to the anti-inflammatory, antioxidant, and antibacterial properties associated to olive leaf extracts [ 17 – 19 ]. Because H. pylori has developed acid acclimation mechanisms to colonize the acidic gastric niche, effective bioactive compounds against this bacterium should keep their stability at gastric conditions. It has been described that the individual stability of the main phenolic compounds present in olive leaf extracts may be affected during gastrointestinal digestion by the combined effect of enzymatic activity and pH changes [ 20 – 22 ]. Since the modification of bioactive compounds during gastric digestion has been mainly related to the acid conditions of the media, it is relevant to consider that the specific values of acid pH of the gastric environment are quite dynamic depending on the type and amount of the intake. In general, gastric pH increases from around 1–2 (fasted state) to around 5–7 (fed state), just after food ingestion, and it depends on the buffering capacity or the solid/liquid state of the meal [ 23 ]. Therefore, the evaluation of the impact of gastric digestion on the bioactivity of olive leaf extracts would be relevant in order to establish the efficacy of these extracts at the specific point of action, but also to further evidence the optimal intake patterns to take full advantage of its effectiveness. In this sense, different static in vitro models simulating gastrointestinal conditions have been developed as suitable, inexpensive, and simple alternatives to in vivo methods to assess the effect of the digestion process on the stability of bioactive compounds. In static in vitro digestion models, samples are subjected to a sequential digestion in which the oral, gastric, and intestinal phases are simulated. Foods 2022,11, 1832 3 of 15 Briefly, these are performed by including the experimental product in a buffer solution containing different salts and the digestive components of each phase, mainly enzymes (amylase for oral, pepsin and lipase for gastric, and a pool of amylase, protease, and lipase for intestinal digestions), as well as a bile solution in case of intestinal digestion. The buffer solution allows to keep the specific pH for each phase and to simulate the main aqueous environment of the gastrointestinal tract. Then, conditions of shaking or mixing and physiological temperatures are reproduced for a variable time, depending on the phase (up to 2 min for oral, 1–3 h for gastric, or 1–5 h for intestinal digestion) [ 24 – 26 ]. Additionally, the versatility of these models allows to easily test the digestion process under different physiological conditions, as fed and fasted states might be. Therefore, the aim of the present work was to evaluate the impact of the gastric digestion of two olive leaf extracts with different phenolic compositions on the anti-inflammatory, antioxidant, and antibacterial activity against H. pylori. With this purpose, in vitro gastric digestions were simulated under typical pH values of fed (pH 5) and fasted (pH 2) states and the subsequent changes of the phenolic and secoiridoid profiles and bioactivities were established. 2. Materials and Methods 2.1. Olive Leaf Extracts and Chemicals Extract E1 was standardized in 4% elenolic acid and its derivates (Isenolic ® ) and extract E2 was standardized in 20% of oleuropein (Olivactive ® ). They were provided by Pharmactive Biotech Products S.L. (Madrid, Spain). The following reference substances trans4,5-DCQA (trans-4,5-dicaffeoylquinic acid) (>95%), quercetin (>95%), 4-HPE-EA-glucoside (ligustroside) (>96.2%), and 3,4-DHPE-EA-glucoside (oleuropein) (>98%) were acquired from Merck (Dramstadt, Germany). Elenolic acid (EA) (>98%) and luteolin (>95%) were purchased from Toronto Research Chemicals (Toronto, ON, Canada). The reference substances 3,4-DHBA (protocatechuic acid) (>90%), 4-HPE (tyrosol) (>95%), trans-3,4-DHCA (trans-caffeic acid) (>99%), trans-4-HCA (trans-4-coumaric acid) (>98%), trans-3-M,4-HCA (trans-ferulic acid) (>98%), quercetin 3-O-rhamnoside (quercitrin) (>93.3%), luteolin 3 0 ,7-diO-glucoside (>97%), eriodictyol-7-O-rutinoside (>98%), eriodictyol 7-O-glucoside (>98%), luteolin 7-O-glucoside (>98%), and 3,4-DHPE caffeoyl glucoside (verbascoside) (>95%) were obtained from Extrasynthese (Genay, France). EA 2-glucoside (oleoside 11-methyl ester) (>98%), EA monoaldehyde form (EMA) 2-glucoside (secoxyloganin) (>99%), 3,4-DHPE (Hydroxytyrosol) (>90%), quercetin 3-O-glucoside (isoquercitrin) (>99%), apigenin 7-Oglucuronide (>90%), and luteolin 4 0 -methyl ether 7-O-glucoside (diosmin) (>90%) were purchased from PhytoLab GmbH & Co. KG (Vestenbergsgreuth, Germany). Apigenin 6,8-di-C-glucoside (>95%) was obtained from Glentham Life Sciences (Corsham, UK). Apigenin 7-O-rutinoside (isorhoifolin) (>99.9%) was obtained from Biosynth AG (Staad, Switzerland). The substance 3,4-dihydroxyphenylglycol (3,4-DHPG) (75%) was provided by Prof. Juan Fernández-Bolaños from Instituto de la Grasa (CSIC) (Sevilla, Spain). Monohydrate extra pure (99.5%) reference 3,4,5-THBA (gallic acid) was purchased from Scharlab (Barcelona, Spain). 2.2. In Vitro Simulated Gastric Digestion of Olive Leaf Extracts The in vitro simulated gastric digestion was based on the gastrointestinal digestion model described by Navarro del Hierro et al. with minor modifications [ 27 ]. Briefly, extracts (E1 and E2) were solubilized in 10 mg/mL of gastric solution (150 mM NaCl, 6 mM CaCl 2 ) adjusted at pH 2 (fasted state) (DE1 and DE2 pH2) or pH 5 (fed state) (DE1 and DE2 pH5) with HCl (4 mM). This gastric solution contained pepsin at 0.85 mg/mL, which was previously prepared by stirring for 10 min. Digestions were performed in an orbital incubator (Titramax 1000, Heidolph Instruments, Schwabach, Germany) at 37 ◦ C and the gastric digestion was performed for 120 min at 250 rpm. Once finished, samples were stored at − 20 ◦ C until use. Digestions for each extract at each pH were performed Foods 2022,11, 1832 4 of 15 in duplicate (n = 2). Control digestion media at each tested pH and in absence of extracts were also prepared for the bioactivity assays. A graphical flowchart summarizing the main steps of the experimental procedure applied is provided in Figure 1. Foods 2022, 11, x FOR PEER REVIEW 4 of 15 gastric digestion was performed for 120 min at 250 rpm. Once finished, samples were stored at −20 °C until use. Digestions for each extract at each pH were performed in duplicate (n = 2). Control digestion media at each tested pH and in absence of extracts were also prepared for the bioactivity assays. A graphical flowchart summarizing the main steps of the experimental procedure applied is provided in Figure 1. Figure 1. Flowchart summarizing the main experimental procedures for evaluation of the bioactive properties of olive leaf extracts and their gastric digests. 2.3. Chemical Characterization of Olive Leaf Extracts and Their Gastric Digests Solutions of 10 mg/mL of water soluble E1 and alcohol soluble E2 extracts and their gastric digests (DE1 and DE2) at different pH were prepared in duplicate (n = 2), in water and methanol, respectively, and were analyzed by reverse-phase high performance liquid chromatography (RP-HPLC), coupled sequentially to photodiode array detector (PAD) and mass spectrometry (MS) with electrospray ionization source (RP-HPLC-PADMS(ESI)), as described by Silvan et al. [13]. The 3,4-DHBA, 3,4-DHPE, 4-HPE, 3,4-DHPE-EA-glucoside, 3,4-DHPE caffeoyl glucoside, quercetin, quercetin 3-O-glucoside, apigenin 7-O-glucuronide, apigenin 6,8-di-Cglucoside, luteolin, luteolin 3′,7-di-O-glucoside, luteolin 7-O-glucoside, luteolin 4′-O-methyl ether 7-O-glucoside, eriodictyol 7-O-rutinoside, EA, EA 2-glucoside, EMA 2-glucoside, trans-3,4-DHCA, trans-4-HCA, trans-3-M,4-HCA, trans-4,5-DCQA, and 3,4,5-THBA were identified unambiguously by co-elution and comparison with their retention time, order of elution, UV spectra, and pseudo-molecular and fragment ion masses of the corresponding reference substances, and quantified according to the calibration curves of each of them. The glucosides of 3,4-DHBA, 3,4-DHPE, and 3,4,5-THBA were identified tentatively by using their corresponding retention time, order of elution, UV spectra, pseudo-molecular, diagnostic fragment ion masses, and bibliographic data [13]. The 3,4Figure 1. Flowchart summarizing the main experimental procedures for evaluation of the bioactive properties of olive leaf extracts and their gastric digests. 2.3. Chemical Characterization of Olive Leaf Extracts and Their Gastric Digests Solutions of 10 mg/mL of water soluble E1 and alcohol soluble E2 extracts and their gastric digests (DE1 and DE2) at different pH were prepared in duplicate (n = 2), in water and methanol, respectively, and were analyzed by reverse-phase high performance liquid chromatography (RP-HPLC), coupled sequentially to photodiode array detector (PAD) and mass spectrometry (MS) with electrospray ionization source (RP-HPLC-PAD-MS(ESI)), as described by Silvan et al. [13]. The 3,4-DHBA, 3,4-DHPE, 4-HPE, 3,4-DHPE-EA-glucoside, 3,4-DHPE caffeoyl glucoside, quercetin, quercetin 3-O-glucoside, apigenin 7-O-glucuronide, apigenin 6,8-di-Cglucoside, luteolin, luteolin 3 0 ,7-di-O-glucoside, luteolin 7-O-glucoside, luteolin 40-O-methyl ether 7-O-glucoside, eriodictyol 7-O-rutinoside, EA, EA 2-glucoside, EMA 2-glucoside, trans-3,4-DHCA, trans-4-HCA, trans-3-M,4-HCA, trans-4,5-DCQA, and 3,4,5-THBA were identified unambiguously by co-elution and comparison with their retention time, order of elution, UV spectra, and pseudo-molecular and fragment ion masses of the corresponding reference substances, and quantified according to the calibration curves of each of them. The glucosides of 3,4-DHBA, 3,4-DHPE, and 3,4,5-THBA were identified tentatively by using their corresponding retention time, order of elution, UV spectra, pseudo-molecular, diagnostic fragment ion masses, and bibliographic data [ 13 ]. The 3,4-DHBA glucoside was quantified as equivalents of 3,4-DHBA, the three 3,4-DHPE glucosides were quantified as equivalents of 3,4-DHPE, and the two 3,4,5-THBA glucosides were quantified as equivalents of 3,4,5-THBA. Foods 2022,11, 1832 5 of 15 2.4. Helicobacter pylori, Growth Media and Culture Conditions H. pylori strain (HpCIAL2) was obtained from MICROBIO bacterial collection (CIALCSIC) by isolation from a gastric biopsy in the Princesa Universitary Hospital, Madrid, Spain. H. pylori strain was stored at − 80 ◦ C in Brucella Broth (BB) (Becton, Dickinson, & Co., Madrid, Spain) plus 20% glycerol. The agar-plating medium used was Müeller-Hinton agar supplemented with 5% defibrinated sheep blood (MHB) (Becton, Dickinson, & Co, Madrid, Spain). Liquid medium BB supplemented with 10% horse serum (HS) (Biowest, Barcelona, Spain) was used. The inoculum for H. pylori strain was prepared as described by Silvan et al. (2020) [ 11 ] and it can be summarized as follows: frozen strain was reactivated by inoculation (200 µ L) in MHB plate and incubation in a Variable Atmosphere Incubator (VAIN) (85% N 2 , 10% CO 2 and 5% O 2 ) (MACS-VA500, Don Whitley Scientific, Bingley, UK) at 37 ◦ C for 72 h. Bacterial grown from one MHB plate was suspended in 2 mL of BB + 10% HS or culture medium cell (~1 × 10 8 colony forming units (CFU/mL)) and used as bacterial inoculum in the different assays. 2.5. Human Gastric Epithelial Cell Cultures AGS cells (human gastric epithelial cell line) were obtained from the American Type Culture Collection (ATCC). Cells were cultured in Dulbecco’s Modified Eagle’s Medium F12 (DMEM/F12) (Lonza, Madrid, Spain) supplemented with 10% fetal bovine serum (FBS) (Hyclone, GE Healthcare, Logan, UK) and 1% penicillin/streptomycin (5000 U/mL) (Lonza). Cell cultures and subcultures were prepared as described by Silvan et al. [ 12 ]. Briefly, cells were plated at densities of ~1 × 10 6 cells in 75 cm 2 culture flasks (Sarstedt, Barcelona, Spain) and maintained at 37 ◦ C under 5% CO 2 in a humidified incubator until 90% of cell confluence. The cell culture medium was changed every 2 days. Before a confluent monolayer appeared, cell sub-culturing was carried out. All experiments were carried out between passage 10 and passage 30 to ensure cell uniformity and reproducibility. 2.6. Cell Viability Previously to the antioxidant and anti-inflammatory assays, cytotoxicity of olive leaf extracts and gastric digested samples was evaluated. AGS cell viability was determined by MTT (3,4,5-dimethylthiazol-2,5-diphenyl-tetrazolium bromide) (Sigma, Madrid, Spain) reduction assay following a protocol previously described [ 13 ]. Briefly, confluent cell cultures (~90%) were trypsinized (Trypsin/EDTA 170,000 U/L) (Lonza) and cells were seeded (~5 × 10 4 cells per well) in 96-well plates (Sarstedt) and incubated in culture medium at 37 ◦ C under 5% CO 2 in a humidifier incubator for 24 h. Cell culture medium was replaced with serum-free medium containing the non-digested and digested extracts (at 2 mg/mL final concentration) and cells were incubated at 37 ◦C under 5% CO2for 2 h. Control cells (non-treated) were incubated in serum-free medium without extracts. Thereafter, cells were washed with phosphate buffered saline (PBS) (Lonza), the medium was replaced by 200 µ L of serum-free medium, and 20 µ L of MTT solution in PBS (5 mg/mL) was added to each well for the quantification of the living metabolically active cells after 1-h incubation. During this period, MTT is reduced to purple formazan in the mitochondria of living cells. Formazan crystals in the wells were solubilized in 200 µ L dimethyl sulfoxide (Sigma, Madrid, Spain). Finally, absorbance was measured at 570 nm wavelength using a microplate reader Synergy HT (BioTek Instruments Inc., Winooski, VT, USA). The viability was calculated considering controls containing serum-free medium (non-treated cells) as 100% viable. Results were obtained from three independent experiments (n = 3). 2.7. Determination of Anti-Inflammatory Activity of Olive Leaf Extracts and Their Gastric Digests after Infection of AGS Human Gastric Epithelial Cells with H. pylori The assay was carried out following the procedure described by Silvan et al. [ 28 ]. Briefly, human gastric AGS cells were seeded (~5 × 10 4 cells/well) in 24-well plates (Sarstedt) and incubated in cell culture medium at 37 ◦ C under 5% CO 2 in a humidifier incubator until a monolayer was formed. Cells were incubated with non-digested (E1 Foods 2022,11, 1832 6 of 15 and E2) and digested olive leaf extracts (DE1 and DE2 at pH 2 and pH 5) at 37 ◦ C under 5% CO 2 for 2 h. The inflammatory response was determined using 2 mg/mL of each extract, since this concentration results as non-cytotoxic for AGS cells during viability test (data not shown). Cells were washed twice with PBS and infected with 0.5 mL/well of H. pylori inoculum in serum-antibiotics-free medium (~1 × 10 8 CFU/mL for all tested strains). The cells and bacteria were incubated at 37 ◦ C under 5% CO 2 for 24 h to allow the bacteria to adhere to the gastric cells. Uninfected cells were included in the experiment as a control. At the end of incubation, the cell supernatants were collected, particulate material was removed by centrifugation (12,000 rpm, 10 min), and samples were stored at − 20 ◦ C until analysis were performed. The amounts of secreted IL-8 cytokine in the collected supernatant of gastric AGS cell samples were determined by ELISA assay. Commercially available ELISA kit (Diaclone) for the quantitation of IL-8 cytokine was used as described in the manufacturer’s instructions. The absorbance was measured at 450 nm using a microplate reader Synergy HT (BioTek Instruments Inc. Winooski, VT, USA). Such as in the absence of bacteria, gastric cells release small amounts of IL-8 [ 28 ], so titers of cytokine released by AGS cells were determined experimentally. Results were expressed as IL-8 production (pg/mL) of three independent experiments (n = 3). 2.8. Determination of Antioxidant Activity of Olive Leaf Extracts and Their Gastric Digests against Intracellular Reactive Oxygen Species (ROS) Production in AGS Cells Human gastric AGS cells were used for the evaluation of oxidative stress induced by H. pylori infection. Intracellular ROS were measured by the DCFH-DA (carboxy-2 0 ,7 0 - dichloro-dihydrofluorescein diacetate) (Sigma) assay as was previously reported by Silvan et al. [ 13 ]. Cells were seeded (5 × 10 4 cells per well) in 24-well plates and grown until they reached 70% of confluence. Cells were incubated with non-digested (E1 and E2) and digested olive leaf extracts (DE1 and DE2 at pH 2 and pH 5) (2 mg/mL) dissolved in serum-free medium at 37 ◦ C under 5% CO 2 for 2 h. After that, cells were washed with PBS and incubated with 20 µ M DCFH-DA at 37 ◦ C under 5% CO 2 for 30 min. Then, cells were washed twice with PBS to remove the unabsorbed probe and infected with H. pylori inoculum strain suspended in serum-antibiotics-free medium (~1 × 10 8 CFU/mL). ROS production was immediately monitored for 180 min in a fluorescent microplate reader Synergy HT (BioTek Instruments Inc. Winooski, VT, USA) using a λex 485 nm and λem 530 nm. After being oxidized by intracellular oxidants, DCFH-DA changes to dichlorofluorescein (DCF) and emits fluorescence. Non-treated but infected cells were used as oxidative control (100% of intracellular ROS production). All samples were analyzed in triplicate (n = 3). Results were expressed as % of inhibition of ROS production. 2.9. Determination of Antibacterial Activity of Olive Leaf Extracts and Their Gastric Digests against H. pylori The antibacterial activity of non-digested (E1 and E2) and digested olive leaf extracts (DE1 and DE2 at pH 2 and pH 5) against H. pylori strain was evaluated following the procedure described by Silvan et al. [ 29 ]. Briefly, 1 mL of extracts (at 2 mg/mL final concentration) was transferred into different flasks containing 4 mL of BB supplemented with 10% HS. Bacterial inoculum (100 µ L of ~1 × 10 8 CFU/mL) was then inoculated into the flasks under aseptic conditions. The cultures were incubated under stirring (150 rpm) in a microaerophilic atmosphere using a VAIN at 37 ◦ C for 24 h. Growth controls were prepared by transferring 1 mL of sterile water to 4 mL of BB supplemented with 10% HS and 50 µ L of bacterial inoculum. After incubation, serial decimal dilutions of cultures were prepared in saline solution (0.9% NaCl) and they were plated (20 µ L) onto fresh MHB agar, and incubated in a microaerophilic atmosphere using a VAIN at 37 ◦ C for 72 h. The number of CFU was assessed after incubation. The results of antibacterial activity were expressed as log CFU/mL (n = 3). Foods 2022,11, 1832 7 of 15 2.10. Statistical Analysis The results were reported as mean value ± standard deviations (SD) performed at least in triplicate (n = 3). Statistical analyses of the concentration of each quantified compound in both olive leaf extracts were performed by t-test. Significant differences in the anti-inflammatory and antioxidant activity results were estimated by applying analysis of variance (ANOVA). The Tukey’s least significant differences (HSD) test was used to evaluate the significance of these values. In all cases, differences were considered significant at p< 0.05 . All statistical tests were performed with IBM SPSS software statistics for Windows, Version 26.0 (IBM Corp., Armonk, NY, USA). 3. Results and Discussion 3.1. Effect of In Vitro Gastric Digestion on the Chemical Composition of Olive Leaf Extracts Results of the chemical composition and quantitative HPLC-PAD-MS determination of phenolic and secoiridoid compounds and their derivatives in the non-digested and gastric digested extracts are shown in Table 1(date is in Supplementary Figure S1 and S2). Table 1. Content of identified phenolic and secoiridoid compounds and their derivatives present in the non-digested (E1 and E2) and gastric digested extracts (DE1 and DE2) at fasted (pH 2) and fed state (pH 5) conditions. Results are expressed as mean value ± standard deviation (mg/100 g of dry matter). Compounds E1 DE1-pH2 DE1-pH5 E2 DE2-pH2 DE2-pH5 Hydroxybenzoic acids and glycoside derivatives 3,4-DHBA (protocatechuic acid) 11.4 ±1.0 B7.3 ±0.1 A13.3 ±0.1 B6.2 ±0.2 b4.8 ±0.2 a5.5 ±0.1 b 3,4-DHBA glucoside 9.2 ±0.2 B7.9 ±0.1 A9.4 ±0.3 BND ND ND 3,4,5-THBA (gallic acid) ND 86.1 ±0.9 B37.2 ±1.9 AND ND ND 3,4,5-THBA glucoside 1 (galloyl glucoside 1) 24.0 ±0.1 BND 23.4 ±0.1 AND ND ND 3,4,5-THBA glucoside 2 (galloyl glucoside 2) 23.2 ±0.1 BND 22.2 ±0.1 AND ND ND ΣHydroxybenzoic acids and glycoside derivatives 67.8 101 105 6.2 4.8 5.5 Hydroxycinnamic acids and derivatives trans-3,4-DHCA (trans-caffeic acid) 140 ±4B117 ±8A140 ±1B7.4 ±0.2 b6.7 ±0.1 a6.8 ±0.1 a,b trans-4-HCA (trans-4-coumaric acid) 177 ±1B132 ±11 A180 ±1B3.2 ±0.4 b1.9 ±0.1 a2.0 ±0.1 a trans-3-M,4-HCA (trans-ferulic acid) 113 ±3B88.9 ±7.9 A114 ±1B5.4 ±0.3 b4.6 ±0.2 a4.8 ±0.2 a,b trans-4,5-DCQA (trans-4,5-dicaffeoylquinic acid) ND ND ND 28.0 ±1.3 b21.4 ±0.4 a28.1 ±1.1 b ΣHydroxycinnamic acids and derivatives 430 338 434 44.0 34.6 41.7 Phenylethanols and glycoside derivatives 3,4-DPHG (3,4-dihydroxy-phenylglycol) 38.6 ±0.2 B31.8 ±1.3 A38.4 ±0.5 B20.8 ±0.3 a33.9 ±1.6 b19.7 ±0.1 a 3,4-DHPE (hydroxytyrosol) + 3,4-DHPE glucoside 1 13,516 ±81 B11,566 ±21 A13,496 ±63 B196 ±8a2631 ±66 b176 ±1a 3,4-DHPE glucoside 2 + 3 1040 ±12 B837 ±1A1025 ±4B159 ±7a176 ±1b145 ±1a 4-HPE (tyrosol) 642 ±1B511 ±1A639 ±1B11.1 ±0.5 a30.8 ±1.4 b9.6 ±0.1 a ΣPhenylethanols and glycoside derivatives 15,237 12,946 15,198 387 2872 350 Secoiridoids and glycoside derivatives EA (elenolic acid) 97.6 ±3.8 C79.6 ±3.7 B34.5 ±1.8 AND ND ND EA 2-glucoside (oleoside 11-methyl ester) 1407 ±1B1100 ±8A1413 ±7B177 ±3c50 ±1a103 ±1b EMA 2-glucoside (secoxyloganin) 1021 ±21 B744 ±3A990 ±7BND ND ND ΣSecoiridoids and glycoside derivatives 2526 1924 2438 177 50 103 Secoiridoid phenylethanols 3,4-DHPE-EA-glucoside (oleuropein) 346 ±1B228 ±4A342 ±6B21,419 ±1909 b7353 ±24 a18,431 ±274 b 4-HPE-EA-glucoside (ligustroside) 147 ±2B87.0 ±8.0 A126 ±7B344 ±4c157 ±6a296 ±7b ΣSecoiridoid phenylethanols 493 315 468 21,763 7510 18,727 Cinnamoyl phenyethanol glycoside derivatives 3,4-DHPE caffeoyl glucoside (verbascoside) 308 ±6B258 ±8A262 ±5A7255 ±315 c5196 ±202 a6310 ±44 b ΣCinnamoyl phenylethanol glycoside derivatives 308 258 262 7255 5196 6310 Foods 2022,11, 1832 8 of 15 Table 1. Cont. Compounds E1 DE1-pH2 DE1-pH5 E2 DE2-pH2 DE2-pH5 Flavones Apigenin 6,8-di-C-glucoside 316 ±9B252 ±21 A321 ±2B34.3 ±0.1 a33.1 ±0.5 a33.4 ±0.2 a Apigenin 7-O-rutinoside (isorhoifolin) 129 ±1B84.7 ±7.8 A125 ±5B124 ±4b77.0 ±1.0 a116 ±7b Apigenin 7-O-glucuronide 70.1 ±0.5 B31.0 ±3.9 A62.5 ±11.9 B115 ±3c30.0 ±0.3 a47.4 ±1.9 b Luteolin 30,7-di-O-glucoside 39.4 ±0.3 B34.5 ±0.8 A40.0 ±0.7 B31.2 ±0.4 b28.2 ±0.7 a30.2 ±0.5 a,b Luteolin 7-O-glucoside 702 ±2B385 ±6A695 ±7B517 ±11 b274 ±4a500 ±4b Luteolin 40-methyl ether 7-O-glucoside (diosmin) 84.2 ±0.1 B68.4 ±0.5 A83.6 ±0.8 B85.9 ±1.5 b51.7 ±5.8 a56.7 ±4.0 a Luteolin ND ND ND 17.0 ±0.5 b9.55 ±0.31 a13.9 ±2.2 a,b ΣFlavones 1341 857 1327 924 504 798 Flavonols Quercetin 3-O-glucoside (isoquercitrin) ND ND ND 15.6 ±0.3 b6.9 ±0.3 a12.8 ±2.4 b Quercetin 3-O-rhamnoside (quercitrin) ND ND ND 7.4 ±0.7 b3.3 ±0.2 a6.6 ±0.9 b Quercetin ND ND ND 24.2 ±0.1 a25.8 ±0.1 b24.4 ±0.1 a ΣFlavonols ND ND ND 47.2 36.0 43.8 Flavanones Eriodictyol 7-O-rutinoside ND ND ND 14.9 ±0.1 b8.3 ±0.61 a13.8 ±0.1 b Eriodictyol 7-O-glucoside ND ND ND 24.5 ±0.3 aND 25.9 ±0.6 a ΣFlavanones ND ND ND 39.4 8.3 39.7 ΣPhenolic and secoiridoid compounds 20,403 16,739 20,233 30,643 16,216 26,419 ND: not detected; 3-M,4-HCA: 3-methoxy-4-hydroxycinnamic acid; DCQA: dicaffeoylquinic acid; DHBA: dihydroxybenzoic acid; DHCA: dihydroxycinnamic acid; DHPE: dihydroxyphenylethanol; EMA 2-glucoside: EA monoaldehyde isomer 2-glucoside; HCA: hydroxycinnamic acid; HPE: hydroxyphenylethanol; THBA: trihidroxybenzoic acid. A–C Values in the same row marked with different uppercase letters indicate significant differences between non-digested and gastric digested E1 extracts by ANOVA post hoc Tukey test (p ≤ 0.05). a–c Values in the same row marked with different lowercase letters indicate significant differences between non-digested and gastric digested E2 extracts by ANOVA post hoc Tukey test (p≤0.05). A whole of 24 phenolic, secoiridoid, and their derivative compounds were identified in extract E1 and its gastric digested extracts (DE1) at pH 2 and pH 5. They could be divided into seven groups of compounds: hydroxybenzoic acids, hydroxycinnamic acids, phenylethanols, secoiridoids, secoiridoid phenylethanols, cinnamoyl phenylethanols, and flavones. One of the most remarkable characteristics of olive leaf extract E1 was its high content in phenylethanols and their glycoside derivatives (15,237 mg/100 g), such as hydroxytyrosol and three of its glucosides (14,556 mg/100 g), as well as relevant amounts of elenolic acid and its glucoside derivatives (2526 mg/100 g), and flavones (1341 mg/100 g), such as luteolin 7-O-glucoside (702 mg/100 g). Gastric digestion of extract E1 at pH 2 (DE1-pH2) showed an overall decrease of most of the identified compounds, which was low to moderate for the protocatechuic acid glucoside (14%), all hydroxycinnamic acids and derivatives (16–25%), all phenylethanols and glycoside derivatives (14–21%), all secoiridoids and glycoside derivatives (18–27%), verbascoside (16%), and for some flavones, such as both apigenin (20%) and luteolin (12%), diglucosides, and luteolin 4 0 - methyl ether 7-O-glucoside (19%). A greater decrease of the contents of protocatechuic acid (36%), oleuropein (34%), and ligustroside (41%), and some flavones, such as apigenin 7-O-rutinoside (34%), apigenin 7-O-glucuronide (51%), and luteolin 7-O-glucoside (45%), was obtained. The largest drop was found for the contents of both galloyl glucosides ( 1 and 2 ), which were completely hydrolyzed at pH 2 to free gallic acid. In this case, the amount of gallic acid raised up to 86.1 mg/100 g. The total concentration of phenolic and secoiridoid compounds in extract E1 dropped by 18% during gastric digestion at pH 2. This value was rather lower than that reported by others (35–70%) for extracts derived from olive oil by-products [ 30 , 31 ]. This may be due to the fact that hydroxytyrosol was the major compound in extract E1 (66% of total phenolic and secoiridoid compounds), which together with its glucoside derivatives are quite stable to acidic gastric digestion conditions [ 32 ]. Gastric digestion of extract E1 at pH 5 (DE1-pH5) did not produce significant differences ( p> 0.05 ) for the contents of most of the identified compounds. Nevertheless, a slight Foods 2022,11, 1832 9 of 15 decrease of verbascoside content (15%) and a greater decrease of elenolic acid content (65%) was observed. Similar to DE1-pH2, gallic acid appeared in DE1-pH5 (37.2 mg/100 g). The partial hydrolysis of the labile linkage between the gallic acid and glucose moiety of both galloyl glucosides at the moderate acidic conditions of DE1-pH5 [ 33 ] could partly explain the emergence of gallic acid. Extraction of negative m/z 331 ion of the non-digested extract E1 that corresponds to the mass of monogalloyl glucosides gave six peaks with different intensities (data not shown) and extraction of ions at m/z 483 and 493, corresponding to digalloyl glucosides and monogalloyl diglucosides, respectively, also gave two peaks. In this work, only two monogalloyl glucosides were identified and quantified, but the other unidentified galloyl compounds should also contribute to the total amount of gallic acid in extract DE1-pH5. On the other hand, 25 phenolic, secoiridoid, and their derivative compounds were identified in olive leaf extract E2 and its gastric digested extracts (DE2) at pH 2 and pH 5, which can be divided into nine groups: hydroxybenzoic acids, hydroxycinnamic acids, phenylethanols, secoiridoids, secoiridoid phenylethanols, cinnamoyl phenylethanols, flavones, flavonols, and flavanones. Extract E2 was characterized mainly by its high content in oleuropein (21,419 mg/100 g) and verbascoside (7255 mg/100 g). Flavones were also detected in relevant amounts (924 mg/100 g), mainly luteolin 7-O-glucoside (517 mg/100 g) and apigenin 7-O-rutinoside (124 mg/100 g). It also contained several flavonol and flavanone compounds that were not detected in extract E1. Galloyl glucosides and some secoiridoids, such as elenolic acid and secoxyloganin, were not detected in extract E2. Gastric digestion of extract E2 at pH 2 (DE2-pH2) increased significantly (p< 0.05) the content of all phenylethanols and their glucoside derivatives. Among them, hydroxytyrosol and tyrosol sharply rose with 1342% and 277%, respectively. This behavior is related to the hydrolysis of oleuropein and ligustroside [ 34 ], whose contents decreased by 66% and 54%, respectively. This result may confirm the possible hydrolysis of oleuropein and ligustroside and probably other isomers and derivatives that were not identified in the present work. Oleuropein, which is usually the major component of many other olive leaf extracts, was reported as highly sensitive to digestive degradation [ 35 , 36 ]. However, a proportional increase in the amounts of elenolic acid and/or its glucosides was not observed, suggesting that this increment could be related with the partial hydrolysis of verbascoside or other hydroxytyrosol/tyrosol containing unidentified compounds, such as oleuropein/ligustroside isomers and hydroxytyrosol/tyrosol elenolate [ 37 ], also known as oleuropein/ligustroside aglycons. A small increase of quercetin (6%) was also observed, due most probably to a possible acid hydrolysis of the flavonols quercitrin and isoquercitrin [ 38 ]. Similar to gastric digestion of E1 extract, most of the identified compounds decreased significantly (p< 0.05) during digestion at pH 2. Protocatechuic acid (23%), most of the hydroxycinnamic acids (9–24%), and luteolin 3 0 ,7-di-O-glucoside (10%) decreased slightly to moderately. The most affected compounds were trans-4-coumaric acid (40%), oleoside 11-methyl ester (72%), verbascoside (28%), most flavones (38–74%), both flavonol glycosides, quercitrin (55%) and isoquercitrin (56%), as well as both flavanones, eriodictyol 7-O-rutinoside and eriodictyol 7-O-glucoside (44% and 100%, respectively). Gastric digestion of extract E2 at pH 5 (DE2pH5) had a similar behavior to extract E1. It was observed no significant (p> 0.05) changes or a little decrease in the contents of protocatechuic, most of the hydroxycinnamic acids, phenylethanols, oleuropein, ligustroside, verbascoside, most flavones, all flavonols, and both flavanones, eriodictyol 7-O-rutinoside and eriodictyol 7-O-glucoside. However, the content of some compounds, such as trans-4-coumaric acid (38%), oleoside 11-methyl ester (42%), apigenin 7-O-glucuronide (59%), and luteolin 4 0 -methyl ether 7-O-glucoside (34%), were significantly (p< 0.05) decreased. 3.2. Effect of In Vitro Gastric Digestion of Olive Leaf Extracts on Their Anti-Inflammatory Properties in H. pylori-Infected AGS Cells As shown in Figure 2, when AGS cells were pretreated for 2 h with the non-digested olive leaf extracts (E1 and E2) and subsequently gastric cells were infected with H. pylori,