Phosphatidyl Derivative of Hydroxytyrosol. In Vitro Intestinal

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Phosphatidyl Derivative of Hydroxytyrosol. In Vitro Intestinal Digestion, Bioaccessibility, and Its Effect on Antioxidant Activity Diana Martin,*,†,‡ Maria I. Moran-Valero,†,‡ Víctor Casado,†,‡ Guillermo Reglero,†,‡,§ and Carlos F. Torres†,‡ †

Departamento de Producción y Caracterización de Nuevos Alimentos, Instituto de Investigación en Ciencias de la Alimentación (CIAL) (CSIC−UAM), 28049 Madrid, Spain ‡ Sección Departamental de Ciencias de la Alimentación, Facultad de Ciencias, Universidad Autónoma de Madrid, 28049 Madrid, Spain § Imdea-Food Institute, CEI UAM+CSIC, 28049 Madrid, Spain ABSTRACT: Intestinal digestion of phosphatidyl derivatives of HT (PHT) and its bioaccessibility under in vitro conditions was performed. First, an in vitro intestinal digestion model for phospholipids was developed. The impact of digestion in the antioxidant ability of PHT was also assayed. PHT was progressively hydrolyzed to lyso-PHT. However, digestion was slower than the phospholipid control. Nevertheless, most hydrolysis products were found at the micellar phase fraction, meaning a high bioaccessibility. Either PHT or digested PHT showed lower antioxidant activity than HT. However, PHT improved its antioxidant ability after digestion, likely related to lyso-PHT. As a summary, the synthetic phosphatidyl derivative of HT as PHT is recognized by phospholipases during simulation of intestinal digestion, although less efficiently than analogous phospholipids. Nevertheless, taking into account the bioaccessibility and the antioxidant activity of digested PHT, the potential of carriers of HT under the form of phospholipids might be of interest. KEYWORDS: hydroxytyrosol, phospholipids, in vitro digestion, bioaccessibility, phenolipids, antioxidants

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INTRODUCTION The popularity of phenolic compounds in general as bioactive natural antioxidants is currently well-known. Within polyphenolic compounds, hydroxytyrosol (HT) has been an attractive molecule in the last decades that has shown a great bioactivity and antioxidant power, which has been related to antiatherogenic, antiplatelet aggregation, anti-inflammatory, antimicrobial, and antitumor effects, or aging regulation.1−4 Although HT is well absorbed at the gastrointestinal tract, the fact is that its bioavailability is poor because it is rapidly metabolized in enterocyte and liver to minor metabolites, and is only found at minor levels in plasma and tissues.5−7 Taking this evidence into account, the production of HT derivatives to enhance its access to cells and tissues, or increase its systemic half-life, has been an approach of intense research in the last years.8−10 In this respect, the production of “phenolipids”, namely, lipophilized phenolics resulting from the union of a lipid to the phenolic moiety,11 has been explored by diverse authors to obtain carriers of HT. HT esters with acyl chains has been the most frequently applied strategy to modify HT, and most of the obtained derivatives have shown improved bioactivities. As example, Trujillo et al.10 showed that HT derivatives as long-chain esters had a higher protective effect against oxidative damage in an ex vivo brain homogenate model. Recent derivates of HT as ethyl ethers12 exhibited stronger intestinal anticarcinogenic activity than HT13 and were more efficiently absorbed than HT.14 Concerning novel derivatives of phenolic compounds, Casado, Reglero, and Torres15 have recently synthesized phospholipid derivatives of HT with phosphatidylcholine, © XXXX American Chemical Society

where the phenolic compound was included in the polar head of the phospholipid by replacing the choline by enzymatic transphosphatidylation. A successful antioxidant activity of this new molecule for edible oils has been shown recently.4 This new molecule (phosphatidylhydroxytyrosol, PHT) was proposed as a potential vehicle of HT but, at the same time, taking the additional interest of the PL as a backbone. This is because phospholipids are also bioactive molecules by themselves. They are well-known as essential molecules for the maintenance of living cells as major constituents of cell membranes. Additionally, evidence has pointed out a positive impact of dietary phospholipids on human health, such as relevant implications in hypercholesterolemia, atherosclerosis, cardiovascular disease, inflammation and immunity, liver disorders, brain development, as well other chronic diseases.16 On the other hand, due to their amphiphilic nature and surface-active properties, phospholipids are well-known as emulsifier ingredients in food, pharmaceutical, or cosmetic industry. The emulsifying properties are also related to the role of phospholipids to enhance the digestion and absorption of hydrophobic molecules at the intestinal level, because they contribute to emulsification of lipid drops in the aqueous media for the proper action of pancreatic lipases, and they form mixed micelles, necessary for the vehiculization of the lipid products to enterocytes.17 Received: July 21, 2014 Revised: September 12, 2014 Accepted: September 12, 2014

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dx.doi.org/10.1021/jf503477h | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry

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Germany). Sodium sulfate anhydrous, sodium chloride, calcium chloride, and ethanol absolute were purchased from Panreac (Barcelona, Spain). Ortho-phosphoric acid was purchased from Scharlab (Sentmenat, Barcelona). All solvents used were of HPLC grade from Lab-Scan (Dublin, Ireland). In Vitro Lipid Digestion. A previously developed model for TG19 was adapted to simulate the simultaneous presence of dietary TG, dietary phospholipids, and endogenous biliary phospholipids. Briefly, the adapted model consisted of a sample of 0.5 g of olive oil and 0.1 g of phospholipid which were mixed with 16 mL of Trizma−maleate buffer 0.1 M pH 7.5. In the case of control digestions and optimization of the in vitro model, the sample was the same phospholipid used for the synthesis of PHT (Phospholipon 90H, PL). In the case of PHT, this was added at the same equivalent weight used for the digestion of PL. The prepared medium was pre-emulsified by homogenization for 2 min at 3500 rpm. On the other hand, a solution trying to simulate biliary secretion was prepared by mixing 0.1 g of PC, 0.25 g of bile salts, 0.02 g of cholesterol, 0.5 mL of a 325 mM CaCl2 solution, 1.5 mL of a 3.25 mM NaCl solution, and 10 mL of Trizma−maleate buffer, and this mixture was homogenized for 2 min at 3500 rpm. Then, the pre-emulsified sample and the simulated biliary secretion were mixed and homogenized together for 2 min at 3500 rpm. The whole media was placed in a thermostatically controlled vessel at 37 °C and continuously stirred by a magnetic stir bar at 1000 rpm. The simulation of intestinal digestion was started by the addition of fresh pancreatin extract (0.5 g of pancreatine in 3 mL of Trizma−maleate buffer, stirred for 10 min and centrifuged at 1600 × g for 15 min). The enrichment with PLA2 was carried out by addition of 5 mg of a food grade PLA2 from Streptomyces violaceoruber (103 U/mg) from Nagase Chemtex Corporation (Fukuchiyama Factory, Kyoto, Japan). After addition of enzymes, reaction was continued for 60 min. In order to study the evolution of lipid products throughout the hydrolytic process, aliquots were taken at 0, 5, 10, 30, and 60 min of reaction. Additional digestions were performed in order to compare the bioaccessibility of PHT with that of HT, or with that of a premixed HT with a dose of dietary PL (HT+PL). In both cases, HT was added to the media at equivalent weights as those added by PHT. Furthermore, in both cases, the coexistence with olive oil was kept. In vitro digestion of each experiment was performed at least in triplicate. Separation of Phases after in Vitro Lipid Digestion. At the end of digestion, the medium was submitted to centrifugation at 4000 rpm for 40 min at 37 °C (5810R Eppendorf Iberica, Madrid, Spain) according to Soler-Rivas et al.20 After centrifugation, an upper oily phase (OP), a lower aqueous or micellar phase (MP), and a minor precipitated phase (PP) were obtained. The lipid composition of each phase was analyzed. In the case of the MP, aliquots were collected for quantification of micellar structures by using a light microscope and a Neubauer cell counter chamber (Brand, Germany).20 Lipid Extraction. The total lipids from samples were extracted twice by a mixture of chloroform/methanol/ortho-phosphoric acid (100:80:4, v/v/v) at a ratio of solvent to sample of 3:1 (v/v). Orthophosphoric acid was necessary for a proper recovery of the lyso-PL species. The mixture was vortexed for 1 min and centrifuged for 10 min at 13500 rpm (ScanSpeed mini, Micro Centrifuge). The organic phase containing the separated lipids was collected, and anhydrous sodium sulfate was added before further analysis. Analysis of Lipid Products. Polar Lipids. The polar lipid composition was determined on a Luna 5 μm HILIC diol column (250 mm, 4.6 mm, Phenomenex, Torrance, California, USA) coupled to an HPLC Agilent 1200 Series containing a thermostatized column compartment, a quaternary pump, an autosampler, a vacuum degasser, and an evaporative light scattering detector. The used method was based on Casado et al.15 with brief modifications. The flow rate was 1.5 mL min−1. A splitter valve was used after the thermostatized column compartment and part of the mobile phase was directed through the detector (3.5 bar and 55 °C). The column temperature was maintained at 55 °C. The mobile phase utilized consisted of a ternary gradient of (A) hexane/2-propanol/acetic acid/triethylamine (815/

However, one of the questions that arise when phenolic compounds are esterified as the polar head of phospholipids would be whether the derived structure would be digested and bioaccesible during gastrointestinal digestion, as the first step before any other physiological action beyond intestinal tissues. The in vitro intestinal models of lipid digestion are an interesting tool for obtaining preliminary and valuable information concerning digestion of lipid species. Due to their utility, the use of in vitro digestion models in the study of bioactive compounds has increased, becoming a well-accepted analytical tool.18 However, as far as we know, all the models of in vitro lipid digestion have been developed for the major lipid dietary forms, namely, triglycerides (TG), whereas the in vitro digestion models focused on phospholipids have not been extensively investigated. In general, we consider that the development of a model that closely simulates the in vivo conditions is especially essential when performing the digestion of novel or unknown lipids under in vitro conditions, in order to accurately understand obtained results and to avoid misinterpretations due to the own methodology used. Therefore, a reliable in vitro digestion model for phospholipids should be previously developed before the application of the model to the experimental molecule PHT. Concerning the general digestion process of phospholipids, this is mainly catalyzed by the enzyme phospholipase A2 (PLA2) at the intestinal level, which hydrolyzes fatty acids from the sn-2 location almost completely to release lysophospholipids and free fatty acids (FFA).17 After hydrolysis, a high bioaccessibility has been described for phospholipids, which means that their lipid products are easily solubilized within the micellar phase for absorption.17 Together with the simulation of these events and results, an in vitro model of intestinal digestion of phospholipids should also take into account the coexistence of dietary exogenous phospholipids together with endogenous biliary phospholipids. Furthermore, the simultaneous presence of other dietary fats together with phospholipids should also be considered, in order to mimick a real intake of dietary lipids, where phospholipids are minor compounds compared to TG. Therefore, the aim of the present research was to perform a simulation of the intestinal digestion of PHT under in vitro conditions. First, the in vitro intestinal digestion model was optimized for the digestion of phospholipids, in order to be certain that the model reflected physiological intestinal digestion for these compounds, and to reject that any artifact or the own conditions of the digestion method would not interfere with the results obtained for PHT. Furthermore, the bioaccessibility of HT esterified as PHT after digestion was compared with that of free HT, or HT premixed with phospholipids. Finally, the impact of digestion on the antioxidant ability of PHT compared to HT was also assayed.

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MATERIALS AND METHODS

Reagents and Materials. HT (purity higher than 90% w/w) was acquired from Seprox Biotech (Madrid, Spain). PHT (86% PHT, 8% HT, and 6% fully hydrogenated phosphatidylcholine) was synthesized by enzymatic transphosphatidilation of HT and phosphatidylcholine (Phospholipon 90H, Lipoid, Switzerland). The detailed procedure of the synthesis of PHT was already described.15 A commercial olive oil was used as a representative of dietary typical oil. Trizma, maleic acid, pancreatin from porcine pancreas, bile salts, and ammonium hydroxide solution 30% were from Sigma-Aldrich Chemie GmbH (Steinheim, Germany). Phosphatidylcholine (PC) from egg yolk (98%) was purchased from Lipoid (Ludwigshafen, B



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compounds of vegetable oils was combined with the method of Carelli et al.23 to separate classes of phospholipids of vegetable oils. Initially, the procedure of Mateos et al.22 was followed. Briefly, a 500 mg diol-bonded phase cartridge (Isolute, Biotage, UK) was conditioned with 6 mL of methanol and 6 mL of hexane, consecutively. The lipid sample (1 mL, 40 mg/mL in chloroform) was added to the cartridge. Most glycerides (mainly MG, and residual DG and TG in case) were first eluted with 6 mL of hexane. Then, most FFA were eluted with 4 mL of hexane: ethyl acetate (90:10, v/v). The next step of the method of Mateos et al.22 used methanol for elution of phenolic compounds. However, we modified this step by mixing methanol with ammonium hydroxide (15 mL of methanol/ ammonium hydroxide solution 99.5:0.5, v/v) for the proper elution of the polar lipids (lyso-PHT, lyso-PL, and residual PHT), according to the procedure of Carelli et al.23 This modification was necessary because it was tested that only methanol was not useful to elute lysoPHT, although it effectively eluted HT. Only the mixture with ammonium hydroxide allowed a proper recuperation of lyso-PHT. Curiously, the complete method of Carelli et al.23 (without combination with the method of Mateos et al.22) was not useful for recovering lyso-PHT, although we tested that it was effective for lysoPL purification. Finally, we found that the combination of the first steps of the method of Mateos et al.22 with the last step of Carelli et al.23 were the most proper conditions to a successful recovery of lysoPHT. At the end of the procedure, the solvent fraction containing polar lipids was evaporated under N2 at 40 °C and redissolved in chloroform/methanol (2:1 v/v) for subsequent analysis. The purity of the lyso-PHT product obtained by the developed protocol was closer to 80%, together with minor levels of FFA, MG, and lyso-PL. Antioxidant Activity of Compounds by DPPH Test. The antioxidant activity of the intestinal medium containing PHT or HT was measured at 0 and 60 min of digestion by DPPH test. An aliquot (50 μL) of ethanol solution containing 5−30 μg/mL of intestinal media was added to 1950 μL of DPPH in ethanol (23.5 μg/mL). The reaction was completed after 90 min at room temperature and darkness, and absorbance was measured at 517 nm. The remaining DPPH concentration in the reaction medium was estimated by proper calibration curves of DPPH. The scavenging activities were expressed as 50% of inhibitory concentration (EC50), which denotes the concentration of HT (mM) (expressed as the equivalent concentration of HT in PHT) required for giving a 50% reduction of DPPH concentration relative to that of a DPPH control. Control experiments of the digestion media in the absence of the tested compounds were also performed, and the lack of change in DPPH concentration was measured. Statistical Analysis. Statistical analyses were performed by means of the general linear model procedure of the SPSS 17.0 statistical package (SPSS Inc., Chicago, IL, USA) by one-way analysis of variance. Differences were considered significant at p ≤ 0.05. Posthoc Tukey’s tests were performed in order to establish significant differences.

170/15/0.5 v/v/v/v), (B) 2-propanol/Milli-Q H2O/acetic acid/ triethylamine (840/140/15/0.5 v/v/v/v), and (C) hexane. The method starts at 50% A and 50% C increasing up to 100% A in 5.1 min, and reaching 12% B and 88% A in 20 min. This percentage is maintained for 15 min, and then is changed up to 40% B and 60% A in 0.5 min. Finally, the gradient returns to initial conditions and is maintained for 20 min. This methodology was used to analyze phospholipid and lysophospholipid species, HT, and bile salts. Identification and quantification was carried out by using standards for each lipid class. In the case of lyso-PHT, a purification procedure of this standard was previously necessary as described later. In order to minimize error when using HPLC with ELSD, rigorous calibration curves utilizing the appropriated standards were developed for each set of samples injected, since the detector response was nonlinear and specific to each compound. Neutral Lipids. The neutral lipids composition was determined on an Agilent poroshell 120 (2.7 μm, 100 × 4.6 mm2) coupled to an HPLC Agilent 1200 Series (Avondale, PA) containing a thermostated column compartment (35 °C), a quaternary pump, an autosampler, a vacuum degasser, and an evaporative light scattering detector (ELSD). Conditions of the ELSD were 3.5 bar and 35 °C. A split valve was used after the column, and only 50% of the mobile phase was directed through the detector. The column temperature was maintained at 35 °C. The ternary gradient has already been detailed by Vazquez, Fernandez, Martin, Reglero, and Torres.21 This methodology was used to analyze TG, diglycerides (DG), monoglycerides (MG), and FFA. Identification and quantification was carried out by using standards for each lipid class. In order to minimize error when using HPLC with ELSD, rigorous calibration curves utilizing appropriated commercial standards were developed for each set of samples injected, since the detector response was nonlinear and specific to each compound. Purification of Lyso-PHT for Analysis of Digestion Products. For a proper quantification of digestion products from PHT, the major hydrolysis product lyso-PHT was necessary as pure as possible to perform the corresponding standard calibration curves. To obtain this standard, it was necessary to (1) develop a protocol to hydrolyze PHT to lyso-PHT up to enough amounts necessary for the study and (2) develop a protocol for the subsequent purification of the released lysoPHT. Hydrolysis Reaction of PHT to Lyso-PHT. Taking the digestion model of PL as a reference, diverse modifications were performed in order to reach as complete hydrolysis as possible of PHT up to lysoPHT. Preliminary assays showed that either other lipids in the medium, as well as other surface active agents, such as bile salts or MG, were necessary for a proper hydrolysis of PL and PHT in general. Therefore, olive oil was kept in the reaction, and MG was used instead of bile salts. This was done because the later separation of bile salts from lyso-PHT and lyso-PL in general during the purification procedure was complicated, contrary to isolation from MG. Briefly, the hydrolysis medium consisted of 80 mg of PHT mixed with 80 mg of olive oil, 60 mg of MG, 0.4 mL of CaCl2 (325 mM), 1.2 mL of NaCl (3.25 mM), and 20.6 mL of Trizma−maleate buffer pH 7.5. The mixture was homogenized (Ultra-Turrax IKA T18) for 4 min at 3500 rpm. The reaction was started with the addition of fresh pancreatin extract (0.3 g of pancreatin in 1.8 mL of Trizma−maleate buffer pH 7.5, stirred for 10 min and centrifuged at 1600 g for 15 min) and PLA2 solution (0.2 g of PLA2 in 2 mL of Trizma−maleate buffer pH 7.5, vortexed for 1 min). The hydrolysis reaction was performed in a thermostatically controlled shaker (IKA KS 4000 ic control) at 37 °C and 250 rpm for 120 min. Finally, the total lipids from the reaction were extracted by chloroform/methanol (2:1, v/v) for subsequent analysis and purification. Under these conditions, around 95% of PHT was hydrolyzed to lyso-PHT and FFA. Purification of Lyso-PHT by SPE. After the hydrolysis reaction, the obtained mixture of lipid compounds consisted of FFA (45%) and lyso-PHT (35%) as major products, together with minor levels of residual PHT, lyso-PL, and MG. The purification of lyso-PHT was performed by SPE. The method of Mateos et al.22 to separate phenolic

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RESULTS AND DISCUSSION Development of an in Vitro Intestinal Model for Digestion of Phospholipids. Previous to in vitro digestion of PHT, an intestinal digestion model for phospholipids was first developed, in order to be certain that the model reflected the physiological intestinal lipid digestion of these lipids, and to reject that any artifact or the own conditions of the digestion method would not interfere with the results obtained for the PHT hydrolysis. One of the main problems to optimize a reliable in vitro model for phospholipids is the imprecise data in the scientific literature on basic and general parameters such as the typical hydrolysis degree of these lipids, the habitual amount of luminal phospholipids and PLA2, or the exact degree of bioaccessibility. Therefore, as a reference of physiological criteria, we considered the data of Borgströ m 17 that stated that

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phospholipids are absorbed efficiently, that conversion to lysophospholipids is essential for absorption to occur, and that the luminal concentration of PLA2 is high enough for hydrolysis to lysophospholipids. A previously developed model for TG19 was adopted as a starting point. Such a model was considered useful because we already demonstrated that it closely reflected the physiological results found in the intraluminal phase in man during fat digestion, namely, TG. However, some modifications would be necessary in order to simulate a proper digestion for phospholipids. The developed model for intestinal digestion of TG uses the pancreatin as the source of digestive enzymes. Pancreatin is a pancreatic extract that contains most of the digestive enzymes responsible for digestion of major macromolecules at intestinal lumen. In the specific case of lipid digestion, pancreatic lipase is the major enzyme found in pancreatin. Although pancreatic lipase may partially hydrolyze phospholipids, most hydrolysis is catalyzed by PLA2. However, both the presence and the level of PLA2 in pancreatin for proper hydrolysis are not stated in the product specifications. On the other hand, the developed digestion model for TG was adapted to simulate the simultaneous presence of dietary exogenous phospholipids and TG, as well as endogenous biliary phospholipids. Concerning exogenous dietary phospholipids, those were included at a proper ratio to TG. This is difficult data to establish, because the dietary intake of phospholipids reported in the scientific literature is quite variable, taking into account the variable amount of phospholipids that can be found depending on a meal. Thus, ratios from 1 to 10% of total daily fat intake has been reported.16,24 A high-phospholipid meal was simulated in the model. Concerning endogenous biliary phospholipids, the included dose was proportional to bile salts and cholesterol, taking into account the gallbladder bile lipid secretion during a fed state.25 Furthermore, a separation of the preparation of both fractions of phospholipids at different stages was considered. This is because exogenous phospholipids are found mixed with TG in the aqueous digestion medium, where the phospholipids are found as a surface component of the TG emulsion.17 On the other hand, the endogenous phospholipids are found as a dispersion in bile salts as lamellar aggregates or mixed micellar aggregates.17 Therefore, we considered that such different phases should be prepared separately, trying to simulate the pre-emulsion of dietary lipids that would enter later the intestinal lumen to be mixed with the preformed biliary secretion. Figure 1a shows the in vitro digestion initially performed by the use of pancreatin. As shown, the level of pancreatin effectively hydrolyzed TG forms up to the physiologically considered level at duodenal lumen; namely, around 75% was degraded to the main hydrolysis products (FFA and MG). On the contrary, both the hydrolysis of phospholipids and the level of the product lysophospholipids were minor, suggesting that pancreatin does not have enough hydrolytic activity to effectively digest phospholipids. Therefore, the enrichment with PLA2 was considered necessary. As far as we know, previous information on typical physiological levels of PLA2 has not been clearly reported, so we considered as criteria of PLA2-dose optimization the statement of Borgstrom,17 who suggested that the luminal concentration of PLA2 is high enough for a complete hydrolysis of phospholipids to lysophospholipids. Therefore, several

Figure 1. Course of in vitro intestinal digestion by (a) pancreatin or (b) PLA2-enrichment of pancreatin.

commercial PLA2’s added at variable levels were tested (data not shown) up to reaching a quantitative hydrolysis level of phospholipids. As shown in Figure 1b, the final optimized method with a proper dose of PLA2 progressively hydrolyzed phospholipids up to almost complete digestion, releasing lysophospholipid as the major hydrolysis product, together with residual nonhydrolyzed phospholipids. Furthermore, the digestion of the rest of the lipids was not influenced by the presence of PLA2. These results were closer to in vivo physiological digestion of phospholipids,17 so the in vitro model that was developed was considered useful to test the intestinal digestion of the experimental molecule PHT. The detailed procedure of the in vitro model after performing all the explained modifications was that described in the Materials and Methods section. In Vitro Intestinal Digestion of Phosphatidylhydroxytyrosol. To perform the digestion of PHT, it was not as easy as replacing total phospholipids of the media by PHT. This is because the fraction of phospholipids from biliary secretion should be kept in order to reproduce a physiological situation. Therefore, the PHT was included in the media as dietary phospholipid, but it coexisted together with the endogenous phospholipid of the medium during the hydrolysis process. The evolution of in vitro intestinal digestion of PHT is shown in Figure 2. It can be observed that PHT was progressively hydrolyzed to lyso-PHT. However, the rate of digestion was slower than the phospholipid control sample (Figure 1b). Thus, at the end of digestion, only around 50% of PHT was hydrolyzed. To compare the digestion of PHT in the presence and in the absence of endogenous phospholipids, additional experiments D



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differences on these factors is unknown but might be considered as potential reasons of the different results on the rate of hydrolysis. According to Scott et al.27 on the contribution of the sn-3 substituent on the PLA2 binding affinity, these authors stated that the own nature of the phosphatidyl ester affects the physical chemistry of the aggregates, such as surface charge distribution or the state of aggregation. In this respect, it is important to remark that PL and PHT might show a different surface charge distribution, taking into account that the former would be a neutral PL, whereas PHT should be an anionic phospholipid due to the removal of choline. Related to the different state of aggregation, the numbers of micellar structures formed after in vitro digestion of PL and PHT were determined. According to Figure 4, a lower number of micellar structures were detected for PHT digestion when compared to PL, which seemed to be in agreement with the lower hydrolysis of PHT.

Figure 2. Course of in vitro intestinal digestion of PHT.

were performed. Thus, PHT as the only form of phospholipid substrate for PLA2 was added to the media, which means that biliary PC was removed and that PHT was added both as dietary and biliary phospholipid. For comparative purposes, the same procedure was performed for PL. The release of lysophospholipid species was considered to compare the rate of hydrolysis. As shown in Figure 3, the rate of digestion of

Figure 4. Hydrolysis of PL and PHT versus the number of micellar structures.

Bioaccessibility of Phosphatidylhydroxytyrosol. During intestinal digestion of dietary fat, it has been shown that the intraluminal content is structured as an oily phase (OP) dispersed in a micellar bile salt solution (MP).29 This OP mainly contains undigested TG and DG, whereas the MP contains bile salts and the end products of enzymatic hydrolysis, namely, MG, FFA, and lysophospholipids. All of these lipid products are aggregated as mixed micelles, micelles, vesicles, or emulsion droplets.30,31 Absorption of lipid products takes place supported by this MP, which enhances the transport of lipid products to the enterocytes throughout the unstirred water layer close to the microvillous membrane, where they are absorbed.32 Furthermore, a minor fraction of insoluble calcium soaps of fatty acids liberated during the pancreatic digestion can be formed, which are not absorbable, tend to precipitate, and are wasted in faeces. The analysis of lipid products of these three phases, MP, OP, and PP, contributes to the study of bioaccessibility, defined as the fraction of a compound that is released from its matrix in the gastrointestinal tract and thus becomes available for intestinal absorption.33 According to Figure 5, most hydrolysis products, including neutral and polar lipids, were found distributed within the bioaccessible fraction MP, suggesting a high bioaccessibility. In the case of the control PL samples, a significant OP was isolated (16% of total lipid products), and a minor PP was also detected. The specific distribution of total lipid products within the three phases is detailed in Figure 6. As shown in Figure 6a,

Figure 3. Lyso-PL or lyso-PHT releasing during in vitro intestinal digestion of PL or PHT, respectively, without coexistence of other phospholipids in the media.

PHT was slower than the hydrolysis of the control PL. Therefore, this experiment showed that the slower digestion of PHT would be related to the own molecule PHT. The explanation of this result is complex, because previous information on the digestion of phospholipids with a modified polar-head with so atypical structures is scarce. Nevertheless, it has been described that there are different factors that determine the rate and extent of enzymatic hydrolysis of any phospholipid in general. According to scientific literature, the aggregation of the substrate under the form of mixed micelles, lamellar structures, liposomes, or emulsions is necessary, since this appears to facilitate the interaction of the sn-2 fatty acid ester with the catalytic site of the enzyme.26−28 However, the specific type of aggregation determines the hydrolysis rate. Furthermore, the presence of other lipids which influences the packing of the fatty acid chains and the headgroup are also related to the action of PLA2.28 Whether PHT determined E



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the ability to reproduce in vivo physiological results on bioaccessibility of phospholipids, and lipids in general. Some differences were observed in the case of the sample containing PHT. As shown in Figure 5, the distribution of lipid products within the OP was lower (around 12% of total lipid products), and the PP increased instead (around 9% of total lipid products precipitated) compared to the PL sample. At any case, most lyso-PHT and residual PHT were found within the bioaccessible fraction MP. As detailed in Figure 6b, the observed increase in the PP fraction was mainly due to an increased precipitation of FFA and a fraction of nondigested PHT. In fact, the bioaccessibility of the FFA changed from 92% in the case of PL samples to 80% in the case of PHT samples. This unexpected a priori result is difficult to explain, attending to the fact that both PL and PHT samples had the same amount and fatty acid profile. In general, any precipitation of FFA during intestinal digestion should be related to a limited solubilization within micellar structures and formation of calcium soaps that cannot be solubilized in the aqueous media. Taking into account that the assays of PHT samples produced a lower amount of micellar structures than PL samples (Figure 4), this might determine the differences on nonmicellated fatty acids between both samples. It was also significant that around 8% of total PHT was precipitated

Figure 5. Partition of total lipid products between the isolated fractions MP, OP, and PP after 60 min of in vitro intestinal digestion.

the polar lipids from hydrolysis of PL (lyso-PL and residual nondigested PL) were totally included in the MP, suggesting a high bioaccessibility of PL. These results were in agreement with the expected physiological data of phospholipids, so these obtained results also validated the in vitro model developed on

Figure 6. Partition of individual lipid products between the isolated fractions MP, OP, and PP after 60 min of in vitro intestinal digestion. F



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mucosa for absorption and subsequent metabolism, and its validation as a potential vehicle of HT. Effect of in Vitro Digestion on the Antioxidant Activity of Phosphatidylhydroxytyrosol. In a recent study, we have successfully shown a significant antioxidant activity of PHT in diverse edible oils, which was comparable or even superior to HT.4 Taking such evidence of antioxidant ability of PHT into account, the antioxidant effect was explored in the current study when it was affected by the intestinal digestion process. This was considered of interest, especially taking into account that PHT was mainly degraded to lysoPHT after intestinal digestion, and this would be the major bioaccessible molecule. Furthermore, the modification of the antioxidant activity of diverse phenolic compounds after the process and conditions of gastrointestinal digestion has been previously described, including HT;34 hence, the interest in producing carriers of these compounds that could protect their activity during the gastrointestinal transit. As shown in Figure 8, either PHT or digested PHT showed a lower antioxidant activity than HT at any moment of digestion.

together with FFA, whereas nondigested PL did not precipitate in the case of PL sample. A minor fraction of precipitated bile salts was also measured for the PHT sample contrary to the PL sample. Therefore, the particular molecule PHT might be related to these results. In order to evaluate the magnitude of PHT as a carrier of HT, its bioaccessibility as the esterified form was compared to that of HT. Furthermore, a comparative study with a premixture HT+PL was considered of interest in order to compare the bioaccessibility of HT when it coexisted with a dose of dietary PL, with that of HT under the esterified form as PL (PHT). According to Figure 7, most HT was included

Figure 7. Partition of total HT between the isolated fractions MP, OP, and PP after 60 min of in vitro intestinal digestion.

within the bioaccessible fraction MP for the three forms of HT addition, suggesting a high bioaccessibility. Nevertheless, the portion of HT within the MP under the form of PHT was slightly superior to the other forms of addition of HT. Such a difference was due to a fraction of HT within the OP that was not detected for the PHT sample. These results would suggest that a portion of HT would not be so easily dispersed in the aqueous media when this phenolic compound was free or premixed with PL, contrary to the esterified forms of HT as PHT. Probably, the amphiphilic properties of PHT as a phospholipid allowed its dispersion in the aqueous media, which in turn indirectly allowed the dispersion of the vehiculated HT. Furthermore, a minor precipitated fraction of HT was found for all samples, which was not significantly different. These results contributed to the study of the potential of PHT as well as to the general behavior of the phenolic HT. Previous detailed information on the fractionation of HT, or combinations HT+PL, between the different phases of the intestinal medium under in vitro conditions was not found. Concerning the bioaccessibility of PHT, it should be noted that Borgström17 stated that conversion of phospholipids to lysophospholipids is essential for absorption to occur. Therefore, according to the obtained results, despite that most of the digested PHT was found within the bioaccessible MP fraction, PHT would be only partially absorbed as lyso-PHT. However, this fact remains unclear and should be elucidated, since it has also been reported that there is no abnormality in phospholipid absorption in the case of deficiency in PLA2 (nonhydrolyzed phospholipids).24 Therefore, further studies concerning bioavailability of PHT would be of interest in order to validate whether PHT would be effectively recognized by intestinal

Figure 8. Scavenging activity of HT and equivalent concentration of HT under the form of PHT, before and after digestion.

Furthermore, the antioxidant activity of the control HT did not seem to be affected by the digestion process. An interesting finding was that the EC50 value of digested PHT was significantly inferior to that observed before digestion. This result might suggest that the digestion products, mainly the major lyso-PHT, might show better antioxidant ability than the former PHT. This could be considered reasonable, taking into account the simpler and more hydrophilic molecule of lysoPHT compared to PHT. Thus, after intestinal digestion, a closer value of EC50 between digested PHT and HT was achieved (0.6 and 0.5 mM, respectively). As a summary, the present study showed that the synthetic phosphatidyl derivative of HT is recognized by phospholipases during the simulation of intestinal digestion but less efficiently than the analogous PL. At any case, both the major hydrolysis product, namely, lyso-PHT, and most of the nondigested PHT were found within the bioaccessible aqueous fraction; hence, most of the esterified HT was potentially bioaccessible. On the other hand, the digested PHT might be a more superior antioxidant than the nonhydrolyzed PHT. Nevertheless, the evaluation of the potential bioactivity of PHT and its digested G



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product would be necessary in further studies, in order to validate the carriers of HT under the form of phospholipids.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +34 910017930. E-mail: [email protected]. Funding

This work was supported by the Ministerio de Economiá y Competitividad (INNSAOLI, project number IPT-2011-1248060000, Subprograma INNPACTO) and the Comunidad de Madrid (ALIBIRD, project number S2009/AGR-1469). The contract of M.I.M.-V. was also supported by the INNSAOLI project and is also acknowledged. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED DG, diglycerides; FFA, free fatty acids; HT, hydroxytyrosol; MG, monoglycerides; MP, micellar phase; OP, oily phase; PC, phosphatidylcholine; PHT, phosphatidylhydroxytyrosol; PL, Phospholipon 90H; PLA2, phospholipase A2; PP, precipitated phase; TG, triglycerides

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REFERENCES

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Phosphatidyl Derivative of Hydroxytyrosol. In Vitro Intestinal

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