Phosphatidyl Derivative of Hydroxytyrosol. In Vitro IntestinalDigestion, Bioaccessibility, and Its Effect on Antioxidant ActivityDiana Martin,*,†,‡ Maria I. Moran-Valero,†,‡ Víctor Casado,†,‡ Guillermo Reglero,†,‡,§

and Carlos F. Torres†,‡

†Departamento de Produccion y Caracterizacion de Nuevos Alimentos, Instituto de Investigacion en Ciencias de la Alimentacion(CIAL) (CSIC−UAM), 28049 Madrid, Spain‡Seccion Departamental de Ciencias de la Alimentacion, Facultad de Ciencias, Universidad Autonoma 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 wasperformed. First, an in vitro intestinal digestion model for phospholipids was developed. The impact of digestion in theantioxidant ability of PHT was also assayed. PHT was progressively hydrolyzed to lyso-PHT. However, digestion was slower thanthe phospholipid control. Nevertheless, most hydrolysis products were found at the micellar phase fraction, meaning a highbioaccessibility. Either PHT or digested PHT showed lower antioxidant activity than HT. However, PHT improved itsantioxidant ability after digestion, likely related to lyso-PHT. As a summary, the synthetic phosphatidyl derivative of HT as PHTis 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 HTunder the form of phospholipids might be of interest.

KEYWORDS: hydroxytyrosol, phospholipids, in vitro digestion, bioaccessibility, phenolipids, antioxidants

■ INTRODUCTION

The popularity of phenolic compounds in general as bioactivenatural antioxidants is currently well-known. Within poly-phenolic compounds, hydroxytyrosol (HT) has been anattractive molecule in the last decades that has shown a greatbioactivity and antioxidant power, which has been related toantiatherogenic, 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 rapidlymetabolized in enterocyte and liver to minor metabolites, and isonly found at minor levels in plasma and tissues.5−7 Taking thisevidence into account, the production of HT derivatives toenhance its access to cells and tissues, or increase its systemichalf-life, has been an approach of intense research in the lastyears.8−10 In this respect, the production of “phenolipids”,namely, lipophilized phenolics resulting from the union of alipid to the phenolic moiety,11 has been explored by diverseauthors to obtain carriers of HT. HT esters with acyl chains hasbeen the most frequently applied strategy to modify HT, andmost of the obtained derivatives have shown improvedbioactivities. As example, Trujillo et al.10 showed that HTderivatives as long-chain esters had a higher protective effectagainst oxidative damage in an ex vivo brain homogenatemodel. Recent derivates of HT as ethyl ethers12 exhibitedstronger intestinal anticarcinogenic activity than HT13 and weremore efficiently absorbed than HT.14

Concerning novel derivatives of phenolic compounds,Casado, Reglero, and Torres15 have recently synthesizedphospholipid derivatives of HT with phosphatidylcholine,

where the phenolic compound was included in the polarhead of the phospholipid by replacing the choline by enzymatictransphosphatidylation. A successful antioxidant activity of thisnew molecule for edible oils has been shown recently.4 Thisnew molecule (phosphatidylhydroxytyrosol, PHT) was pro-posed as a potential vehicle of HT but, at the same time, takingthe additional interest of the PL as a backbone. This is becausephospholipids are also bioactive molecules by themselves. Theyare well-known as essential molecules for the maintenance ofliving cells as major constituents of cell membranes. Addition-ally, evidence has pointed out a positive impact of dietaryphospholipids on human health, such as relevant implicationsin hypercholesterolemia, atherosclerosis, cardiovascular disease,inflammation and immunity, liver disorders, brain development,as well other chronic diseases.16 On the other hand, due to theiramphiphilic nature and surface-active properties, phospholipidsare well-known as emulsifier ingredients in food, pharmaceut-ical, or cosmetic industry. The emulsifying properties are alsorelated to the role of phospholipids to enhance the digestionand absorption of hydrophobic molecules at the intestinal level,because they contribute to emulsification of lipid drops in theaqueous media for the proper action of pancreatic lipases, andthey form mixed micelles, necessary for the vehiculization of thelipid products to enterocytes.17

Received: July 21, 2014Revised: September 12, 2014Accepted: September 12, 2014

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However, one of the questions that arise when phenoliccompounds are esterified as the polar head of phospholipidswould be whether the derived structure would be digested andbioaccesible during gastrointestinal digestion, as the first stepbefore any other physiological action beyond intestinal tissues.The in vitro intestinal models of lipid digestion are an

interesting tool for obtaining preliminary and valuableinformation concerning digestion of lipid species. Due totheir utility, the use of in vitro digestion models in the study ofbioactive compounds has increased, becoming a well-acceptedanalytical tool.18 However, as far as we know, all the models ofin vitro lipid digestion have been developed for the major lipiddietary forms, namely, triglycerides (TG), whereas the in vitrodigestion models focused on phospholipids have not beenextensively investigated. In general, we consider that thedevelopment of a model that closely simulates the in vivoconditions is especially essential when performing the digestionof novel or unknown lipids under in vitro conditions, in order toaccurately understand obtained results and to avoid misinter-pretations due to the own methodology used. Therefore, areliable in vitro digestion model for phospholipids should bepreviously developed before the application of the model to theexperimental 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 fromthe sn-2 location almost completely to release lyso-phospholipids and free fatty acids (FFA).17 After hydrolysis, ahigh bioaccessibility has been described for phospholipids,which means that their lipid products are easily solubilizedwithin the micellar phase for absorption.17 Together with thesimulation of these events and results, an in vitro model ofintestinal digestion of phospholipids should also take intoaccount the coexistence of dietary exogenous phospholipidstogether with endogenous biliary phospholipids. Furthermore,the simultaneous presence of other dietary fats together withphospholipids should also be considered, in order to mimick areal intake of dietary lipids, where phospholipids are minorcompounds compared to TG.Therefore, the aim of the present research was to perform a

simulation of the intestinal digestion of PHT under in vitroconditions. First, the in vitro intestinal digestion model wasoptimized for the digestion of phospholipids, in order to becertain that the model reflected physiological intestinaldigestion for these compounds, and to reject that any artifactor the own conditions of the digestion method would notinterfere with the results obtained for PHT. Furthermore, thebioaccessibility of HT esterified as PHT after digestion wascompared with that of free HT, or HT premixed withphospholipids. Finally, the impact of digestion on theantioxidant ability of PHT compared to HT was also assayed.

■ MATERIALS AND METHODSReagents 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 synthesizedby enzymatic transphosphatidilation of HT and phosphatidylcholine(Phospholipon 90H, Lipoid, Switzerland). The detailed procedure ofthe synthesis of PHT was already described.15 A commercial olive oilwas 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-AldrichChemie GmbH (Steinheim, Germany). Phosphatidylcholine (PC)from egg yolk (98%) was purchased from Lipoid (Ludwigshafen,

Germany). Sodium sulfate anhydrous, sodium chloride, calciumchloride, and ethanol absolute were purchased from Panreac(Barcelona, Spain). Ortho-phosphoric acid was purchased fromScharlab (Sentmenat, Barcelona). All solvents used were of HPLCgrade 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 gof phospholipid which were mixed with 16 mL of Trizma−maleatebuffer 0.1 M pH 7.5. In the case of control digestions and optimizationof the in vitro model, the sample was the same phospholipid used forthe synthesis of PHT (Phospholipon 90H, PL). In the case of PHT,this was added at the same equivalent weight used for the digestion ofPL. The prepared medium was pre-emulsified by homogenization for 2min at 3500 rpm.

On the other hand, a solution trying to simulate biliary secretionwas prepared by mixing 0.1 g of PC, 0.25 g of bile salts, 0.02 g ofcholesterol, 0.5 mL of a 325 mM CaCl2 solution, 1.5 mL of a 3.25 mMNaCl solution, and 10 mL of Trizma−maleate buffer, and this mixturewas homogenized for 2 min at 3500 rpm. Then, the pre-emulsifiedsample and the simulated biliary secretion were mixed andhomogenized together for 2 min at 3500 rpm. The whole mediawas placed in a thermostatically controlled vessel at 37 °C andcontinuously stirred by a magnetic stir bar at 1000 rpm. Thesimulation of intestinal digestion was started by the addition of freshpancreatin extract (0.5 g of pancreatine in 3 mL of Trizma−maleatebuffer, stirred for 10 min and centrifuged at 1600 × g for 15 min). Theenrichment with PLA2 was carried out by addition of 5 mg of a foodgrade PLA2 from Streptomyces violaceoruber (103 U/mg) from NagaseChemtex Corporation (Fukuchiyama Factory, Kyoto, Japan). Afteraddition of enzymes, reaction was continued for 60 min. In order tostudy the evolution of lipid products throughout the hydrolyticprocess, aliquots were taken at 0, 5, 10, 30, and 60 min of reaction.

Additional digestions were performed in order to compare thebioaccessibility of PHT with that of HT, or with that of a premixedHT with a dose of dietary PL (HT+PL). In both cases, HT was addedto 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 intriplicate.

Separation of Phases after in Vitro Lipid Digestion. At theend of digestion, the medium was submitted to centrifugation at 4000rpm for 40 min at 37 °C (5810R Eppendorf Iberica, Madrid, Spain)according to Soler-Rivas et al.20 After centrifugation, an upper oilyphase (OP), a lower aqueous or micellar phase (MP), and a minorprecipitated phase (PP) were obtained. The lipid composition of eachphase was analyzed. In the case of the MP, aliquots were collected forquantification of micellar structures by using a light microscope and aNeubauer cell counter chamber (Brand, Germany).20

Lipid Extraction. The total lipids from samples were extractedtwice 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). Ortho-phosphoric acid was necessary for a proper recovery of the lyso-PLspecies. The mixture was vortexed for 1 min and centrifuged for 10min at 13500 rpm (ScanSpeed mini, Micro Centrifuge). The organicphase containing the separated lipids was collected, and anhydroussodium sulfate was added before further analysis.

Analysis of Lipid Products. Polar Lipids. The polar lipidcomposition was determined on a Luna 5 μm HILIC diol column(250 mm, 4.6 mm, Phenomenex, Torrance, California, USA) coupledto an HPLC Agilent 1200 Series containing a thermostatized columncompartment, a quaternary pump, an autosampler, a vacuum degasser,and an evaporative light scattering detector. The used method wasbased on Casado et al.15 with brief modifications. The flow rate was 1.5mL min−1. A splitter valve was used after the thermostatized columncompartment and part of the mobile phase was directed through thedetector (3.5 bar and 55 °C). The column temperature wasmaintained at 55 °C. The mobile phase utilized consisted of a ternarygradient of (A) hexane/2-propanol/acetic acid/triethylamine (815/

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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. Themethod starts at 50% A and 50% C increasing up to 100% A in 5.1min, and reaching 12% B and 88% A in 20 min. This percentage ismaintained for 15 min, and then is changed up to 40% B and 60% A in0.5 min. Finally, the gradient returns to initial conditions and ismaintained for 20 min.This methodology was used to analyze phospholipid and

lysophospholipid species, HT, and bile salts. Identification andquantification was carried out by using standards for each lipid class.In the case of lyso-PHT, a purification procedure of this standard waspreviously necessary as described later. In order to minimize errorwhen using HPLC with ELSD, rigorous calibration curves utilizing theappropriated standards were developed for each set of samplesinjected, since the detector response was nonlinear and specific to eachcompound.Neutral Lipids. The neutral lipids composition was determined on

an Agilent poroshell 120 (2.7 μm, 100 × 4.6 mm2) coupled to anHPLC Agilent 1200 Series (Avondale, PA) containing a thermostatedcolumn compartment (35 °C), a quaternary pump, an autosampler, avacuum degasser, and an evaporative light scattering detector (ELSD).Conditions of the ELSD were 3.5 bar and 35 °C. A split valve was usedafter the column, and only 50% of the mobile phase was directedthrough 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 wascarried out by using standards for each lipid class. In order to minimizeerror when using HPLC with ELSD, rigorous calibration curvesutilizing appropriated commercial standards were developed for eachset of samples injected, since the detector response was nonlinear andspecific to each compound.Purification of Lyso-PHT for Analysis of Digestion Products.

For a proper quantification of digestion products from PHT, the majorhydrolysis product lyso-PHT was necessary as pure as possible toperform the corresponding standard calibration curves. To obtain thisstandard, it was necessary to (1) develop a protocol to hydrolyze PHTto lyso-PHT up to enough amounts necessary for the study and (2)develop a protocol for the subsequent purification of the released lyso-PHT.Hydrolysis Reaction of PHT to Lyso-PHT. Taking the digestion

model of PL as a reference, diverse modifications were performed inorder to reach as complete hydrolysis as possible of PHT up to lyso-PHT. Preliminary assays showed that either other lipids in themedium, 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 insteadof bile salts. This was done because the later separation of bile saltsfrom lyso-PHT and lyso-PL in general during the purificationprocedure 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), 1.2mL of NaCl (3.25 mM), and 20.6 mL of Trizma−maleate buffer pH7.5. The mixture was homogenized (Ultra-Turrax IKA T18) for 4 minat 3500 rpm. The reaction was started with the addition of freshpancreatin extract (0.3 g of pancreatin in 1.8 mL of Trizma−maleatebuffer 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 bufferpH 7.5, vortexed for 1 min). The hydrolysis reaction was performed ina thermostatically controlled shaker (IKA KS 4000 ic control) at 37 °Cand 250 rpm for 120 min. Finally, the total lipids from the reactionwere extracted by chloroform/methanol (2:1, v/v) for subsequentanalysis and purification. Under these conditions, around 95% of PHTwas 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%) andlyso-PHT (35%) as major products, together with minor levels ofresidual PHT, lyso-PL, and MG. The purification of lyso-PHT wasperformed by SPE. The method of Mateos et al.22 to separate phenolic

compounds of vegetable oils was combined with the method of Carelliet al.23 to separate classes of phospholipids of vegetable oils.

Initially, the procedure of Mateos et al.22 was followed. Briefly, a 500mg diol-bonded phase cartridge (Isolute, Biotage, UK) wasconditioned 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 residualDG 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 forelution of phenolic compounds. However, we modified this step bymixing methanol with ammonium hydroxide (15 mL of methanol/ammonium hydroxide solution 99.5:0.5, v/v) for the proper elution ofthe polar lipids (lyso-PHT, lyso-PL, and residual PHT), according tothe procedure of Carelli et al.23 This modification was necessarybecause it was tested that only methanol was not useful to elute lyso-PHT, although it effectively eluted HT. Only the mixture withammonium hydroxide allowed a proper recuperation of lyso-PHT.Curiously, the complete method of Carelli et al.23 (withoutcombination with the method of Mateos et al.22) was not useful forrecovering lyso-PHT, although we tested that it was effective for lyso-PL purification. Finally, we found that the combination of the firststeps of the method of Mateos et al.22 with the last step of Carelli etal.23 were the most proper conditions to a successful recovery of lyso-PHT. At the end of the procedure, the solvent fraction containingpolar lipids was evaporated under N2 at 40 °C and redissolved inchloroform/methanol (2:1 v/v) for subsequent analysis. The purity ofthe lyso-PHT product obtained by the developed protocol was closerto 80%, together with minor levels of FFA, MG, and lyso-PL.

Antioxidant Activity of Compounds by DPPH Test. Theantioxidant activity of the intestinal medium containing PHT or HTwas measured at 0 and 60 min of digestion by DPPH test. An aliquot(50 μL) of ethanol solution containing 5−30 μg/mL of intestinalmedia was added to 1950 μL of DPPH in ethanol (23.5 μg/mL). Thereaction was completed after 90 min at room temperature anddarkness, and absorbance was measured at 517 nm. The remainingDPPH concentration in the reaction medium was estimated by propercalibration curves of DPPH. The scavenging activities were expressedas 50% of inhibitory concentration (EC50), which denotes theconcentration of HT (mM) (expressed as the equivalent concentrationof HT in PHT) required for giving a 50% reduction of DPPHconcentration relative to that of a DPPH control.

Control experiments of the digestion media in the absence of thetested compounds were also performed, and the lack of change inDPPH concentration was measured.

Statistical Analysis. Statistical analyses were performed by meansof the general linear model procedure of the SPSS 17.0 statisticalpackage (SPSS Inc., Chicago, IL, USA) by one-way analysis ofvariance. Differences were considered significant at p ≤ 0.05. PosthocTukey’s tests were performed in order to establish significantdifferences.

■ RESULTS AND DISCUSSIONDevelopment of an in Vitro Intestinal Model for

Digestion of Phospholipids. Previous to in vitro digestion ofPHT, an intestinal digestion model for phospholipids was firstdeveloped, in order to be certain that the model reflected thephysiological intestinal lipid digestion of these lipids, and toreject that any artifact or the own conditions of the digestionmethod would not interfere with the results obtained for thePHT hydrolysis.One of the main problems to optimize a reliable in vitro

model for phospholipids is the imprecise data in the scientificliterature on basic and general parameters such as the typicalhydrolysis degree of these lipids, the habitual amount of luminalphospholipids and PLA2, or the exact degree of bioaccessibility.Therefore, as a reference of physiological criteria, weconsidered the data of Borgstrom17 that stated that

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phospholipids are absorbed efficiently, that conversion tolysophospholipids is essential for absorption to occur, and thatthe luminal concentration of PLA2 is high enough forhydrolysis to lysophospholipids.A previously developed model for TG19 was adopted as a

starting point. Such a model was considered useful because wealready demonstrated that it closely reflected the physiologicalresults found in the intraluminal phase in man during fatdigestion, namely, TG. However, some modifications would benecessary in order to simulate a proper digestion forphospholipids.The developed model for intestinal digestion of TG uses the

pancreatin as the source of digestive enzymes. Pancreatin is apancreatic extract that contains most of the digestive enzymesresponsible for digestion of major macromolecules at intestinallumen. In the specific case of lipid digestion, pancreatic lipase isthe major enzyme found in pancreatin. Although pancreaticlipase may partially hydrolyze phospholipids, most hydrolysis iscatalyzed by PLA2. However, both the presence and the level ofPLA2 in pancreatin for proper hydrolysis are not stated in theproduct specifications.On the other hand, the developed digestion model for TG

was adapted to simulate the simultaneous presence of dietaryexogenous phospholipids and TG, as well as endogenous biliaryphospholipids. Concerning exogenous dietary phospholipids,those were included at a proper ratio to TG. This is difficultdata to establish, because the dietary intake of phospholipidsreported in the scientific literature is quite variable, taking intoaccount the variable amount of phospholipids that can be founddepending on a meal. Thus, ratios from 1 to 10% of total dailyfat intake has been reported.16,24 A high-phospholipid meal wassimulated in the model. Concerning endogenous biliaryphospholipids, the included dose was proportional to bilesalts and cholesterol, taking into account the gallbladder bilelipid secretion during a fed state.25

Furthermore, a separation of the preparation of bothfractions of phospholipids at different stages was considered.This is because exogenous phospholipids are found mixed withTG in the aqueous digestion medium, where the phospholipidsare found as a surface component of the TG emulsion.17 Onthe other hand, the endogenous phospholipids are found as adispersion in bile salts as lamellar aggregates or mixed micellaraggregates.17 Therefore, we considered that such differentphases should be prepared separately, trying to simulate thepre-emulsion of dietary lipids that would enter later theintestinal lumen to be mixed with the preformed biliarysecretion.Figure 1a shows the in vitro digestion initially performed by

the use of pancreatin. As shown, the level of pancreatineffectively hydrolyzed TG forms up to the physiologicallyconsidered level at duodenal lumen; namely, around 75% wasdegraded to the main hydrolysis products (FFA and MG). Onthe contrary, both the hydrolysis of phospholipids and the levelof the product lysophospholipids were minor, suggesting thatpancreatin does not have enough hydrolytic activity toeffectively digest phospholipids. Therefore, the enrichmentwith PLA2 was considered necessary.As far as we know, previous information on typical

physiological levels of PLA2 has not been clearly reported, sowe considered as criteria of PLA2-dose optimization thestatement of Borgstrom,17 who suggested that the luminalconcentration of PLA2 is high enough for a complete hydrolysisof phospholipids to lysophospholipids. Therefore, several

commercial PLA2’s added at variable levels were tested (datanot shown) up to reaching a quantitative hydrolysis level ofphospholipids. As shown in Figure 1b, the final optimizedmethod with a proper dose of PLA2 progressively hydrolyzedphospholipids up to almost complete digestion, releasinglysophospholipid as the major hydrolysis product, togetherwith residual nonhydrolyzed phospholipids. Furthermore, thedigestion of the rest of the lipids was not influenced by thepresence of PLA2. These results were closer to in vivophysiological digestion of phospholipids,17 so the in vitro modelthat was developed was considered useful to test the intestinaldigestion of the experimental molecule PHT. The detailedprocedure of the in vitro model after performing all theexplained modifications was that described in the Materials andMethods section.

In Vitro Intestinal Digestion of Phosphatidylhydrox-ytyrosol. To perform the digestion of PHT, it was not as easyas replacing total phospholipids of the media by PHT. This isbecause the fraction of phospholipids from biliary secretionshould be kept in order to reproduce a physiological situation.Therefore, the PHT was included in the media as dietaryphospholipid, but it coexisted together with the endogenousphospholipid 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 progressivelyhydrolyzed to lyso-PHT. However, the rate of digestion wasslower than the phospholipid control sample (Figure 1b). Thus,at the end of digestion, only around 50% of PHT washydrolyzed.To compare the digestion of PHT in the presence and in the

absence of endogenous phospholipids, additional experiments

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

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

PHT was slower than the hydrolysis of the control PL.Therefore, this experiment showed that the slower digestion ofPHT 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 modifiedpolar-head with so atypical structures is scarce. Nevertheless, ithas been described that there are different factors thatdetermine the rate and extent of enzymatic hydrolysis of anyphospholipid in general. According to scientific literature, theaggregation of the substrate under the form of mixed micelles,lamellar structures, liposomes, or emulsions is necessary, sincethis appears to facilitate the interaction of the sn-2 fatty acidester with the catalytic site of the enzyme.26−28 However, thespecific type of aggregation determines the hydrolysis rate.Furthermore, the presence of other lipids which influences thepacking of the fatty acid chains and the headgroup are alsorelated to the action of PLA2.28 Whether PHT determined

differences on these factors is unknown but might beconsidered as potential reasons of the different results on therate of hydrolysis. According to Scott et al.27 on thecontribution of the sn-3 substituent on the PLA2 bindingaffinity, these authors stated that the own nature of thephosphatidyl ester affects the physical chemistry of theaggregates, such as surface charge distribution or the state ofaggregation. In this respect, it is important to remark that PLand 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 theremoval of choline. Related to the different state of aggregation,the numbers of micellar structures formed after in vitrodigestion of PL and PHT were determined. According toFigure 4, a lower number of micellar structures were detectedfor PHT digestion when compared to PL, which seemed to bein agreement with the lower hydrolysis of PHT.

Bioaccessibility of Phosphatidylhydroxytyrosol. Dur-ing intestinal digestion of dietary fat, it has been shown that theintraluminal content is structured as an oily phase (OP)dispersed in a micellar bile salt solution (MP).29 This OPmainly contains undigested TG and DG, whereas the MPcontains bile salts and the end products of enzymatichydrolysis, namely, MG, FFA, and lysophospholipids. All ofthese lipid products are aggregated as mixed micelles, micelles,vesicles, or emulsion droplets.30,31 Absorption of lipid productstakes place supported by this MP, which enhances the transportof lipid products to the enterocytes throughout the unstirredwater layer close to the microvillous membrane, where they areabsorbed.32 Furthermore, a minor fraction of insoluble calciumsoaps of fatty acids liberated during the pancreatic digestion canbe formed, which are not absorbable, tend to precipitate, andare wasted in faeces. The analysis of lipid products of thesethree phases, MP, OP, and PP, contributes to the study ofbioaccessibility, defined as the fraction of a compound that isreleased from its matrix in the gastrointestinal tract and thusbecomes available for intestinal absorption.33

According to Figure 5, most hydrolysis products, includingneutral and polar lipids, were found distributed within thebioaccessible fraction MP, suggesting a high bioaccessibility. Inthe case of the control PL samples, a significant OP was isolated(16% of total lipid products), and a minor PP was alsodetected. The specific distribution of total lipid products withinthe three phases is detailed in Figure 6. As shown in Figure 6a,

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

Figure 3. Lyso-PL or lyso-PHT releasing during in vitro intestinaldigestion of PL or PHT, respectively, without coexistence of otherphospholipids in the media.

Figure 4. Hydrolysis of PL and PHT versus the number of micellarstructures.

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the polar lipids from hydrolysis of PL (lyso-PL and residualnondigested PL) were totally included in the MP, suggesting ahigh bioaccessibility of PL. These results were in agreementwith the expected physiological data of phospholipids, so theseobtained results also validated the in vitro model developed on

the ability to reproduce in vivo physiological results onbioaccessibility 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 lipidproducts within the OP was lower (around 12% of total lipidproducts), and the PP increased instead (around 9% of totallipid products precipitated) compared to the PL sample. At anycase, most lyso-PHT and residual PHT were found within thebioaccessible fraction MP.As detailed in Figure 6b, the observed increase in the PP

fraction was mainly due to an increased precipitation of FFAand a fraction of nondigested PHT. In fact, the bioaccessibilityof the FFA changed from 92% in the case of PL samples to 80%in the case of PHT samples. This unexpected a priori result isdifficult to explain, attending to the fact that both PL and PHTsamples had the same amount and fatty acid profile. In general,any precipitation of FFA during intestinal digestion should berelated to a limited solubilization within micellar structures andformation of calcium soaps that cannot be solubilized in theaqueous media. Taking into account that the assays of PHTsamples produced a lower amount of micellar structures thanPL samples (Figure 4), this might determine the differences onnonmicellated fatty acids between both samples. It was alsosignificant that around 8% of total PHT was precipitated

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

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

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together with FFA, whereas nondigested PL did not precipitatein the case of PL sample. A minor fraction of precipitated bilesalts was also measured for the PHT sample contrary to the PLsample. Therefore, the particular molecule PHT might berelated to these results.In order to evaluate the magnitude of PHT as a carrier of

HT, its bioaccessibility as the esterified form was compared tothat of HT. Furthermore, a comparative study with apremixture HT+PL was considered of interest in order tocompare the bioaccessibility of HT when it coexisted with adose of dietary PL, with that of HT under the esterified form asPL (PHT). According to Figure 7, most HT was included

within the bioaccessible fraction MP for the three forms of HTaddition, suggesting a high bioaccessibility. Nevertheless, theportion of HT within the MP under the form of PHT wasslightly superior to the other forms of addition of HT. Such adifference was due to a fraction of HT within the OP that wasnot detected for the PHT sample. These results would suggestthat a portion of HT would not be so easily dispersed in theaqueous media when this phenolic compound was free orpremixed with PL, contrary to the esterified forms of HT asPHT. Probably, the amphiphilic properties of PHT as aphospholipid allowed its dispersion in the aqueous media,which in turn indirectly allowed the dispersion of thevehiculated HT. Furthermore, a minor precipitated fraction ofHT was found for all samples, which was not significantlydifferent. These results contributed to the study of the potentialof PHT as well as to the general behavior of the phenolic HT.Previous detailed information on the fractionation of HT, orcombinations HT+PL, between the different phases of theintestinal medium under in vitro conditions was not found.Concerning the bioaccessibility of PHT, it should be noted

that Borgstrom17 stated that conversion of phospholipids tolysophospholipids is essential for absorption to occur. There-fore, according to the obtained results, despite that most of thedigested 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 hasalso been reported that there is no abnormality in phospholipidabsorption in the case of deficiency in PLA2 (nonhydrolyzedphospholipids).24 Therefore, further studies concerning bio-availability of PHT would be of interest in order to validatewhether PHT would be effectively recognized by intestinal

mucosa for absorption and subsequent metabolism, and itsvalidation as a potential vehicle of HT.

Effect of in Vitro Digestion on the AntioxidantActivity of Phosphatidylhydroxytyrosol. In a recentstudy, we have successfully shown a significant antioxidantactivity of PHT in diverse edible oils, which was comparable oreven superior to HT.4 Taking such evidence of antioxidantability of PHT into account, the antioxidant effect was exploredin the current study when it was affected by the intestinaldigestion process. This was considered of interest, especiallytaking into account that PHT was mainly degraded to lyso-PHT after intestinal digestion, and this would be the majorbioaccessible molecule. Furthermore, the modification of theantioxidant activity of diverse phenolic compounds after theprocess and conditions of gastrointestinal digestion has beenpreviously described, including HT;34 hence, the interest inproducing carriers of these compounds that could protect theiractivity 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.

Furthermore, the antioxidant activity of the control HT did notseem to be affected by the digestion process. An interestingfinding was that the EC50 value of digested PHT wassignificantly inferior to that observed before digestion. Thisresult might suggest that the digestion products, mainly themajor lyso-PHT, might show better antioxidant ability than theformer PHT. This could be considered reasonable, taking intoaccount the simpler and more hydrophilic molecule of lyso-PHT compared to PHT. Thus, after intestinal digestion, acloser value of EC50 between digested PHT and HT wasachieved (0.6 and 0.5 mM, respectively).As a summary, the present study showed that the synthetic

phosphatidyl derivative of HT is recognized by phospholipasesduring the simulation of intestinal digestion but less efficientlythan the analogous PL. At any case, both the major hydrolysisproduct, namely, lyso-PHT, and most of the nondigested PHTwere found within the bioaccessible aqueous fraction; hence,most of the esterified HT was potentially bioaccessible. On theother hand, the digested PHT might be a more superiorantioxidant than the nonhydrolyzed PHT. Nevertheless, theevaluation of the potential bioactivity of PHT and its digested

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

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

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

■ AUTHOR INFORMATIONCorresponding Author*Phone: +34 910017930. E-mail: diana.martin@uam.es.

FundingThis work was supported by the Ministerio de Economia yCompetitividad (INNSAOLI, project number IPT-2011-1248-060000, Subprograma INNPACTO) and the Comunidad deMadrid (ALIBIRD, project number S2009/AGR-1469). Thecontract of M.I.M.-V. was also supported by the INNSAOLIproject and is also acknowledged.

NotesThe authors declare no competing financial interest.

■ ABBREVIATIONS USEDDG, 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, precipitatedphase; TG, triglycerides

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