Aurélia Malapert

Valérie Tomao

Olivier Dangles

Emmanuelle Reboul
email: emmanuelle.reboul@univ-amu.fr

Effect of foods and β-cyclodextrin on the bioaccessibility and the uptake by Caco-2 cells of hydroxytyrosol from either a pure standard or alperujo

Keywords: Olive pomace, Bioavailability, Biophenols, Digestion, Intestinal absorption, Enterocyte

Hydroxytyrosol bioaccessibility and absorption by the intestinal cells have been studied using an in vitro digestion model and Caco-2 TC7 monolayers cells in culture, in the presence or absence of β-cyclodextrin and foods. Hydroxytyrosol was either provided as a pure standard or in an alperujo powder. The presence of foods significantly decreased hydroxytyrosol bioaccessibility and absorption (-20% and -10%, respectively), while β-cyclodextrin had no effect. Moreover, the presence of other compounds from alperujo in the intestine compartment reduced hydroxytyrosol absorption by Caco-2 cells compared to pure standard (-60%). The final bioavailability of hydroxytyrosol, defined as its quantity at the basolateral side of cultured cell monolayers compared to the initial amount in the test meal, was 6.9±0.4%, 31.1±1.1% and 40.9±1.5% when hydroxytyrosol was from alperujo, or a standard administered with or without food, respectively.

Our results show that conversely to foods, β-cyclodextrin does not alter hydroxytyrosol bioavailability.

Introduction

Two-phase olive pomace, also called alperujo, is one of the most abundant industrial Mediterranean pollutants. It is produced in large quantity in two-phase centrifuge mills and is composed of olive vegetation water and solid olive pieces [START_REF] Niaounakis | Olive processing waste management: literature review and patent survey[END_REF] . Two-phase centrifuge mills allow reducing the water consumption and then the quantity of olive mill wastes compared to both traditional and three-phase centrifuge systems. Alperujo pollutant character is especially due to its high phenolic content. Interestingly, previous studies have demonstrated the great interest of these phenolic compounds because of their high health benefits [START_REF] Dermeche | Olive mill wastes: Biochemical characterizations and valorization strategies[END_REF] .

In order to valorize these co-products, the phenolic composition of alperujo has been extensively characterized, thus confirming that it can be an interesting source of valuable compounds for the nutraceutical, cosmetic and food industries. The major phenolic compounds identified into olive mill wastes were hydroxytyrosol (HT) and tyrosol, both belonging to the phenyl alcohol family, as well as p-hydroxycinnamic acids such as caffeic acid and derivatives [START_REF] Ghanbari | Valuable nutrients and functional bioactives in different parts of olive (Olea europaea L.)-a review[END_REF][START_REF] Leouifoudi | Identification and characterisation of phenolic compounds extracted from Moroccan olive mill wastewater[END_REF] . These molecules display a catechol unit that confers them a reducing (electron-donating) character tightly related to their bioactivity (e.g., their antioxidant potential) [START_REF] Malapert | An overview of the analysis of phenolic compounds found in olive mill by-products[END_REF] . HT has received a health claim by the European Food Safety Agency (EFSA) due to its high ability to scavenge reactive oxygen species and to reduce the risks of cardiovascular disease [START_REF] Giordano | 3-O-Hydroxytyrosol glucuronide and 4-O-hydroxytyrosol glucuronide reduce endoplasmic reticulum stress in vitro[END_REF][START_REF] Jemai | Antidiabetic and antioxidant effects of hydroxytyrosol and oleuropein from olive leaves in alloxan-diabetic rats[END_REF][START_REF] Visioli | Extra virgin olive oil's polyphenols: biological activities[END_REF] . However, the electron-donating properties of olive phenols make them sensitive to oxidation. Hence, investigating the influence of the food matrix, including food ingredients used for formulation purposes, on the stability and bioavailability of olive phenols is a relevant issue.

Cyclodextrins (CDs) are natural cyclic oligosaccharides made of D-glucose units bound by α-1,4 linkages and mainly used in the agro-food and pharmaceutical industries to form inclusion complexes (IC) with bioactive compounds, to enhance their stability and solubility [START_REF] Iacovino | beta-Cyclodextrin inclusion complex to improve physicochemical properties of pipemidic acid: characterization and bioactivity evaluation[END_REF][START_REF] Patil | Inclusion complex system; a novel technique to improve the solubility and bioavailability of poorly soluble drugs: a review[END_REF] . β-CD (7 D-glucose units) is the most used CD because of its low price, its availability and its ability to form inclusion complexes with a large range of medium-sized compounds (MM < 800 g/mol) [START_REF] Szejtli | Past, present, and future of cyclodextrin research[END_REF] .

β-CD can also be used to facilitate polyphenol extraction from plants, such as resveratrol from grape pomace [START_REF] Munin | Encapsulation of natural polyphenolic compounds; a review[END_REF][START_REF] Pinho | Cyclodextrins as encapsulation agents for plant bioactive compounds[END_REF][START_REF] Ratnasooriya | Extraction of phenolic compounds from grapes and their pomace using beta-cyclodextrin[END_REF] , and were suggested to be suitable to extract bioactives such as triterpenes from alperujo [START_REF] Lopez-Miranda | Complexation between oleanolic and maslinic acids with native and modified cyclodextrins[END_REF] . However, its ability to interact with polyphenol bioavailability is not known.

In vitro digestion studies can be carried out to assess the bioaccessibility of a given compound, i.e. the fraction of the ingested dose that is transferred from the food matrix to the aqueous phase or to mixed micelles (combining bile acids and lipid digestion products). This fraction is considered as available for subsequent absorption by the enterocytes, which can be investigated using the Caco-2 cell model. Bioaccessibility and intestinal absorption are two critical steps governing a compound bioavailability, i.e. the fraction of the ingested dose (native forms + metabolites) that reaches the general blood circulation and/or target tissues [START_REF] Rein | Bioavailability of bioactive food compounds: a challenging journey to bioefficacy[END_REF] .

In this work, we investigated the effects of β-CD, alperujo matrix and foods (represented by a test meal containing pureed potatoes, minced beef and refined olive oil) on the bioaccessibility and the intestinal absorption of HT (from a standard powder and from a local alperujo).

Materials and Methods

Supplies

β-CD was given from Roquette Freres (Lestern, France). HT (purity > 98%) was kindly provided by Pr. Francesco Visioli (IMDEA, Madrid, Spain). Tyrosol and gallic acid were supplied from Sigma-Aldrich Co (St Louis, USA). Pepsin, porcine pancreatin, porcine bile extract, water, formic acid, ethanol and acetonitrile were purchased from Sigma-Aldrich (Fontenay sous Bois, France).

Dulbecco's modified Eagle's medium (DMEM) containing 4.5 g/L glucose and trypsin-EDTA (500 mg/L and 200 mg/L, respectively), non-essential amino acids, penicillin/streptomycin and PBS were purchased from Life Technologies (Illkirch, France). Fetal bovine serum (FBS) came from PAA (Vélizy Villacoublay, France). Olive pomace was collected from the Castelas mill equipped with a two-phase centrifuge system (Baux-de-Provence, France). Foods were purchased from a local supermarket.

Preparation of the alperujo sample

Alperujo (Aglandau variety, 72% of moisture, stored in cheesecloth canvas) was manually pressed, then filtered on celite and passed through 0.45µm and 0.2µm paper filters (VWR). Ethanol was added to a final proportion of 42% to precipitate proteins (removed by centrifugation). After ethanol evaporation under vacuum, the protein-free aqueous phase from alperujo was frozen at -20°C.

Preparation of the inclusion complexes

In aqueous solution, HT is known to bind β-CD with a 1:1 stoichiometry and a binding constant ≈ 40 M -1 17 . Hence, the inclusion complex was prepared from an equimolar solution of HT and β-CD (5 mM) in water. The concentration was chosen high enough to allow a substantial formation of the inclusion complex (≈ 20%), despite the relatively low affinity of HT for β-CD in aqueous solution. The equimolar HT:β-CD solution was stirred at 200 rpm for 1h at room temperature, then freeze-dried. The inclusion complex in a solid form (powder) was kept in amber flask at -20°C until use.

The total phenol concentration of the protein-free aqueous phase of alperujo was assessed using Folin-Ciocalteau method and diluted to reach a total phenol concentration of 5 mM in gallic acid equivalent [START_REF] Singleton | Colorimetry of Total Phenolics with Phosphomolybdic-Phosphotungstic Acid Reagents[END_REF] . Then, β-CD was added to the sample in the same concentration and the solution was stirred at 200 rpm for 1h at room temperature. After freeze-drying, the aqueous phase of alperujo + β-CD sample was stored as a powder at -20°C in amber glass. For comparison, an alperujo extract without β-CD was also freeze-dried and store as a powder in similar conditions.

Simulated digestion

The test meal, when present, was composed of pureed potatoes (6.7 g), cooked minced beef (1.2 g) and refined olive oil (0.2 g). HT and alperujo samples with or without β-CD were added so as to reach 7 mg of HT in the meal. The simulated digestion was carried out as described previously (Figure 1) [START_REF] Goncalves | Fatty acids affect micellar properties and modulate vitamin D uptake and basolateral efflux in Caco-2 cells[END_REF] . All analyses were run in quadruplicate. Aliquots from oral, gastric and duodenal steps were taken up and frozen at -80°C until use.

Cell culture and uptake experiments

The human colon adenocarcinoma cell line Caco-2 TC7 was cultured on transwell membrane (sixwell plate, 1 mm pore size polycarbonate membrane; Becton Dickinson) to obtain confluent and differentiated cell monolayers as previously described [START_REF] Goncalves | Pinoresinol of olive oil decreases vitamin D intestinal absorption[END_REF] .

Cytotoxicity of digestion samples on Caco-2 TC7 was primarily evaluated to determine the suitable dilution of the phenolic aqueous fractions from in vitro digestion in HBSS before adding them to the apical side of cell monolayers. These results showed that 1/20 and 1/10 dilutions were required for HT and alperujo samples, respectively. To avoid any interference with DMEM or serum components, the phenolic aqueous fractions were diluted in HBSS and Caco-2 cells received HBSS in both chambers 12h before the experiments. At the beginning of each experiment, cell monolayers were washed twice with 1 mL of PBS and received 1 mL of diluted aqueous fraction.

Finally, cell monolayers were incubated at 37°C for 2h, 4h and 6h. After the incubation period, apical and basolateral solutions were harvested. Cell monolayers were washed twice with 1 mL of PBS and scraped in 0.5 mL of PBS. All samples were stored at -80°C until use.

2.6. Analyses of HT and alperujo samples 2.6.1. Extraction of HT and alperujo samples Phenolic compounds were extracted from salivary, gastric and duodenal steps as follows: 0.3 mL ethanol containing the internal standard was added to 0.2 mL of sample. The internal standards were tyrosol and gallic acid for HT and alperujo samples, respectively, as gallic acid was not found in the alperujo extracts [START_REF] Malapert | Direct and Rapid Profiling of Biophenols in Olive Pomace by UHPLC-DAD-MS[END_REF] . n-Hexane (0.2 mL) was then added and the mixture was homogenized for 10 min using a vortex blender at maximal speed. After centrifugation (2500 rpm for 10 min at 4°C), the lower phase was collected and the sediment further extracted with 0.3 mL ethanol and additional vortexing for 10 min. The two ethanol phases were pooled, evaporated to dryness using a Speed-Vac®, and the dried extracts dissolved in 200 µL H 2 O and frozen at -80°C before analysis.

Apical media from cell culture experiments were directly injected into UHPLC-DAD-MS system for analysis. Basolateral media (1900 µL) were primarily dried using a Speed-Vac® and the residues dissolved in 80 µL H 2 O.

The PBS fractions containing harvested cells (500 µL) were sonicated with 50 µL of internal standard for 10 min at room temperature and centrifuged at 7000 rpm for 10 min at 4°C 22 .

Supernatants were recovered and evaporated to dryness, then dissolved in 50 µL H 2 O and frozen at -80°C before analysis.

Chromatographic analysis

All extracts were analyzed by UHPLC-DAD-MS using an Acquity UPLC® system linked to both a diode array detector and a Bruker Daltonics HCT Ultra Ion Trap mass spectrometer equipped with an Electron Spray Ionization (ESI) source operating in negative mode. The separation was performed on an Acquity C18 BEH column (50x2.1 mm i.d., 1.7 µm). The solvents were (A) water/formic acid (99.5/0.5) and (B) acetonitrile. For alperujo analyses, the proportions of solvent B used were: 0-10 min: 1-20%, 10-12 min: 20-30%, 12-14 min: 30-100%. The injection volume was 1 µL for all samples and 10 µL for cells and basolateral extracts. The column temperature was kept at 35°C. Along the 3 steps of the gradient, the flow rate was 0.30, 0.35 and 0.40 mL/min.

Chromatograms were acquired at 280 nm. The spectroscopic detection was performed in the range 200-800 nm with a resolution of 1.2 nm. HT concentrations were estimated from a calibration curve (peak area vs. concentration) constructed with HT standard with R² values greater than 0.99.

Homovanillyl alcohol and homovanillyl alcohol glucuronide were quantified as HT equivalent [START_REF] Malapert | Direct and Rapid Profiling of Biophenols in Olive Pomace by UHPLC-DAD-MS[END_REF] .

For HT analyses, the same conditions were used and the flow rate was constant at 0.30 mL/min. The proportions of B were: 0 -2.4 min: 1-30%, 2.4 -3 min: 30-100%.

Calculation and statistics

All the in vitro experiments were run in quadruplicate. Results were expressed as means and standard deviations. Differences between means were assessed using ANOVA followed by the post-hoc Tukey test for parametric data. P values under 0.05 were considered significant. The bioavailability was assessed by the ratio between the amount of phenolic compounds in the basolateral side and the initial amount added to the apical side or to the meal.

Results

HT bioaccessibility in the oral, gastric and duodenal compartments

The digestion of the HT in the 3 compartments was assessed with free HT and β-CD-bound HT (HTCD). The influence of the meal on HT bioaccessibility was also evaluated with the HTCD complex. Figure 2 shows the percentage of remained HT in each compartment. In the mouth compartment, β-CD seems to act as a protective agent for HT within the meal, HT recovery being 87.6 % (± 1.2) and 96.4 % (± 1.1) for HT and HTCD respectively (p < 0.0001). In the absence of food (HTCD-FF), β-CD provided a weaker but still significant protective effect. In the gastric compartment, HT recoveries in this step were 91.3 % (± 2.7), 94.9 % (± 1.9) and 97.7 % (± 2.7) for HT, HTCD and HTCD-FF, respectively. No significant benefit of β-CD was observed.

Conversely, the presence of food had a negative effect on HT recovery (HTCD vs HTCD-FF, p < 0.05). Finally, except for the HTCD condition, the stability of HT was not significantly different in the duodenal compartment at pH 6 compared to the gastric one. The total apparent losses in the aqueous fractions were 37.2 % (± 1.0), 33.6 % (± 0.9) and 13.5 % (± 3.2) for HT, HTCD and HTCD-FF respectively. There was thus no difference regarding HT bioaccessibility between free HT and its β-CD complex. However, the absence of food significantly improved HT bioaccessibility compared to the other conditions (p < 0.0001).

Figure 3 presents the bioaccessibility of HT from an alperujo powder during the same digestion steps. There was no significant influence of β-CD on HT recovery from alperujo in the different compartments. As for pure HT, no degradation was observed in the mouth while an important loss was observed in the duodenal compartment. HT recovery from alperujo samples in the gastric compartment and in the aqueous phase decreased from 77.2 % (± 2.8) to 52.6 % (± 2.3) and from 76.4 % (± 2.7) to 50.3 % (± 1.2) for alperujo and alperujo-CD, respectively. Overall, HT recovery from alperujo samples decreased along digestion (p < 0.0001). The stability of HT into the alperujo samples appeared less important than for pure HT (p < 0.001).

HT absorption by Caco-2 TC7 cells

The absorption and metabolism of HT were studied using differentiated Caco-2 TC7 cell monolayers. Bioavailability was determined as the quantity of targeted compounds in the basolateral side vs. the initial amount added to the meal. The aqueous fractions obtained from the precedent digestion studies were used to study HT absorption into the enterocytes after a 1/10 or a 1/20 dilution for alperujo and HT samples, respectively. Thus, the cells received about 5.1-5.3, 6.0 and 6.7 µg per well of HT from meals containing HT, HTCD or alperujo and from HTCD-FF, respectively. Chromatograms of the different fractions are presented on Figure 4A. UPLC-DAD-MS analyses allowed to identify homovanillyl alcohol (HVA) as a O-methylether metabolite of HT, giving a parent ion [M-H] -at m/z 167 and a fragment ion at m/z 153 (HT). Figure 4 (B,C,D, E panels) shows the quantity of HT and HVA recovered at the apical and basolateral sides of the cells. Each quantity was expressed as a percentage of the initial HT concentration at the apical side. For all conditions, a significant decrease of the HT content was observed at the apical side (more than 85%, p > 0.05) after 6h incubation. Concomitantly, a significant increasing quantity of HT was recovered at the basolateral side (>40%). The curve profiles highlight the time-dependent transport of HT from the apical to basolateral side. No significant difference was observed at the basolateral side between the three HT samples. HVA was the only HT metabolite observed in our conditions. It was mainly recovered at the apical side reaching about 32.8± 1.2%, 37.5± 4.1% and 40.3± 2.5% of the initial HT content for the HTCD-FF, HTCD and HT conditions, respectively.

The lower percentage of HVA that appeared in the aqueous fraction without meal was due to the higher initial quantity of HT in this condition.

In the case of HTCD-FF, the initial HT amount was higher (+26%) compared to other conditions. The final HT amounts at the basolateral side were about 2.2 µg for HT and HTCD and 2.9 µg for HTCD-FF, respectively. Thus, the absorption of native HT from HTCD-FF increased over 30% (p < 0.0001) compared to conditions containing foods. The transport of HT through the intestinal barrier was also concentration-dependent. The same amount of HVA was approximately produced whatever the initial HT concentration. The amount of native HT at the basolateral side reached 65.7± 2.1%, 63.9± 1.4% of the initial apical content for HT and HTCD, and 71.0± 0.8% for HTCD-FF. The analysis of cell contents revealed neither HT nor HT metabolite: more than 99% of HT and its metabolite were recovered in the apical and basolateral compartments.

The absorption of HT from alperujo samples by Caco-2 cells was also evaluated, as shown in Figure 5. Chromatograms of the different fractions are presented on Figure 5A. The initial HT amount was around 6 µg per well at the apical side of the cells. The general curve profile indicates a time-dependent transport of HT. No significant difference regarding the amounts of both HT and its metabolite was found between alperujo and alperujo-CD samples in all culture media over time. About 0.88 ± 0.05 µg and 0.91 ± 0.04 µg of HT were recovered at the basolateral side for alperujo and alperujo-CD conditions, respectively. So, 14.4 ± 0.8% and 15.0 ± 0.5% of the initial apical HT amount from alperujo samples crossed the cell monolayers (p > 0.05). Despite the higher HT load in the alperujo conditions (about 6 µg vs 5.1-5.3 µg for alperujo conditions, HT and HTCD), the amount of HT absorbed was about 2.5-fold less important than for HT and HTCD conditions, i.e. the absorption rate decreases by more than 60% (p < 0.0001). Homovanillyl alcohol glucuronide (HVA-GlcU) was identified according to its molecular ion [M-H] -at m/z 343 and its fragment ions [M-H-GlcU] -at m/z 167 (HVA) and 153 (HT), characteristic of the homovanillyl moiety. This HT metabolite was only found in the basolateral compartment and was estimated at 6.7 ± 0.8% and 7.3± 0.9% of the initial apical HT amount for alperujo and alperujo-CD, respectively. The total amount of unmetabolized HT at the basolateral side was 68.8 ± 1.5% and 69.8 ± 2.6 % for alperujo and alperujo-CD, respectively. So, β-CD had no significant effect on the HT metabolism rate. In these conditions and conversely to the standard samples, a low amount of HT was recovered into Caco-2 cells. Moreover, the total recovery of HT in these three compartments exceeds 110 % after 6h of incubation (p < 0.0001). This may be explained by the fact that a partial metabolization of other compounds from alperujo could generate HT.

Discussion

This work evaluated for the first time in a comprehensive manner the effect of the presence of β-CD and/or foods on both HT bioaccessibility and HT uptake by intestinal cells.

The first step of HT digestion occurs in the mouth. Mastication favors interactions between the phenolic compounds, food, saliva and dioxygen. In this compartment, the protective effect of β-CD observed in our study may be linked to its ability to build a protective shell around HT, thereby limiting its contact with potential food prooxidants such as iron species. In the stomach, food disintegration intensifies due to the periodic and synchronized contractions of its wall, the acidic environment and the enzymatic activity [START_REF] Drechsler | Modelling the breakdown mechanics of solid foods during gastric digestion[END_REF] . The in vitro digestion of HT standard confirmed that HT is stable in the acidic conditions of the gastric compartment, which is in accordance with previous data [START_REF] Gómez-Romero | Metabolism and Bioavailability of Olive Oil Polyphenols[END_REF] . The recovery of HT after the gastric step was almost total, in agreement with Pereira-Caro et al. who obtained a recovery rate higher than 99 % in their in vitro digestion study without food [START_REF] Pereira-Caro | Digestive stability of hydroxytyrosol, hydroxytyrosyl acetate and alkyl hydroxytyrosyl ethers[END_REF] . In our work, the small loss (lower than 5%) in the gastric compartment may be due to interactions between HT and the food matrix.

In our duodenal conditions, pure HT recovery did not significantly decrease. Several studies have shown that HT was not stable in neutral or mildly alkaline conditions. In their work on the digestion of phenolic compounds from olive oil, Soler et al. observed a loss of HT in alkaline conditions [START_REF] Soler | Digestion stability and evaluation of the metabolism and transport of olive oil phenols in the human small-intestinal epithelial Caco-2/TC7 cell line[END_REF] . Corona et al. also observed that the amounts of HT and its derivatives progressively decreased during digestion by pancreatin (pH 7.5), reaching a total apparent loss of 20.3% after 2h for HT. This value took into account the formation of 3,4-dihydroxyphenylacetic acid (DOPAC) as a side-product of HT [START_REF] Corona | The fate of olive oil polyphenols in the gastrointestinal tract: Implications of gastric and colonic microflora-dependent biotransformation[END_REF] . In our cases, no DOPAC formation was observed. We suggest that the total apparent losses in the aqueous fractions were the result of the partition of HT after the centrifugation step.

The comparative study of the HT standard and the HTCD sample showed a slight effect of β-CD on the final bioaccessibility of HT. β-CD is mainly used in the pharmaceutical industry to protect bioactive compounds and increase their water solubility and consequently their bioavailability [START_REF] Fang | Encapsulation of polyphenols -a review[END_REF] .

As a cyclic starch derivative, β-CD may be partially hydrolyzed during digestion. However in vivo, β-CD only partly digested in the upper gastrointestinal tract and can reach the large intestine where it is metabolized by the microflora fermentation [START_REF] Flourie | Fate of betacyclodextrin in the human intestine[END_REF][START_REF]Safety evaluation of certain food additives World Health Organization[END_REF] . Besides possible β-CD digestion, dilution is the major factor triggering the release of the guest compound from a CD complex [START_REF] Shimpi | Cyclodextrins: application in different routes of drug administration[END_REF] . This factor should be very important in our study because HT has only a weak affinity for b-CD (binding constant < 10 2 M -1 17 , data not shown). Overall, the bioaccessibility of HT from HTCD was not significantly increased compared to the free HT standard without β-CD.

In the absence of food, the bioaccessibility of HT was increased by almost 20% compared to the same sample in the presence of food. Many macromolecular food components can bind phenolic compounds and retain them within the food matrix. In this work, potato is a source of starch, which is known to retain phenolic compounds [START_REF] Manach | Polyphenols: food sources and bioavailability[END_REF] . Similarly, beef is rich in proteins, which have a general affinity for phenols [START_REF] Bohn | Dietary factors affecting polyphenol bioavailability[END_REF][START_REF] Jakobek | Interactions of polyphenols with carbohydrates, lipids and proteins[END_REF] . The clear influence of the food matrix on the bioaccessibility of dietary plant phenols such as HT outlines the importance of including real meal components in in vitro digestion studies.

The study with alperujo samples was carried out in the presence of food. In each compartment, HT from alperujo samples was generally less bioaccessible than from free standard. Indeed, although alperujo samples are protein-free, they contain fibers and sugars, which could interact with the meal components and the digestive enzymes (possibly slowing down protein and starch digestion), thereby reducing the bioaccessibility of phenolic compounds 2 . The apparent loss of HT bioaccessibility from alperujo powders (compared to the standard) was also higher (+10%).

Again, no significant effect of β-CD was observed.

Caco-2 TC7 cells were then chosen as a suitable model to follow the absorption of target compounds through the intestinal barrier. HT was brought to the cells as an aqueous fraction obtained from our previous digestion study. The study of HT and its b-CD complex within a meal revealed that HT was largely absorbed through Caco-2 cells and partly metabolized into homovanillyl alcohol due to the catechol-O-methyltransferase (COMT) activity of the enterocytes.

Most of the HT recovered at the basolateral side was unmetabolized (over 60%) and HVA was recovered in the two culture media, especially in the apical compartment. These data are in agreement with previous results. Indeed, Manna et al. (2000) observed that 25% of HT reached the basolateral side of Caco-2 cells after 1h, 90% of which being unmetabolized. They also identified HVA as a metabolite. They determined that HT reaches the basolateral side through passive diffusion and that this transport was time-and concentration-dependent [START_REF] Manna | Transport mechanism and metabolism of olive oil hydroxytyrosol in Caco-2 cells[END_REF] . Corona et al. also observed 90% of unmetabolized HT at the basolateral side of the cells after the phenolic compounds were added to the apical side from a standard buffered solution [START_REF] Corona | The fate of olive oil polyphenols in the gastrointestinal tract: Implications of gastric and colonic microflora-dependent biotransformation[END_REF] . Finally, Mateos et al. observed that 59% of HT from apical side (initial concentration = 50 µM) reached the basolateral side after 4h of incubation, with almost 20% recovered as O-methylether and 80% as unmetabolized HT [START_REF] Mateos | Acetylation of hydroxytyrosol enhances its transport across differentiated Caco-2 cell monolayers[END_REF] . In our study, for HT, HTCD and HTCD-FF conditions, the transport of unmetabolized HT from the apical to the basolateral side of Caco-2 cells ranged from 25.4 ± 1.5% to 42.3 ± 0.8% over 2 to 6h. The difference regarding HT absorption rates in our study compared to previous data is likely explained by the fact that HT was just dissolved in an aqueous buffer (HBSS or PBS) in previous studies, while we used a more complex mixture obtained from in vitro digestion.

In the case of HT from alperujo powders, we observed the appearance of HVA-GlcU as a HT metabolite. Moreover, the transport of HT from the apical to basolateral side ranged from 8.4 ± 0.6% to 14.7± 0.5% over 2 to 6h. This lower transport rates compared to HT standard conditions can be the result of a competition between HT and other alperujo components to cross the intestinal cells. The same phenomenon was observed when comparing the absorption of pure diosmetin and diosmetin from a rosemary extract [START_REF] Perez-Sanchez | Evaluation of the intestinal permeability of rosemary (Rosmarinus officinalis L.) extract polyphenols and terpenoids in Caco-2 cell monolayers[END_REF] . Interestingly, a small amount of HT from alperujo was recovered into the harvested Caco-2 cells and the total recovery of HT exceeded 100%. If metabolized, HT-glucoside that is also present in alperujo extract could be a source of HT, which would explain this result.

Finally, it is interesting to compare two methods to calculate the in vitro bioavailability of HT, by making a ratio with either the initial HT apical content during the absorption experiments (Table 1) or the initial HT amount brought via the meal (Table 2). The second method allows to correct the common overestimation of the in vitro bioavailability when considering uniquely the absorption step and not the whole digestion process. The second values were decreased by more than 25% and 50% for HT standard and alperujo samples, respectively. This analysis confirmed that HT was more bioavailable when it was brought as a pure standard form than as a plant extract.

The absence of food also participated in increasing the final HT bioavailability. HT bioavailability from the alperujo samples was lower, likely because of possible interactions between HT and other alperujo components and/or competition between them for absorption by Caco-2 cells [START_REF] Soler | Digestion stability and evaluation of the metabolism and transport of olive oil phenols in the human small-intestinal epithelial Caco-2/TC7 cell line[END_REF] .

In summary β-CD, which can be used to enhance phenol stability during storage, did not modified the bioaccessibility and the bioavailability of HT from alperujo, in the presence or in the absence of foods. It would be interesting to extend this result to modified β-CDs such as 2-hydroxypropylβ-CD, which can also be used to complex bioactive compounds [START_REF] Lopez-Miranda | Complexation between oleanolic and maslinic acids with native and modified cyclodextrins[END_REF] . Conversely to β-CD, interactions with food components (probably potato starch and beef proteins) were shown to decrease HT bioaccessibility. These interactions had a strong impact on HT final bioavailability, the HT amount absorbed by the intestinal cells being strongly dependent on the bioaccessible HT content. Besides, HT was more bioaccessible and better absorbed by enterocytes from a pure form than from an alperujo powder, in which it likely competes with other phenolic compounds at different steps of the digestion-absorption process. HVA was the only metabolite observed when HT was from a pure standard and HVA-GlcU was detected when HT was provided via alperujo powders. The low bioavailability of HT reflected its high metabolization in the intestine.

Our data have dietary significance as plant phenol supplements are usually consumed within a meal and under the form of complex mixtures, rather than individual supplements taken at fast.

The authors have declared no conflicts of interest.

Abbreviations: β-CD, β-cyclodextrin; HT, hydroxytyrosol; HTCD: HT:β-CD inclusion complex; HTCD-FF: HT:β-CD inclusion complex-food free condition; HVA: homovanillyl alcohol; HVA-GlcU: homovanillyl alcohol glucuronide with cell culture and Dr. B. Gleize (UMR408 SQPOV, France) for her help with statistical analyses.

Funding source: This work was supported by SFR TERSYS, University of Avignon. Values are expressed as mean ± SD of quadruplicate measurements.

Tables

2. 6

 6 .3. Mass spectrometry ESI mass spectra were obtained in the following conditions: ionization energy = 50 or 100 eV, capillary voltage = 2 kV, source temperature = 365°C. The drying gas was introduced at a flow rate of 10 L/min and the skimmer voltage was 40V. Scans were performed in the m/z range 100 -2000.

Figure 1 .

 1 Figure 1. In vitro digestion procedure

Figure 2 .

 2 Figure 2. Bioaccessibility of hydroxytyrosol from standard powder in each digestive compartment

Figure 3 .

 3 Figure 3. Bioaccessibility of hydroxytyrosol from alperujo powder in each digestive compartment

Table 1 . Bioavailability of phenolic compounds as percentage of HT initial apical amount.

 1 Values are expressed as mean ± SD of quadruplicate measurements.

	Samples	HT	HVA	HVA-GlcU
	HT	42.6 ± 2.5	22.2 ± 2.5
	HTCD	41.4 ± 3.0	23.4 ± 2.0
	HTCD-FF	43.0 ± 0.7	17.5 ± 0.6
	Alperujo	14.4 ± 0.8		6.7 ± 0.8
	Alperujo-CD	15.0 ± 0.5		7.3 ± 0.9

Table 2 . Bioavailability of the phenolic compounds as percentage of HT initial amount in the test meal.

 2

	Samples	HT	HVA	HVA-GlcU
	HT	31.1 ± 1.1	16.2 ± 1.3
	HTCD	30.9 ± 1.8	17.5 ± 1.4
	HTCD-FF	40.9 ± 1.5	16.7 ± 0.6
	Alperujo	6.9 ± 0.4		3.2 ± 0.3
	Alperujo-CD	7.3 ± 0.3		3.6 ± 0.4

Acknowledgment:

The authors thank Pr F. Visioli (Madrid, Spain), Moulin de Castelas (Baux de Provence, France) and Roquettes Frères (Lesterm, France) for providing us hydroxytyrosol, alperujo and β-cyclodextrin, respectively. The authors are also grateful to M. Nowicki for her help