C Dessal

L Martínez

C Maheu

T Len

F Morfin

J L Rousset

E Puzenat

P Afanasiev

M Aouine

L Soler

J Llorca
email: jordi.llorca@upc.edu

L Piccolo

Influence of Pt particle size and reaction phase on the photocatalytic performances of ultradispersed Pt/TiO 2 catalysts for hydrogen evolution

Keywords: Photocatalysis, Hydrogen evolution reaction, Pt/TiO2, Metal ultradispersion, Size effects

Influence of Pt particle size and reaction phase on the photocatalytic performances of ultradispersed Pt/TiO2

catalysts for hydrogen evolution

Introduction

It is urgent to develop alternatives to fossil fuels. Sunlight is a renewable, environment-friendly energy source and hydrogen is one of the most promising solar fuels [START_REF] Montoya | Materials for solar fuels and chemicals[END_REF]. Three photocatalytic reactions can produce hydrogen: photocatalytic water splitting [START_REF] Takanabe | Photocatalytic Water Splitting: Quantitative Approaches toward Photocatalyst by Design[END_REF], photocatalytic dehydrogenation and photocatalytic reforming. The second and the third processes may use biomass-derived alcohols such as methanol or ethanol [START_REF] Kondarides | Hydrogen Production by Photo-Induced Reforming of Biomass Components and Derivatives at Ambient Conditions[END_REF][START_REF] Daskalaki | Efficient production of hydrogen by photo-induced reforming of glycerol at ambient conditions[END_REF][START_REF] Murdoch | The effect of gold loading and particle size on photocatalytic hydrogen production from ethanol over Au/TiO2 nanoparticles[END_REF][START_REF] Bahruji | New insights into the mechanism of photocatalytic reforming on Pd/TiO2[END_REF][START_REF] Kandiel | Mesoporous TiO2 nanostructures: a route to minimize Pt loading on titania photocatalysts for hydrogen production[END_REF][START_REF] Kandiel | Photonic efficiency and mechanism of photocatalytic molecular hydrogen production over platinized titanium dioxide from aqueous methanol solutions[END_REF][START_REF] Navarro | The effect of Pt characteristics on the photoactivity of Pt/TiO2 for hydrogen production from ethanol[END_REF][START_REF] Ahmad | Hydrogen from photo-catalytic water splitting process: A review[END_REF][START_REF] Puga | Photocatalytic production of hydrogen from biomass-derived feedstocks[END_REF][START_REF] Kennedy | Hydrogen generation by photocatalytic reforming of potential biofuels: Polyols, cyclic alcohols, and saccharides[END_REF].

The reference photocatalytic material, TiO 2 semiconductor, must be supplemented by a co-catalyst to increase the photocatalytic hydrogen evolution reaction (PHER) efficiency. The role of the co-catalyst, which can be a metal [START_REF] Al-Azri | The roles of metal cocatalysts and reaction media in photocatalytic hydrogen production: Performance evaluation of M/TiO2 photocatalysts (M=Pd, Pt, Au) in different alcohol-water mixtures[END_REF][START_REF] Herrmann | From catalysis by metals to bifunctional photocatalysis[END_REF] or a semiconductor [START_REF] Girel | On the photocatalytic and electrocatalytic hydrogen evolution performance of molybdenum sulfide supported on TiO2[END_REF], is to enhance the charge career separation through the collection of photoelectrons, and thereby avoid electron-hole recombination [START_REF] Al-Azri | The roles of metal cocatalysts and reaction media in photocatalytic hydrogen production: Performance evaluation of M/TiO2 photocatalysts (M=Pd, Pt, Au) in different alcohol-water mixtures[END_REF][START_REF] Chen | Semiconductor-based Photocatalytic Hydrogen Generation[END_REF][START_REF] Li | Critical roles of co-catalysts for molecular hydrogen formation in photocatalysis[END_REF].

Although noble metals such as Au and Pd -or combinations-have also proven to be efficient [START_REF] Murdoch | The effect of gold loading and particle size on photocatalytic hydrogen production from ethanol over Au/TiO2 nanoparticles[END_REF][START_REF] Bahruji | New insights into the mechanism of photocatalytic reforming on Pd/TiO2[END_REF][START_REF] Al-Azri | The roles of metal cocatalysts and reaction media in photocatalytic hydrogen production: Performance evaluation of M/TiO2 photocatalysts (M=Pd, Pt, Au) in different alcohol-water mixtures[END_REF][START_REF] Bowker | Hydrogen production by photoreforming of biofuels using Au, Pd and Au-Pd/TiO2 photocatalysts[END_REF][START_REF] Yang | Photo-catalytic production of hydrogen form ethanol over M/TiO2 catalysts (M=Pd, Pt or Rh)[END_REF][START_REF] Bonmatí | Hydrogen Photoproduction from Ethanol-Water Mixtures Over Au-Cu Alloy Nanoparticles Supported on TiO2[END_REF], Pt/TiO 2, with a Pt loading as low as 0.2 wt%, is the most popular system [START_REF] Kandiel | Mesoporous TiO2 nanostructures: a route to minimize Pt loading on titania photocatalysts for hydrogen production[END_REF][START_REF] Navarro | The effect of Pt characteristics on the photoactivity of Pt/TiO2 for hydrogen production from ethanol[END_REF][START_REF] Chen | Semiconductor-based Photocatalytic Hydrogen Generation[END_REF][START_REF] Li | A self-assembled 3D Pt/TiO2 architecture for high-performance photocatalytic hydrogen production[END_REF].

As metal particle size influences the number and type of active sites, but also the overall electronic structure in the case of small particles (clusters of size < 2 nm) due to quantum size confinement, Pt nanoparticle size is expected to influence the Pt/TiO 2 activity. For example, a Pt cluster size effect in PHER was evidenced in combination with CdS and a triethanolamine/water mixture by Schweinberger et al., with a maximum activity for Pt 46 clusters [START_REF] Schweinberger | Cluster Size Effects in the Photocatalytic Hydrogen Evolution Reaction[END_REF]. This effect was attributed to the size-dependent electronic properties of the metal clusters with respect to the band edges of the semiconductor. From theoretical calculations, Wang et al. predicted 1 nm to be the optimum size for titania-supported Pt particles in PHER, as it fits the requirements for both electron transfer and H-H coupling [START_REF] Wang | Revealing the Size Effect of Platinum Cocatalyst for Photocatalytic Hydrogen Evolution on TiO2 Support: A DFT Study[END_REF]. In contrast to these findings, from investigations of Pt/TiO 2 catalysts exhibiting single Pt cations, two groups have recently concluded that these atomically dispersed species are more active than metallic Pt nanoparticles for PHER from methanol aqueous solution [START_REF] Xing | Active sites on hydrogen evolution photocatalyst[END_REF][START_REF] Xing | Stable Isolated Metal Atoms as Active Sites for Photocatalytic Hydrogen Evolution[END_REF][START_REF] Sui | Atomically dispersed Pt on specific TiO2 facets for photocatalytic H2 evolution[END_REF].

In this paper, we investigate the photocatalytic dehydrogenation of ethanol in water over a variety of Pt/TiO 2 systems. Two commercial and three homemade shape-controlled titania supports as well as various Pt impregnation post-treatments and two Pt loadings have been employed in combination with thorough pre-and post-reaction analysis of the samples by scanning transmission electron microscopy (STEM) in an image-aberration-corrected microscope. Moreover, we have compared the photocatalytic performances between gas-phase and liquid-phase conditions. Our results suggest that Pt particle size is a key parameter driving PHER, and that nanosized metallic clusters are more active and much more stable under the reaction conditions than single Pt cations.

Experimental

Materials and catalyst preparation

Shape-controlled TiO2 samples (nanotubes, nanowires and nanobelts) were prepared following literature methods [START_REF] Kasuga | Formation of Titanium Oxide Nanotube[END_REF][START_REF] Martínez | Effect of TiO2 nanoshape on the photoproduction of hydrogen from water-ethanol mixtures over Au3Cu/TiO2 prepared with preformed Au-Cu alloy nanoparticles[END_REF]. In addition, two commercial TiO 2 materials (Evonik Aeroxide, purity >99.5%) were used in this study, P25 and P90. Table 1 reports the main characteristics of all the considered titania supports.

Scanning transmission electron microscopy

The annular-dark-field STEM experiments (ADF-STEM) were performed with a FEI Titan ETEM G2 electron microscope equipped with a Cs image aberration corrector and operated at 300 keV. The extraction voltage, camera length, acceptance angles, STEM resolution and probe current were 4500 V, 245 mm, 29.2-146 °, 0.14 nm and <0.1 nA, respectively. For sample preparation, the powder was crushed and sonicated in ethanol, dropped onto a TEM grid, and dried by a lamp. To avoid contamination during analysis and remove all residual carbon, the samples were Ar plasma-cleaned for 20 s (Plasma Prep 5, GaLa Instrumente). A simple-tilt FEI sample holder and holey carbon-coated images. Particle size histograms were obtained from the analysis of several images with the help of ImageJ software. The average particle diameter was calculated from the mean diameter frequency distribution with the formula: d = Σn idi/Σni, where ni is the number of particles with particle diameter d i in a certain range. The diameter is that of the disk having the same area as the projected STEM image of the object. The error on particle size corresponds to standard deviation.

Photocatalytic ethanol dehydrogenation

Photocatalysts tests in the gas phase

The photocatalytic tests were performed at RT and atmospheric pressure in a tubular glass photoreactor [START_REF] Molins | Au/TiO2 Lyogels for Hydrogen Production[END_REF][START_REF] Martínez | Preparation and photocatalytic activity of Au/TiO2 lyogels for hydrogen production[END_REF]. An argon stream [START_REF] Bonmatí | Hydrogen Photoproduction from Ethanol-Water Mixtures Over Au-Cu Alloy Nanoparticles Supported on TiO2[END_REF] The membrane was then placed between the glass tubes of the photoreactor, with the side that contains the photocatalyst facing towards the UV-light source. The reactor tubes and the impregnated membrane were sealed using an O-ring, parafilm and a clamp. The photoreactor was connected to a GC (micro gas chromatograph Agilent 490) equipped with MS 5Å, Plot U and Stabilwax columns for a complete analysis of the photoreaction products, which were monitored on-line every 4 minutes.

Before the photocatalytic tests, the system was purged with argon for one hour, and the absence of oxygen was checked by GC.

Photocatalysts tests in the liquid phase

The photocatalytic tests were performed in a double-wall semi-batch slurry reactor with 600 rpm stirring. Its temperature was regulated to 20 °C through thermostated water recirculation in the double-wall. The reactor was filled with 50 mg of catalyst dispersed in 50 mL of a mixture of ethanol and distilled water (50:50 vol%). Before irradiation, the system was purged with an argon flow for 30 min. The gases were analyzed by micro GC with thermal conductivity detectors (Agilent 300A equipped with MS 5A, PLOT U, OV-1 and Alumina columns). A 125 W high-pressure mercury polychromatic lamp was used to illuminate the reactor through a 20 cm 2 surface area. The incident photon intensity likely to be absorbed by the photocatalyst was measured with a radiatometer device composed of a spectrometer (Avantes, AvaSpec-2018) coupled with a seven-fiber optics probe (Avantes, FCR-7UV-400-2-ME).

In both gas-and liquid-phase conditions, only H 2 and acetaldehyde were produced. No CO2, CO or other secondary products were formed, meaning that the only observed reaction was primary ethanol dehydrogenation:

C 2H5OH → C2H4O + H2 (1)
The photonic yield writes as follows [START_REF] Braslavsky | Glossary of terms used in photocatalysis and radiation catalysis (IUPAC Recommendations 2011)[END_REF]:

𝑃𝑃𝑃𝑃 = 𝑟𝑟 𝐻𝐻 2 𝛷𝛷 𝑝𝑝ℎ𝑜𝑜𝑜𝑜
where rH2 is the dihydrogen formation rate and 𝛷𝛷 𝑝𝑝ℎ𝑜𝑜𝑜𝑜 is the absorbable photon flux. In order to facilitate comparison of measured PY with the literature, we also refer to the apparent quantum yield, which writes as [START_REF] Chen | Semiconductor-based Photocatalytic Hydrogen Generation[END_REF][START_REF] Castedo | Effect of temperature on the gas-phase photocatalytic H2 generation using microreactors under UVA and sunlight irradiation[END_REF]:

𝐴𝐴𝐴𝐴𝑃𝑃 = 2𝑟𝑟 𝐻𝐻 2 𝛷𝛷 𝑝𝑝ℎ𝑜𝑜𝑜𝑜

𝑜𝑜𝑜𝑜𝑜𝑜

In gas-phase conditions, 𝛷𝛷 𝑝𝑝ℎ𝑜𝑜𝑜𝑜 = 𝛷𝛷 𝑝𝑝ℎ𝑜𝑜𝑜𝑜 𝑜𝑜𝑜𝑜𝑜𝑜 = 2120 µmol/h (diode power 760 W/m², Sopt = 2.54 cm², λ = 365 nm). In liquid-phase conditions, the UV lamp is polychromatic with a total photon flux of 𝛷𝛷 𝑝𝑝ℎ𝑜𝑜𝑜𝑜 𝑜𝑜𝑜𝑜𝑜𝑜 = 6600 µmol/h (power 220 W/m², S opt ≈ 20 cm²). The TiO2 samples absorbing only in the 290 -410 nm range (see irradiance spectrum, Fig. S0), the flux of absorbable photons is 𝛷𝛷 𝑝𝑝ℎ𝑜𝑜𝑜𝑜 = 1500 µmol/h. Note that in the case of polychromatic irradiation (as in the liquid-phase conditions used here), PY and AQY should be called "photonic efficiency" and "apparent quantum efficiency", respectively [START_REF] Braslavsky | Glossary of terms used in photocatalysis and radiation catalysis (IUPAC Recommendations 2011)[END_REF][START_REF] Qureshi | Insights on Measuring and Reporting Heterogeneous Photocatalysis: Efficiency Definitions and Setup Examples[END_REF].

However, for the sake of simplicity, only PY and AQY terms are used herein. For gas-phase conditions, the experimental error on H 2 formation rate was evaluated to 10% in standard deviation by testing 4 samples from the same batch (0.2 wt% Pt/TiO 2-P90 pre-reduced at 300 °C). For liquid-phase conditions, it was estimated to 12%.

Results

Structural characterization

A series of 13 samples, listed in Table 2, was investigated, as well as bare TiO2-P90 and Pt(NH 3)4(NO3)2/P90 impregnates (without thermal treatment). The samples are named according to the target loading in wt%Pt, the TiO 2 type, the thermal treatment type, and the maximum temperature in °C. For example, 0.2Pt/P90 C300R300 denotes a catalyst supported on P90, loaded with 0.2 wt% Pt, calcined in air at up to 300 °C, and finally reduced in H 2 at up to 300 °C. The TiO2 types were commercial P25 and P90, and shape-controlled nanotubes (NT), nanowires (NW) and nanobelts (NB), the main characteristics of which are reported in Table 1. While calcination was always performed at 300 °C, reduction was carried out between 200 °C and 600 °C. The minimal temperatures of thermal treatments were chosen on the basis of thermal analysis results, which show that weight loss, heat release and water desorption occur below ca. 300 °C for calcination and below ca. 200 °C for reduction (Fig. S2).

Figure 1 shows STEM images of the samples, together with the corresponding size histograms. The calcination treatment leads to ca. 100% Pt dispersion (i.e., all the Pt atoms are exposed to the surface), with mostly single atoms for 0.2Pt C300 and a mixture of single atoms and small clusters (0.65 ± 0.30 nm) for 1Pt C300. The reduction and calcination-reduction treatments lead to a majority of Pt clusters.

For shape-controlled 0.2Pt/TiO 2 R300, the average Pt cluster size is 1.4-1.7 nm, while it is 1.2 nm for 0.2Pt/P25 and 0.9-1.0 nm for 0.2Pt/P90 samples reduced at up to 400 °C. b For the commercial catalysts, the slight decrease in Pt dispersion can be ascribed to the decrease in TiO 2 surface area from P90 (114 m 2 /g) to P25 (50 m 2 /g). However, this does not hold for shape-controlled anatase titania samples, which exhibit either surface areas higher than 250 m²/g (nanotubes and nanowires) or small surface area (nanobelts, 35 m²/g). In those cases, the Pt particle size might also be controlled by the titania texture and surface structure. In particular, nanotubes and nanobelts expose predominantly {100} facets whereas nanowires expose {001} facets [START_REF] Martínez | Effect of TiO2 nanoshape on the photoproduction of hydrogen from water-ethanol mixtures over Au3Cu/TiO2 prepared with preformed Au-Cu alloy nanoparticles[END_REF]. For the P90 support, the Pt loading and the b For 0.2Pt/P90 R300, the size is calculated to be 1.3 ± 0.3 nm, but from 51 particles only (Fig. S9).

MANUSCRIPT -7

reduction temperature were increased to 1 wt% and 450-600 °C, respectively, in order to increase the Pt particle size. As a result, the 1Pt/P90 R450 and 1Pt/P90 R600 samples display average sizes of 1.3 nm and 1.6 nm, respectively. In situ X-ray diffraction performed under H 2 flow on a 1 wt% Pt/P90 sample prepared by IWI and direct reduction at 200 °C shows that the titania crystallites start to grow and anatase starts to convert into rutile above 500 °C (Fig. S3). This leads to a loss of surface area, the catalyst treated at 600 °C having a BET surface area of 43 m 2 /g. Besides, high-magnification STEM images (Fig. S4) do not reveal the occurrence of strong metal-support interaction (SMSI), i.e. diffusion of titanium oxide over the Pt particles upon reducing thermal treatment at high temperature [START_REF] Tauster | Strong metal-support interactions. Group 8 noble metals supported on titanium dioxide[END_REF]. MANUSCRIPT -8

Gas-phase dehydrogenation of ethanol

First, experimental conditions were screened with a given photocatalyst, 0.2Pt/P90 R300.

Figures S5 reports the rate of hydrogen production for various catalyst weights and ethanol concentrations in water. Figure S5a shows that the H 2 production rate is roughly constant between 10% and 100% ethanol in the gas phase. Similarly, a previous kinetic study of PHER from water-ethanol mixtures on Au/TiO 2 catalysts has shown that the reaction is near-zero order in ethanol for high ethanol concentrations. This behavior could be well described with a Langmuir-Hinshelwood mechanism [START_REF] Castedo | Kinetic analysis and CFD simulations of the photocatalytic production of hydrogen in silicone microreactors from water-ethanol mixtures[END_REF].

Figure S5b shows that the rate increases with the mass of catalyst, and starts to reach a plateau above ca. 2.5 mg, which is due to photon transfer limitations [START_REF] Qureshi | Insights on Measuring and Reporting Heterogeneous Photocatalysis: Efficiency Definitions and Setup Examples[END_REF][START_REF] Taboada | Dynamic photocatalytic hydrogen production from ethanol-water mixtures in an optical fiber honeycomb reactor loaded with Au/TiO2[END_REF]. This led us to choose 10% ethanol concentration and 1.8 ± 0.2 mg catalyst weight as standard parameters. wt% Pt, R300) and (b) various thermal treatments (P90, 0.2 or 1 wt% Pt). Figure S6 reports the full dataset, which includes the results for 0.2Pt/P90 C300, C300R300, and R300C300. In addition, a 20 hlong test is shown in Figure S7 for the 0.2Pt/P90 R300 catalyst. In terms of catalytic stability, for the pre-reduced samples, the rate reaches a maximum within 10 min and then stabilizes or slowly decreases. The catalyst treated at the lowest temperature, 0.2Pt/P90 R200, is the one that deactivates most. A different behavior is displayed by the two C300 catalysts, whose rates show an initial induction period (up to 1 h) before stabilization. Thus, low-temperature reduction and/or calcination seem to generate Pt species relatively unstable under the reaction conditions.

From Figure 2a, the activity order between the TiO 2 samples is: NB < NT < NW < P25 < P90. Notably, P90 provides a higher activity than the widely used P25. This trend might relate to the differences in the TiO 2 polymorph phase, the specific surface area and/or the Pt cluster size, and these parameters may influence each other. According to Figure 2b, the pre-calcined catalyst is nearly twice less active than the pre-reduced catalysts (0.2Pt/P90 R200-R400). The highest activities are obtained after reduction in H 2 below 450 °C. For example, in the case of 0.2Pt/P90 R300, the Pt co-catalyst amplification factor with respect to bare TiO 2 reaches 750, which is much higher than previously reported [START_REF] Ahmad | Hydrogen from photo-catalytic water splitting process: A review[END_REF][START_REF] Puga | Photocatalytic production of hydrogen from biomass-derived feedstocks[END_REF][START_REF] Li | A self-assembled 3D Pt/TiO2 architecture for high-performance photocatalytic hydrogen production[END_REF].

Figure S6 shows that, for the 0.2-1 wt% range, the Pt loading has no significant effect on the photocatalytic performance for Pt/P90 C300. This result is consistent with that of Xing et al. for Pt supported on anatase or P25 TiO 2 for photocatalytic methanol dehydrogenation. The authors found an increase of the activity with the Pt loading up to 0.2 wt%, followed by a plateau between 0.2 wt% and 1 wt% [START_REF] Xing | The size and valence state effect of Pt on photocatalytic H2 evolution over platinized TiO2 photocatalyst[END_REF]. For a higher surface-area mesoporous TiO 2 (209 m²/g against 50 m²/g for P25), Kandiel et al. even evidenced an activity optimum for 0.2 wt% Pt [START_REF] Kandiel | Mesoporous TiO2 nanostructures: a route to minimize Pt loading on titania photocatalysts for hydrogen production[END_REF]. Alternatively, the above result may be due to the existence of an optimal Pt loading comprised between 0.2 wt% and 1 wt%, as found by Li et al.

for the same reaction on nanostructured catalysts [START_REF] Li | A self-assembled 3D Pt/TiO2 architecture for high-performance photocatalytic hydrogen production[END_REF].

Another interesting result (Fig. S6) is that the 0.2Pt/P90 R300C300 has a similar activity as its R300 and C300R300 counterparts, which implies that, for comparable average Pt particles sizes, the initial oxidation state of Pt has little influence on the catalytic activity. Indeed, as compared to reduction, air calcination obviously increases Pt oxidation, but under reaction conditions the same steady active state of Pt appears to be formed for pre-reduced and pre-calcined samples.

MANUSCRIPT -10

Plotting the photonic yield of H2 production after 1 h on stream as a function of the Pt cluster size for all the reduced samples suggests an inverse correlation between the photonic yield and the size (Fig. 3). Note that Pt particle size, especially in the range below 2 nm, influences the amount, the geometry, and the electronic structure of surface Pt active sites. As the Pt loading has little effect on the photocatalytic activity, this behavior cannot be ascribed to an influence of the number of available surface Pt sites, and instead points to an intrinsic size effect. The support and the treatment temperature would thus play an indirect role in tuning the Pt cluster size. These results corroborate the recent theoretical investigation by Wang et al. predicting that a Pt particle size of 1 nm, corresponding to quasi-bilayer clusters, would be the best compromise between the efficiency of the photoelectron transfer from TiO 2 to Pt (more favorable at smaller sizes) and that of the H-H coupling at the Pt surface (more favorable at larger sizes) [START_REF] Wang | Revealing the Size Effect of Platinum Cocatalyst for Photocatalytic Hydrogen Evolution on TiO2 Support: A DFT Study[END_REF]. Moreover, our hypothesis on size effect is supported by the results of Li et al., who have shown that the crystal facets of TiO 2 do not play a critical role in PHER [START_REF] Li | Critical roles of co-catalysts for molecular hydrogen formation in photocatalysis[END_REF]. However, the Pt/TiO 2 activity might also be influenced by parameters such as the TiO 2 phase and surface area, and the Pt particle shape and number density. In addition, since a reducing treatment at 600 °C initiates TiO 2 modification (Fig. S3) and may also induce SMSI effect (although not evidenced in Fig. S4), the data point for Pt/P90 R600 in Figure 3 should be considered with caution.

While Pt/NB is by far the least active catalyst, Pt/P90 R200 is initially the most active. However, this sample is also the most prone to deactivation (Fig. 2). This may originate from the initially incomplete Pt precursor decomposition, although thermal analysis shows that most features are present below 200 °C (Fig. S2). This led us to test the activity of as-impregnated Pt(NH 3)4(NO3)2/TiO2-P90 ("fresh") samples. Figure S8 shows that these samples (0.2 wt% and 1 wt% Pt loadings) slowly reach activities similar to that of the 0.2Pt/P90 R300 catalyst. This suggests an autocatalytic process, in which formed H 2 gradually reduces the impregnate. The maximum activities are reached in 26 min and ca. 40 min for 0.2 wt% and 1 wt% Pt loadings, respectively. The activity of the 0.2Pt/P90 fresh sample then slowly decreases (similarly to 0.2Pt/P90 R200), whereas the activity of the 1Pt/P90 fresh sample stabilizes.

This difference in behavior recalls that of the two Pt/P90 C300 catalysts (Fig. S6) and may correspond to a two-step process, with Pt reduction followed by agglomeration. The latter likely depends on the Pt loading via the Pt particle surface density.

To explain the differences in time evolution of the H 2 production rates, selected catalysts were analyzed by STEM after the photocatalytic tests. Figure S9 shows representative STEM images and Figure 4 displays the evolution of the Pt particle size histograms from pre-to post-reaction state. The catalysts supported on commercial TiO 2 (P90 and P25) and pre-reduced (R300) exhibit little change:

while in the P90 case the size distribution is almost unaffected, for P25 it shows a positive shift of 0.3 nm. The increase is stronger for 0.2Pt/NT R300, with a positive shift of the average size by 1.0 nm and a broadening of the size distribution by 0.4 nm. Finally, the 1Pt/P90 C300 catalyst, which initially contained a large fraction of single atoms coexisting with subnanometric clusters, is deeply affected by the reaction medium, which mostly generates 2-3 nm-sized nanoparticles. Sintering of Pt species was also observed for 0.2Pt/P90 C300 (Fig. S9). The instability of these pre-oxidized catalysts can be ascribed to photoreduction as well as to the reducing effect of hydrogen, which is known to destabilize metal cations initially anchored on oxides [START_REF] Sinkler | Aberration-corrected transmission electron microscopy and in situ XAFS structural characterization of Pt/γ-Al2O3 nanoparticles[END_REF][START_REF] Gänzler | Tuning the Structure of Platinum Particles on Ceria In Situ for Enhancing the Catalytic Performance of Exhaust Gas Catalysts[END_REF][START_REF] Liu | Evolution and stabilization of subnanometric metal species in confined space by in situ TEM[END_REF][START_REF] Duan | Stability investigation of a high number density Pt1 /Fe2O3 single-atom catalyst under different gas environments by HAADF-STEM[END_REF][START_REF] Dessal | Atmosphere-dependent stability and mobility of Pt single atoms and clusters on γ-Al2O3[END_REF]. The 0.2Pt/P90 and 1Pt/90 fresh impregnates were also analyzed by STEM after the photocatalytic tests. They exhibit nanoparticles of 2.1 ± 0.4 nm and 1.4 ± 0.4 nm in size, respectively, confirming the in situ decomposition of the Pt precursor and the subsequent aggregation of Pt atoms. The larger particle size at lower loading, although counterintuitive, is consistent with the lower activity of the 0.2Pt sample with respect to the 1Pt sample (Fig. S8), when one takes into account the size-activity relationship displayed in Figure 3.

Indeed, in spite of the sintering of most catalysts on stream, the inverse correlation between the photonic yield and the Pt particle size is still valid when one considers the post-reaction size, as shown in Figure S10.

Liquid-phase photocatalytic dehydrogenation of ethanol

In order to confirm the generality of the above results, a selection of the P90-supported catalysts was tested for the same reaction, but in liquid-phase conditions. In this case, the experimental conditions were not screened, but were chosen not to limit the kinetics by the amount of catalyst or ethanol, accordingly to literature works [START_REF] Bahruji | New insights into the mechanism of photocatalytic reforming on Pd/TiO2[END_REF][START_REF] Navarro | The effect of Pt characteristics on the photoactivity of Pt/TiO2 for hydrogen production from ethanol[END_REF][START_REF] Al-Azri | The roles of metal cocatalysts and reaction media in photocatalytic hydrogen production: Performance evaluation of M/TiO2 photocatalysts (M=Pd, Pt, Au) in different alcohol-water mixtures[END_REF][START_REF] Herrmann | Heterogeneous photocatalysis: state of the art and present applications[END_REF].

Figure 5 displays the hydrogen production rate as a function of time. Several observations can be made. For a given Pt loading (0.2 wt%), the activity order is C300 < C300R300 < R300, which is in line with the gas-phase results. More surprising is the effect of the Pt loading, which negatively affects the activity of the C300 catalyst, whereas it has little effect under gas-phase conditions (Fig. 2 and Fig. S6). As pointed out by Li et al., this can be attributed to excessive Pt on TiO 2, which increases the opacity and light scattering of the suspension, thereby reducing the photon absorption of TiO 2.

Moreover, Pt at high concentration may act as a charge recombination center, resulting in the decreased activity [START_REF] Li | A self-assembled 3D Pt/TiO2 architecture for high-performance photocatalytic hydrogen production[END_REF]. The threshold loadings above which these phenomena occur may be different in gaseous and liquid-phase conditions.

Similarly to the gas-phase conditions under which the pre-calcined catalyst activities slowly evolved, in the liquid phase these catalysts show an activation over the whole testing period. This is most likely due to the reduction/aggregation phenomenon previously evoked for the gas phase. Another striking similarity between the two conditions lies in the behavior of the 0.2Pt/P90 and 1Pt/P90 fresh impregnates (see Fig. S11 for liquid phase versus Fig. S8 for gas phase). In both cases, the activities reach their maximum after an induction period, but only 0.2Pt gradually deactivates after reaching its maximum activity, and this activity is lower than that of 1Pt. STEM measurements after the photocatalytic tests show that the Pt particle size in reduced samples is essentially unchanged, whereas again the pre-calcined samples are deeply affected: starting from atomically dispersed Pt, the average Pt particle sizes after the reactions are 2.1 ± 0.4 nm and 2.4 ± 0.6 nm for 0.2Pt/P90 C300 and 1Pt/P90 C300, respectively (Fig. S12). The "fresh" impregnates contain, after the tests, a mixture of Pt species ranging from single Pt atoms to large aggregates (size 2.8 ± 1.1 nm for 0.2Pt/P90 fresh and 11± 4 nm for 1Pt/P90 fresh). Therefore, similarly to gas-phase conditions, in the liquid phase only the reduced catalysts are structurally stable.

Discussion

Figure 6 reports the photonic yield in liquid phase versus that in gas phase. The 0.2Pt/P90 C300 catalyst, i.e. the sample initially composed mainly of single Pt atoms, seems to be favored by liquidphase conditions. Besides, under our conditions, the co-catalytic effect of Pt with respect to bare TiO 2 is lower in the liquid phase: the maximum ratio between the photonic yields for Pt/TiO 2 and TiO2 is of 130, versus 960 for 0.2Pt/P90 R200 in the gas phase. C300 catalyst (see Fig. 5).

Table 3 summarizes the performances of the 0.2Pt/P90 R300 catalyst in liquid and gaseous phases. The H 2 production rates are of the order or superior to the values reported so far for Pt/TiO2 catalysts [START_REF] Ahmad | Hydrogen from photo-catalytic water splitting process: A review[END_REF][START_REF] Al-Azri | The roles of metal cocatalysts and reaction media in photocatalytic hydrogen production: Performance evaluation of M/TiO2 photocatalysts (M=Pd, Pt, Au) in different alcohol-water mixtures[END_REF][START_REF] Chen | Semiconductor-based Photocatalytic Hydrogen Generation[END_REF][START_REF] Li | A self-assembled 3D Pt/TiO2 architecture for high-performance photocatalytic hydrogen production[END_REF][START_REF] Xing | Active sites on hydrogen evolution photocatalyst[END_REF][START_REF] Xing | Stable Isolated Metal Atoms as Active Sites for Photocatalytic Hydrogen Evolution[END_REF][START_REF] Sui | Atomically dispersed Pt on specific TiO2 facets for photocatalytic H2 evolution[END_REF][START_REF] Xing | The size and valence state effect of Pt on photocatalytic H2 evolution over platinized TiO2 photocatalyst[END_REF][START_REF] Huang | Photocatalytic properties of redox-treated Pt/TiO2 photocatalysts for H2 production from an aqueous methanol solution[END_REF][START_REF] Yu | Hydrogen Production by Photocatalytic Water Splitting over Pt/TiO2 Nanosheets with Exposed (001) Facets[END_REF][START_REF] Lian | Pt-Enhanced Mesoporous Ti 3+ /TiO2 with Rapid Bulk to Surface Electron Transfer for Photocatalytic Hydrogen Evolution[END_REF]. Nevertheless, such comparisons should be taken with caution as the experimental conditions (nature of the alcohol, gas or liquid phase, presence of photon-transfer and/or mass-transfer limitations, UV irradiation power, etc.) can have dramatic effects on the photocatalytic activity. Note, however, that the activities of Pt/TiO 2 for PHER from methanol and ethanol aqueous solutions were previously found to be similar [START_REF] Al-Azri | The roles of metal cocatalysts and reaction media in photocatalytic hydrogen production: Performance evaluation of M/TiO2 photocatalysts (M=Pd, Pt, Au) in different alcohol-water mixtures[END_REF][START_REF] Yang | Photo-catalytic production of hydrogen form ethanol over M/TiO2 catalysts (M=Pd, Pt or Rh)[END_REF]. The co-catalyst factor is the ratio between Pt/TiO2 and TiO2 activities measured in the same conditions. a Calculated for 0.5 mg of catalyst (Fig. S5b) to minimize photon-transfer and mass-transfer limitation effects.

Conclusions

The focus of this work was on the relationship between the platinum initial state, the Pt cluster size and the resulting PHER activity of the Pt/TiO 2 system. In order to disentangle the effects under investigation from the influence of TiO 2 properties, we studied catalysts supported on several titania types. A series of Pt/TiO 2 photocatalysts was prepared by incipient wetness impregnation of Pt(NH 3)4(NO3)2 onto commercial or homemade shape-controlled titania followed by calcination and/or reduction thermal treatments. While calcination-reduction or direct reduction generates Pt clusters of 10-20 Å in size, air calcination leads to atomically dispersed Pt. For the catalysts pre-reduced in H 2, independently of the TiO 2 material, the steady-state gas-phase PHER rate from water-ethanol mixture shows a possible reverse relationship with the Pt cluster size. In particular, the higher surface area of TiO 2-P90 with respect to TiO2-P25 leads to higher Pt dispersion, which would explain the superior activity in the P90 case. For Pt clusters supported on this form of titania, the Pt loading (0.2 or 1 wt%) and the initial treatment (oxidizing vs. reducing) have little influence on the catalytic activity. However, the atomically dispersed catalysts exhibit ca. twice less PHER activity than their pre-reduced counterparts. From post-reaction STEM, it appears that the former undergo extensive sintering on stream, whereas cluster catalysts are structurally more stable.

Complementary photocatalytic tests in liquid-phase conditions confirm the superiority of catalysts containing Pt nanoclusters over atomically dispersed Pt. However, pre-calcined low-loaded Pt/TiO 2-

Figure 1 .

 1 Figure 1. ADF-STEM images and Pt particle size histograms of the as-prepared Pt/TiO2 samples. The calcined samples contain majority (0.2Pt/P90 C300) and minority (1Pt/P90 C300) fractions of single Pt atoms (arrows), in coexistence with small clusters (squares in 1Pt/P90 C300 image, size 0.65±0.30 nm). NT, NW and NB stand for TiO2 nanotubes, nanowires and nanobelts, respectively.

Figure 2 .

 2 Figure 2. H2 production rate versus time in gas-phase conditions: (a) effect of TiO2 type; (b) effect of Pt loading and thermal treatment. The formation of acetaldehyde follows similar kinetics as H2 formation (not shown).

Figure 2

 2 Figure 2 reports the rate of H2 production vs. time for Pt/TiO2 catalysts with (a) several TiO2 types (0.2

Figure 3 .

 3 Photonic yield in gas phase (%)

Figure 4 .

 4 Figure 4. Pt particle size distributions (arbitrary units) determined from STEM before (black) and after (red) the gas-phase catalytic tests. Single atoms and small clusters remaining in 1Pt/P90 C300 after reaction are not taken into account in the corresponding histogram.

Figure 5 .

 5 Figure 5. H2 production rate versus time in liquid-phase conditions for Pt/P90 catalysts.

Figure 6 .

 6 Photonic yield in liquid phase (%)

Table 1 .

 1 Characteristics of TiO2 phases

	TiO2 sample	TiO2 phases	SBET (m 2 /g)
	P25	80% anatase, 20% rutile	50
	P90	92% anatase, 8% rutile	114
	Nanotubes	anatase	274
	Nanowires	75% anatase, 25% TiO2(B)	250
	Nanobelts	91% TiO2(B), 9% anatase	35

SBET denotes the Brunauer-Emmett-Teller surface area, as measured by N2 adsorption volumetry (Micromeritics ASAP 2020) at -196 °C, with pre-outgassing at 300 °C for 3 h (commercial samples) or 500 °C for 10 h (nanoshaped samples) under secondary vacuum.

Pt/TiO 2 samples were prepared by incipient wetness impregnation with an aqueous solution of Pt(NH 3)4(NO3)2 precursor (Sigma-Aldrich, >50%), followed by drying at 60 °C for 4 h and calcination (synthetic air) and/or reduction (pure H 2) treatment (gas flow 30 mL/min, standard temperature 300 °C, heating rate 2 °C/min, plateau 2 h). Pt loadings were measured by inductively coupled plasmaoptical emission spectroscopy (ICP-OES Activa from Horiba Jobin Yvon) after the samples were prereduced at 450 °C in H 2 flow and dissolved in an acid mixture at 300 °C. The actual Pt loadings corresponding to 0.2 wt% Pt and 1.0 wt% Pt target loadings were 0.15 ± 0.04 wt% and 0.78 ± 0.22 wt%, respectively (see Table

2

in Section 3.1).

Table 2 .

 2 Characteristics of Pt/TiO2 photocatalysts

	Sample	Pt loading (wt%)	Average Pt cluster size (Å)
	0.2Pt/P25 R300	0.13	12 ± 3
	0.2Pt/P90 C300	0.14	Atomic dispersion
	0.2Pt/P90 C300R300	0.12	9 ± 2
	0.2Pt/P90 R200	0.11	10 ± 3
	0.2Pt/P90 R300	0.13	13 ± 3
	0.2Pt/P90 R300C300	0.13	10 ± 3
	0.2Pt/P90 R400	0.11	10 ± 3
	1Pt/P90 C300	0.58	Atomic dispersion
	1Pt/P90 R450	0.75	13 ± 3
	1Pt/P90 R600	1.02	16 ± 4
	0.2Pt/NT R300	0.19	15 ± 4
	0.2Pt/NW R300	0.25	14 ± 3
	0.2Pt/NB R300	0.14	17 ± 8

C and R denote calcination and reduction, respectively. NT, NW and NB stand for TiO2 nanotubes, nanowires and nanobelts, respectively. Pt loadings and average cluster sizes were determined by ICP-OES and STEM, respectively.

Table 3 .

 3 Performances of the 0.2Pt/P90 R300 catalyst.

	Conditions	H2 prod. rate (µmol/min)	H2 prod. rate (mmol h -1 gcat -1 )	Photonic yield (%)	App. quantum yield (%)	Co-catalyst factor
	Gas phase	2.5	170 a	7.0	14	750
	Liquid phase	5.2	6.2	21	9.5	130

Acknowledgement

Région Auvergne-Rhône-Alpes (CMIRA 2016 project), Agence Nationale de la Recherche (UltraCat project ANR-17-CE06-0008 and LABEX iMUST ANR-10-LABX-0064/ANR-11-IDEX-0007), MINECO/FEDER ENE2015-63969-R and GC 2017 SGR 128 are acknowledged for financial support. F. Bosselet, P. Mascunan, N. Cristin, and B. Jouguet (IRCELYON) are acknowledged for analyses. The Consortium Lyon-Saint-Etienne de Microscopie (CLYM) is acknowledged for access to the Titan microscope. JL is a Serra Húnter Fellow and is grateful to ICREA Academia program. LM is grateful to CONACYT México for the PhD grant no. 409809.


Javier Marhuenda

Sonia Medina

Pedro Martínez-Hernández

Simon Arina

Pilar Zafrilla

Juana Mulero

Camille Oger

Jean-Marie Galano

Thierry Durand

Antonio Solana

Federico Ferreres

Juan José López-García

Angel Gil-Izquierdo
email: angelgil@cebas.csic.es

Effect of the dietary intake of melatonin-and hydroxytyrosol-rich wines by healthy female volunteers on the systemic lipidomic-related oxylipins

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Lipid peroxidation products have been investigated in order to determine their use as biomarkers of oxidative status in different plant and mammalian systems. Linolenic acid (ALA), arachidonic acid (AA), adrenic acid (AdA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are commonly studied fatty acids [START_REF] Miller | Isoprostanes and neuroprostanes as biomarkers of oxidative stress in neurodegenerative diseases[END_REF][START_REF] Imbusch | Formation of isoprostane F2-like compounds ( phytoprostanes F1) from α-linolenic acid in plants[END_REF][START_REF] Medina | A ultra-pressure liquid chromatography/triple quadrupole tandem mass spectrometry method for the analysis of 13 eicosanoids in human urine and quantitative 24 hours values in healthy volunteers in a controlled constant diet[END_REF] which result in phytoprostanes (PhytoPs), [START_REF] Imbusch | Formation of isoprostane F2-like compounds ( phytoprostanes F1) from α-linolenic acid in plants[END_REF][START_REF] Marhuenda | Dependency of Phytoprostane Fingerprints of Must and Wine on Viticulture and Enological Processes[END_REF] isoprostanes (IsoPs), [START_REF] Miller | Isoprostanes and neuroprostanes as biomarkers of oxidative stress in neurodegenerative diseases[END_REF][START_REF] Medina | A ultra-pressure liquid chromatography/triple quadrupole tandem mass spectrometry method for the analysis of 13 eicosanoids in human urine and quantitative 24 hours values in healthy volunteers in a controlled constant diet[END_REF] F 2 -dihomo-isoprostanes (F 2 -dihomo-IsoPs), F 3 -neuroprostanes (F 3 -NeuroPs) and F 4neuroprostanes (F 4 -NeuroPs), respectively. [START_REF] Miller | Isoprostanes and neuroprostanes as biomarkers of oxidative stress in neurodegenerative diseases[END_REF] IsoPs have been established as general and reliable "in vivo" oxidative stress biomarkers, and their presence in several clinical physiological and pathological conditions such as aging, cardiovascular disorders, cancer, and neurological pathologies has been established. [START_REF] Medina | A ultra-pressure liquid chromatography/triple quadrupole tandem mass spectrometry method for the analysis of 13 eicosanoids in human urine and quantitative 24 hours values in healthy volunteers in a controlled constant diet[END_REF][START_REF] Kadiiska | Biomarkers of Oxidative Stress Study II: Are oxidation products of lipids, proteins, and DNA markers of CCl4 poisoning?[END_REF][START_REF] Durand | Isoprostanes and phytoprostanes: Bioactive lipids[END_REF] They are prostaglandin-like compounds, produced by the non-enzymatic peroxidation of AA, normally catalyzed by free radicals. [START_REF] Durand | Isoprostanes and phytoprostanes: Bioactive lipids[END_REF][START_REF] Morrow | Free radical-induced generation of isoprostanes in vivo: evidence for the formation of D-ring and E-ring isoprostanes[END_REF] Other metabolites derived from the oxidation of AA are prostaglandins (PGs); however, they are formed enzymatically (in contrast to IsoPs). Their formation is regulated by cyclooxygenase (COX) from fatty acids like AA. [START_REF] Vane | Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs[END_REF] The rates of action of PGs are large since they are able to exert physiological effects related to inflammation, fever or pain, thanks to prostaglandin signaling through specific G protein-coupled receptors. [START_REF] Medina | Assessment of oxidative stress markers and prostaglandins after chronic training of triathletes[END_REF][START_REF] Funk | Prostaglandins and leukotrienes: Advances in eicosanoid biology[END_REF] Both PG and IsoPs are defined as eicosanoids, considered as lipid mediators, involved in the physiopathology of all organs, tissues, and cells, being associated with homeostasis, biological functions and inflammation. [START_REF] Galano | Non-enzymatic cyclic oxygenated metabolites of adrenic, docosahexaenoic, eicosapentaenoic and α-linolenic acids; bioactivities and potential use as biomarkers[END_REF] A Mediterranean diet (MD) has been proved to exert several benefits in order to maintain the redox balance. Antioxidants, mainly polyphenols, have been thought to be mainly responsible for the antiradical effects related to MD. [START_REF] Guerrero | Wine, resveratrol and health: A review[END_REF] Wine is a representative alcoholic component of the MD, considered responsible for the "French paradox". [START_REF] Renaud | Wine, alcohol, platelets, and the French paradox for coronary heart disease[END_REF] Red wine polyphenols have demonstrated in vivo and in vitro antioxidant capacity, [START_REF] Guerrero | Wine, resveratrol and health: A review[END_REF][START_REF] Bertelli | Grapes, wines, resveratrol, and heart health[END_REF] leading to the protection against cardiovascular and degenerative diseases, besides some cancers and inflammation processes. [START_REF] Mushtaq | Polyphenols and human health-A review[END_REF] In fact, ethanol has been considered to exert antioxidant capacity in vivo, [START_REF] Renaud | Wine, alcohol, platelets, and the French paradox for coronary heart disease[END_REF][START_REF] Schröder | Alcohol consumption is associated with high concentrations of urinary hydroxytyrosol[END_REF][START_REF] De La Torre | Is dopamine behind the health benefits of red wine?[END_REF] so alcohol could be partially responsible for the effects of red wine.

Despite these beneficial effects, polyphenols have demonstrated a very low bioavailability, both in vivo and in vitro. [START_REF] Guerrero | Wine, resveratrol and health: A review[END_REF][START_REF] Mushtaq | Polyphenols and human health-A review[END_REF][START_REF] Mulero | Antioxidant activity and phenolic compounds in organic red wine using different winemaking techniques[END_REF] Concerning the low absorption of polyphenols, other compounds such as melatonin (MEL) and hydroxytyrosol (OHTyr) and its metabolites from colonic microflora metabolism could be partially responsible for the antioxidant benefits of red wine. [START_REF] Fernández-Mar | Bioactive compounds in wine: Resveratrol, hydroxytyrosol and melatonin: A review[END_REF] Both MEL and OHTyr act as radical scavengers, regulating the oxidation status of the organism. [START_REF] Fernández-Mar | Bioactive compounds in wine: Resveratrol, hydroxytyrosol and melatonin: A review[END_REF][START_REF] Hardeland | Melatonin, a potent agent in antioxidative defense: Actions as a natural food constituent, gastrointestinal factor, drug and prodrug[END_REF][START_REF] Vilaplana Peréz | Hydroxytyrosol and potential uses in cardiovascular diseases, cancer and AIDS[END_REF] They are not only able to scavenge oxidants, but they have also demonstrated the endogenous enzymatic activation of the antioxidant defense system and cardioprotective properties. [START_REF] Fernández-Mar | Bioactive compounds in wine: Resveratrol, hydroxytyrosol and melatonin: A review[END_REF][START_REF] Hardeland | Melatonin, a potent agent in antioxidative defense: Actions as a natural food constituent, gastrointestinal factor, drug and prodrug[END_REF][START_REF] Martin | Hydroxytyrosol induces antioxidant/detoxificant enzymes and Nrf2 translocation via extracellular regulated kinases and phosphatidylinositol-3-kinase/ protein kinase B pathways in HepG2 cells[END_REF] They are even able to trespass physiological barriers, crossing the blood-brain barrier. Therefore, MEL and OHTyr can act as local and general antioxidants, exerting antiradical protection when needed. [START_REF] Srinivasan | Role of melatonin in neurodegenerative diseases[END_REF][START_REF] Wu | Measurement of free hydroxytyrosol in microdialysates from blood and brain of anesthetized rats by liquid chromatography with fluorescence detection[END_REF] The present research aims to clarify the state of the art about the systemic lipid peroxidation markers and enzymatic lipid oxidation in the human body related to the effect of the intake of a cup of red wine, paying attention to its bioactive compounds in general and OHTyr/MEL in particular, by conducting an in vivo study with 18 female volunteers.

Materials and methods

Chemicals and reagents

HPLC-grade ACN and methanol were purchased from Scharlau Chemie (Barcelona, Spain). Ultrapure water (Milli-Q) was obtained from Milli-Q Gradient A10 system (Millipore, Bedford, MA). β-Glucuronidase, type H2 from Helix pomatia and BIS-TRIS (bis-(2-hydroxyethyl)-amino-tris(hydroxymethyl)methane) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Strata X-AW cartridges, 100 mg 3 mL -1 , were obtained from Phenomenex (Torrance, CA, USA). 15-F 2t -IsoP, 15-epi-15-F 2t -IsoP, ent-15-epi-15-F 2t -IsoP, 2,3-dinor-15-F 2t -IsoP, 2,3-dinor-15-epi-15-F 2t -IsoP, 5-F 2t -IsoP, 5-epi-5-F 2t -IsoP, and 8-F 3t -IsoP were synthesized according to our published procedures. [START_REF] Guy | Oxygenated Metabolites of n-3 Polyunsaturated Fatty Acids as Potential Oxidative Stress Biomarkers: Total Synthesis of 8-F3t-IsoP, 10-F4t-NeuroP and [D4]-10-F4t-NeuroP[END_REF][START_REF] Durand | Total Synthesis of (15 R)-and (15 S)-F2t-Isoprostanes by a Biomimetic Process Using the Cyclization of Acyclic Dihydroxylated Octa-5, 7-dienyl Radicals[END_REF][START_REF] Durand | Syntheses and preliminary pharmacological evaluation of the two epimers of the 5-F 2t-isoprostane[END_REF] The other isoprostanes and prostaglandins were purchased from Cayman Chemical (Ann Arbor, MI, USA). These compounds were: PGDM, PGD 2 , Tetranor-PGDM lactone, 11β-PGF 2α , 2,3-dinor-11β-PGF 2α , Tetranor-PGDM, Tetranor-PGJM, PGE 2 , 20-OH-PGE 2 , Tetranor-PGEM, Tetranor-PGAM, Tetranor-PGFM, 15-keto-PGF 2α , 20-OH-PGF 2α , 19(R)-OH-PGF 2α , 2,3dinor-6-keto-PGF 1α , 6-keto-PGF 1α , ent-PGF 2α , 15-keto-15-F 2t -IsoP, 9-epi-15-F 2t -IsoP, 15-keto-15-E 2t-IsoP, 15-epi-15-E 2t -IsoP, 11-dh-TXB 2 , 17-trans-PGF 3α , 8-epi-8-F 3t -IsoP, PGE 1 , PGF 1α , 15-E 1t -IsoP, 15-F 1t -IsoP, U-44619, and U-44069.

Red wine samples assayed in this clinical and nutritional trial

The description of red wines and their primary musts was provided in our previous studies. [START_REF] Marhuenda | Melatonin and hydroxytyrosol-rich wines influence the generation of DNA oxidation catabolites linked to mutagenesis after the ingestion of three types of wine by healthy volunteers[END_REF][START_REF] Marhuenda | Melatonin and hydroxytyrosol protect against oxidative stress related to the central nervous system after the ingestion of three types of wine by healthy volunteers[END_REF] The red wines were provided by the Baigorri winery (Bodegas Baigorri S.A.U, Samaniego, Álava). With the aim to compare different vinification and aging procedures, three different wines were chosen and named as follows: "Baigorri carbonic maceration 2010", "Baigorri aged 2007" and "Baigorri high expression 2010". Wines were stored between 12 °C and 14 °C after bottling, leading to a correct maintenance. The primary musts of the wine samples were stored at -20 °C after the harvest of the grapes, avoiding fermentation. The use of the primary musts of the wine samples allowed the determination of the differences between the wines arising from the differences in the winemaking processes, avoiding any differences in the grape variety or harvesting procedure from must to wine.

Study design

The characteristics of the present study have been described in our previous reports, [START_REF] Marhuenda | Melatonin and hydroxytyrosol-rich wines influence the generation of DNA oxidation catabolites linked to mutagenesis after the ingestion of three types of wine by healthy volunteers[END_REF][START_REF] Marhuenda | Melatonin and hydroxytyrosol protect against oxidative stress related to the central nervous system after the ingestion of three types of wine by healthy volunteers[END_REF] being previously approved by the Bioethics Committee of the Catholic University San Antonio de Murcia (UCAM). The study included a total of 18 (N = 18) healthy female volunteers, between 18 and 27 years of age (the characteristics are shown in Table 1). Importantly, all individuals in this study were nonsmokers and during the study the women were not in their menstrual days. Informed and signed consent of each individual involved in the trial was obtained. The development of the study was double-blind, crossover and placebo-controlled, carried out in four stages. At the beginning of each stage, the volunteers (N = 18) were divided into two equal groups (N 1 = 9 and N 2 = 9) in order to facilitate the crossover of the study. The structure of the stages consisted of 48 hours of urine collection since the beginning of the treatment, followed by a 72 hour washout period. Finally, after the washout period, the crossover was developed, collecting the urine of the volunteers in the following 48 hours. Afterwards, each stage was in turn separated by washout periods of 72 hours too. The graphical distribution of the different stages developed in the present study is shown in Fig. 1. Urine samples were obtained equally in all the stages of the study (graphically represented in Fig. 1). At 0 hours, the first urine in the morning was obtained both before and after the crossover. Additionally, urine samples were collected from 0 to 48 hours in order to establish the in vivo effects of the intake of the different beverages. Importantly, although the measurements were performed in the urine of 48 hours, the data obtained are expressed in µg 24 h -1 in order to facilitate the comparison with previously reported data.

Extraction and determination of melatonin and phenolics by UHPLC-QqQ-MS/MS

The methods used for the characterization of the samples were described in our previous reports. [START_REF] Marhuenda | Melatonin and hydroxytyrosol-rich wines influence the generation of DNA oxidation catabolites linked to mutagenesis after the ingestion of three types of wine by healthy volunteers[END_REF][START_REF] Marhuenda | Melatonin and hydroxytyrosol protect against oxidative stress related to the central nervous system after the ingestion of three types of wine by healthy volunteers[END_REF] The different phenolic compounds detected in the samples of wine and must were identified by their UV-Vis spectra and chromatographic comparison with authentic commercial markers. [START_REF] Mulero | Antioxidant activity and phenolic compounds in organic red wine using different winemaking techniques[END_REF][START_REF] Cantos | Postharvest UV-C-irradiated grapes as a potential source for producing stilbene-enriched red wines[END_REF] Anthocyanins were quantified at 520 nm by comparison with malvidin-3glucoside (LOD = 0.074 ppm, LQD = 0.240 ppm) and flavonols were quantified at 360 nm by comparison with quercetin-3-rutinoside (LOD = 0.123 ppm, LQD = 0.409 ppm). Hydroxycinnamic acid derivatives were quantified at 320 nm by comparison with caffeic acid (LOD = 0.064 ppm, LQD = 0.212 ppm) and finally, E-stilbenes were quantified by comparison with trans-resveratrol (LOD = 0.024 ppm, LQD = 0.074 ppm).

For the determination of OHTyr and its metabolites, a UHPLC coupled to a 6460 QqQ-MS/MS (Agilent Technologies, Waldbronn, Germany) was used, following the method designed by Domínguez-Perles et al. (2015). [START_REF] Domínguez-Perles | Gender Differences on Plasma and Urine metabolites from Sprague-dawley Rats after Oral Administration of Normal and High Dose of Hydroxytyrosol, Hydroxytyrosol Acetate, and DOPAC[END_REF] For the analysis of MEL and serotonin, the extraction procedure for the wines and musts was as previously described. [START_REF] Fernández-Pachón | Alcoholic fermentation induces melatonin synthesis in orange juice[END_REF] The separation and determination of MEL and serotonin was performed using a UHPLC coupled to a 6460 QqQ-MS/MS (Agilent Technologies, Waldbronn, Germany) and a Waters Acquity UHPLC BEH C 18 1.7 µm 2.1 × 50 mm column.

Sample preparation for eicosanoid analysis

The extraction of eicosanoids in urine was carried out following the method developed by ref. 33. All solutions were stored at -80 °C. The eicosanoid concentrations were calculated from standard curves freshly prepared each day.

Fig. 1 Graphical representation of the in vivo assay, divided into four stages. Stages 2, 3 and 4 were similar but the beverage drunk changed between stages. A period of 72 hours was included both between stages and before the crossover. CMW/CMM: carbonic maceration wine/must; AW/AM: aged wine/must; HEW/HEM: high expression wine/must.

Food & Function Paper

This For the enzymatic hydrolysis to remove the glucuronide and sulfate conjugates, the enzyme β-glucuronidase from Helix pomatia was used according to ref. 33. Before the analysis by UHPLC-QqQ-MS/MS, the samples were subjected to solid phase extraction (SPE) using a Strata X-AW cartridge (100 mg 3 mL -1 ) according to the procedure described by Medina et al. (2015). [START_REF] Medina | Dihomo-isoprostanes -non-enzymatic metabolites of AdA-are higher in epileptic patients compared to healthy individuals by a new UHPLC-QqQ-MS/MS method[END_REF] The analytes were eluted with 1 mL of MeOH and dried under vacuum using a SpeedVac concentrator (Savant SPD121P, Thermo Scientific, MA, USA). The dry extracts were reconstituted with A : B phases (90 : 10, v : v) and filtered through a 0.45 μm filter (Millipore, MA, USA). Samples were finally injected and analyzed by UHPLC-QqQ-MS/MS.

UHPLC-QqQ-MS/MS analysis of urinary eicosanoids

LC-MS/MS has demonstrated to be the most specific and versatile method to determine the biomarkers of lipid peroxidation. [START_REF] Vigor | Non-enzymatic lipid oxidation products in biological systems: ASSESSMENT of the metabolites from polyunsaturated fatty acids[END_REF] Eicosanoids were separated using a UHPLC system coupled to a 6460 triple quadrupole tandem mass spectrometer (Agilent Technologies, Waldbronn, Germany). Chromatographic separation was carried out on an ACQUITY BEH C 18 column (2.1 × 50 mm, 1.7 μm pore size) (Waters, MA, USA), and the column temperatures were 6 °C both left and right. Sample analysis was carried out following the method previously reported by Medina et al. (2015); [START_REF] Medina | Dihomo-isoprostanes -non-enzymatic metabolites of AdA-are higher in epileptic patients compared to healthy individuals by a new UHPLC-QqQ-MS/MS method[END_REF] the multiple reaction monitoring (MRM) mode was performed using the negative electrospray ionization (ESI) mode. The mobile phases used were A (water) and B (methanol), both containing 0.01% of acetic acid (v : v). The operating conditions for MS parameters and flow rate were defined by Medina et al. (2015). [START_REF] Medina | Dihomo-isoprostanes -non-enzymatic metabolites of AdA-are higher in epileptic patients compared to healthy individuals by a new UHPLC-QqQ-MS/MS method[END_REF] The injection volume was 20 μL in UHPLC-QqQ-MS/ MS. Data acquisition and processing was performed using MassHunter software version B.04.00 (Agilent Technologies, Walbronn, Germany). Finally, the amount of eicosanoids in urine was calculated from the area ratio of the ion peaks of the compounds over the corresponding standards.

Statistical analysis

The analysis of variance (ANOVA; Duncan) was applied to establish significant differences between the means obtained during the fermentation process. In order to understand the changes observed in the different samples and the influence of the different bioactive compounds, a statistical procedure was developed that has been described in our previous studies. [START_REF] Marhuenda | Melatonin and hydroxytyrosol-rich wines influence the generation of DNA oxidation catabolites linked to mutagenesis after the ingestion of three types of wine by healthy volunteers[END_REF][START_REF] Marhuenda | Melatonin and hydroxytyrosol protect against oxidative stress related to the central nervous system after the ingestion of three types of wine by healthy volunteers[END_REF] Simple regression was performed and the results obtained were compared with the results of multiple regression analysis in stepwise mode, in order to establish a realistic relationship. These analyses were performed with SPSS version 21 software (SPSS Inc., Chicago, IL, USA).

Results

Qualitative and quantitative analysis of melatonin and phenolics in wines and musts

The results relating to the content of bioactive compounds in different wine and must samples have been reported and discussed in our previous studies. [START_REF] Marhuenda | Melatonin and hydroxytyrosol-rich wines influence the generation of DNA oxidation catabolites linked to mutagenesis after the ingestion of three types of wine by healthy volunteers[END_REF][START_REF] Marhuenda | Melatonin and hydroxytyrosol protect against oxidative stress related to the central nervous system after the ingestion of three types of wine by healthy volunteers[END_REF] In order to help understand the study, the results are summarized in the present paper. The concentrations of the different bioactive compounds are shown in Table 2 (describing the content of MEL, OHTyr and its metabolites) and Table 3 (showing the content of phenolic compounds).

Briefly, the wines showed a higher content of MEL, while the musts contained very small amounts of this compound. In wines, the highest MEL concentration was found in carbonic maceration wine (CMW) (161.83 ± 7.14 ng mL -1 ), while aged wine (AW) (26.31 ± 1.35 ng mL -1 ) and high expression wine (HEW) (19.63 ± 1.14 ng mL -1 ) showed minor contents ( p < 0.01) but similar concentrations ( p > 0.05).

Musts showed high amounts of HV-Acid, but minor contents of HV-Alc and a very slight presence of OHTyr. HV-Alc followed a trend opposite to that of HV-Acid, with its concentration increasing after winemaking for CMW and AW, but not HEW (Table 2). The most important difference between the wines and musts was the increment of OHTyr after vinification ( p < 0.05). As shown in Table 2, HEW showed the highest OHTyr concentration of all the beverages (35.11 ± 2.51 ng mL -1 ), followed by AW (29.65 ± 0.46 ng mL -1 ) and CMW (16.13 ± 2.98 ng mL -1 ).

The concentration of phenolic compounds from the wines and musts is shown in Table 3. Anthocyanins were the major phenolic compounds found in the wines and musts, underlining malvidin-3-O-glucoside as the major anthocyanin both in the wines and musts ( p < 0.05), followed by delphinidin-3-Oglucoside and petunidin-3-O-glucoside which also highlighted from the rest of the anthocyanins. The most abundant hydroxycinnamic acid derivatives were caftaric acid and coutaric acid, mainly found in the wines. On the contrary, p-coumaric acid and fertaric acid were minor compounds in the wines and undetectable in the musts. Moreover, quercetin-3-O-rutinoside and kaempferol-3-O-glucoside were identified as the major flavonols. Finally, the major stilbene was trans-resveratrol, both in the wines and musts.

Qualitative analysis of isoprostanes in urine from healthy volunteers

The qualitative profile of the IsoPs analyzed in this study is shown in Table 4. F 2 -IsoPs are reliable oxidative stress markers and they are considered gold standard markers by the European Food Safety Authority (EFSA). [START_REF] Medina | A ultra-pressure liquid chromatography/triple quadrupole tandem mass spectrometry method for the analysis of 13 eicosanoids in human urine and quantitative 24 hours values in healthy volunteers in a controlled constant diet[END_REF][START_REF] Kadiiska | Biomarkers of Oxidative Stress Study II: Are oxidation products of lipids, proteins, and DNA markers of CCl4 poisoning?[END_REF] IsoPs found in every 48 h urine sample for the whole treatment are as follows: 15-F 1t -IsoP, 5-epi-15-F 2t -IsoP, 15-F 2t -IsoP, 9-epi-15-F 2t -IsoP, 5-F 2t -IsoP, 5-epi-5-F 2t -IsoP, ent-15-epi-15-F 2t -IsoP, ent-PGF 2α , 15-E 1t - IsoP, 8-F 3t -IsoP, 2,3-dinor-15-F 2t -IsoP, and 2,3-dinor-15-epi-15-F 2t -IsoP. These markers helped us to describe the protection grade against systemic oxidative stress (in vivo antioxidant capacity) after the intake of the different musts and wines by healthy volunteers included in this study. Other IsoPs were not found in any urine sample: 15-keto-15-F 2t -IsoP, 15-keto-15-E 2t -IsoP, 15-epi-15-E 2t -IsoP, and 8-epi-8-F 3t -IsoP.

Quantitative analysis of isoprostanes after the intake of red wines by healthy volunteers Total urinary IsoPs are shown in Fig. 2. As can be observed, the control period largely exhibited the major total IsoP content of all stages (198.46 ± 45.43 μg 24 h -1 ). Statistically, minor urinary concentrations of total IsoPs were found after the intake of the three assayed wines ( p < 0.05). Therefore, the IsoP content decreased until 102.43 ± 25.77 μg 24 h -1 , 98.38 ± 20.72 μg 24 h -1 , and 85.21 ± 16.48 μg 24 h -1 upon the administration of a cup of carbonic maceration wine (CMW), aged wine (AW), and high expression wine (HEW), respectively.

The administration of an ethanol controlmarked by the intake of white rum (WR)also presented a minor concentration of total IsoPs (158.22 ± 40.75 μg 24 h -1 ) compared to that in the control period. However, contrary to the intake of the wines, the differences were not statistically significant ( p > 0.05).

A similar trend was found after the intake of the three primary musts assayed in the current trial. All three musts led to a minor total IsoP content compared with that in the control period. However, they did not reach the lowest total IsoP concentration, noticeable for the wine treatments ( p > 0.05). So, the intake of the primary musts for carbonic maceration wine (CMM), aged wine (AM) and high expression wine (HEM) led to 157.39 ± 36.62 μg 24 h -1 , 150.81 ± 25.36 μg 24 h -1 and 141.85 ± 22.41 μg 24 h -1 respectively.

Therefore, ethanol intake and must treatments showed antioxidant protection observed by total IsoP excretion. However, they exhibited minor antioxidant protection compared to the treatments with their respective wines.

The disparity of individual IsoPs between the control stage and other periods has also been studied. A total of 12 IsoPs were identified in urine (Table 4). The most abundant compounds were 2,3-dinor-15-F 2t -IsoP, 5-epi-5-F 2t -IsoP and ent-15epi-15-F 2t -IsoP. As expected, the trend followed by total IsoPs was concomitant but more deeply marked than that of the individual IsoP content.

Largely, the higher content of every individual IsoP was mainly found after the control period. However, different trends could be observed in the different IsoPs. Two IsoPs were found to have similar urinary contents over the whole study. Both 5-F 2t -IsoP and 5-epi-5-F 2t -IsoP did not present significant differences after any of the treatments ( p > 0.05). The content of 2,3-dinor-15-F 2t -IsoP in urine at baseline was 27.40 ± 6.24 μg 24 h -1 , presenting a statistical decrease after the intake of every beverage of the study ( p < 0.05). After the control period, 15-F 1t -IsoP (0.79 ± 0.09 μg 24 h -1 ), ent-15-epi-15F 2t -IsoP (21.96 ± 5.96 μg 24 h -1 ), 2,3-dinor-15-F 2t -IsoP (88.89 ± 20.74 μg 24 h -1 ) and 2,3-dinor-15-epi-15-F 2t -IsoP (7.71 ± 1.89 μg 24 h -1 ) were detected at higher amounts than after the intake of CMW, AW and HEW ( p < 0.01). In the control period, the 9-epi-15-F 2t -IsoP and ent-PGF 2α contents were 6.93 ± 1.55 μg 24 h -1 and 1.96 ± 0.45 μg 24 h -1 , respectively, but after the intake of AW and HEW, these decreased significantly ( p < 0.05). As reflected by the results, the wine treatments were able to considerably reduce the IsoP content marked by the control period. In fact, only 15-epi-15-F 2t -IsoP was found in a minor concentration after the ingestion of AM and HEM compared to the control period ( p < 0.05) (Table 6).

The differences between the treatments with wines and their main musts were also studied. The intake of AW led to a smaller urinary content of 15-F 1t -IsoP, 15-epi-15-F 2t -IsoP, 15-E 1t -IsoP, 8-F 3t -IsoP and 15-F 2t -IsoP than the intake of AM ( p < 0.05). The intake of HEW also reported a lower amount of some IsoPs than its corresponding must. 15-E 1t -IsoP, 8-F 3t -IsoP and 15-F 2t -IsoP were also found in a lower quantity after the intake of HEW compared to the intake of HEM ( p < 0.05).

Finally, WR intake only caused a significant reduction in the case of 2,3-dinor-15-F 2t -IsoP. In fact, after the intake of CMW and AW, 15-F 1t -IsoP showed significantly smaller urinary values than that after the intake of WR ( p < 0.05). The same trends were found after the intake of AW and HEW when quantifying 9-epi-15-F 2t -IsoP, ent-15-F 2t -IsoP, ent-PGF 2α and 2,3dinor-15-F 2t -IsoP. The same behavior was found for 8-F 3t -IsoP after the intake of CMW and HEW, and for 2,3-dinor-15-epi-15-F 2t -IsoP after the intake of CMW, AW and HEW.

Qualitative analysis of prostaglandins after the intake of red wines by healthy volunteers

In order to clarify the health effects of the different treatments, PG series were divided into groups, according to their main precursor in the corresponding physiological pathway (Table 5). Therefore, the groups were defined as shown in Table 6. The individual PGs found in the urine samples were:

Quantitative analysis of prostaglandins in 24 h urine from healthy volunteers

Regarding total PG content, Fig. 3 and4 present the variation of the different PG families over the whole clinical trial. Total PG-D series presented a dramatic decrease in their concen-tration after the intake of the different beverages compared with the control period (Fig. 3). The decrease in the urinary excretion of PGs was not common to all PG series. In fact, total PG-E series and total PG-F series did not show a uniform decrease pattern. In contrast, the contents of urinary PG-E and PG-F series were smaller after the control period than those after the intake of all beverages except for HEM (Fig. 4). Total Ω3/6-PG series provided a uniform pattern among all the treatments (Fig. 3). Nonetheless, only the urinary content of total PG-D series showed significant statistical variation. After the intake of CMW and HEW and their corresponding musts (CMM and HEM), the urinary content of total PG-D series was smaller during these treatments than that at the baseline ( p < 0.05).

The individual content of the different prostaglandin series was also studied (Table 6). The urinary excretion of PGs did not vary widely. In fact, only the urinary content of 2,3-dinor-11β-PGF 2 , tetranor PGEM, PGE 1 and 17-trans-PGF 3α presented statistically significant modifications. After the control period, 2,3-dinor-11-β-PGF 2 showed a higher urinary content (100.95 ± 22.09 μg 24 h -1 ) than after the intake of the three musts and their finished wines ( p < 0.05). Therefore, the urinary content of 2,3-dinor-11-β-PGF 2 after the intake of musts was 37.09 ± 5.77 μg 24 h -1 for CMM, 44.21 ± 8.64 μg 24 h -1 for AM and 29.22 ± 7.04 μg 24 h -1 for HEM. Finally, the content of 2,3dinor-11-β-PGF 2 after the intake of the wines was 32.92 ± 6.71 μg 24 h -1 for CMW, 35.68 ± 7.20 μg 24 h -1 for AW and 26.53 ± 3.86 μg 24 h -1 for HEW.

Two PGs -PGE 1 and 17-trans-PGF 3αfrom the enzymatic oxidation of Ω3/6 fatty acids were the last individual PGs which showed a statistical variation with respect to the control period. The intake of all three wines was able to increase the urinary content of PGE 1 ( p < 0.05), with respect to the control period (0.24 ± 0.11 μg 24 h -1 ). After the intake of CMW, the PGE 1 content was 2.10 ± 0.58 μg 24 h -1 and it was 1.23 ± 0.25 μg 24 h -1 and 1.44 ± 0.52 μg 24 h -1 after the intake of AW and HEW, respectively. 17-trans-PGF 3α seemed to be modified by the intake of HEM and its corresponding wine, HEW. After the control period, the content of 17-trans-PGF 3α was 3.26 ± 0.81 μg 24 h -1 . However, after the intake of HEM, its content was 1.37 ± 0.26 μg 24 h -1 , achieving similar results for HEW (1.33 ± 0.30 μg 24 h -1 ).

Finally, despite some individual PGs showing variations (cited above) after the intake of the beverages, no variations related to the intake of WR were found in any of the cases.

Discussion

Moderate consumption of red wine has been related to the cardioprotective and antioxidant benefits for human beings, mainly attributed to polyphenols. [START_REF] Mulero | Antioxidant activity and phenolic compounds in organic red wine using different winemaking techniques[END_REF] However, the scientific literature has widely shown their poor absorption at the intestinal level, [START_REF] Bouayed | Bioaccessible and dialysable polyphenols in selected apple varieties following in vitro digestion vs. their native patterns[END_REF][START_REF] Bouayed | Total phenolics, flavonoids, anthocyanins and antioxidant activity following simulated gastro-intestinal digestion and dialysis of apple varieties: Bioaccessibility and potential uptake[END_REF][START_REF] Tagliazucchi | In vitro bio-accessibility and antioxidant activity of grape polyphenols[END_REF] limiting their activity in vivo. However, anthocyanins, which are the most important polyphenol in wines, are directly absorbed in the intestine, exerting the major benefits. [START_REF] Talavéra | Anthocyanin metabolism in rats and their distribution to digestive area, kidney, and brain[END_REF] In the present work, the individual intake of hydroxytyrosol and total polyphenolsnot including colon derivatives (only hydroxytyrosol microflora metabolites)has been related to the formation of lipid peroxidation products and the enzymatic formation of oxylipins. Moreover, other anti-inflammatory compounds such as phytoprostanes are well absorbed in the intestine, showing health benefits despite colonic metabolites not being reported yet. [START_REF] Marhuenda | Dependency of Phytoprostane Fingerprints of Must and Wine on Viticulture and Enological Processes[END_REF][START_REF] Barden | Flaxseed oil supplementation increases plasma F1-phytoprostanes in healthy men[END_REF] Therefore, this preliminary intervention may help to understand the role of certain wine compounds in the oxidation of AA and the formation of enzymatic oxylipins.

Isoprostanes after the intake of wine and must by healthy volunteers

The urinary content of IsoPs decreased after the ingestion of the three types of wine. Red wine has shown in vivo protection against oxidative stress (related to antioxidant capacity). However, this was mainly attributed to its content of bioactive compounds and their antioxidant capacity. [START_REF] Basli | Wine polyphenols: Potential agents in neuroprotection[END_REF][START_REF] Rodríguez-Naranjo | Melatonin is synthesised by yeast during alcoholic fermentation in wines[END_REF][START_REF] Gómez-Serranillos | Study of red wine neuroprotection on astrocytes[END_REF] Not only wines, but also the intake of musts proved to be able to reduce the excretion of IsoPs. Finally, the oral administration of ethanolshown as WR in the figures and tablesalso reduced the urinary excretion of IsoPs, emphasizing the in vivo antioxidant capacity of ethanol.

However, the amount of bioactive compounds present in the wine is greater and more varied than that observed in their musts. Moreover, the intake of wine leads to the ingestion of ethanol (similar amount to WR). Red wines provide a varied amount of bioactive compounds; the intake of CMW provides a greater amount of MEL, while the intake of AW and HEW provides a higher content of OHTyr. Given the antioxidant capacity of both compounds, the intake of every wine led to an increase in the protection against oxidative stress by the accumulation of antioxidant compounds. Due to the greater amount of bioactive compounds and the presence of ethanol, red wine intake decreased the urinary excretion of IsoPs more than the intake of the primary musts (without ethanol) and the intake of WR (non-bioactive compounds).

The characterization of the samples allowed the establishment of a relationship between bioactive compounds and the urinary excretion of lipid peroxidation markers, and vascular function and inflammation markers. The total urinary excretion of IsoPs was individually associated with the different bioactive compounds and TPC of the samples, as shown in Table 6. OHTyr has shown its antioxidant capacity in vivo. [START_REF] Fernández-Mar | Bioactive compounds in wine: Resveratrol, hydroxytyrosol and melatonin: A review[END_REF][START_REF] Vilaplana-Pérez | Hydroxytyrosol and Potential Uses in Cardiovascular Diseases, Cancer, and AIDS[END_REF] Indeed, the simple regression analysis showed an inverse relationship between the content of OHTyr of wines and musts, and the urinary excretion of IsoPs. OHTyr is responsible for 33.4% of the observed change in the excretion of IsoPs. Moreover, the HV-Alc administration was also inversely related to the urinary excretion of IsoPs (25.4% of the effect). Although less marked, TPC intake was also shown to be inversely related to IsoP excretion, accounting for 10.1% of the change observed. Finally, the oral administration of MEL and HV-Acid was also inversely correlated with the total IsoP excretion, accounting for 6% and 2.9% of the observed changes, respectively.

Finally, multiple regression analysis showed that 42.4% of the change in the urinary excretion of IsoPs can be explained by the combined intake of the different compounds present in wine and must. However (as shown in Table 6), when analyzing the data simultaneously, the content of OHTyr seems to be the key factor producing the observed changes in the excretion of IsoPs. OHTyr was responsible for 40.5% of the observed variations in the IsoP urinary excretion, when it was simultaneously compared with the other compounds.

Prostaglandins after the intake of wine and must by healthy volunteers

The biological functions of PGs have been extensively studied. Depending on the studied family, a wide variety of actions can be attributed. [START_REF] Ricciotti | Prostaglandins and inflammation[END_REF] PG functions depend on the family to which they belong. PG-D series can exert both vasoconstrictor effects and vasodilator effects in the organism. [START_REF] Funk | Prostaglandins and leukotrienes: Advances in eicosanoid biology[END_REF][START_REF] Ricciotti | Prostaglandins and inflammation[END_REF][START_REF] Lewis | Prostaglandin D2 generation after activation of rat and human mast cells with anti-IgE[END_REF][START_REF] Urade | The major source of endogenous prostaglandin D2 production is likely antigen-presenting cells. Localization of glutathione-requiring prostaglandin D synthetase in histiocytes, dendritic, and Kupffer cells in various rat tissues[END_REF][START_REF] Smith | Enzymes of the cyclooxygenase pathways of prostanoid biosynthesis[END_REF][START_REF] Gilroy | Inducible cyclooxygenase may have anti-inflammatory properties[END_REF] PG-I series could not be quantified in urine because they were not detected during this study; however, they are capable of exerting platelet, antiplatelet and vasodilator effects, with the consequent prevention of cardiovascular diseases. [START_REF] Noda | Neuroprotective role of bradykinin because of the attenuation of pro-inflammatory cytokine release from activated microglia[END_REF][START_REF] Libby | Interleukin 1: A mitogen for human vascular smooth muscle cells that induces the release of growth-inhibitory prostanoids[END_REF] Another family of PGs, PG-E series, demonstrated neuroprotective capacity, as well as pro-inflammatory and anti-inflammatory actions. [START_REF] Trebino | Impaired inflammatory and pain responses in mice lacking an inducible prostaglandin E synthase[END_REF][START_REF] Thorén | Coordinate up-and downregulation of glutathione-dependent prostaglandin E synthase and cyclooxygenase-2 in A549 cells: Inhibition by NS-398 and leukotriene C4[END_REF][START_REF] Moriyama | Sensitization of TRPVI by EP1 and IP reveals peripheral nociceptive mechanism of prostaglandins[END_REF] Finally, PG-F-series could be partly responsible for the pro-inflammatory effects of PGs. [START_REF] Ricciotti | Prostaglandins and inflammation[END_REF] Urinary excretion of the PG-D family was affected to a greater extent than that of the PG-H family. The intake of three wines and three musts caused a decrease in the excretion of the PGs of series D (Fig. 3). Particularly, 2,3-dinor-11-β-PGF 2α decreased significantly ( p < 0.01) within the PG-D family (Table 5). The biological activity of 2,3-dinor-11-β-PGF 2α is very similar to that of 11-β-PGF 2α , increasing bronchoconstriction [START_REF] Pollock | Thromboxane-induced phosphatidate formation in human platelets. Relationship to receptor occupancy and to changes in cytosolic free calcium[END_REF][START_REF] Coleman | Comparison of the actions of U-46619, a prostaglandin H2-analogue, with those of prostaglandin H2 and thromboxane A2 on some isolated smooth muscle preparations[END_REF] and inhibiting adipose differentiation. [START_REF] Coleman | Prostanoid-induced contraction of human bronchial smooth muscle is mediated by TP-receptors[END_REF][START_REF] Lepak | Inhibition of adipose differentiation by 9α, 11β-prostaglandin F2α[END_REF] The levels of the PG-E family in urine remained stable throughout the study except after the ingestion of AM (Fig. 4), resulting in a particularly important increase in the excretion of tetranor PGEM (Table 5). The biological action of tetranor PGEM has not been investigated enough, but it is a representative marker of the synthesis of PGE 1 and PGE 2 . PGE 1 exerts vasodilator effects, while the effects of PGE 2 are variable, and depending on the receiver, it can exert vasoconstriction or vasodilatation, as well as bronchoconstriction and bronchodilator effects. [START_REF] Greenhough | The COX-2/PGE2 pathway: Key roles in the hallmarks of cancer and adaptation to the tumour microenvironment[END_REF] Therefore, an increase in the excretion of tetranor PGEM can be representative of a vasodilator effect exerted by PGE 1 and PGE 2 .

From the two remaining PG families, PG-F levels remained constant ( p > 0.05) in urine throughout the study (Fig. 4). However, the excretion of -the Ω3/6-PG series presented marked differences. After the intake of HEM, the levels of PGF 1α and 17-trans-PGF 3α in urine decreased considerably. Finally, the urinary excretion of PGE 1 increased considerably after the ingestion of the three types of wine (Table 6). The biological activity of PGE 1 is relevant, as already mentioned, acting as a vasodilator, and it may act as an important compound for the cardiovascular balance. [START_REF] Olley | Prostaglandins and the ductus arteriosus[END_REF][START_REF] Cole | Prolonged prostaglandin E1 infusion: Histologic effects on the patent ductus arteriosus[END_REF][START_REF] Rabbani | ProstaglandinE1 for the medical management of erectile dysfunction[END_REF] The effects of PGs as individual compounds may be even more diverse. They can exert anti-or pro-inflammatory effects, depending upon the compound and the receptor, in addition to depending upon the tissue where the action occurs. [START_REF] Ricciotti | Prostaglandins and inflammation[END_REF] Thus, the intake of red wines was able to decrease the urinary excretion of pro-inflammatory prostaglandin (acting as pro-aggregating compounds and vasoconstrictors) such as 2,3dinor-11-β-PGF 2α . In turn, wine intake increased the excretion of another vasodilator prostaglandin, PGE 1 . These results strengthen the cardioprotective effects of red wine intake in particular and an ingredient of MD in general.

In response to these findings, statistical analysis was performed to describe the relationship of the bioactive compounds in red wine and musts and the actions described above. In this way, it was found that the relationship of PGs with bioactive compounds was widely distributed, and some relationships are noteworthy.

OHTyr intake also showed a relationship with the urinary excretion of some PGs. The specific PGs that were inversely related ( p < 0.05) to the contents of OHTyr were 11-β-PGF 2α , tetranor PGJM, tetranor PGAM, 20-OH-PGF 2α and 17-trans-PGF 3α .

Based on these results, statistical relationships were established in the same way as that used for studying the relationship with IsoPs. Thus, it was found that the ratio of PG with bioactive compounds was very irregular, and there is no pattern to define it. However, the statistical relationship of PGE 1 , which, as described above, can exert vasodilator capacity, [START_REF] Olley | Prostaglandins and the ductus arteriosus[END_REF][START_REF] Cole | Prolonged prostaglandin E1 infusion: Histologic effects on the patent ductus arteriosus[END_REF][START_REF] Rabbani | ProstaglandinE1 for the medical management of erectile dysfunction[END_REF] with the different compounds present in wines and musts was noticeable (Table 6).

From simple regression, the urinary excretion of PGE 1 was dependent on a 53.8% by the intake of MEL, 13.9% by OHTyr, 6.2% by HV-Acid, 39% by HV-Alc and 40.7% by TPC. In turn, the study of multiple regression showed that the observed variations in the excretion of PGE 1 are dependent in a 60.9% by the whole of the compounds studied simultaneously. However, the intake of MEL and OHTyr was determinant, both being responsible for 59.9% of the observed increase in the excretion of PGE1.

The cardioprotective effects of PGE 1 , and the direct relationship found between the ingestion of bioactive compounds and their excretion in urine may be important in the understanding of the cardioprotective effects of red wine. On the other hand, the intake of musts and WR ( providing bioactive compounds and ethanol, respectively) cannot provide the cardioprotective effects as provided by the intake of red wines (contributing ethanol and bioactive compounds) measured by urinary PGE 1 .

These results reinforce the findings of the previous publications, and exalt the role of red wine as a supporter of the beneficial actions of MD. Finally, it is apparent that the content of OHTyr and MEL is a key factor in the antioxidant protection of red wines. Furthermore, the cardioprotective capacity of red wine can be related to the increase of PGE 1 .

Conclusions

Wine treatments are more effective in reducing in vivo oxidative stress than the treatments with their corresponding musts and/or white rum (ethanol). Specifically, HEW showed to be the most effective wine reducing total IsoPs.

The capacity of wine and musts to decrease the concentration of IsoPs is statistically related to their content of OHTyr. Therefore, the wine showing a higher OHTyr concentration was able to provide a higher reduction of the oxidative status of the organism. To a small extent ( p > 0.05), MEL was also related to the antioxidant protection of the treatments, so that the treatments which provided more MEL led to higher protection against oxidative stress.

Both alcohol and bioactive compounds seem to be partly responsible for the antioxidant capacity of the beverages. Red wines containing both bioactive compounds and alcohol became the best treatment for reducing the organism's oxidative status measured by total IsoPs ( p < 0.05) compared to musts and alcohol. In fact, the correlation between the different bioactive compounds present in the three wines and the drop in total IsoPs is statistically significant. In contrast, alcohol and bioactive compounds separately are not enough to reduce lipid peroxidation. In addition, the intake of red wine led to cardioprotective effects by reducing the urinary excretion of the pro-inflammatory prostaglandin 2,3-dinor-11-β-PGF 2α , besides increasing the vasodilator prostaglandin PGE 1 , mediated by the melatonin ( p < 0.05) and hydroxytyrosol ( p < 0.05) contents.

Therefore, MEL and OHTyr and their derivatives can be important compounds in wine and must for the reduction of oxidative injury. However, other compounds such as poly-phenol colon derivatives and newly discovered phytoprostanes may also be effective compounds for reducing oxidative damage. Moreover, chemical reactions that occur during the winemaking process (oxidation or polymerization) make the comparison between wine musts and wine more difficult, because the changes produced in their chemical and phenolic composition make the two beverages completely different from each other. [START_REF] Mulero | Antioxidant activity and phenolic compounds in organic red wine using different winemaking techniques[END_REF][START_REF] Cantos | Postharvest UV-C-irradiated grapes as a potential source for producing stilbene-enriched red wines[END_REF] Therefore, further studies will help to understand the effect of other bioactive compounds on reducing lipid peroxidation and prostaglandin formation.

Fig. 2

 2 Fig. 2 Total IsoPs found in urine. Means are expressed in μg 24 h -1 . WR: white rum; CMW/CMM: carbonic maceration wine/must; AW/AM: aged wine/must; HEW/HEM: high expression wine/must. *Represents statistical differences.

Fig. 3

 3 Fig. 3 Total PG-H and PG-G found in urine. Means are expressed in μg 24 h -1 . WR: white rum; CMW/CMM: carbonic maceration wine/must; AW/AM: aged wine/must; HEW/HEM: high expression wine/must. *Represents statistical differences.

  This journal is © The Royal Society of Chemistry 2017 Food Funct., 2017, 8, 3745-3757 | 3751 Published on 05 September 2017. Downloaded by University of Montpellier on 18/10/2017 10:43:14.

Fig. 4

 4 Fig. 4 Total PG-E and PG-F found in urine. Means are expressed in μg 24 h -1 . WR: white rum; CMW/CMM: carbonic maceration wine/must; AW/AM: aged wine/must; HEW/HEM: high expression wine/must.

Table 1

 1 Physical and clinical parameters of the participants (N = 18)

	Parameters	Mean	SD
	Age (years)	22.5	6.3
	Weight (kg)	64.3	16.4
	Height (cm) BMI (kg m -2 ) Glucose (mg dL -1 ) Urea (mg dL -1 ) Creatinine (mg dL -1 ) Uric acid (mg dL -1 ) Cholesterol (mg dL -1 ) Triglycerides (mg dL -1 ) HDL-Cholesterol (mg dL -1 ) LDL-Cholesterol (mg dL -1 )	164.1 23.54 87.2 28.7 0.7 3.8 190.1 87.5 70.9 101.1	17.7 1.07 7.7 6.6 0.1 0.8 31.7 39.3 18.8 24.7

Table 2

 2 Characterization of the bioactive compounds of red wines and musts and the white rum ethanol content

	Hydroxytyrosol metabolites (ng mL -1 )

WR: white rum; CMW/CMM: carbonic maceration wine/must; AW/AM: aged wine/must; HEW/HEM: high expression wine/must. *It is considered as the total dose ingested during 48 hours: 74 mL of white rum and 200 mL of wine and must.

Table 3

 3 Polyphenolic content of red wines and musts

		CMW	CMM	AW	AM	HEW	HEM
	Delphinidin-3-O-glucoside	55.97 ± 2.18 a	12.11 ± 0.56 c,d	39.81 ± 0.48 b	12.96 ± 1.10 c,d	11.36 ± 0.47 d	14.63 ± 1.14 c
	Cyanidin-3-O-glucoside	2.45 ± 0.05 b,c	4.03 ± 0.17 b	3.44 ± 0.09 b,c	3.69 ± 0.08 b	0.60 ± 0.04 c	7.29 ± 0.88 a
	Petunidin-3-O-glucoside	54.73 ± 1.26 a	9.13 ± 0.59 d	30.21 ± 0.27 b	9.12 ± 0.98 d	8.17 ± 0.20 d	13.85 ± 0.95 c
	Peonidin-3-O-glucoside	11.77 ± 1.04 b	7.36 ± 1.52 c	10.96 ± 0.35 b	7.38 ± 0.74 c	3.37 ± 0.04 d	14.66 ± 1.39 a
	Malvidin-3-O-glucoside	236.87 ± 10.00 a	35.80 ± 2.30 d	90.10 ± 5.09 b	38.53 ± 20.00 d	35.19 ± 0.61 c	74.16 ± 3.29 d
	Total anthocyanins	361.79 ± 11.27 a	68.43 ± 3.71 d	174.53 ± 5.98 b	71.68 ± 3.25 d	58.68 ± 0.74 c	124.59 ± 4.56 d
	Caftaric acid	45.09 ± 4.52 b,c	0.59 ± 0.02 d	48.81 ± 0.03 a,b	0.31 ± 0.02 d	55.70 ± 4.73 a	2.35 ± 0.12 d
	p-Coumaric acid	1.30 ± 0.07 c	ND	10.59 ± 0.73 b	ND	14.42 ± 1.04 a	ND
	Fertaric acid	0.76 ± 0.04 c	ND	2.40 ± 0.08 b	ND	5.20 ± 0.30 a	ND
	Coutaric acid	40.82 ± 1.75 a	0.39 ± 0.06 d	13.69 ± 0.32 c	0.28 ± 0.02 d	20.54 ± 0.71 b	1.13 ± 0.10 d
	Total hydroxycinnamic derivatives	87.96 ± 6.39 a	0.98 ± 0.08 c	75.50 ± 1.15 b	0.59 ± 0.04 c	95.85 ± 6.77 a	3.48 ± 0.22 c
	Myricetin-3-O-glucoside	3.96 ± 0.06 a	1.06 ± 0.04 c	4.21 ± 0.23 a	1.00 ± 0.04 c	2.84 ± 0.26 b	1.22 ± 0.05 c
	Quercetin-3-O-glucoside	2.33 ± 0.21 c	0.63 ± 0.10 d	3.36 ± 0.12 b	0.60 ± 0.09 d	6.88 ± 0.25 a	0.73 ± 0.12 d
	Quercetin-3-O-rutinoside	33.37 ± 1.88 b	2.15 ± 0.14 d	24.93 ± 3.32 c	2.03 ± 0.14 d	49.77 ± 5.24 a	2.49 ± 0.17 d
	Kaempferol-3-O-glucoside	10.19 ± 1.27 b	2.54 ± 0.13 d	6.49 ± 0.34 c	2.39 ± 0.12 d	18.82 ± 1.28 a	2.93 ± 0.15 d
	Myricetin	5.08 ± 0.16 a	1.83 ± 0.17 d	2.30 ± 0.14 c	1.73 ± 0.16 d	3.71 ± 0.18 b	2.12 ± 0.19 c,d
	Quercetin	3.23 ± 0.13 b	1.79 ± 0.24 c,d	1.25 ± 0.03 d	1.69 ± 0.23 c,d	4.82 ± 0.16 a	2.06 ± 0.28 c
	Total flavonols	58.15 ± 1.31 b	10.01 ± 0.32 c	42.54 ± 4.18 b	9.43 ± 0.30 c	86.84 ± 4.22 a	11.55 ± 0.36 c
	Piceatannol	0.26 ± 0.02 b	0.08 ± 0.01 c	0.20 ± 0.01 b	0.08 ± 0.01 c	0.57 ± 0.04 a	0.27 ± 0.03 b
	trans-Resveratrol	4.85 ± 1.03 a	0.84 ± 0.06 d	2.10 ± 0.15 c	0.79 ± 0.08 d	3.37 ± 0.25 b	0.82 ± 0.06 d
	Total E-stilbenes	5.12 ± 1.05 a	0.92 ± 0.07 c	2.30 ± 0.16 b	0.87 ± 0.09 c	3.94 ± 0.29 a	1.09 ± 0.09 b,c
	Total phenolic compounds (TPC)	513.02 ± 20.02 a	80.34 ± 4.18 c.d	294.87 ± 11.47 b	82.57 ± 3.68 c.d	245.31 ± 12.02 b	140.71 ± 5.23 c

WR: white rum; CMW/CMM: carbonic maceration wine/must; AW/AM: aged wine/must; HEW/HEM: high expression wine/must. Results are expressed in μg mL -1 .

Table 4

 4 Isoprostane content in urine measured after the different treatments Published on 05 September 2017. Downloaded by University of Montpellier on 18/10/2017 10:43:14.

		Control	WR	CMW	CMM	AW	AM	HEW	HEM
	15-F 1t -IsoP	0.79 ± 0.09	0.70 ± 0.20	0.34 ± 0.09	0.59 ± 0.15	0.23 ± 0.03	0.65 ± 0.12	0.39 ± 0.06	0.62 ± 0.09
	5-epi-15-F 2t -IsoP	4.82 ± 1.70	4.36 ± 1.17	2.30 ± 0.60	4.72 ± 1.03	2.42 ± 0.61	5.36 ± 0.89	2.05 ± 0.42	4.39 ± 0.9
	15-F 2t -IsoP	2.95 ± 0.52	2.66 ± 0.37	1.69 ± 0.41	2.21 ± 0.58	1.72 ± 0.29	3.46 ± 0.58	1.55 ± 0.28	2.84 ± 0.39
	9-epi-15-F 2t -IsoP	6.93 ± 1.55	5.97 ± 1.84	4.00 ± 1.07	6.34 ± 0.88	2.02 ± 0.38	4.15 ± 0.70	2.23 ± 0.40	3.47 ± 0.50
	5-F 2t -IsoP	15.55 ± 3.30	14.07 ± 4.07	10.36 ± 2.68 14.33 ± 3.33	16.41 ± 2.39 16.34 ± 2.88 10.14 ± 2.85 15.93 ± 2.43
	5-epi-5-F 2t -IsoP	33.05 ± 7.82	32.54 ± 9.59	22.93 ± 5.63 27.83 ± 6.57	23.63 ± 5.68 32.33 ± 6.44 20.43 ± 4.40 26.70 ± 4.46
	ent-15-epi-15-F 2t -IsoP	21.96 ± 5.26	17.70 ± 5.23	10.62 ± 2.85 17.37 ± 3.35	7.83 ± 1.72 14.30 ± 2.24	7.56 ± 1.68 14.57 ± 2.73
	ent-PGF 2α	1.96 ± 0.45	1.70 ± 0.30	1.24 ± 0.32	1.64 ± 0.46	0.65 ± 0.18	1.17 ± 0.13	0.53 ± 0.12	1.01 ± 0.17
	15-E 1t -IsoP	11.25 ± 1.58	9.39 ± 1.43	8.35 ± 1.96	9.13 ± 1.32	7.43 ± 1.53 12.42 ± 1.78	7.15 ± 1.13 12.66 ± 1.57
	8-F 3t -IsoP	2.61 ± 0.54	2.40 ± 0.30	1.32 ± 0.34	2.24 ± 0.44	1.48 ± 0.30	2.82 ± 0.39	1.19 ± 0.13	2.25 ± 0.36
	2,3-Dinor-15-F 2t -IsoP	88.89 ± 20.74 59.35 ± 14.21 35.81 ± 8.94 64.52 ± 18.94 52.24 ± 6.87 31.51 ± 5.32 29.24 ± 4.52 52.32 ± 7.96
	2,3-Dinor-15-epi-15-F 2t -IsoP	7.71 ± 1.89	7.37 ± 2.06	3.47 ± 0.87	6.46 ± 1.59	5.15 ± 0.72	5.15 ± 0.88	5.15 ± 0.47	5.15 ± 0.77

Means are expressed in μg 24 h -1 . WR: white rum; CMW/CMM: carbonic maceration wine/must; AW/AM: aged wine/must; HEW/HEM: high expression wine/must. This journal is © The Royal Society of Chemistry 2017 Food Funct., 2017, 8, 3745-3757 | 3749

Table 6

 6 Determination coefficients derived from the study of simple regression and multiple regression of the urinary excretion of total IsoPs and PGE 1

		Total IsoPs	PGE 1
	Simple regression (R 2 )
	MEL	0.060	0.538
	OHTyr	0.334	0.139
	HV-Acid	0.029	0.062
	HV-Alc	0.254	0.390
	TPC	0.101	0.407
	Multiple regression
	R 2 Tot.	0.424	0.609
	R 2 Select.	0.405 (OHTyr)	0.599 (MEL, OHTyr)

MEL: melatonin; OHTyr: hydroxytyrosol; HV-Acid: homovanillic acid; HV-Alc: homovanillic alcohol; TPC: total phenolic compounds. R 2 Tot.: coefficient of regression which expresses the statistical significance of all the compounds studied with respect to each biomarker. R 2 Select.: coefficient of determination of compounds selected by the statistical robustness model. All these values have to be treated as % of influence on the final concentration.

Table 5

 5 Prostaglandin content in urine measured after the different treatments ± 1.72 10.74 ± 1.65 6.16 ± 1.51 8.46 ± 2.38 Tetranor-PGDM 40.16 ± 9.44 41.88 ± 9.61 35.05 ± 7.35 34.80 ± 5.82 34.12 ± 7.17 34.23 ± 7.59 24.59 ± 6.27 23.22 ± 4.39 2,3-Dinor-11-β-PGF 2 100.95 ± 22.09 56.40 ± 10.27 32.92 ± 6.71 37.09 ± 5.77 39.64 ± 7.20 44.21 ± 8.64 26.53 ± 3.86 29.22 ± 7.04 Tetranor-PGDM lactone 46.16 ± 11.86 45.17 ± 12.50 33.60 ± 8.51 25.81 ± 7.01 39.67 ± 7.44 39.41 ± 6.82 35.24 ± 6.55 47.80 ± 10.19 PGF 2α 36.91 ± 10.10 26.45 ± 3.62 44.29 ± 16.6 52.94 ± 15.44 27.13 ± 7.84 41.11 ± 15.53 27.45 ± 8.60 40.29 ± 11.25 19(R)-OH-PGF 2α 54.60 ± 11.87 45.71 ± 7.68 57.92 ± 13.25 48.19 ± 2.28 55.45 ± 10.88 68.00 ± 16.80 45.09 ± 8.6 44.58 ± 7.48

		Control	WR	CMW	CMM	AW	AM	HEW	HEM
	PGs SERIE D
	11-β-PGF 2α	2.03 ± 0.38	2.42 ± 0.62	1.85 ± 0.51	2.12 ± 0.44	1.82 ± 0.41	2.61 ± 0.57	1.77 ± 0.38 2.24 ± 0.44
	PGDM 8.34 Tetranor-PGJM 7.88 ± 2.15 8.36 ± 2.35 8.66 ± 2.40 6.60 ± 1.75 0.88 ± 0.19 1.48 ± 0.34 0.86 ± 0.16 0.90 ± 0.18 0.64 ± 0.12	1.13 ± 0.21	0.75 ± 0.18 0.89 ± 0.11
	PGs SERIE E
	Tetranor-PGAM	5.69 ± 1.13	5.02 ± 0.76	4.67 ± 1.20	6.87 ± 1.67	4.54 ± 0.98	7.04 ± 1.40	4.43 ± 0.85 6.70 ± 0.75
	Tetranor-PGEM	9.01 ± 2.53	8.50 ± 2.14 10.10 ± 1.82	8.89 ± 2.44 16.11 ± 3.39 23.17 ± 4.05	7.75 ± 1.86 10.26 ± 0.17
	20-OH-PGE 2	10.56 ± 1.71 14.20 ± 3.62 14.58 ± 3.84 11.10 ± 2.66 11.25 ± 2.38 12.42 ± 4.05 11.52 ± 2.56 13.39 ± 3.67
	PGE 2	0.81 ± 0.21	1.11 ± 0.29	0.96 ± 0.25	0.82 ± 0.17	0.59 ± 0.14	0.96 ± 0.20	0.75 ± 0.15 0.88 ± 0.17
	PGs SERIE F
	20-OH-Tetranor-PGFM	9.03 ± 2.34	8.25 ± 2.06	7.69 ± 2.03	7.33 ± 1.83	8.56 ± 2.12	9.22 ± 1.51	7.74 ± 2.00 7.82 ± 2.01
	PGs SERIE Ω3/6
	PGF 1α	2.36 ± 0.53	2.79 ± 0.68	2.00 ± 0.49	1.71 ± 0.38	1.90 ± 0.45	2.91 ± 0.58	2.88 ± 0.61 1.80 ± 0.44
	PGE 1	0.24 ± 0.11	0.78 ± 0.21	2.10 ± 0.58	0.73 ± 0.13	1.90 ± 0.45	0.71 ± 0.10	1.44 ± 0.52 0.76 ± 0.17
	17-trans-PGF 3α	3.26 ± 0.81	2.55 ± 0.70	1.65 ± 0.46	2.41 ± 0.36	1.42 ± 0.32	2.11 ± 0.37

1.33 ± 0.30 1.37 ± 0.26 Means are expressed in μg 24 h -1 . WR: white rum; CMW/CMM: carbonic maceration wine/must; AW/AM: aged wine/must; HEW/HEM: high expression wine/must.

This journal is © The Royal Society of Chemistry 2017Published on 05 September 2017. Downloaded by University of Montpellier on 18/10/2017 10:43:14.View Article Online

Acknowledgements

This work has been partially funded by the "Fundación Séneca de la Región de Murcia" Grupo de Excelencia 19900/GERM/15. The authors are grateful to UCAM for the support provided during the development of the assay for the study. Sonia Medina is grateful to the CICYT for a research contract (AGL2011-23690). Special thanks to David Walker for the revision and correction of English grammar.

Author contribution

Javier Marhuenda carried out the analytical processes and wrote and discussed the present paper. Sonia Medina supervised the analytical processes and developed the discussion of the paper. Pedro Martinez-Hernández and Simón Arina provided the beverages of the study and supervised the article. Pilar Zafrilla and Juana Mulero developed the randomized controlled trial. Hans Camille Oger, Jean-Marie Galano and Thierry Durand provided the markers for the study and helped with the review of the manuscript. Federico Ferreres helped with the experimental procedures linked to UHPLC-QqQ-MS/ MS. Ángel Gil-Izquierdo and Sonia Medina designed, supervised, and discussed this research work.

Conflicts of interest

The authors declare that there is no conflict of interest associated with this manuscript.