Red Wine Tannins Fluidify and Precipitate Lipid Liposomes and

Article pubs.acs.org/Langmuir

Red Wine Tannins Fluidify and Precipitate Lipid Liposomes and Bicelles. A Role for Lipids in Wine Tasting? Aurélien L. Furlan,† Aurore Castets,† Frédéric Nallet,‡ Isabelle Pianet,§ Axelle Grélard,† Erick J. Dufourc,† and Julie Géan*,† †

Institute of Chemistry and Biology of Membranes and Nano-objects, UMR 5248, CNRS, University of Bordeaux, IPB, F-33600 Pessac, France ‡ CRPP, UPR 8641, CNRS, University of Bordeaux, F-33600 Pessac, France § ISM, UMR 5255, CNRS, University of Bordeaux, IPB, F-33400 Talence, France S Supporting Information *

ABSTRACT: Sensory properties of red wine tannins are bound to complex interactions between saliva proteins, membranes taste receptors of the oral cavity, and lipids or proteins from the human diet. Whereas astringency has been widely studied in terms of tannin−saliva protein colloidal complexes, little is known about interactions between tannins and lipids and their implications in the taste of wine. This study deals with tannin−lipid interactions, by mimicking both oral cavity membranes by micrometric size liposomes and lipid droplets in food by nanometric isotropic bicelles. Deuterium and phosphorus solid-state NMR demonstrated the membrane hydrophobic core disordering promoted by catechin (C), epicatechin (EC), and epigallocatechin gallate (EGCG), the latter appearing more efficient. C and EGCG destabilize isotropic bicelles and convert them into an inverted hexagonal phase. Tannins are shown to be located at the membrane interface and stabilize the lamellar phases. These newly found properties point out the importance of lipids in the complex interactions that happen in the mouth during organoleptic feeling when ingesting tannins.

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INTRODUCTION

Notably, it is interesting to describe and analyze the effects of tannins on lipids in the frame of wine tasting. Physicochemical studies suggest that the affinity of catechins for the lipid membrane may be governed by the catechin chemical structure and also by the electric charge of the lipid membrane or of the medium.8,10,11 Association of catechins to lipid membranes is suggested to depend on their octanol−water partition coefficients, Kow. Measured values for C, EC, EGC, ECG, and EGCG are respectively 2.4, 2.4, 0.3, 48.0, and 12.112 and are consistent with a thermodynamically favorable interaction with the membrane.10,13−16 The effects of catechins on the membrane structure and organization are still subject to debate. Galloylated catechins (ECG, EGCG) show a sealing effect at very low concentration (below 1 nM) on Egg PC SUV,8 whereas they promote leakage at micromolar concentrations on LUV composed of a mixture of Egg PC and PG, a negatively charged phospholipid.13 On the other hand, controversial effects of catechins on membrane fluidity have been reported. Tsuchiya et al. have concluded from polarization

Condensed tannins or proanthocyanidins are the most abundant polyphenols in red wines. They are derived from the polymerization of flavan-3-ol units such as catechin (C), epicatechin (EC), epigallocatechin (EGC), epicatechin gallate (ECG), and epigallocatechin gallate (EGCG). Condensed tannins from grape seeds and skins are known to play an important gustative role since they contribute to red wine astringency, a dry and rough sensation in the mouth during red wine tasting. This feeling results from the formation of precipitating colloidal complexes between tannins and proline-rich salivary proteins.1−6 Moreover, tannins are also known to interact with lipid bilayers,7−9 an association that is very likely to influence wine tasting. Lipid polymorphism is very important (lamellar phases, multilamellar liposomes, micelles, cubic, sponge or hexagonal phases, bicelles, monolayers, etc.), and two main lipid organizations may be thought to play key roles in modulating the interaction: lipids as membrane components of the oral cavity and/or lipids as colloidal components in fatty foods. The influence of food on the sensory perception of wine is well-known from the oenological viewpoint, but little is known from a molecular viewpoint. © 2014 American Chemical Society

Received: February 6, 2014 Revised: April 18, 2014 Published: May 2, 2014 5518

dx.doi.org/10.1021/la5005006 | Langmuir 2014, 30, 5518−5526

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measurements that catechins cause a fluidity decrease of DPPC and DOPC liposomes; galloylated molecules were reported to be more efficient.9,17 Conversely, Caturla et al. have reported also by fluorescence anisotropy and infrared spectroscopy that galloylated catechins induce a tight packing of acyl chains.13 Yu et al. have deduced from a 31P and 2H solid-state NMR study that EGCG might increase the fluidity of acyl chains.18 A similar effect at the membrane surface was also described for EGCG and EGC by Ulrih et al. using EPR on POPC/POPE/ POPS/Chol MLV.19 From previously published work, it can be concluded that an interaction between tannins and lipids exists, but the localization of tannins in the lipid membrane remains unclear and few studies focused on the dynamics and organization of membrane lipids at the molecular level.15,18−20 Furthermore, none of the studies found in the literature consider tannins− lipids interactions in the biological context of red wine tasting as it has been done for tannins−proteins interactions.5 Indeed, it is worth noting that most of the results quoted above have been obtained on membrane models made by cosolubilizing catechins and lipids within liposomes, forcing the tannins to interact with the lipids. In this work, we have investigated the effects of red wine catechins on lipids by noninvasive methods, 31P and 2H NMR, and by considering the interaction between catechin (C), epicatechin (EC), and epigallocatechin gallate (EGCG) and two model lipid systems, multilamellar vesicles (MLV) and isotropic bicelles (B) (see structures in Figure S1 of the Supporting Information). In the context of wine tasting, micrometric MLV of DMPC and nanometric DMPC/DCPC isotropic bicelles with q = [DMPC]/[DCPC] = 0.5 have been used. Both systems, though crude, are first approaches to mimic respectively the oral cavity membranes, which lipid bilayers are mainly composed of zwitterionic phospholipids like DMPC composing MLV,21−24 and the food lipid droplets having a hydrophobic interior,25 such as the isotropic bicelles.26 The three tannin monomers were externally added to both lipid systems at low tannin/lipid molar ratios that are reached in the mouth after the consumption of a glass of red wine with or without fatty food. Because red wines can contain up to 4 g of tannins per liter,27,28 high tannin/lipid ratios have been also explored. 2H and 31P NMR experiments have been performed to study the structural and dynamic properties of lipids at the membrane surface and down in the hydrophobic core by incorporating chain deuterium-labeled phospholipids.29 In this paper, we will first describe the disordering effect of tannins on MLV at low tannin/lipid ratios. In a second part, we will report a strong perturbation of the lipid phase, at high tannin/lipid ratio, which leads, in the case of isotropic bicelles, to the formation of a hexagonal lipid phase. This further behavior will be explored using water dispersions of DPoPE that are prone to form hexagonal phases.

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(France). Ultrapure water with a nominal resistivity of 18.2 MΩ·cm (Milli-Q, Millipore, France) was used for lyophilization. Sample Preparation. MLV containing tannins were prepared in two ways: cohomogenization and external addition of a stock tannin solution to preformed MLV. Homogenization was performed in water by mixing of 11.1 mg of DMPC with an appropriate amount of dry tannin powder (0.27 or 0.38 mg for C, EC, or EGCG and 0.63 or 1 mg for C, EC, or EGCG, respectively) to obtain a tannin/lipid ratio of 1/ 20 and 1/8, respectively. After lyophilization, the mixture was hydrated by deuterium-depleted water (100 μL) and homogenized by 5 cycles of vigorous shaking in a vortex mixer (3000 rpm), freezing (−196 °C, liquid nitrogen, 2 min), and thawing (40 °C, 10 min). This procedure leads to a milky fluid suspension of micrometer size MLV at a lipid concentration of 157 mM. External addition of tannins to preformed MLV was also carried out and gave essentially the same results as for the cohomogenization procedure. The mixed tannin/MLV system at a tannin/lipid ratio of 1/1.2 and at a final lipid concentration of 60 mM was obtained by addition of a tannin stock solution (100 mM for C) on a DMPC MLV suspension (120 mM) prepared from the hydration of 24.4 mg of lipid powder as described above. Isotropic bicelles were prepared at two concentrations according to a reported procedure.30 DMPC (2.8 or 16.7 mg) and DCPC (3.6 or 21.7 mg) were mixed in appropriated quantities such as the molar content of DMPC vs DCPC, q, was equal to 0.5. Deuterated water (200 or 600 μL) was added to obtain 94−97% hydration (w/w). The solution was then submitted 5 times to the following cycle of sample equilibration: freezing in liquid nitrogen for 2 min, warming up in a 40 °C water bath for 10 min, 10 min of centrifugation at 3000 rpm. A transparent bicelle suspension (visual observation) was obtained for a total lipid concentration of 60 and 120 mM; the 60 mM preparation was used as reference. An appropriate amount of tannin stock solution (100 or 170 mM for C or EGCG) was added to the bicelle solution (120 mM) to obtain a tannin/lipid molar ratio of 1/8, 1/1.2, and 1/0.7 and a final lipid concentration of 60 mM. Labeled MLV and bicelles were prepared in the same way as for nonlabeled systems, with deuterium-depleted water and by substituting a fraction (20, 25, or 100%, molar) of DMPC by DMPC-2H27. For experiments with DPoPE, 13.1 mg of lipids was hydrated by deuterated water or with a catechin deuterated water stock solution. Since no 2H NMR was performed on this sample, the D2O allowed using the spectrometer 2H-lock. Two catechin concentrations have been prepared, 6 and 60 mM, respectively, below and above the critical micelle concentration, CMC, of catechin (9 ± 2 mM).31 This corresponds to a catechin/lipid molar ratio of 1/33 and 1/3.3 for a DPoPE final concentration of 196 mM. Solid-State NMR Spectroscopy. NMR experiments were carried out using Bruker Avance II 300 MHz WB (7.05 T), Avance II 400 MHz SB (9.4 T), and Avance II 500 MHz WB (11.75 T). 31P NMR spectra were acquired at 202 MHz using a phase cycled Hahn-echo pulse sequence (90°x-τ-180°x/y-τ-acq) with gated broadband proton decoupling.32 2H NMR experiments on 2H-labeled DMPC were performed at 46 and 76 MHz by means of a phase-cycled quadrupolar echo pulse sequence (90°x-τ-90°y-τ-acq).33 Typical acquisition parameters were as follows: spectral window of 250 kHz for 31P NMR, 500 kHz for 2H NMR, π/2 pulse widths of 5.75 μs for 31P and ranged from 2.1 to 7.5 μs for 2H, interpulse delays τ were of 30 μs for 31 P, and 40−60 μs for 2H, recycled delays were 5 s for 31P NMR and 2 s for 2H NMR. 2k acquisitions were recorded for phosphorus NMR and 4k−16k scans for deuterium NMR depending on samples. A Lorentzian line broadening of 50 Hz for 31P and 100 Hz for deuterium spectra was applied before Fourier transform from the top of the echo. Quadrature detection was used in all cases. Samples were allowed to equilibrate at least 15 min at a given temperature prior to data acquisition. Phosphorus chemical shifts were referenced to 85% H3PO4 (0 ppm), and the reference for solid-state deuterium powder patterns was set to zero and the position of the carrier placed in the middle of the symmetric Pake pattern (powder spectrum). Theoretical Basis. 2H NMR is a useful tool for measuring the degree of order in deuterated lipid systems. In fluid biomembranes, phospholipids exist in a liquid-crystalline state and undergo fast axially

EXPERIMENTAL SECTION

Materials. 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dicaproyl-sn-glycero-3-phosphocholine (DCPC), 1-myristoyl(d27)2-myristoyl-sn-glycero-3-phosphocholine (DMPC- 2 H 27 ), 1,2dicaproyl(d22)-sn-glycero-3-phosphocholine (DCPC-2H22), and 1,2dipalmitoleoyl-sn-glycero-3-phosphoethanolamine (DPoPE) were purchased from Avanti Polar Lipids (Alabaster, AL). (+)-Catechin (C), (−)-epicatechin (EC), and (−)-epigallocatechin gallate (EGCG) were provided by Sigma-Aldrich (St. Louis, MO). Deuterated water (99.9%), deuterium-depleted water, and sodium 3-trimethylsilyl2,2,3,3-2H4-propionate (TMSP, 99.8%) were obtained from Eurisotop 5519


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symmetric reorientation around the bilayer normal. In lamellar phases, average orientations of C−D bonds can be described in terms of orientational order parameters, SCD, related to the observed quadrupolar splitting, ΔνQ, according to34,35 3 ⎛ 3 cos2 θ − 1 ⎞ AQ ⎜ ⎟SCD 2 2 ⎝ ⎠

ΔvQ (θ ) =

coupled with Goebel Mirrors generates a highly parallel and monochromatic Cu Kα (0.154 nm) radiation. The scattering pattern was recorded with a Bruker gas-based Hi-Star 2D detector (1024 × 1024 pixels, pixel size ca. 105 μm) at a distance of 25 cm of the sample (0.4 ≤ q ≤ 8.0 nm−1). The experimental data were analyzed with the FIT2D program (A.P. Hammersley/ESRF) and expressed as a function of q, the scattering vector in the reciprocal space.

(1)

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2

where AQ = (e qQ)/h is the static quadrupolar coupling constant equal to 167 kHz for deuterated methylene or methyl groups36 and θ is the angle between the bilayer normal and the magnetic field. For hexagonal phases, quadrupolar splitting is twice smaller than those for lamellar phases accounting for the change in symmetry due to axial rotation around the cylinder axes; eq 1, left member, must be then divided by a factor 2.37 The order parameter is related to the angular fluctuations of the C−D bond vector with respect to the lipid long axis. If β denotes the instantaneous angle between the C−D bond vector and the bilayer normal (lamellar phases) or the lipid long axis (hexagonal phases), then SCD is given by SCD =

1 ⟨3 cos2 β − 1⟩ 2

RESULTS Tannin Effect on Oral Membrane Mimics (MLV). The effect of the three tannins of our study (C, E, EGCG) on MLV was followed in a first series of experiments at a tannin/lipid molar ratio of 1/8. In order to explore different temperature ranges, experiments were carried out in 5 °C steps from 10 to 40 °C on each side of the DMPC gel−fluid phase transition temperature.42 Figure 1 shows selected 2H NMR spectra of DMPC-2H27 MLV alone (Figure 1A) and in the presence of catechin (Figure 1B), epicatechin (Figure 1C), or EGCG (Figure 1D). In the absence of tannins and below 20 °C, an axially asymmetric 2H NMR spectrum is detected, characteristic of Lβ′ gel phases. In contrast, at higher temperatures (25 °C and

(2)

where the brackets represent the averaging over all motions that are fast compared to the static quadrupolar splitting constant. Because MLV consist of a large number of lipids that laterally diffuse very slowly and where the molecular axes are randomly oriented with respect to the magnetic field, the 2H NMR spectrum is a powder spectrum resulting from the superposition of doublets with quadrupolar splitting representative of all possible orientations. When using perdeuterated lipid chains, as with DMPC-2H27, it may therefore be difficult to extract individual bond order parameters, SkCD (k = 2−14 for a C14 chain), due to insufficiently resolved powder spectra. SkCD may sometimes be determined using oriented-like spectra, for example choosing θ = 90°, calculated from powder spectra using the “de-Pake-ing” procedure.38,39 In cases where the resolution is good enough 2H NMR spectral simulations can alternatively be performed using an in-house FORTRAN program (Dufourc, unpublished) to extract the complete set of SkCD. This requires the input of individual quadrupolar splittings, ΔvkQ, individual line width, δvk1/2, and the number of deuterons per labeled position k. Liposome deformation as induced by the magnetic field can also be implemented. Initial values of ΔvkQ and δvk1/2 are obtained as far as possible from de-Paked spectra. All parameters are then optimized to simulate as better as possible the experimental 2 H NMR spectra. On some occurrences, the determination of all SkCD may be difficult due to a low S/N ratio or to a nonaxially symmetric experimental spectrum; a more global estimate of perdeuterated chain ordering, ⟨SCD⟩chain, may be then obtained using the first spectral moment, M1, according to the expression40,41 ⟨SCD⟩chain =

3 M1 πA Q

(3)

Because DMPC possesses a perdeuterated chain, angular brackets are used to represent the average over all labeled positions. The first moments were calculated by a homemade routine (Buchoux, unpublished material). To “translate” first moments into average chain ordering, the quantity 2⟨SCD⟩chain was also plotted on a double-y axis.41 The 2 factor accounts here for the fact that the average orientation of almost all C−D bonds is 90° with respect to the lipid long axis. This statement is not entirely true for the methyl terminal and for deuterons at the 2-position of the sn2 chain, where an additional geometrical factor must be used, but the approximation allows gross comparison of ordering for the entire chain. Using this definition allows to easily compare completely disordered systems (2⟨SCD⟩chain = 0) to fully rigid systems (2⟨SCD⟩chain = 1). Small-Angle X-ray Scattering. X-ray scattering experiments were performed on samples placed in glass capillaries using a NANOSTAR (Bruker, Billerica, MA), equipped with a Cu anode X-ray source operated at 40 kV and 35 mA. The three-pinhole collimation system

Figure 1. Tannin effect on DMPC MLV. 2H NMR spectra (scaled to each spectrum maximum intensity) of DMPC-2H27 MLV with or without tannin at T/L = 1/8 and at selected temperatures below and above 21.5 °C, the gel-to-fluid transition phase temperature of pure DMPC-2H27. (A) MLV, (B) C/MLV, (C) EC/MLV, (D) EGCG/ MLV. [L]MLV = 157 mM. Number of acquisitions: 3k−4k for EGCG/ MLV and 6k−8k in other cases. A Lorentzian line broadening of 200 Hz for EGCG/MLV and 100−150 Hz in other cases was applied prior to Fourier transformation. 5520


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lipid,43 whereas in the presence of tannins, Tm is 5 °C lower. At 10 °C however there is no detectable effect of tannins, within the experimental error. At the opposite, for high temperature, first moments are smaller in the presence of tannins and the smallest values are observed in the case of EGCG. Spectral dePake-ing and spectral simulations as described above were applied to high temperature spectra and quadrupolar splittings hence obtained (Table S1 in the Supporting Information). A minute description of chain ordering can therefore be achieved using eq 1 by plotting the quantity 2|SCD| as a function of labeled carbon position (Figure 2B). The order parameter profile of pure DMPC MLV is characterized by a plateau region (constant value of 0.44 up to position 8), followed by a second region where order parameters decrease rapidly toward the very low order of the methyl terminal in the middle of the bilayer. In the presence of tannins, the shape of the order parameter profile remains the same, but the values are smaller for all positions except for the methyl terminal. Plateau values are more affected than the values near the bilayer center. Again, it appears that EGCG is the tannin that induces the greater disordering effect. The progressive addition of catechin was also followed. Molar ratios of 1/20, 1/8, and 1/1.2 were prepared. The 1/20 sample behaved like the 1/8 sample as described above, i.e., lowering of the phase transition temperature and disordering of the fluid phase, but to a much lower extent. As a side observation, important for the following, an eye inspection of the MLV after addition of catechin at 1/20 and 1/8 molar ratios did not change the fluid milky aspect of the suspension, at room temperature (ca. 23 °C). However, for very high doses of tannins (T/L = 1/1.2), a precipitate occurs. A solid phase coexists with a clear solution. The precipitate was recovered after centrifugation of the sample at 5000 rpm and removal of the clear liquid phase. Analysis of the clear solution by 1H NMR shows that there are only a few tannin signals (not shown); the lipid signals are not detected, indicating that mostly all lipids are in the precipitate. Analysis of the latter was performed by solid-state 31P and 2H NMR at 37 °C. Spectra can be seen in Figure 3A; they resemble those of a classical fluid lamellar phase. The 31P NMR spectrum shows a typical powder pattern with an axially symmetric line shape characteristic of a Lα lamellar phase.44,45 The chemical shielding anisotropy, Δσ, is of ca. 33 ppm, i.e., much lower than the 45 ppm observed for pure MLV of DMPC (data not shown). The 2H NMR spectrum also displays an axially symmetric powder pattern with many resolved quadrupolar splittings. Of notice, the spectrum is much narrower that those already observed at lower tannin/lipid ratios (Figure 1). Order parameters can be obtained via spectral deconvolution and simulation as described above. An example of very good fitting between experimental and simulated spectra can be seen in Figure S2 of the Supporting Information. Order parameters along the acyl chain are hence determined and plotted in Figure 4, at 37 °C. It can be seen that the progressive addition of catechin leads to a progressive decrease of ordering for all labeled positions. The effect is more important for positions near the interface than at the bilayer core. Tannin Effects on Lipid Droplet Mimics (Isotropic Bicelles). Addition of low amounts of tannins (ratios of 1/20 and 1/8) on isotropic bicelles leaves the solution as clear as in their absence. Solution NMR can therefore be applied because the entire system remains isotropic. Changes in isotropic chemical shifts can then be observed (Figure S3A in the

above), the spectrum is much narrower and shows resolved individual splitting as in Lα fluid phases.29,40,41 Addition of polyphenols at a 1/8 tannin/lipid ratio does not lead to detectable changes on spectra at 15 °C and below. At 20 °C, significant changes in line shapes are observed in the presence of tannins whatever their nature: narrower axially symmetric spectra appear where several quadrupolar splittings can be measured. The presence of tannins thus decreases the gel-to-fluid phase transition temperature. It is noteworthy that the more the temperature increases, the narrower the spectra, i.e., the greater the increase in membrane disorder. The temperature dependence can be followed by calculating the first spectral moment, M1 (Figure 2). The temperature of the order−disorder (gel-to-fluid) phase transition of the lipids, Tm, can be read at the inflection point of the curve M1 = f(T) (Figure 2A). For pure DMPC MLV, Tm is found at ca. 22 ± 2 °C as expected for a partially deuterated

Figure 2. Tannin effect on membrane thermotropism and ordering. (A) Thermal variations of the first moment, M1, from 2H NMR spectra of Figure 1 at T/L = 1/8. On double y-axis is also plotted twice the chain order parameter. (B) 2|SCD| order parameter of DMPC-2H27 acyl chains as a function of the labeled carbon position. (▲) MLV, (■) C/ MLV, (◆) EC/MLV, (●) EGCG/MLV. [L]MLV = 157 mM, T = 30 °C. 5521


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Figure 4. Small-angle X-ray scattering (SAXS) spectrum obtained for the precipitate of C/bicelles (T/L = 1/1.2). The curve (intensity vs the scattering vector q) is obtained by circular integration along the diffraction angle on the recording image plate detector. [L]bicelles = 60 mM, T = 20 °C.

catechin/bicelles system by 31P solid-state NMR (Figure S4 in the Supporting Information). Indeed, at a low dose (T/L = 1/ 8), a sharp and isotropic line is detected. At a higher dose (T/L = 1/1.2), a 31P NMR powder pattern spectrum appears below an isotropic line. It can straightforwardly be assigned to the precipitate. Using an external reference of known tannin concentration, the amount of tannin in the precipitate was respectively found to be 70 and 60% of the total catechin or EGCG added initially. Experiments to study this solid phase were realized above the precipitation threshold, at a tannin/ lipid molar ratio of 1/1.2 and 1/0.7 for catechin and EGCG, respectively. The precipitate was recovered after centrifugation of the sample at 5000 rpm to remove the liquid phase and then analyzed by 31P and 2H solid-state NMR (Figure 3). Precipitates were also observed when applying catechin at similar tannin/lipid molar ratio on MLV and added to Figure 3 for comparison. Pure isotropic bicelles spectra, as controls in the absence of tannins, are also added to Figure 3. In the presence of catechin (Figure 3B), the precipitate obtained when starting from an initial state of isotropic bicelles is characterized by a 31P NMR spectrum representing a powder pattern twice as small and of reversed chemical shielding anisotropy (Δσ of ca. −16 ppm) compared to that measured for the catechin/MLV precipitate (Figure 3A). This is a strong indication of the presence of a hexagonal phase (vide inf ra). The 2H NMR spectrum in Figure 3B obtained with DMPC-2H27 shows a powder pattern with a narrow triangular shape compared to the much larger and well-resolved spectrum obtained in Figure 3A for the lamellar phase precipitate. This triangular shape has been already assigned by Thurmond and co-workers to a HII inverted hexagonal phase.46,47 Using labeled DCPC (DCPC-2H22) on the same system (Figure S5 in the Supporting Information) demonstrates that both DMPC and DCPC are present in the precipitate. The phase structure of the precipitate was confirmed by the SAXS spectrum reported in Figure 4. Indeed, the observed Bragg peaks located successively at scattering vectors q0, √3q0, and √7q0 are characteristic of a

Figure 3. 31P and 2H NMR spectra of tannin/lipid system precipitates (scaled to individual spectrum maximum intensity). (A) Precipitate of C/MLV (T/L = 1/1.2). (B) Precipitate of C/bicelles (T/L = 1/1.2). (C) Precipitate of EGCG/bicelles (T/L = 1/0.7). (D) Pure isotropic bicelles as control. [L]bicelles = [L]MLV = 60 mM, T = 37 °C. Precipitates are obtained after centrifugation at 5000 rpm for 5 min and removal of the liquid supernatant. For 31P NMR spectra, the number of acquisitions was 128 for bicelles alone and 2k in other cases. A Lorentzian line broadening of 20 Hz for bicelles and 50 Hz in other cases was applied prior to Fourier transform. For 2H NMR spectra, the number of acquisitions was 12k for bicelles and the C/ MLV precipitate and 26k−67k for both tannin/bicelles precipitates. A Lorentzian line broadening of 50 Hz was applied prior to Fourier transform. The spectrum center was arbitrarily set to 0 Hz.

Supporting Information) and used to titrate the effect of tannin progressive addition to lipids (association constants, stoichiometry, etc.). This will be reported elsewhere (Furlan, Dufourc, Géan, in preparation). Increasing the tannin content above a catechin/lipid ratio of 1/1.6 and a EGCG/lipid ratio of 1/1 leads to the observation of a phase separation: a precipitate coexists with a clear solution. Increasing further the tannin content leads to a decrease of the spectrum intensity that can be recorded using solution 1H NMR (Figure S3B in the Supporting Information): the solid precipitate gives rise to a solid-state NMR feature that cannot be recorded using solution-NMR techniques. No precipitate could be observed with epicatechin due to the low solubility of this tannin, thus preventing exploring high tannin/lipid molar ratios. The above visual observations could be backed up in the case of the 5522


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hexagonal order (the 2q0 position included in the form factor is not visible on the spectrum).48 In the presence of EGCG, the isotropic bicelle system leads also to a precipitate. The corresponding 31P and 2H NMR spectra are shown in Figure 3C. Both spectral shapes and widths are reminiscent of those of Figure 3B, i.e., possibly depicting the presence of a HII phase. However, the line shapes are much broader, and the deuterium spectrum suggests an isotropic line (as for the pure isotropic bicelles, Figure 3D) exchanging with a broader power pattern. NMR spectra in Figure 3A,B (deuterium part) are sufficiently resolved to allow de-Pake-ing and spectral simulations (Figure S2 and Table S2 in the Supporting Information). Figure 5 reports the order

Figure 6. 31P NMR spectra of DPoPE (absolute intensity) with or without catechin below and above 33 °C, the DPoPE lamellar-tohexagonal phase transition temperature. (A) Pure DPoPE, (B) C/ DPoPE (T/L = 1/33) ratio, (C) C/DPoPE (T/L = 1/3.3). [DPoPE] = 196 mM. Number of acquisitions: 1k. A Lorentzian line broadening of 80 Hz was applied prior to Fourier transformation. Figure 5. SCD order parameters of DMPC-2H27 lipid systems as a function of the labeled carbon position. SCD values are obtained from de-Pake-ing and simulation of 2H NMR powder spectra (see text). (□) MLV, (■) C/MLV (T/L = 1/20), (◆) C/MLV (T/L = 1/8), (▼) precipitate of C/MLV (T/L = 1/1.2), (●) precipitate of C/ bicelles (T/L = 1/1.2) after centrifugation and removal of liquid supernatant. [L]bicelles = 60 mM, [L]MLV = 157 mM, T = 37 °C.

a hexagonal phase appears superimposed on that of a lamellar one. At 36 °C and above, a pure hexagonal phase is detected through the reduction of Δσ by half and the inversion of the chemical shielding anisotropy. In the presence of low doses of catechin (T/L = 1/33, Figure 6B), the spectrum characteristic of the hexagonal phase only appears at 42 °C, whereas at higher doses (T/L = 1/3.3, Figure 6C), the spectrum of the lamellar phase is stabilized over the whole temperature range investigated. Thus, catechin delays the appearance of the DPoPE HII phase in a concentration-dependent manner and rather stabilizes the lamellar phase.

parameters profile for each labeled carbon position on the acyl chain obtained from 2H NMR spectra of Figure 3. Order parameter values are given in Table S3 of the Supporting Information. For the catechin/bicelles precipitate, where the hexagonal phase is detected, the order profile is very different than those obtained with MLV: all SCD values are much lower and decrease quasi-linearly vs labeled carbon position. Tannin Effects on a Lipid Hexagonal Phase. As it has been reported above, catechin is capable of inducing a hexagonal phase when applied at elevated doses onto isotropic bicelles. It is therefore interesting to investigate whether tannins may alter the morphology of hexagonal lipid phases and alter for instance the lamellar-to-hexagonal phase transition of a PE lipid (DPoPE) that is prone at generating hexagonal phases. Figure 6 represents 31P NMR spectra of DPoPE with or without catechin at 1/33 and 1/3.3 tannin/lipid ratio below and above the DPoPE transition phase temperature (ca. 33 °C). For the pure system (Figure 6A), at 24 °C, 31P NMR spectra indicate a lamellar phase. At 32 °C, a spectrum characteristic of

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DISCUSSION The effects of wine tannins on crude models for membranes of cells covering the oral cavity and lipid droplets contained in fatty food provide two important results in the context of wine tasting. (i) Tannins promote a great disorder of the hydrophobic interior of lipid vesicles and stabilize the lamellar topology, and (ii) lipid droplets as modeled by isotropic lipid bicelles are destabilized and a new lipid−tannin topology is evidenced, the inverted hexagonal phase. These two findings will tentatively be discussed in the context of wine tasting. Tannins Disorder Micrometer Size Liposomes and Stabilize Planar Membrane Geometry. Catechin, epicatechin, and epigallocatechin gallate clearly disorder the 5523


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bicelles. Isotropic bicelles are in fact highly curved oblong micelles of ca. 10−20 nm size obtained by mixing in excess water DMPC (C14 chains) and DCPC (C6 chains) (molar ratio of 1:2). As described above for large liposomes, the effect of tannins can be also considered as chaotropic on isotropic bicelles: by sitting near the interface but in the hydrophobic region, it contributes increasing the hydrophobic volume, which, according to Israelashvili’s theory, reduces the highly positive curvature of bicelles edges and promotes the bicelle-tohexagonal transition.54 However, the impact of tannins is higher on bicelles that are highly curved and much disordered structures43 than on liposomes where line tension is smaller. Thus, with MLV, tannins do not change the lamellar topology whereas with bicelles, the system goes to a more stable morphology, the hexagonal phase, where line tension is reduced. It is interesting here to comment on the detergent effect that has been reported by Sun and co-workers.55 Production of micelles can be readily seen by solid-state NMR: powder patterns representative of large liposomal membranes are converted into sharp isotropic lines due to the fast tumbling of nanometer-sized objects.56−58 We never detected such sharp lines, strongly indicating that the tannins of our study are not acting as detergents on phosphatidylcholine membranes. Hypothetical Implications for Wine Tasting. Wine perception is a very complex phenomenon that is still poorly understood. Progresses have been nonetheless accomplished recently concerning the dryness effect called astringency by oenologists.59−61 Different mechanisms involving the formation of colloidal complexes between tannins and proline-rich saliva proteins have been evidenced. For tannin concentrations above the critical micelle concentration, CMC, precipitates may be observed that completely cluster proteins that are otherwise needed for palate lubrication, hence, the transient feeling for dryness.6 Findings in this work clearly evidence a strong interaction between tannins and lipids. Although our model for lipid droplets in food is very crude, it nonetheless points out that tannins can precipitate lipids under colloidal morphology to form hexagonal phases. This indicates that eating during drinking may diminish the availability of tannins for saliva proteins and hence modulate the astringency feeling. Accurate determination of tannin−lipid binding constants is currently under way in the laboratory and will be compared in another report to available binding constants between tannins and proline-rich proteins.5 Another interesting finding is the disordering effect produced by tannins on liposomes of phosphatidylcholine lipids mimicking the oral cell membranes. Although our model is very simplified, it points out a dosedependent fluidifying effect of tannins: the more galloylated, the stronger the effect. This first result will be strengthened by using model membranes more representative of the lipid composition of oral cell membranes in future studies. Nevertheless, it suggests that the interaction between tannins and lipids in particular those surrounding the taste receptors present throughout the mouth could disturb taste receptors. However, to validate this assumption, binding constants between tannins and receptors must be determined and compared to those between tannins and lipids, which are currently under determination in the laboratory.

hydrophobic core of DMPC liposomes (Figure 2). The effect is more pronounced from the bilayer interface (the glycerol backbone) to the middle of fatty acyl chains and is dosedependent (Figure 5). The bilayer center, which is already very mobile, is little affected in terms of further disordering. Interestingly, the galloylated tannin (EGCG) induces a greater effect compared to the other two non-galloylated tannins: catechin and epicatechin. This may be related to a greater affinity for membranes because of its greater hydrophobic character as it has been previously proposed.10,13 Indeed, EGCG is the most nonpolar tannin (Kow = 12.1), and it is expected to interact more with the nonpolar acyl chain region and affect the membrane fluidity further than the more hydrophilic catechin and epicatechin (Kow = 2.4).12 This result is in agreement with results obtained by EPR and NMR spectroscopic techniques,18,19 and questions opposite results found by fluorescence measurements.9,13,17 In the latter one may suspect specific interactions between tannins and the externally added fluorescent molecules to report on ordering. Another interesting effect is the induction of a 5 °C decrease in the gel-to-fluid phase transition, Tm, of DMPC multilamellar vesicles. The stabilization of the lamellar fluid phase occurs independently of the tannin structure and again suggests that tannins are well inserted in the hydrophobic part of the bilayer to prevent their close chain packing when the temperature is decreased below Tm. Tannins thus influence the thermal behavior of the zwitterionic DMPC in a manner similar to chaotropics (water-structure breakers) such as I−, SCN−, ClO−, EtOH, etc.49,50 This result can be linked to the effect of tannins on lipids that undergo lamellar-to-hexagonal phase transitions, such as phosphatidylethanolamines. Indeed, we have shown that catechin clearly expands the temperature range of the fluid lamellar phase at the expense of the hexagonal phase. This supports the chaotropic character of polyphenols investigated in this study. According to Tenchov and co-workers, a chaotropic substance stabilizes the lamellar fluid phase by increasing its concentration at interfaces in comparison to its concentration in bulk watera property that we already evidenced from inspection of the order parameter profile as a function of labeled carbon position along the lipid fatty acyl chain (vide supra). Reported chaotropic substances are the wellknown magnesium chloride, guanidine hydrochloride, urea, short chain alcohols (butanol, propanol, ethanol), etc. Interestingly, phenol is reported as a strongly chaotropic compound.52 It is therefore not surprising that polyphenols promote similar effects. Tannins Destabilize Phospholipid Nanometer-Sized Bicelles To Generate Hexagonal Phases. In order to mimic tannin effect on lipid droplets present in fatty foods, we used, as a first and crude model, isotropic lipid bicelles that similarly have a pure hydrophobic interior. It is very clear from experiments that the small nanometer bicelles are completely destabilized by high amounts of C and EGCG and that a new phase precipitates out from the bicellar colloid suspension, the hexagonal phase. The 1/1.2 tannin/lipid molar ratio, corresponding to around 15−20 g/L in tannins depending on samples, may be considered high compared to average total polyphenol concentrations in red wines that can raise 6 g/ L.27,53 This concentration does not necessarily reflect classical concentrations, although such high concentrations may locally be reached during wine ingestion. Nevertheless, from a physicochemical point of view, looking at the effect of high doses highlights the potential destabilizing effect of tannins on 5524


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(6) Cala, O.; Dufourc, E. J.; Fouquet, E.; Manigand, C.; Laguerre, M.; Pianet, I. The colloidal state of tannins impacts the nature of their interaction with proteins: The case of salivary proline-rich protein/ procyanidins binding. Langmuir 2012, 28, 17410−17418. (7) Kitano, K.; Nam, K.-Y.; Kimura, S.; Fujiki, H.; Imanishi, Y. Sealing effects of (−)-epigallocatechin gallate on protein kinase C and protein phosphatase 2A. Biophys. Chem. 1997, 65, 157−164. (8) Hashimoto, T.; Kumazawa, S.; Nanjo, F.; Hara, Y.; Nakayama, T. Interaction of tea catechins with lipid bilayers investigated with liposome systems. Biosci. Biotechnol. Biochem. 1999, 63, 2252−2255. (9) Tsuchiya, H. Effects of green tea catechins on membrane fluidity. Pharmacology 1999, 59, 34−44. (10) Kajiya, K.; Kumazawa, S.; Nakayama, T. Steric effects on interaction of tea catechins with lipid bilayers. Biosci. Biotechnol. Biochem. 2001, 65, 2638−2643. (11) Kajiya, K.; Kumazawa, S.; Nakayama, T. Effects of external factors on the interaction of tea catechins with lipid bilayers. Biosci. Biotechnol. Biochem. 2002, 66, 2330−2335. (12) Hagerman, A. E.; Rice, M. E.; Ritchard, N. T. Mechanisms of protein precipitation for two tannins, pentagalloyl glucose and epicatechin16 (4→8) catechin (procyanidin). J. Agric. Food Chem. 1998, 46, 2590−2595. (13) Caturla, N.; Vera-Samper, E.; Villalain, J.; Mateo, C. R.; Micol, V. The relationship between the antioxidant and the antibacterial properties of galloylated catechins and the structure of phospholipid model membranes. Free Radical Biol. Med. 2003, 34, 648−662. (14) Kajiya, K.; Kumazawa, S.; Naito, A.; Nakayama, T. Solid-state NMR analysis of the orientation and dynamics of epigallocatechin gallate, a green tea polyphenol, incorporated into lipid bilayers. Magn. Reson. Chem. 2008, 46, 174−177. (15) Scheidt, H. A.; Pampel, A.; Nissler, L.; Gebhardt, R.; Huster, D. Investigation of the membrane localization and distribution of flavonoids by high-resolution magic angle spinning NMR spectroscopy. Biochim. Biophys. Acta 2004, 1663, 97−107. (16) Sirk, T. W.; Brown, E. F.; Friedman, M.; Sum, A. K. Molecular binding of catechins to biomembranes: Relationship to biological activity. J. Agric. Food Chem. 2009, 57, 6720−6728. (17) Tsuchiya, H. Stereospecificity in membrane effects of catechins. Chem. Biol. Interact. 2001, 134, 41−54. (18) Yu, X.; Chu, S.; Hagerman, A. E.; Lorigan, G. A. Probing the interaction of polyphenols with lipid bilayers by solid-state NMR spectroscopy. J. Agric. Food Chem. 2011, 59, 6783−6789. (19) Ulrih, N. P.; Ota, A.; Sentjurc, M.; Kure, S.; Abram, V. Flavonoids and cell membrane fluidity. Food Chem. 2010, 121, 78−84. (20) Uekusa, Y.; Kamihira, M.; Nakayama, T. Dynamic behavior of tea catechins interacting with lipid membranes as determined by NMR spectroscopy. J. Agric. Food Chem. 2007, 55, 9986−9992. (21) Squier, C. A.; Cox, P.; Wertz, P. W. Lipid content and water permeability of skin and oral mucosa. J. Invest. Dermatol. 1991, 96, 123−126. (22) Law, S.; Wertz, P. W.; Swartzendruber, D. C.; Squier, C. A. Regional variation in content, composition and organization of porcine epithelial barrier lipids revealed by thin-layer chromatography and transmission electron microscopy. Arch. Oral Biol. 1995, 40, 1085− 1091. (23) Thompson, I. O. C.; van der Bijl, P.; van Wyk, C. W.; van Eyk, A. D. A comparative light-microscopic, electron-microscopic and chemical study of human vaginal and buccal epithelium. Arch. Oral Biol. 2001, 46, 1091−1098. (24) Diaz-del Consuelo, I.; Jacques, Y.; Pizzolato, G.-P.; Guy, R. H.; Falson, F. Comparison of the lipid composition of porcine buccal and esophageal permeability barriers. Arch. Oral Biol. 2005, 50, 981−987. (25) Dalgleish, D. G. Food emulsionstheir structures and structure-forming properties. Food Hydrocolloids 2006, 20, 415−422. (26) Andersson, A.; Mäler, L. Size and shape of fast-tumbling bicelles as determined by translational diffusion. Langmuir 2006, 22, 2447− 2449.

ASSOCIATED CONTENT

S Supporting Information *

Additional tables and figures as described in the text. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected] (J.G.). Present Address

A.C.: Université de Pau et des pays de l’Adour - CNRS, IPREM/ECP, UMR 5254, Hélioparc -2 Avenue du Président Pierre Angot, 64053 Pau Cedex 9, France. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge financial support of the Conseil Interprofessionnel du Vin de Bordeaux (CIVB, 1, Cours du XXX juillet, F-33075 Bordeaux Cedex). The research was carried out with the IECB NMR platform equipment purchased thanks to the financial support of the Conseil Régional d’Aquitaine and the TGE RMN THC FR 3050. We particularly acknowledge Axelle Grélard (CBMN) for her help on NMR spectrometers.

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ABBREVIATIONS C, (+)-catechin; CMC, critical micelle concentration; Chol, cholesterol; DCPC, 1,2-dicaproyl-sn-glycero-3-phosphocholine; DCPC-2H22, 1,2-dicaproyl(2H22)-sn-glycero-3-phosphocholine; DMPC, 1,2-dimyristoyl-sn-glycero-3-phosphocholine; DMPC-2H27, 1-myristoyl(2H27)-2-myristoyl-sn-glycero-3-phosphocholine; DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; DPoPE, 1,2-dipalmitoleoyl-sn-glycero-3-phosphoethanolamine; DPPC, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; EC, (−)-epicatechin; ECG, (−)-epicatechin gallate; EGC, (−)-epigallocatechin; EGCG, (−)-epigallocatechin gallate; Kow, octanol−water partition coefficient; LUV, large unilamellar vesicles; MLV, multilamellar vesicles; NMR, nuclear magnetic resonance; PC, phosphocholine; PG, phosphoglycerol; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; POPE, 1palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine; POPS, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (sodium salt); T/L, tannin/lipid molar ratio; TMSP, sodium 3trimethylsilyl-2,2,3,3-2H4-propionate; SUV, small unilamellar vesicles.

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REFERENCES

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NOTE ADDED AFTER ASAP PUBLICATION This article was published ASAP on May 2, 2014. The caption to Figure 5 has been modified. The correct version was published on May 5, 2014.

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Red Wine Tannins Fluidify and Precipitate Lipid Liposomes and

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