Interactions between Pectic Compounds and Procyanidins are

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Interactions between Pectic Compounds and Procyanidins are Influenced by Methylation Degree and Chain Length Aude A. Watrelot,*,†,‡ Carine Le Bourvellec,†,‡ Anne Imberty,§ and Catherine M. G. C. Renard†,‡ †

INRA, UMR408 Sécurité et Qualité des Produits d’Origine Végétale, F-84000 Avignon, France Université d’Avignon et des Pays de Vaucluse, UMR408 Sécurité et Qualité des Produits d’Origine Végétale, F-84000 Avignon, France § Centre de Recherches sur les Macromolécules Végétales, CERMAV-CNRS (affiliated with Université de Grenoble), B.P. 53, F-38041 Grenoble, France ‡

S Supporting Information *

ABSTRACT: The interactions between procyanidins and pectic compounds are of importance in food chemistry. Procyanidins with low (9) and high (30) average degrees of polymerization (DP9 and DP30) were extracted from two cider apple varieties. Commercial apple and citrus pectins, as well as three pectin subfractions (homogalacturonans, partially methylated homogalacturonans with degree of methylation 30 and 70) at 30 mM galacturonic acid equivalent, were titrated with the two procyanidin fractions (at 30 mM (−)-epicatechin equivalent) by isothermal titration calorimetry and UV−vis spectrophotometry. Slightly stronger affinities were recorded between commercial apple or citrus pectins and procyanidins of DP30 (Ka = 1460 and 1225 M−1 respectively, expressed per monomer units) compared to procyanidins of DP9 (Ka = 1240 and 1085 M−1, respectively), but stoichiometry and absorbance maxima differed between apple and citrus pectins. It was proposed that methylated homogalacturonans interacted with procyanidins DP30 mainly through hydrophobic interactions. The stronger association was obtained with the longer procyanidin molecules interacting with highly methylated pectins.

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INTRODUCTION Polyphenols are natural constituents from plants, some of which are characterized by their high propensity to bind to macromolecules. Polyphenols interact with proteins, hence, their name of “tannins”, but also with polysaccharides. For example, proanthocyanidins (also known as “condensed tannins”) have been demonstrated to bind strongly to pectin, a major constituent of most plant cell walls.1−3 The affinity between tannins and proteins is involved in both tanning effect and haze formation and is responsible for perception of bitterness and astringency of beverages such as apple juice, wine, beer, or cider.4 Interaction between tannins and proteins may be involved in the beneficial effects of polyphenols for human health, and they have therefore been extensively characterized.5 Recently, isothermal titration calorimetry (ITC), a powerful method that provides direct thermodynamic information in solution, has been used to determine the influence of both polyphenol and protein structures on their interaction.6−11 Much less information is available on the interactions between polyphenols and polysaccharides, although plant polyphenols are biosynthesized in a polysaccharide-rich environment. Extensive contacts between plant polysaccharides and polyphenols occur during destruction of plant tissues, either by food processing, chewing, or digestion. Most data available on the interactions between polyphenols and polysaccharides have been obtained using biphasic systems with polysaccharides under the semisolid form. The character© 2013 American Chemical Society

istics of the interaction between polyphenols and dextran gels are similar to those observed for polyphenols interacting with bovine serum albumin.12 Data obtained with suspension of apple cell walls in aqueous buffer demonstrated their noncovalent binding to oligomeric and polymeric procyanidins.1,13 This binding is fast and spontaneous and involves weak interactions such as hydrogen bonds and hydrophobic contacts. Among cell wall polysaccharides, pectin displays the strongest affinity to procyanidins, whatever their average degree of polymerization (DPn).1,2 The use of dynamic light scattering (DLS) demonstrated the aggregation of polyphenols in the presence of a fraction of pectin containing a high amount of rhamnogalacturonan II.14 When titration calorimetry is used,15 it has been shown that affinity between procyanidins of DP9 and pectins is at least 2 orders of magnitude lower than tannin−polyproline interactions but 1 order of magnitude higher than tannin−BSA interactions.9,11,16 However, pectin is a very heterologous polymer with different structural regions, and the influence of the fine structure of the polysaccharide on the interaction with procyanidins has not been determined. Pectins are constituted of homogalacturonans (also known as “smooth region”) and rhamnogalacturonans: RGI often called “hairy region” and RG II.17−20 Homogalacturonans are the main constituent of pectins (about 60% of pectins in the plant Received: November 20, 2012 Revised: January 16, 2013 Published: January 17, 2013 709

dx.doi.org/10.1021/bm301796y | Biomacromolecules 2013, 14, 709−718

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a Whatmann 4−7 GF/D filter. They were injected on a 20 × 5 cm column of Lichrospher 100 RP-18 (12 μm; Merck, Darmstadt, Germany) and purified as described in Renard et al., 2001. The purified procyanidin fractions are designated as DP9 (from “Marie Ménard”) and DP30 (from “Avrolles”). Homogalacturonans Preparation. Homogalacturonans were prepared as described by Renard and Jarvis29 using 15 g/L of apple pectins saponified at 4 °C, pH > 12, and left overnight. The solution was then brought to pH 6 and precipitated with three volumes of ethanol/water/hydrochloric acid mixture (96:3:1, v/v/v) under stirring at 4 °C and left overnight. This suspension was filtered on a G0 sintered glass filter. After filtration, the pectinic acid precipitate was washed extensively with an ethanol/water mixture (70:30, v/v) until any remnant of chloride ions was eliminated (as verified by silver nitrate precipitation). The resulting powder was dried by solvent exchange (ethanol 96% and acetone), followed by drying at 40 °C in an oven overnight. The pectinic acid was dissolved (6 g/L) and hydrolyzed by hydrochloric acid (0.25 N) during 72 h at 80 °C.21 Homogalacturonans precipitate were separated by centrifugation (8400 g during 35 min) and then freeze-dried and named HG. Methylation of homogalacturonans was carried out as described previously.29 An aqueous solution of homogalacturonans (20 g/L) was neutralized by tetrabutyl ammonium hydroxide (TBA−OH) 10% up to pH 6.5. After freeze-drying, the homogalacturonan−TBA salts were dissolved in DMSO (10 mL for 100 mg of galacturonic acid) overnight under stirring. Volumes of iodo-methane (CH3I) calculated to obtain molar ratio CH3I/galacturonic acid of 0.6 and 10 were added. The mixture was left to react overnight under stirring. The reaction mixture was dialyzed for 5 days against water until any remnant of DMSO was eliminated, then dialyzed against hydrochloric acid (0.1 M) and sodium chloride (0.2 M) in order to eliminate TBA, and then against water. The dialysis retentate was freeze-dried. Two homogalacturonan fractions characterized by their degree of methylation were obtained: a homogalacturonan fraction with a degree of methylation (DM) of 30% named HG 30% and a homogalacturonan fraction with a DM of 70% named HG 70%.29 Phase Diagram. A spectrophotometric method was used to identify aggregates formation during pectin−procyanidin interactions. Systematic variation of concentrations and relative amounts was carried out on a 96-wells “microplate” at 25 °C. Concentrations of procyanidins were varied along the lines (0, 0.03, 0.06, 0.117, 0.23, 0.47, 0.94, 1.875, 3.75, 7.5, 15, and 30 mM (−)-epicatechin equivalent) and concentrations of pectic compounds along the columns (0, 0.03, 0.06, 0.117, 0.47, 1.875, 7.5, and 30 mM galacturonic acid equivalent). Solutions were prepared in citrate/phosphate buffer at pH 3.8 and ionic strength 0.1 mol/L. Equal amounts (50 μL) of procyanidins and pectic subfractions solutions were mixed before each spectra measurement during 20 s. Absorbance spectra (200− 650 nm) were recorded and absorbance at 650 nm were analyzed for each solution using a SAFAS flx-Xenius XM spectrofluorimeter (SAFAS, Monaco). Control spectra were obtained using wells containing only pectic compounds or procyanidins in buffer. After spectra recording, microplates were centrifuged 10 min at 2100 g. The speed was defined by the limit of microplate centrifugation and the time by the pellet obtained. After this centrifugation step, clear supernatants could be obtained with the pellet separated. Supernatants of control

cell wall). They consist of a long chain of 1→ 4 linked galacturonic acids (α-D-GalpA) that can be methyl esterified and/or acetylated.21,22 The level of methylation influences strongly the physicochemical properties of pectins. Because it affects the hydrophobicity of the polymer, the degree of methylation of pectins can modulate their interaction with other biomolecules. Proanthocyanidins (also known as condensed tannins) are polyphenols, derived from the oligomerization of flavan-3-ol units. Procyanidins are a subclass of proanthocyanidins characterized by their constitutive units, their interflavanic linkages, and their degree of polymerization.23,4 Specifically, apple procyanidins are characterized by the predominance of (−)-epicatechin and of C4−C8 interflavanic linkages.24−26 In cider apples, procyanidins have a large distribution of size with DPn varying from 4.5 to 50 for varieties Jeanne Renard and Avrolles, respectively.25 The aim of this study was to quantify the binding of a specific class of proanthocyanidins, the procyanidins, to pectins and homogalacturonans with different degrees of methylation (DM). Turbidity and UV−visible spectroscopy were used to detect aggregation. We present the use of isothermal titration calorimetry to probe the thermodynamic nature of interactions between pectins and homogalacturonans with procyanidins of intermediate (DP9) and high (DP30) number average degree of polymerization.

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MATERIAL AND METHODS Chemicals. Methanol, acetonitrile, and acetone of chromatographic quality were provided by Biosolve (Distribio, Evry, France). Hexane (Merck, Darmstadt, Germany) was of analytical quality. Ethanol and acetone were provided by Fisher Scientific (Strasbourg, France). Silver nitrate was provided by Merck (Darmstadt, Germany). Sodium borohydride (NaBH4), N-methyl imidazole, acetic anhydride, dimethylsulfoxide (DMSO), tetrabutylammonium hydroxide (10% solution), apple and citrus pectins (degrees of methylation ∼75%), toluene-α-thiol, and iodomethane were provided by SigmaAldrich (Deisenhofen, Germany). Chlorogenic acid, (+)-catechin, and (−)-epicatechin were obtained from Sigma-Aldrich. 4-Coumaric acid was obtained from Extrasynthese (Lyon, France). Phloridzin was obtained from Fluka (Buchs, Switzerland). Sugar standards were from Fluka (Buchs, Switzerland). Methanol-d3 was from Acros Organics (Geel, Belgium). Plant Material. Apple fruits (Malus domestica Borkh.) of the Avrolles and Marie Ménard varieties were harvested at commercial maturity during the 2000 season in the experimental orchard of the Centre Technique des Productions Cidricoles (CTPC, Sées, France). Fruits were mechanically peeled and cored as previously described by Guyot et al.,27 and cortex tissues were freeze-dried and stored at −20 °C until extraction. Procyanidins Extraction and Purification. Hexane, methanol, and aqueous acetone extracts of apple polyphenols were obtained by successive solvent extractions of “Marie Ménard” or “Avrolles” freeze-dried pulps.28 Hexane and methanol extracts were discarded as they did not contain the required procyanidin fraction. Aqueous acetone extracts containing procyanidins were pooled and concentrated on a rotary evaporator prior to freeze-drying. The freeze-dried aqueous acetone extracts were dissolved in acidified water (water/acetic acid 97.5:2.5, v/v), centrifuged (16800 g, 15 min), and filtered successively on a 10 μm porosity filter (Millipore) and then on 710


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in Le Bourvellec et al.31 conditions. The DPn of procyanidins was measured by calculating the molar ratio of all the flavan-3ol units (thioether adducts plus terminal units) to (−)-epicatechin and (+)-catechin corresponding to terminal units. Neutral sugars, galacturonic acid, and methanol were analyzed as described previously.32 The molecular weight distribution of polysaccharides was determined using a high pressure size exclusion chromatography (HPSEC) system involving a Jasco LC-NET II/ADC interface, a Jasco PU-2080 plus intelligent HPLC pump, a Jasco RI-2031 plus intelligent RI detector, a degasser, and controlled by the ChromNav Software (Jasco, Tokyo, Japan). Separations were achieved using two columns in series: a (8.0 mm ID × 300 mm) OH-pack SB-802 HQ column (Showa Denko Europe, Munich, Germany) with a (300 × 7.8 mm ID) TSK- Gel PWXL column (Tosohaas, Stuttgart, Germany) at 35 °C and a guard column (40 × 6.0 mm id) TSK-Gel PWXL (Tosohaas, Stuttgart, Germany). Solutions (20 μL) of the extracts (10 g/L) were injected and eluted with 0.4 M sodium acetate buffer, pH 3.5, at 0.8 mL/min. Dextrans T500, T70, T40, and T10 (Pharmacia BioProcess Technology, Uppsala, Sweden) and glucose (Sigma-Aldrich, Deisenhofen, Germany) were used to calibrate the column system. To allow comparison between the different pectic substrates and given the polydispersity in molecular weight for both pectic fractions and procyanidins, it was chosen to express all concentrations relative to the main monomers, that is, galacturonic acid for pectins and (−)-epicatechin for procyanidins. Statistical Analysis. Results are presented as mean values, and the reproducibility of the results is expressed as pooled standard deviation. Pooled standard deviations were calculated for each series of replicates using the sum of individual variances balanced by the sum of individual degrees of freedom.33 Multifactor ANOVA analysis was realized on phase diagram results by STATGRAPHICS Centurion XVI software.

wells (pectic compounds at 30 mM in buffer (named S1A) and procyanidins at 30 mM in buffer (named S1B)) and supernatants of wells containing procyanidins at a concentration of 30 mM with pectic compounds at a concentration of 30 mM (named S2) were analyzed by high pressure size exclusion chromatography (HPSEC) and high-performance liquid chromatography-diode array detection (HPLC-DAD) to define qualitative changes in partition coefficient (ΔKav = S2− S1A) of pectic fractions and in number average degree of polymerization of flavan-3-ols (DPn = S2−S1B). Isothermal Titration Calorimetry. To measure enthalpy changes associated with pectin−tannin interactions at 25 °C, a VP-ITC microcalorimeter (Microcal, GE Healthcare, Chalfont St. Giles, United Kingdom) was used. Procyanidins and pectin fractions were dissolved in the same citrate/phosphate buffer pH 3.8, ionic strength 0.1 mol/L, and filtered on 0.45 μm membrane. All solutions were degassed prior to measurements. The reference cell was filled by water. To obtain a hyperbole curve, as recommended for low affinity systems,30 different concentrations of compounds were tested. The pectin solution was placed in the 1.448 mL sample cell of the calorimeter and the procyanidin solution was loaded into the injection syringe and titrated into the sample cell by 30 injections of 10 μL aliquots. The duration of each injection was 20 s, with separating delay of 5 min. The contents of the sample cell were stirred throughout the experiment at 307 rpm to ensure mixing. Raw data obtained as a plot of heat flow (microcalories per second) against time (minutes) are then integrated peak-bypeak and normalized to obtain a plot of observed enthalpy change per mole of injectant (ΔH, kcal·mol−1) against the molar ratio (epicatechin/galacturonic acid). Peak integration is performed using Microcal Origin 7.0 (Microcal Software, GE Healthcare). Control experiments include the titration of procyanidin fractions into buffer and are subtracted from titration experiments. The experimental data are fitted to a theoretical titration curve using Microcal Origin 7.0, with ΔH (enthalpy change), Ka (association constant), and n (number of binding sites per molecule) as adjustable parameters, from the relationship Qi =

⎡ nPtΔHV0 ⎢ At 1 ⎢1 + nP + nK P − 2 t a t ⎣

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RESULTS Composition of the Interacting Species. Pectins and Their Subfractions. The composition of the different pectic materials and their partition coefficients are shown in Table S1. Commercial pectins from apple and from citrus peel were selected for simplicity of use; their main constituent was galacturonic acid (74.6% (w/w) of the monosaccharides), while main neutral sugars were galactose (11.6%) and glucose (8.3%) in apple pectins. However, in citrus pectins, glucose content (5.3%) was lower and rhamnose (3.2%) was higher. The degree of methyl-esterification was 73% (apple pectins) and 79% (citrus pectins) corresponding to high-methoxyl pectins. The composition was in agreement with known data on commercial apple pectin34,21 but different of known data on commercial citrus pectin35,36 in terms of rhamnose and galactose contents. Rhamnose content was twice that in Dronnet et al.36 and galactose content was three and half times than in Axelos et al.35 The partition coefficients of commercial pectins were similar and displayed low values, in agreement with their expected high molecular weights (Kav 0.02 and 0.04 for apple and citrus pectins, respectively).21 Homogalacturonans were characterized by high galacturonic acid content (866 mg·g−1 dry matter) and very low neutral sugar content, as expected as the acid hydrolysis cleaves preferentially glycosidic bonds between neutral sugars.29,37

⎤ 2 ⎛ A A ⎥ 1 ⎞ ⎟ −4 t⎥ ⎜1 + t + nPt nK aPt ⎠ nPt ⎝ ⎦

(1)

where Pt is the total galacturonic acid concentration, At is the total concentration of the ligand, V0 is the volume of the cell, and Qi is the total heat released for injection i. The other thermodynamic parameters (ΔG and ΔS) were calculated from the van’t Hoff equation: ΔG = −RT ln Ka = ΔH − T ΔS

(2)

where ΔG is free enthalpy, Ka is the association constant, ΔH is the enthalpy, and ΔS is the entropy of interaction. Analytical. Polyphenols were measured by HPLC-DAD after thioacidolysis, as described previously.28,31 Analyses were performed using an ultrafast liquid chromatography Shimadzu Prominence system (Kyoto, Japan) including two pumps, LC20AD Prominence liquid chromatograph UFLC, a DGU-20A5 Prominence degasser, a SIL-20ACHT Prominence autosampler, a CTO-20AC Prominence column oven, a SPD-M20A Prominence diode array detector, a CBM-20A Prominence communication bus module, and controlled by a LC solution software (Shimadzu, Kyoto, Japan). Separations were achieved 711


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Figure 1. Thermograms of titration of apple pectin (30 mM galacturonic acid equivalent) by (a) procyanidins DP9 and (b) procyanidins DP30 (30 mM (−)-epicatechin equivalent in both cases): (top) Control data obtained with procyanidins in buffer; (middle) Measurement of heat release during the titration of apple pectins by procyanidins; (bottom) Molar enthalpy change against a procyanidins/apple pectin ratio after peak integration. The one-site fit curve is displayed as a thin line. Experiments were done in duplicate.

exclusive extension unit.24 Purified DP9 and DP30 also contained traces of monomeric flavan-3-ols ((+)-catechin and (−)-epicatechin), hydroxycinnamic acids (caffeoylquinic acid and p-coumaroylquinic acid), and dihydrochalcones (phloridzin and phloretin xyloglucoside). These contaminants were slightly higher in DP9 than in DP30 fraction. The two purified fractions contained procyanidins with homologous structures and contrasted degrees of polymerization. Interaction with Flavan-3-ol Monomers. The titration of commercial apple pectins (30 mM galacturonic acid equivalent) by (−)-epicatechin (30 mM) led to endothermic peaks. After blank ((−)-epicatechin in buffer) subtraction, no curve was obtained and no titration could be observed (data not shown). The same result was obtained for the titration of commercial apple pectins by (+)-catechin. No interaction could therefore be measured for the monomers using ITC. Interactions with Procyanidins of DP 9. Commercial Pectins. A typical thermogram of titration of commercial apple pectins (30 mM galacturonic acid equivalent) by procyanidins DP9 (30 mM (−)-epicatechin equivalent) displayed strong exothermic peaks (Figure 1a). The blank experiment (injections of procyanidins DP9 in buffer) yielded small endothermic peaks that were subtracted before integration. Thermodynamic parameters are shown in Table S3. Stoichiometry values ((−)-epicatechin/galacturonic acid) of 19 and 8.5 were obtained for apple and citrus pectins, respectively, while the association constants were in the same order of magnitude (1.24 × 103 M−1 for apple pectins and 1.08 × 103

They have higher partition coefficients than pectins, as expected due to the decrease in molecular weight caused by acid hydrolysis. Degrees of polymerization between 70 and 100 have been determined for homogalacturonans obtained by the same procedure for pectins from beet, apple, and citrus.21 After methylation of homogalacturonans, low (HG 30%) and high (HG 70%) degrees of methylation were obtained. Impact of the methylation of homogalacturonans on molecular weight was slight, as observed earlier (Table S1):29 the elution time in HPSEC was the same (Kav 0.15, elution time 16.3 min) for homogalacturonans and homogalacturonans of low degree of methylation and only shifted to Kav 0.18, that is elution time 16.6 min, for the highly methylated homogalacturonans. Procyanidins. The two apple varieties had been chosen for the contrasting characteristics of their procyanidins,25,38 which were verified here (Table S2). The preliminary methanol extraction allowed the elimination of most monomeric polyphenols as well as of procyanidins of a low degree of polymerization, so that acetone extraction followed by a simple C18 purification yielded procyanidins of high purity. Procyanidins content in purified aqueous acetone extract of Avrolles variety was high (721.4 mg·g−1 dry matter), with a DPn of 30. The purified Marie Ménard extract contained procyanidins with lower DPn (9) and procyanidins content. (−)-Epicatechin was always the predominant constitutive unit, accounting for more than 95% of total units in Avrolles variety and for 88% in Marie Ménard variety. In both, (+)-catechin was present only as a terminal unit and (−)-epicatechin was the 712


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Figure 2. Absorbance at 650 nm obtained in the phase diagram between pectic fractions and procyanidins DP9. (a) Variation of absorbance of commercial pectins at different concentrations (galacturonic acid equivalent) with procyanidins DP9 (30 mM (−)-epicatechin equivalent). (b) Variation of absorbance of commercial pectins (30 mM galacturonic acid) at different concentrations of procyanidins DP9 ((−)-epicatechin equivalent). (c) Variation of absorbance of homogalacturonans at different concentrations (galacturonic acid equivalent) with procyanidins DP9 (30 mM in (−)-epicatechin equivalent). (d) Variation of absorbance of homogalacturonans (30 mM galacturonic acid) at different concentrations of procyanidins DP9 ((−)-epicatechin equivalent). (■) apple pectin; (▲) citrus pectin; (×) HG 0%; (*) HG 30%; (●) HG 70%.

M−1 for citrus pectins). Analysis of the thermodynamic contributions for the two types of pectins indicated an enthalpy contribution (ΔH = −5.4 kJ·mol−1) related to the exothermic interaction. The interaction was driven mostly by entropy (−TΔS = −11.9 kJ·mol−1 compared to ΔG = −5.4 kJ·mol−1). The origin of the entropy contribution may be rather complex to evaluate for polysaccharide/tannins interaction, with a possible favorable role of entropy of mixing (expansion of polymers) or desolvatation and an unfavorable role of conformational entropy (limitation of degrees of freedom). However, in the ligand-design approach, it is generally accepted that enthalpy-driven interactions are due to hydrogen bonds and entropy-driven ones are due to hydrophobic contacts.46 The presence of many aromatic groups in tannins is in favor of this interpretation and hydrophobic contacts have been previously considered of first importance entropy-driven interaction of tannins with biomolecules.9,16 Differences between the two commercial pectins for their interactions with procyanidins DP9 were confirmed by UV−vis absorbance. Figure 2a shows the variation of optical density (OD) at 650 nm between commercial pectins and procyanidins DP9 at 30 mM (−)-epicatechin equivalent with the increase of concentrations of pectic fractions in galacturonic acid equivalent. Absorbance at 650 nm was chosen as an indicator for formation of cloud and precipitate, as neither pectins nor

procyanidins absorb at this wavelength. Absorbance of commercial pectins with procyanidins DP9 at 30 mM (−)-epicatechin equivalent rose with the increase of concentrations of pectins. This increase was more marked for citrus pectin with absorbance of 0.1 at 30 mM galacturonic acid equivalent than for apple pectin in the same conditions (0.05). Figure 2b shows the variation of absorbance between commercial pectins at 30 mM galacturonic acid equivalent and procyanidins DP9 with the increase of concentrations of procyanidins DP9 (−)-epicatechin equivalent. The decrease of OD between 0 and 0.03 mM of (−)-epicatechin corresponded to a slight turbidity of commercial pectins; addition of a low concentration of procyanidins led to a decrease of the OD. With the increase of procyanidins concentrations, the rise of absorbance of commercial pectins was similar to the Figure 2a. At a high concentration of pectins and procyanidins, interactions led to the formation of haze detected at 650 nm and visually. The formation of haze appeared from the lowest concentrations (0.06 mM) of procyanidins DP9 in the case of citrus pectin. For procyanidins DP9 at 30 mM with apple pectins at 30 mM, haze was not detected visually, though optical density increased slightly. The ΔKav corresponds to the difference of partition coefficient in HPSEC between the pectic compounds (at 30 mM in galacturonic acid equivalent) that had not formed 713


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Figure 3. Absorbance at 650 nm obtained in the phase diagram method between pectic fractions and procyanidins DP30. (a) Variation of absorbance of commercial pectins at different concentrations (galacturonic acid equivalent) with procyanidins DP30 (30 mM (−)-epicatechin equivalent). (b) Variation of absorbance of commercial pectins (30 mM galacturonic acid) at different concentrations of procyanidins DP30 ((−)-epicatechin equivalent). (c) Variation of absorbance of homogalacturonans at different concentrations (galacturonic acid equivalent) with procyanidins DP30 (30 mM in (−)-epicatechin equivalent). (d) Variation of absorbance of homogalacturonans (30 mM galacturonic acid) at different concentrations of procyanidins DP30 ((−)-epicatechin equivalent). (■) apple pectin; (▲) citrus pectin; (×) HG 0%; (*) HG 30%; (●) HG 70%.

onans (30 mM), endothermic peaks were obtained and no titration could be observed (data not shown). Identical results were obtained when titrating methylated homogalacturonans, both with HG 30% and HG 70%, at 30 mM. Therefore no interaction could be detected by ITC between homogalacturonans, methylated or not, and procyanidins DP9. The lack of interaction with HG 0% and HG 30% was confirmed by absorbance (Figure 2c,d). Demethylated and low methyl-esterified homogalacturonans showed a stable absorbance at 650 nm, about 0.015 and 0.03, respectively, whatever the concentrations of procyanidins used (Figure 2d). For highly methyl-esterified homogalacturonans, the OD650 quadrupled between 1.875 mM galacturonic acid equivalent and 7.5 mM for a constant procyanidin concentration of 30 mM (Figure 2c). At 30 mM galacturonic acid equivalent of HG 70%, the same increase of absorbance was obtained (Figure 2d). Visual examination showed the formation of haze. The supernatants of HG 70% after interaction with procyanidins DP9 (S2) and of HG 70% in buffer (S1A) were analyzed by HPSEC and the difference of the two partition coefficients showed a positive value (ΔKav of 0.11). The smaller HG 70% molecules remained in the supernatant while the larger molecules of HG 70% aggregated with procyanidins DP9. The ΔDPn was −1.40: the

aggegates with procyanidins (at 30 mM (−)-epicatechin equivalent) (S2) and the initial pectic compounds in buffer (S1A). After centrifugation of commercial pectins incubated with procyanidins DP9, low values of ΔKav were obtained, of 0.01 for apple pectins and −0.02 for citrus pectins (Table S3). Procyanidins DP9 may have a slight selectivity for the smaller molecules in citrus pectins. The ΔDPn corresponds to the difference of degree of polymerization between procyanidins that had not formed aggregates with pectins (S2) and the initial procyanidins in buffer (S1B). The ΔDPn was −5.15 in the case of apple pectins and −3.84 for citrus pectins. This means that the degree of polymerization of free procyanidins was lower than the initial degree of polymerization of procyanidins, that is, that pectins interacted preferentially with highly polymerized procyanidins. The molecules that remained in solution after pectin−procyanidin aggregation were the smaller procyanidins and to a lesser extent the larger citrus pectins. By differences, it can be deducted that small pectin molecules and large procyanidins were coaggregated or that the aggregates contained the procyanidins of higher DPn and the pectins of smaller hydrodynamic volume. Homogalacturonans. In the ITC experiment, during injection of procyanidins in fully demethylated homogalactur714


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Figure 4. Thermogram of titration of (a) HG 30% and (b) HG 70% (30 mM galacturonic acid equivalent) by procyanidins DP30 (30 mM (−)-epicatechin equivalent). (c) Titration of HG 70% (3 mM galacturonic acod equivalent) by procyanidins DP30 (60 mM (−)-epicatechin equivalent): (top) Control data obtained with procyanidins in buffer; (middle) Measurement of heat release during the titration of HG by procyanidins; (bottom) Molar enthalpy change against procyanidins DP30/HG ratio after peak integration. The one-site fit curve is displayed as a thin line. Experiments were done in duplicate.

degree of polymerization of free procyanidins was lower than the degree of polymerization of initial procyanidins. It seemed that the largest molecules of procyanidins DP9 interact with HG 70%, but the difference was less marked than with apple and citrus pectins. Interaction with Procyanidins Fraction DP30. Commercial Pectins. A typical thermogram of titration of commercial apple pectins 30 mM (galacturonic acid equivalent) by procyanidins DP30 30 mM ((−)-epicatechin equivalent) displayed strong exothermic peaks (Figure 1b). The blank experiment (injections of procyanidins DP30 in buffer) yielded small endothermic peaks that were subtracted before integration. The titration curve corresponds to the saturation of the available binding sites on commercial pectins. For the interaction between commercial apple pectins and procyanidins, an average stoichiometry (n) (−)-epicatechin/galacturonic acid residues of 23 was obtained (Table S3). However, for the interaction between commercial citrus pectins and procyanidins, a n of 10 was observed. The affinity constants (1.46 × 103 M−1 for apple pectins and 1.22 × 103 M−1 for citrus pectins) were of the same order of magnitude. The derived parameters of free energy and entropy are presented in Table S3. Analysis of the thermodynamic contributions indicated an enthalpy contribution (ΔH = −7.8 kJ·mol−1) related to the exothermic interaction. A favorable entropy contribution (−TΔS = −10 kJ·mol−1) indicated that the interaction was driven mostly by entropy which may result from hydrophobic interactions.9,16

After analysis of OD650 of procyanidins DP30 and commercial pectin mixtures, some differences between pectins were observed. In the presence of a high concentration of procyanidins DP30 (30 mM (−)-epicatechin equivalent), both commercial pectins (Figure 3a) showed an increase of absorbance with the increase of concentration (from 0.117 to 30 mM galacturonic acid equivalent) up to 0.22. In the presence of a high concentration of pectins (30 mM galacturonic acid equivalent; Figure 3b), the OD650 increased with the increase of procyanidin DP30 concentration from 1.875 to 30 mM (−)-epicatechin equivalent. This increase was particularly marked with the citrus pectin, from 1.875 mM of galacturonic acid equivalent and from 7.5 mM of (−)-epicatechin equivalent. After visual examination, a slight haze appeared in these conditions. In comparison with the initial pectin solutions, HPSEC of pectins remaining in solution in the presence of procyanidins of DP30 gave a ΔKav for apple pectin of −0.04, whereas for citrus pectin, the ΔKav was −0.02. This means that procyanidins DP30 associated selectively with the smaller molecules in apple pectins but were less selective for citrus pectin. The ΔDPn obtained between procyanidins DP30 and commercial pectins was similar with apple and citrus (−12.89 and −13.53, respectively). This high decrease of the degree of polymerization indicated that highly polymerized procyanidins of fraction DP30 associated selectively with pectins. Homogalacturonans. In ITC experiment, interaction between demethylated homogalacturonans (30 mM in galacturonic acid equivalent) and procyanidins DP30 30 mM 715


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pectins and procyanidins were identified. The degree of polymerization of procyanidins had the highest effect (F ratio: 167.2 with P < 0.001) on interactions between the two compounds. The concentration of procyanidins used had an effect (F ratio: 119.2 with P < 0.001) higher than concentration of pectins (F ratio: 24.1 with P < 0.001) and type of pectins (F ratio: 23.5 with P < 0.001).

(in (−)-epicatechin equivalent) led to small endothermic peaks (data not shown) and no titration could be observed in the conditions used for other experiments. OD650 of HG 0% with procyanidins DP30 (Figure 3c) showed that the absorbance decreases slightly with the increase of concentration of galacturonic acid and whatever the concentration of procyanidins (Figure 3d), no interaction was detected. The titration of HG 30% (30 mM, galacturonic acid equivalent) by procyanidins DP30 is shown in Figure 4a. During the first injections, the titration peaks were exothermic but with much smaller heat release (−1.5 μJ/sec) than with commercial pectins (−40 μJ/sec) in the same conditions. After fitting by one-site model, a stoichiometry of 60 molecules of (−)-epicatechin per galacturonic acid and an association constant of 6.22 × 103 M−1 (Table S3) were determined. Considering that the average chain length for homogalacturonans prepared in this method is of 70−100 GalA units21 and a procyanidin of DP30, one homogalacturonan may interact with one or two procyanidins, while one procyanidin may cross-link a high number of homogalacturonans. The thermodynamic contributions (ΔH = −0.8 kJ·mol−1, −TΔS = −21 kJ.mol−1) indicated that the interaction was driven mostly by entropy, which could result from extensive hydrophobic interactions9,16 between HG 30% and procyanidins DP30. In contrast to ITC results, the OD650 evolution of HG 30%procyanidins DP30 was similar to HG 0%-DP30 that is a decrease of absorbance with the increase of concentrations of galacturonic acid (Figure 3c) and no absorbance whatever the concentration of procyanidins used (Figure 3d). Thus, interactions detected by calorimetry were not confirmed by formation of aggregates. The experimental data observed with HG 70% were quite different. At the beginning of the titration, small exothermic peaks were observed. After additional procyanidins injection, peaks remained exothermic and heat released continued to increase (Figure 4b). However, given the shape of the curve it was not possible to adjust the curve with the model and to determine the thermodynamic parameters. By increasing procyanidins concentration (60 mM) and decreasing HG 70% concentration (3 mM), a different curve was obtained (Figure 4c). The association constant was 1 order of magnitude lower than with HG 30% and stoichiometry was higher, but because of concentrations used, thermodynamic parameters cannot be compared readily with others. Procyanidins DP30 seemed to react more with HG 70% than with HG 30% and HG 0%, showing an impact of the degree of methylation of pectin in their ability to interact with procyanidins. This result was correlated to aggregate formation and to OD650 analysis (Figure 3c,d). OD650 increased from 1.875 mM of galacturonic acid and 7.5 mM of (−)-epicatechin and was remarkably high (1.44) at 30 mM of the two components. This was related to visual examination showing formation of aggregates. After analysis of the supernatant of HG 70% after interaction with procyanidins DP30 (S2) and HG 70% in buffer (S1A) by HPSEC and by HPLC-DAD, the ΔKav was of 0.03 and ΔDPn of −24.4. The ΔKav indicated a slight selectivity of procyanidins DP30 for formation of aggregates with the larger molecules of HG 70%. The very strong ΔDPn indicated that highly polymerized procyanidins of fraction DP30 interacted selectively with homogalacturonans of fraction HG 70%. By multifactor ANOVA statistics, the effect of type (DP) of procyanidins, of type of pectins, and of concentrations of

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DISCUSSION Both isothermal titration calorimetry and optical density at 650 nm indicated that procyanidins formed complexes with pectins or homogalacturonans. In all cases, a favorable entropy contribution suggested that the interaction was driven mostly by entropy, which may result from hydrophobic interactions, but also from other sources (expansion, desolvatation, conformational changes).9,16 Similarly, Poncet-Legrand et al.9 have shown that the interaction between procyanidin-rich fraction (DP4) and poly(L-proline) were entropy-driven. Interactions detected by ITC for commercial pectins with procyanidins of different degrees of polymerization were in agreement with the formation of haze measured by the spectrophotometric method. The absence of titration for HG 0% and HG 30% with procyanidins DP9 measured by ITC was in agreement with the absence of modification of the optical density at 650 nm. However, differences between the two methods appeared for HG 70% with procyanidins DP9. ITC showed no titration, whereas the optical density at 650 nm increased with high concentrations, indicating that the resulting enthalpy was beyond the limit of detection of the calorimetric method. The inverse effect was obtained for interaction between HG 0% and HG 30% with procyanidins DP30, that is, a titration was detected by ITC, while no evolution of optical density at 650 nm was obtained. There is an association with procyanidins DP30 but no aggregates are formed. Procyanidins DP30 showed a similar or higher affinity constant with commercial pectins or homogalacturonans, respectively, than procyanidins DP9. An affinity constant of 104 M−1 was previously obtained for procyanidins DPn 8.7 interacting with pectin extracted from apple cell wall material by boiling,15 which is 10× higher than what is obtained here for commercial pectins−procyanidins DP9 interactions. These pectins were richer in neutral sugars and, most notably, in arabinose. The association constant of pectins with procyanidins DP9 was slightly lower than previously observed between grape seed tannins and bovine serum albumin (BSA) (Table S4).16 The association constants of commercial pectins with procyanidins DP30 were of the same order of magnitude as found between grape seed proanthocyanidins and BSA but lower than grape seed proanthocyanidins/gelatin interactions16 or grape seed proanthocyanidins/poly(L-proline) interactions9,11 (Table S4). Moreover, stoichiometry of apple pectins−procyanidins DP30 was higher than previously observed for grape seed proanthocyanidins/poly(L-proline) interactions (n for DP4/proline unit of 6)9,11 or grape seed proanthocyanidins/BSA interactions (n of 7).16 However, the stoichiometries are not fully comparable as we express them per monomeric unit. These associations could be related to a tanning effect, as in the case of polyphenol/protein or polyphenol/cell wall interactions.15 This tanning effect is a function of both the presence of functional groups able to form hydrogen bonds and hydrophobic interactions with macromolecules and of the molecular weights, that is, the size of the polyphenol. The larger procyanidins have been reported to 716


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bind more strongly to apple cell wall material,1,13,2 to grape flesh cell wall material,3 or to a proline-rich peptide.39 A previous study on globular protein−procyanidins interactions showed that the higher the degree of polymerization, the higher the affinity constant.8 Poncet-Legrand et al.40 showed that low polymerized procyanidins are more susceptible to selfassociation than higher polymerized procyanidins and this self-association tendency could limit the association with other compounds such as pectins. Frazier et al.16 showed that grape seed proanthocyanidins in the presence of BSA revealed very weak interactions and suggested that a lack of flexibility in the proanthocyanidins may have contributed to their low affinity. Moreover, in this study, the propensity of pectins to bind to highly polymerized procyanidins was confirmed by the determination of the ΔDPn. This determination showed a selective partition of procyanidins between pectins and supernatant. The selectivity of pectins was more pronounced with highly polymerized fraction, that is, DP30, because of its high polydispersity.41 This difference confirmed that complexes formed depend on the length of procyanidins, as according to Carn et al.42 The efficiency of the procyanidins binding derives from the fact that procyanidins are multidentate ligands able to bind simultaneously to several regions on the pectin polymer.39 Thus, the higher the DP, the more bonds a procyanidin will be able to form with the pectin. The impact of the degree of methyl esterification of homogalacturonans on association with procyanidins DP30 was demonstrated. The higher the DM, the higher the energy released during titration and the higher the association with procyanidins. This might be due to hydrophobic interactions between methyl groups of pectins and dihydropyran heterocycles (C-rings) of procyanidins. Moreover, Morris et al.43 showed an increasing chain flexibility of citrus pectin with increasing degree of esterification. The association of HG to procyanidins could be made easier by their extended conformation.44 The difference between apple and citrus pectins, of very similar DM, could be related to their neutral sugars composition. Here the pectins from citrus contained more rhamnose than the apple pectins. The inclusion of rhamnose units in the alternating galacturonic acid residues slightly reduces the persistence length of the pectin chain.45 Indeed, rhamnose has a potential role in forming “kinks” and an increasing of rhamnose content in pectins led to a rise of flexibility.34 In addition to a marked effect of DM, the neutral sugars composition also influenced interaction between pectins and procyanidins.

macromolecules. This material is available free of charge via the Internet at http://pubs.acs.org.

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Corresponding Author

*Tel.: +33 (0)432722537. Fax: +33 (0)432722492. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The research leading to these results has received funding from the European Community’s Seventh Framework Programme (FP7/2007-2013) under the Grant Agreement No. FP7-222 654-DREAM.

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REFERENCES

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CONCLUSIONS Binding of procyanidins to pectins was demonstrated both by ITC and haze formation. The intensity of interaction depended upon structural factors of procyanidins, including the size, form, conformational mobility and flexibility, and upon structural and conformational characteristics of the pectins. However the differences observed between commercial pectins suggested that neutral sugars side chains are also implicated in the interactions of pectins with procyanidins.

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AUTHOR INFORMATION

ASSOCIATED CONTENT

S Supporting Information *

Composition of pectins, pectic subfractions and polyphenols, thermodynamic binding parameters of two compounds, and thermodynamic binding parameters of procyanidins with 717


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Interactions between Pectic Compounds and Procyanidins are

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