Accepted Manuscript Title: Neutral sugar side chains of pectins limit interactions with procyanidins Author: Aude A. Watrelot Carine Le Bourvellec Anne Imberty Catherine M.G.C. Renard PII: DOI: Reference:
S0144-8617(13)00878-3 http://dx.doi.org/doi:10.1016/j.carbpol.2013.08.094 CARP 8084
To appear in: Received date: Revised date: Accepted date:
19-7-2013 23-8-2013 24-8-2013
Please cite this article as: Watrelot, A. A., Le Bourvellec, C., Imberty, A., & Renard, C. M. G. C., Neutral sugar side chains of pectins limit interactions with procyanidins, Carbohydrate Polymers (2013), http://dx.doi.org/10.1016/j.carbpol.2013.08.094 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Neutral sugar side chains of pectins limit interactions with procyanidins
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C. Renarda, b
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INRA, UMR408 Sécurité et Qualité des Produits d’Origine Végétale, F-84000,
cr
a
5
Avignon, France. b
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Université d'Avignon et des Pays de Vaucluse, UMR408 Sécurité et Qualité des
Produits d'Origine Végétale, F-84000 Avignon, France.
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c
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Centre de Recherches sur les Macromolécules Végétales, CERMAV-CNRS (affiliated with
Université de Grenoble), B.P. 53, F-38041 Grenoble, France.
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*Corresponding author:
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Aude A. Watrelot
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INRA, UMR408 Sécurité et Qualité des Produits d’Origine Végétale, Domaine
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Saint-Paul, Site Agroparc, 84914 Avignon Cedex 9 Tel: +33 (0)4.32.72.25.37
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Fax: +33 (0)4.32.72.24.92
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Aude A. Watrelota, b*, Carine Le Bourvelleca, b, Anne Imbertyc, Catherine M. G.
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E-mail: [email protected]; [email protected]
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Abstract
22
Interactions between seven hairy regions of pectins, rhamnogalacturonans II and
23
arabinogalactan-proteins and procyanidins with different average degrees of polymerization,
24
low (DP9) and high (DP30), were investigated by isothermal titration calorimetry and
25
absorption analysis to study the impact of neutral sugar side chains of pectins on these
26
associations. Associations between pectic fractions and procyanidins involved hydrophobic
27
interactions and hydrogen bonds. No difference in association constants between various
28
hairy regions and procyanidins DP9 was found. Nevertheless, arabinan chains showed lower
29
association constants, and hairy regions of pectins with only monomeric side chains showed
30
higher association with procyanidin DP30. Only very low affinities were obtained with
31
rhamnogalacturonans II and arabinogalactan-proteins. Aggregation could be observed only
32
with the procyanidins of DP30 and the protein-rich arabinogalactan-protein. Associations
33
were obtained at both degrees of polymerization of the procyanidins, but differed depending
34
on neutral sugar composition and the structure of pectic fractions.
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Highlights
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• Pectic hairy regions-procyanidins interactions has been studied by ITC.
38
• The higher the pectic ramifications, the lower association with procyanidins is.
39
• Structures of both compounds are implicated.
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Keywords
44
Polyphenol, condensed tannins, polysaccharide, rhamnogalacturonan, association, ITC.
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1. Introduction
47
Pectins are complex polysaccharides of plant cell walls, present mostly in the middle
48
lamella and primary wall. They comprise smooth and hairy regions. The “smooth regions”,
49
also known as homogalacturonans (HGs), consist of a long chain of (1→4) linkedα-D-
50
galacturonic acid (GalpA) with various degrees of methylation and/or acetylation. The hairy
51
regions, named rhamnogalacturonans (RGs), are of two types (I and II) (Thomas et al., 1987;
52
De Vries et al., 1982; Caffall & Mohnen, 2009). Rhamnogalacturonans I are formed by the
53
repeating unit →4)-α-D-GalpA-(1→2)-α-L-Rhap-(1→), the rhamnose residues of the RG
54
fractions being decorated by long side-chains composed mostly of galactose and arabinose
55
(Ridley, O’Neill & Mohnen, 2001; Voragen et al., 2009).RG I presents this strictly alternating
56
RG backbone(De Vries et al., 1982; Schols et al., 1990; Renard et al., 1995), while the
57
misleadingly named RG II possesses a backbone of (1→4) linkedα-D-GalpA as
58
homogalacturonans with side chains containing rhamnose and a variety of rare
59
monosaccharides (Pérez et al., 2003). Arabinogalactan-proteins (AGPs) are often associated
60
with pectins because both molecules contain a similar structural motif of type II galactans, i.e.
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(1→3) and (1→6) linked galactans ramified with arabinan oligomers (Voragen et al., 2009).
62
However, in AGPs the arabinogalactan domains are connected via a specific hydroxyproline-
63
rich protein (Clarke, Anderson & Stone, 1979).
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Procyanidins are flavonoids, major polyphenols found in vacuoles of fruits and
65
vegetables. They are a subclass of proanthocyanidins derived from the oligomerization of
66
flavan-3-ols unitsand form the condensed tannins. They are characterized by their constitutive
Page 3 of 35
units, their interflavanic linkages and their degree of polymerization (Hemingway &
68
Karchesy, 1989; Quideau et al., 2011).Specifically, apple procyanidins are characterized by
69
the predominance of (-)-epicatechin and of C4-C8 interflavanic linkages (Guyot et al.,1998;
70
Sanoner et al., 1999; Wojdyło, Oszmianski & Laskowski, 2008). They can be oligomers or
71
polymers and in cider apples, their number-average degree of polymerization range between
72
DP n 4.5 to 50 for varieties Jeanne Renard and Avrolles respectively (Sanoner et al., 1999).
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They have been reported to exhibit several health effects as antioxidants and anticarcinogens,
74
and to protect against cardiovascular diseases.
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Fruit and vegetable processing ruptures cells. This disruption leads to association between
76
constituents of cell walls and vacuoles, with a prominent role of pectins and procyanidins (Le
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Bourvellec, Bouchet & Renard, 2005). The bioavailability of polyphenols is dependent on
78
their structure and their association with other compounds such as sugars (Parada & Aguilera,
79
2007). Procyanidins associated with dietary fiber are not bioaccessible in the upper part of the
80
human intestine, and travel to the colon, where they become fermentable substrates for
81
bacterial microflora (Cheynier et al., 2012; Palafox-Carlos et al., 2011; Saura-Calixto et al.,
82
2010), producing metabolites that are both absorbable and bioactive (Williamson & Clifford,
83
2010).In vitro, the interaction between procyanidins and dietary fiber reduces the
84
polysaccharide fermentation and increases the production of procyanidin colonic metabolites
85
(Saura-Calixto et al., 2010; Aura et al., 2012). To understand the health effect of polyphenols
86
after binding to polysaccharides such as pectins, earlier work dealt with mechanisms of
87
interactions between these two macromolecules. In our previous work (Watrelot et al., 2013),
88
interactions between procyanidins and pectins were analyzed, and the impact of the degree of
89
methylation of smooth regions and of the degree of polymerization of procyanidins could be
90
demonstrated. However, the role of the hairy regions of pectins has not been determined. The
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aim of this study was to quantify the binding of a specific class of proanthocyanidins, the
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procyanidins, to hairy regions of pectins. Isothermal titration calorimetry and a
93
spectrophotometric method were used to probe thermodynamic parameters and aggregation
94
phenomena in interactions between hairy region fractions and procyanidins with different
95
average degrees of polymerization: low (DP9) and high (DP30).
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2. Materials and methods
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2.1.
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Chemicals
Methanol, acetonitrile, and acetone of chromatographic quality were from Biosolve
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(Distribio, Evry, France). Hexane (Merck, Darmstadt, Germany) was of analytical quality.
102
Ethanol and acetone were from Fisher Scientific (Strasbourg, France).
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Silver nitrate was from Merck (Darmstadt, Germany). Sodium borohydride (NaBH4),
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N-methylimidazole, acetic anhydride, toluene-α-thiol and pectin from apple were from Sigma-
105
Aldrich (Deisenhofen, Germany). Chlorogenic acid, (+)-catechin, and (−)-epicatechin were
106
from Sigma-Aldrich. 4-Coumaric acid was from Extrasynthèse (Lyon, France). Phloridzin
107
and sugar standards were from Fluka (Buchs, Switzerland). Methanol-d3 was from Acros
108
Organics (Geel, Belgium). Enzymatic cocktail Endozym polifruit liq+ was from Spindal
109
groupe AEB (Gretz-Armainvilliers, France). Modified hairy regions (MHR1) from apple were
110
kindly supplied and characterized by Dr. M. C. Ralet (INRA-UR1268 Biopolymères
111
Interactions Assemblages, Nantes, France). Modified hairy regions MHR2, MHR3 and
112
MHR4 from pear, onion and soybean (SSPS) respectively were kindly supplied by Dr. Henk
113
Schols
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Rhamnogalacturonans of type II monomer and dimer (mRG-II and dRG-II) and
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(WU
Agrotechnology
&
Food
Sciences,
Wageningen,
Netherlands).
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arabinogalactan-protein (AGP0 and AGP4) were kindly supplied by Dr. Claire Dufour
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(INRA-UMR408 Sécurité et Qualité des Produits d’Origine Végétale, Avignon, France).
117 118
2.2.
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Apple fruits (Malus domestica Borkh.) of the Avrolles and Marie Ménard varieties
120
were harvested at commercial maturity during the 2000 season in the experimental orchard of
121
the Centre Technique des Productions Cidricoles (CTPC, Sées, France). Fruits were
122
mechanically peeled and cored as previously described by Guyot et al. (1997) and cortex
123
tissues were freeze-dried. Apple pomace was from Val de Vire Bioactives (Condé-sur-Vire,
124
France). Fibrex 615 (Sugar beet pulp) was from Nordic Sugar (Copenhagen, Denmark).
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Plant material
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2.3.
127
Procyanidins were extracted from “Marie Ménard” and “Avrolles” freeze-dried pulps
128
by successive solvent extractions and purified as described by Watrelot et al. (2013). The
129
purified procyanidin fractions were designated DP9 (from “Marie Ménard”) and DP30 (from
130
“Avrolles”). In the two fractions, (−)‐epicatechin was the predominant constitutive unit,
131
accounting for more than 95% and 88% in DP30 and DP9 respectively. For details, see
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Watrelot et al. (2013)
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Procyanidin extraction and purification
2.4.
Rhamnogalacturonan preparation
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Rhamnogalacturonans were prepared as described by Renard & Jarvis (1999) using 15
135
g/L of apple pectins saponified at 4 °C, pH > 12 and left overnight. The solution was then
136
brought to pH 6 and precipitated with three volumes of an ethanol/water/hydrochloric acid
137
mixture (96: 3: 1, v/v/v) with stirring at 4 °C and left overnight. This suspension was filtered
138
on a G0 sintered glass filter. After filtration the pectic acid precipitate was washed thoroughly
Page 6 of 35
with an ethanol/water mixture (70:30, v/v) until all remaining chloride ions were eliminated
140
(as verified by silver nitrate precipitation). The resulting powder was dried by solvent
141
exchange (ethanol 96% and acetone), followed by drying at 40 °C in an oven overnight. The
142
pectic acid was dissolved (6 g/L) and hydrolyzed with hydrochloric acid (0.25 N) for 72 h at
143
80 °C (Thibault et al., 1993).The supernatant was concentrated on a rotary evaporator before
144
dialyzing against 0.1 M NaCl followed by water to eliminate monomers and neutral oligomers
145
(galactose and arabinose). The rhamnogalacturonan oligomers (RG) were finally freeze-dried.
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Apple hairy regions were obtained by a modified alkaline peroxide treatment as
147
described by Renard et al. (1997). Apple pomace was suspended overnight at room
148
temperature with stirring at 50 g/L in water, and pH was stabilised at >13. Polyphenols were
149
then eliminated by adding 200 mL of hydrogen peroxide (30%). The medium was left to react
150
with stirring for 2 h. The pH was adjusted to 5 with acetic acid, and enzymatic cocktail
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Endozym polifruit liq+ (1mL) was added and left to act overnight with stirring at room
152
temperature. The suspension was filtered on a nylon membrane (250 µm pore size) under
153
vacuum. The filtrate was dialyzed against 0.1 M NaCl, and then water at 4 °C. Finally, the
154
filtrate was freeze-dried to give the apple hairy region fraction (AHR).
156 157
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Acid-treated hairy regions (HR-H) were obtained from sugar beet pulp as described by
Ralet et al. (2010).
Modified hairy regions (MHR1) from apple were prepared by the method of Schols,
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Posthumus & Voragen, (1990) and were a kind gift from Dr. M.-C. Ralet. Modified hairy
159
regions (MHR2 and MHR3) from pear and onion were prepared by the method of Schols &
160
Voragen, (1994). MHR4 from soybean was prepared by Coenen (2007). MHR2, MH3 and
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MHR4 were a kind gift from Dr. Henk Schols. All MHRs resulting from drastic enzymic
162
hydrolysis of plant cell walls were isolated as high molar mass polymers with a
163
rhamnogalacturonan backbone. However, the structure of the side chain depends on the
Page 7 of 35
botanical origin (Table 1). MHR1 from apple was mostly composed of β-(1→4) linked
165
galactans and arabinans (Schols & Voragen, 1994; Renard et al., 1991). They were
166
characterized by a high xylose content with a molar ratio Xyl: GalA of 0.4, which
167
corresponds to xylogalacturonan chains (Schols et al., 1995). MHR2 from pear was rich in
168
arabinose present as (1→5)-linked chains (with proportion of 71 %). Its galactans were
169
predominantly highly ramified β-(1→3) and β-(1→6) type II galactan and galactose residues
170
were terminally linked (with proportion of 36 %) (Schols & Voragen,1994).MHR3 from
171
onion was rich in galactose with high proportion of terminal galactose residues (58 %) and in
172
β-(1→4) linked type I galactan(19 %) (Schols & Voragen, 1994), as was MHR4 from
173
soybean with the additionnal presence of β-D-Xyl side chains substituted to (1,4)-linked
174
galacturonic acid, i.e. also xylogalacturonans (Coenen et al., 2007; Nakamura et al., 2001).
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Polysaccharides isolated from red wine were kindly donated by Dr. C. Dufour (INRA-
176
UMR408 Sécurité et Qualité des Produits d’Origine Végétale, Avignon, France).
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Rhamnogalacturonans, RG-IIs, were purified as described in Pellerin et al. (1996). Fractions
178
mRG-II and dRG-II were predominantly monomeric (90% pure) and dimeric (87% pure).
179
Monomer and dimer (mRG-II and dRG-II) were rich in arabinose and composed of rare
180
neutral sugars. The dimer was distinguished by the presence of boron.
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Type II arabinogalactan-protein fractions, AGP0 and AGP4, were isolated by anion
182
exchange chromatography according to Pellerin et al. (1995). Fractions were similar in terms
183
of neutral sugar composition except for arabinose and galactose with higher content in AGP0
184
(35.2 % and 57.3 % respectively) than AGP4 (27.1 % and 38.3 %, respectively), while uronic
185
acid content was higher in AGP4 than AGP0. Also, non-reducing arabinose and rhamnose
186
were observed in both AGPs, but rhamnose was 2,4-linked in AGP4. Galactose was 3,6-
187
linked more in AGP4, but 3,4,6-linked more in AGP0 (Pellerin et al., 1995).
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189
2.5.
Phase diagram
To identify aggregate formation during pectin-tannin interactions, a spectrophotometric
191
method was used at 25 °C. Systematic variation of concentrations and relative amounts was
192
carried out on a 96-well “microplate”. This was done by varying the concentrations of
193
procyanidins (0, 0.015, 0.03, 0.06, 0.117, 0.23, 0.47, 0.94, 1.875, 3.75, 7.5, 15 mmol/L (−)-
194
epicatechin equivalent) along the rows, and varying theconcentrations of hairy regions (0,
195
0.12, 0.23, 0.47, 0.94, 1.875, 3.75, 7.5 mmol/Lgalacturonic acid equivalent) along the
196
columns. Solutions were prepared in citrate/phosphate buffer at pH 3.8 and ionic strength
197
0.1 mol/L. Equal amounts (50 µL) of procyanidin and hairy region fraction solutions were
198
mixed before each spectrum measurement for 20 s. Absorbance spectra (200–650 nm) were
199
recorded, and absorbance at 650 nm was analyzed for each solution using a SAFAS flx-
200
Xenius XM spectrofluorimeter (SAFAS, Monaco). Control spectra were obtained using wells
201
containing hairy regions alone in buffer and procyanidins alone in buffer. After spectra
202
recording, microplates were centrifuged for 10 min at 2100g, and supernatants of control
203
wells (hairy regions at 7.5 mmol/L in buffer (S1A) and procyanidins at 15 mmol/L in buffer
204
(S1B)) and supernatants of wells containing procyanidins at a concentration of 15 mmol/L
205
with hairy regions at a concentration of 7.5 mmol/L (S2) were analyzed by high pressure size
206
exclusion chromatography (HPSEC) and high-performance liquid chromatography – diode
207
array detection (HPLC-DAD) to define changes in partition coefficient (ΔKav= S2-S1A) of
208
hairy region fractions and in average degree of polymerization of flavan-3-ols (ΔDPn = S2-
209
S1B).
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210 211
2.6.
212
To measure enthalpy changes associated with hairy region-tannin interactions at
213
25 °C, a VP-ITC microcalorimeter (Microcal®, GE Healthcare, Northampton, MA, USA)
Isothermal Titration Microcalorimetry
Page 9 of 35
was used. To allow comparison between the different pectic substrates, and given the
215
polydispersity in molecular weight for both pectic fractions and procyanidins, all
216
concentrations are expressed relative to the main monomers, i.e. galacturonic acid for pectins
217
and (–)-epicatechin for procyanidins. Procyanidins and hairy region fractions were dissolved
218
in the same citrate/phosphate buffer pH 3.8, ionic strength 0.1 mol/L, and filtered on 0.45 µm
219
membrane. All the solutions were degassed before measurements. The reference cell was
220
filled with water. To obtain a hyperbolic curve as recommended for low affinity
221
systems(Turnbull & Daranas, 2003),different concentrations of compounds were tested. The
222
pectic fraction solution was placed in the 1.448 mL sample cell of the calorimeter, and the
223
procyanidin solution was loaded into the injection syringe and titrated into the sample cell by
224
30 injections of 10 µL aliquots. The duration of each injection was 20 s, with a separating
225
delay of 5 min. The contents of the sample cell were stirred throughout the experiment at 307
226
rpm to ensure mixing.
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Raw data obtained as a plot of heat flow (microcalories per second) against time
228
(minutes) were then integrated peak-by-peak and normalized to obtain a plot of observed
229
enthalpy change per mole of injectant (ΔH, kJ.mol−1) against the molar ratio
230
((−)‐epicatechin/galacturonic acid). Peak integration was performed using Microcal Origin 7.0
231
(Microcal Software, GE Healthcare). Control experiments included the titration of
232
procyanidin fractions into buffer, and were subtracted from titration experiments. The
233
experimental data were fitted to a theoretical titration curve using Microcal Origin 7.0, with
234
ΔH (enthalpy change), Ka (association constant), and n (number of binding sites per molecule)
235
as adjustable parameters, from the relationship
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∆ 2 236
0
1
1
1
1
2
4
, Eq. 1
Page 10 of 35
where Pt is the total galacturonic acid concentration, At is the total concentration of the ligand,
238
V0 is the volume of the cell, and Qi is the total heat released for injection i.
239
The other thermodynamic parameters (ΔG and ΔS) were calculated from the van’t Hoff
240
equation:
241
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ΔG=-RTlnKa= ΔH-TΔS, Eq. 2
where ∆G is free enthalpy, Ka is the association constant, ∆H is the enthalpy and ∆S is the
243
entropy of interaction.
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244 245
2.7.
246
Polyphenols
were
measured
by
an
Analysis methods
high-performance
liquid
chromatography
(HPLC)/diode array detection (DAD) after thioacidolysis as described by Guyot et al. (2001)
248
and Le Bourvellec et al. (2011). Analyses were performed using an ultra fast liquid
249
chromatography Shimadzu Prominence system (Kyoto, Japan) comprising two LC-20AD
250
pumps, UFLCProminence liquid chromatograph, a DGU-20A5 Prominence degasser, a SIL-
251
20ACHT Prominence autosampler, a CTO-20AC Prominence column oven, a SPD-M20A
252
Prominence diode array detector, and a CBM-20A Prominence communication bus module,
253
and was controlled by LC Solution software (Shimadzu, Kyoto, Japan). Separations were
254
achieved in the conditions of Le Bourvellec et al. (2011). The average degree of
255
polymerization (DPn) of procyanidins was measured by calculating the molar ratio of all the
256
flavan-3-ol units (thioether adducts plus terminal units) to (−)-epicatechin and (+)-catechin
257
corresponding to terminal units. Neutral sugars, galacturonic acid and methanol were
258
analyzed as described by Renard & Ginies (2009).
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259
The molecular weight distribution of polysaccharides was determined using a high
260
pressure size exclusion chromatography (HPSEC) system comprising a Jasco LC-NET
261
II/ADC interface, a Jasco PU-2080 plus intelligent HPLC pump, a Jasco RI-2031 plus
Page 11 of 35
intelligent RI detector, and a degasser, and was controlled by ChromNav solftware (Jasco,
263
Tokyo, Japan). Separations were achieved using two columns in series: a 8.0 mm × 300 mm
264
i.d. OH-pack SB-802 HQ column (Showa Denko Europe, Munich, Germany) and a 300 ×
265
7.8 mm i.d. TSK-Gel PWXL column (Tosohaas, Stuttgart, Germany) at 35 °C and a 40 ×
266
6.0 mm i.d. guard column TSK-Gel PWXL (Tosohaas, Stuttgart, Germany). Solutions
267
(20 μL) of the extracts (10 g/L) were injected and eluted with 0.4 mol/Lsodium acetate buffer
268
pH 3.5 at 0.8 mL/min. Dextrans T500, T70, T40 and T10 (Pharmacia BioProcess Technology,
269
Uppsala, Sweden) and glucose (Sigma-Aldrich, Deisenhofen, Germany) were used to
270
calibrate the column system.
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271 272
2.8.
273
Results are presented as mean values, and the reproducibility of the results is
274
expressed as pooled standard deviation. Pooled standard deviations were calculated for each
275
series of replicates using the sum of individual variances weighted by the sum of individual
276
degrees of freedom (Box, Hunter & Hunter, 1978).
278 279
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Statistical analysis
3. Results 3.1.
Composition of the hairy regions
Page 12 of 35
Table 1. Samples (mg/g)
Kav
RG
0.26
49
3
45
AHR
0.28
23
2
251 16
-
30
19
167
HR-H
0.13 143
-
25
-
199
-
437
1.7
0.1 9.6 0.7
236 128 458
0.9
9.5 5.7
4.5
385
MHR1
−0.06
89
9
83 113
-
134
a
MHR2
−0.03
79
-
240 42
-
101
a
MHR3
0.01 132
-
40
MHR4
-
Arabinan Type I galactan Xylogalacturonan
209
Arabinan Type II galactan
nd
41
33 188 52
-
351 27
165
Galactan Xylogalacturonan Type II Arabinogalactan
17
7
208
-
M
b
-
RG with monomeric galactans
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a
Arabinan
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-
10
cr
-
15
Characteristic structure
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Pooled SD
Rha Fuc Ara Xyl Man Gal Glc GalA
283
Type I galactan
c
AGP0
0.08
11
1
358
2
18
583 16
28
c
AGP4
0.02
93
2
250 18
23
354
180
d
280
3
RG, rhamnogalacturonans; AHR, apple hairy regions; HR-H, acid-treated hairy regions; MHR,
282
modified hairy regions (1 from apple, 2 from pear, 3 from onion and 4 from soybean); AGPs,
283
arabinogalactan-proteins; Kav, partition coefficient; Rha, rhamnose; Fuc, fucose; Ara, arabinose; Xyl,
284
xylose; Man, mannose; Gal, galactose; Glc, glucose; GalA, galacturonic acid, Pooled SD: pooled
285
standard deviation, n =11.a: Schols and Voragen 1994; b: Coenen 2007; c: Pellerin et al., 1995.
287
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te
281
The characterization of hairy regions is shown in Table 1. RG contained a high
288
proportion of galacturonic acid, with a molar ratio GalA: Rha > 7, indicating the
289
predominance of homogalacturonans in the backbone. Its main neutral sugars were galactose
290
and glucose. AHR contained a substantial portion of homogalacturonans (GalA: Rha = 6), but
291
unlike RG, it also contained a high proportion of arabinan (molar ratio Ara: Rha > 10). HR-H
292
was the simplest rhamnogalacturonan fraction, with the lowest neutral sugar content. The
Page 13 of 35
molar ratio GalA: Rha of 2.5 is consistent with the rhamnogalacturonic backbone, while the
294
low (Gal+Ara): Rha ratio (< 2) corresponds to very short side chains of arabinogalactan
295
(Voragen et al., 2009).MHR1 and MHR4 contained more homogalacturonic backbone (GalA:
296
Rha = 3.6 and 3.4 respectively) than MHR2 and MHR3 with a molar ratio GalA: Rha of 2.2
297
and 1.8 respectively.
ip t
293
The partition coefficients of RG and AHR were identical (Kav of 0.26 and 0.28
299
respectively with retention times of 17.2 min and 17.4 min), corresponding to small polymers
300
obtained after acid hydrolysis from commercial pectins or after enzymatic reaction from apple
301
pomace. The partition coefficient of HR-H fraction was lower (Kav of 0.13 with a retention
302
time of 16.3 min), indicating a higher hydrodynamic volume. Partition coefficients were close
303
between RG-IIs (Kav of 0.27 for mRG-II and 0.22 for dRG-II) and were in good agreement
304
with calculated molecular weight (Pellerin et al., 1996). Partition coefficients of AGPs were
305
low (Kav of 0.02 for AGP4) and similar to commercial apple pectins (Watrelot et al., 2013).
d
M
an
us
cr
298
308 309
3.2.
Interactions with procyanidins of DPn 9
Ac ce p
307
te
306
3.2.1. Hairy regions
Thermograms of titration of hairy region fractions (3.75 mmol/L galacturonic acid
310
equivalent) by procyanidins DP9 (7.5 mmol/L (−)-epicatechin equivalent) are shown in
311
Figure 1.
312 313 314
Figure 1. Error! Reference source not found.
315
The blank experiment (injections of procyanidins DP9 in buffer) yielded small endothermic
316
peaks that were subtracted before integration. The thermogram of titration of RG by
Page 14 of 35
procyanidins DP9 (Figure 1a) showed small exothermic peaks for the first injections, and
318
stronger endothermic peaks after saturation. The thermogram of AHR titration by
319
procyanidins DP9 (Figure 1b) showed strong exothermic peaks (−8 µJ/sec) with clear
320
titration.
Ac ce p
te
d
M
an
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cr
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317
Page 15 of 35
i RG
AHR
HR-H
0.01
0.02
0.02
2530
1955
∆H (kJ.mol )
−6
∆G (kJ.mol-1)
−19
n -1
Ka (M ) -1
-1
-1
45
MHR1
MHR2
MHR3
MHR4
mRG-II
dRG-II
AGP0
AGP4
0.02
0.02
0.02
-
-
-
0.02
0.02
1850
2105
872
2225
-
-
-
44
57
−3
−18
−5
−16
−26
-
-
-
−67
−38
−19
−19
−19
−17
−19
-
-
-
−9
−10
51
1
47
2
−25
-
-
-
−193
−95
0.000
0.006
0.001
−0.002
−0.001
nd
0.002
−0.001
0.001
0.005
−0.001
Δ DP n DP 30
−1.61
−1.06
−1.07
0.17
0.00
−0.55
nd
0.15
−0.16
−0.22
0.15
RG
AHR
HR-H
MHR1
MHR2
MHR3
MHR4
RGII2
RGII3
AGP0
AGP4
0.02
0.02
0.02
0.02
0.02
0.02
-
0.02
0.02
0.02
0.02
2620
574
5390
1835
875
1170
-
72
62
112
165
∆H (kJ.mol )
−3
−17
−4
−7
−24
−41
-
−16
−14
−54
−31
∆G (kJ.mol-1)
−19
−16
−21
−19
−17
−17
-
−11
−10
−12
−13
56
−4
59
38
−23
−78
-
−18
−12
−142
−61
ΔKav
0.004
0.004
0.015
0.011
0.004
−0.006
nd
0.000
−0.004
0.009
0.011
Δ DP n
−11.82
−10.17
−9.49
−4.89
−5.01
−8.44
nd
0.28
0.27
−3.62
0.53
pt
ΔKav
ce
∆S (J.mol .K )
-1
Ka (M )
Ac
n -1
-1
-1
∆S (J.mol .K )
323
us
cr DP 9
M
(30 mmol/L in (−)-epicatechin equivalent).
ed
322
an
Table 2.Thermodynamic binding parameters of interaction between pectic compounds and procyanidin fractions DP9 and DP30
321
Average of duplicates for each measurement. Stoichiometry corresponds to the molar ratio value of (−)-epicatechin/galacturonic acid. nd: not defined
Page 16 of 35
Thermodynamic parameters are shown in Table 2.Stoichiometry (defined as ratio of (−)-
325
epicatechin/galacturonic acid) was fixed at 0.01 for RG (1 molecule of (−)-epicatechin binds
326
with 100 molecules of galacturonic acid) and 0.02 for other fractions (1 molecule of (−)-
327
epicatechin binds with 50 molecules of galacturonic acid) using a one-site model. The
328
association constant (Ka = 2.53 × 103 M-1) observed for RG and DP9 was of the same order of
329
magnitude as that measured for AHR, HR-H, MHR1 and MHR3 (Ka from 1955 M-1 to 2225
330
M-1). The value of Ka was lower for interaction between MHR2 and procyanidins DP9 (Ka =
331
872 M-1). Analysis of the thermodynamic contributions indicated an enthalpy contribution
332
(∆H = −18 kJ.mol-1,−16 kJ.mol-1 and −26 kJ.mol-1 for HR-H, MHR2 and MHR3 respectively)
333
related to the exothermic interaction. The entropy term between HR-H, MHR2 and
334
procyanidins DP9 was almost null (−T∆S = −0.3 to −0.5 kJ.mol-1 respectively), indicating a
335
weak role of hydrophobic interactions. The analysis of the thermodynamic contributions for
336
MHR3 gave a negative entropy term (−TΔS = 7 kJ.mol-1) indicating that the interaction was
337
driven by enthalpy, which may result from hydrogen bond association. A favorable entropy
338
contribution was observed for RG, AHR and MHR1 (−T∆S ~ −13 kJ.mol-1), indicating that
339
the interaction was driven mostly by entropy, which may result from hydrophobic interactions
340
(Poncet-Legrand et al., 2007; Frazier et al., 2010). The titration of MHR from soybean
341
(MHR4) by procyanidins DP9 (data not shown) displayed strong exothermic peaks, but no
342
thermodynamic parameters could be defined.
Ac ce p
te
d
M
an
us
cr
ip t
324
17
Page 17 of 35
Figure 2.
M
an
us
cr
ip t
343
Ac ce p
te
d
344
345
The absence of differences in affinity between hairy regions and procyanidins DP9
346 347
was confirmed by UV-vis absorbance at 650 nm. Figure 2a, c shows the variation of
348
absorbance at 650 nm between hairy regions (RG, AHR, HR-H, MHR1, MHR2 and MHR3)
349
and procyanidins DP9 at 15 mmol/L (−)-epicatechin equivalent with the increase of
350
concentrations of hairy regions, standardized here in galacturonic acid equivalent. Absorbance
351
at 650 nm was chosen as an indicator for the formation of cloud and precipitate, as neither
352
pectins nor procyanidins absorb at this wavelength. Absorbance of all hairy regions with
353
procyanidins DP9 was low (0.04), quite stable and similar between hairy regions. Only the 18
Page 18 of 35
MHR3 fraction showed an increase in absorption up to 0.08 from 1.875 mmol/L galacturonic
355
acid equivalent. Figure 2b, d shows the variation in absorbance between hairy regions (RG,
356
AHR, HR-H, MHR1, MHR2 and MHR3) at 7.5 mmol/L galacturonic acid equivalent and
357
procyanidins DP9 with the increase in concentrations of procyanidins DP9, given in (−)-
358
epicatechin equivalent. Initially, all hairy regions displayed a slight turbidity, which
359
disappeared with addition of a low concentration of procyanidins. With the increase in
360
procyanidin concentrations, absorbance of hairy regions RG, AHR and HR-H increased
361
slightly (up to 0.04) and in a similar fashion. Among MHRs fractions (Figure 2d), MHR3
362
showed an increase in absorption up to 0.08 from 0.94 mmol/L (−)-epicatechin equivalent,
363
while MHR1 and 2 were stable around 0.01.
an
us
cr
ip t
354
The ∆Kav correspond to the difference in partition coefficient in HPSEC between the
365
pectic hairy regions (at 7.5 mmol/L in galacturonic acid equivalent) that had not formed
366
aggregates with procyanidins (at 15 mmol/L (−)-epicatechin equivalent) (S2) and the initial
367
pectic compounds in buffer (S1A). After centrifugation of hairy regions incubated with
368
procyanidins DP9, values of ∆Kav obtained were close to nil irrespective of the pectic
369
fractions (Table 2). There was no difference in hydrodynamic volume for hairy regions
370
before and after association with procyanidins DP9.The ∆DPn corresponds to the difference in
371
degree of polymerization between procyanidins that had not formed aggregates with pectins
372
(S2) and the initial degree of polymerization of procyanidins in buffer (S1B). The ∆DPn was
373
−1.61 for RG and −1.06 for AHR and HR-H. The degree of polymerization of free
374
procyanidins was slightly lower than the initial degree of polymerization of procyanidins, i.e.
375
hairy regions RG, AHR and HR-H precipitated preferentially with highly polymerized
376
procyanidins. The value of ∆DPn of MHR3 was slightly negative, but there was no difference
377
for MHR2 and MHR1. This means that MHR3, MHR1 and MHR2 were not selective with
378
procyanidins DP9.
Ac ce p
te
d
M
364
19
Page 19 of 35
379
3.2.2. Rhamnogalacturonans II and arabinogalactan-proteins During injection of procyanidins in rhamnogalacturonans type II (mRG-II and dRG-II),
381
peaks were endothermic and heat was absorbed. No titration could be observed. Hence no
382
interaction could be detected by ITC between RGIIs and procyanidins DP9.
ip t
380
A thermogram of titration of arabinogalactan-protein by procyanidins DP9 displayed only
384
exothermic peaks (data not shown). Thermodynamic parameters were calculated from raw
385
data after subtraction of blank experiments (procyanidins DP9 in buffer) (Table 2).
386
Stoichiometry was fixed at 0.02 using a one-site model. Association constants obtained for
387
AGP0 and AGP4 were very weak (Ka = 44 M-1 and 57 M-1 respectively) and correspond to
388
the limit of detection by isothermal titration calorimetry.
M
an
us
cr
383
The variation of absorbance at 650 nm between rhamnogalacturonans II and
390
arabinogalactan-proteins and procyanidins DP9 at 15 mmol/L (−)-epicatechin equivalent with
391
the increase in concentrations of rhamnogalacturonans in galacturonic acid equivalent is
392
shown in Figure 2e. Whatever the pectic fraction, the absorption was stable at around 0.04.
393
The variation of absorbance between RG-IIs and AGPs at 7.5 mmol/L galacturonic acid
394
equivalent and procyanidins DP9 with the increase in concentrations of procyanidins DP9 in
395
(−)-epicatechin equivalent (Figure 2f) was low (0.01) and stable between fractions, but the
396
dimer of RGII (dRG-II) showed absorption at 0.03.
Ac ce p
te
d
389
397
The ΔKav obtained was close to nil for all RGIIs and AGPs. With RGIIs and AGPs, the
398
ΔKav and ∆DPn were very low or nil: there was no selective precipitation of either the
399
procyanidins or the polysaccharides.
400 401
3.3.
Interaction with procyanidins of DPn 30 20
Page 20 of 35
402
3.3.1. Hairy regions ITC measurements were made with 3.75 mmol/L of pectic compound (galacturonic
403
acid equivalent) and 7.5 mmol/L ((−)-epicatechin equivalent) of procyanidins DP30.
405
Figure 3.
406
Error! Reference source not found.
c
ip t
404
Titration of RG, AHR, HR-H and MHR1, MHR2 and MHR3 by procyanidins DP30
408
showed complex curves characterized by strong exothermic peaks (Figure 3a and 3b); to
409
avoid over-fitting, the stoichiometry was fixed at 0.02 for all fractions to allow fit and
410
determination of association parameters. Thermodynamic parameters are presented in Table
411
2. The association constant Ka ranged from 574 M-1 to 5390 M-1 in the order
412
AHR
S0144-8617(13)00878-3 http://dx.doi.org/doi:10.1016/j.carbpol.2013.08.094 CARP 8084
To appear in: Received date: Revised date: Accepted date:
19-7-2013 23-8-2013 24-8-2013
Please cite this article as: Watrelot, A. A., Le Bourvellec, C., Imberty, A., & Renard, C. M. G. C., Neutral sugar side chains of pectins limit interactions with procyanidins, Carbohydrate Polymers (2013), http://dx.doi.org/10.1016/j.carbpol.2013.08.094 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Neutral sugar side chains of pectins limit interactions with procyanidins
1
2
C. Renarda, b
6
INRA, UMR408 Sécurité et Qualité des Produits d’Origine Végétale, F-84000,
cr
a
5
Avignon, France. b
7
Université d'Avignon et des Pays de Vaucluse, UMR408 Sécurité et Qualité des
Produits d'Origine Végétale, F-84000 Avignon, France.
9
c
an
8
M
Centre de Recherches sur les Macromolécules Végétales, CERMAV-CNRS (affiliated with
Université de Grenoble), B.P. 53, F-38041 Grenoble, France.
11
*Corresponding author:
13
Aude A. Watrelot
Ac ce p
INRA, UMR408 Sécurité et Qualité des Produits d’Origine Végétale, Domaine
14 15
Saint-Paul, Site Agroparc, 84914 Avignon Cedex 9 Tel: +33 (0)4.32.72.25.37
16
Fax: +33 (0)4.32.72.24.92
17 18
te
12
d
10
us
4
ip t
Aude A. Watrelota, b*, Carine Le Bourvelleca, b, Anne Imbertyc, Catherine M. G.
3
E-mail: [email protected]; [email protected]
19
20
Page 1 of 35
Abstract
22
Interactions between seven hairy regions of pectins, rhamnogalacturonans II and
23
arabinogalactan-proteins and procyanidins with different average degrees of polymerization,
24
low (DP9) and high (DP30), were investigated by isothermal titration calorimetry and
25
absorption analysis to study the impact of neutral sugar side chains of pectins on these
26
associations. Associations between pectic fractions and procyanidins involved hydrophobic
27
interactions and hydrogen bonds. No difference in association constants between various
28
hairy regions and procyanidins DP9 was found. Nevertheless, arabinan chains showed lower
29
association constants, and hairy regions of pectins with only monomeric side chains showed
30
higher association with procyanidin DP30. Only very low affinities were obtained with
31
rhamnogalacturonans II and arabinogalactan-proteins. Aggregation could be observed only
32
with the procyanidins of DP30 and the protein-rich arabinogalactan-protein. Associations
33
were obtained at both degrees of polymerization of the procyanidins, but differed depending
34
on neutral sugar composition and the structure of pectic fractions.
te
d
M
an
us
cr
ip t
21
36
Ac ce p
35
Highlights
37
• Pectic hairy regions-procyanidins interactions has been studied by ITC.
38
• The higher the pectic ramifications, the lower association with procyanidins is.
39
• Structures of both compounds are implicated.
40
41 42
Page 2 of 35
43
Keywords
44
Polyphenol, condensed tannins, polysaccharide, rhamnogalacturonan, association, ITC.
ip t
45
1. Introduction
47
Pectins are complex polysaccharides of plant cell walls, present mostly in the middle
48
lamella and primary wall. They comprise smooth and hairy regions. The “smooth regions”,
49
also known as homogalacturonans (HGs), consist of a long chain of (1→4) linkedα-D-
50
galacturonic acid (GalpA) with various degrees of methylation and/or acetylation. The hairy
51
regions, named rhamnogalacturonans (RGs), are of two types (I and II) (Thomas et al., 1987;
52
De Vries et al., 1982; Caffall & Mohnen, 2009). Rhamnogalacturonans I are formed by the
53
repeating unit →4)-α-D-GalpA-(1→2)-α-L-Rhap-(1→), the rhamnose residues of the RG
54
fractions being decorated by long side-chains composed mostly of galactose and arabinose
55
(Ridley, O’Neill & Mohnen, 2001; Voragen et al., 2009).RG I presents this strictly alternating
56
RG backbone(De Vries et al., 1982; Schols et al., 1990; Renard et al., 1995), while the
57
misleadingly named RG II possesses a backbone of (1→4) linkedα-D-GalpA as
58
homogalacturonans with side chains containing rhamnose and a variety of rare
59
monosaccharides (Pérez et al., 2003). Arabinogalactan-proteins (AGPs) are often associated
60
with pectins because both molecules contain a similar structural motif of type II galactans, i.e.
61
(1→3) and (1→6) linked galactans ramified with arabinan oligomers (Voragen et al., 2009).
62
However, in AGPs the arabinogalactan domains are connected via a specific hydroxyproline-
63
rich protein (Clarke, Anderson & Stone, 1979).
Ac ce p
te
d
M
an
us
cr
46
64
Procyanidins are flavonoids, major polyphenols found in vacuoles of fruits and
65
vegetables. They are a subclass of proanthocyanidins derived from the oligomerization of
66
flavan-3-ols unitsand form the condensed tannins. They are characterized by their constitutive
Page 3 of 35
units, their interflavanic linkages and their degree of polymerization (Hemingway &
68
Karchesy, 1989; Quideau et al., 2011).Specifically, apple procyanidins are characterized by
69
the predominance of (-)-epicatechin and of C4-C8 interflavanic linkages (Guyot et al.,1998;
70
Sanoner et al., 1999; Wojdyło, Oszmianski & Laskowski, 2008). They can be oligomers or
71
polymers and in cider apples, their number-average degree of polymerization range between
72
DP n 4.5 to 50 for varieties Jeanne Renard and Avrolles respectively (Sanoner et al., 1999).
73
They have been reported to exhibit several health effects as antioxidants and anticarcinogens,
74
and to protect against cardiovascular diseases.
us
cr
ip t
67
Fruit and vegetable processing ruptures cells. This disruption leads to association between
76
constituents of cell walls and vacuoles, with a prominent role of pectins and procyanidins (Le
77
Bourvellec, Bouchet & Renard, 2005). The bioavailability of polyphenols is dependent on
78
their structure and their association with other compounds such as sugars (Parada & Aguilera,
79
2007). Procyanidins associated with dietary fiber are not bioaccessible in the upper part of the
80
human intestine, and travel to the colon, where they become fermentable substrates for
81
bacterial microflora (Cheynier et al., 2012; Palafox-Carlos et al., 2011; Saura-Calixto et al.,
82
2010), producing metabolites that are both absorbable and bioactive (Williamson & Clifford,
83
2010).In vitro, the interaction between procyanidins and dietary fiber reduces the
84
polysaccharide fermentation and increases the production of procyanidin colonic metabolites
85
(Saura-Calixto et al., 2010; Aura et al., 2012). To understand the health effect of polyphenols
86
after binding to polysaccharides such as pectins, earlier work dealt with mechanisms of
87
interactions between these two macromolecules. In our previous work (Watrelot et al., 2013),
88
interactions between procyanidins and pectins were analyzed, and the impact of the degree of
89
methylation of smooth regions and of the degree of polymerization of procyanidins could be
90
demonstrated. However, the role of the hairy regions of pectins has not been determined. The
91
aim of this study was to quantify the binding of a specific class of proanthocyanidins, the
Ac ce p
te
d
M
an
75
Page 4 of 35
procyanidins, to hairy regions of pectins. Isothermal titration calorimetry and a
93
spectrophotometric method were used to probe thermodynamic parameters and aggregation
94
phenomena in interactions between hairy region fractions and procyanidins with different
95
average degrees of polymerization: low (DP9) and high (DP30).
ip t
92
96
2. Materials and methods
cr
97
99
2.1.
us
98
Chemicals
Methanol, acetonitrile, and acetone of chromatographic quality were from Biosolve
101
(Distribio, Evry, France). Hexane (Merck, Darmstadt, Germany) was of analytical quality.
102
Ethanol and acetone were from Fisher Scientific (Strasbourg, France).
103
Silver nitrate was from Merck (Darmstadt, Germany). Sodium borohydride (NaBH4),
104
N-methylimidazole, acetic anhydride, toluene-α-thiol and pectin from apple were from Sigma-
105
Aldrich (Deisenhofen, Germany). Chlorogenic acid, (+)-catechin, and (−)-epicatechin were
106
from Sigma-Aldrich. 4-Coumaric acid was from Extrasynthèse (Lyon, France). Phloridzin
107
and sugar standards were from Fluka (Buchs, Switzerland). Methanol-d3 was from Acros
108
Organics (Geel, Belgium). Enzymatic cocktail Endozym polifruit liq+ was from Spindal
109
groupe AEB (Gretz-Armainvilliers, France). Modified hairy regions (MHR1) from apple were
110
kindly supplied and characterized by Dr. M. C. Ralet (INRA-UR1268 Biopolymères
111
Interactions Assemblages, Nantes, France). Modified hairy regions MHR2, MHR3 and
112
MHR4 from pear, onion and soybean (SSPS) respectively were kindly supplied by Dr. Henk
113
Schols
114
Rhamnogalacturonans of type II monomer and dimer (mRG-II and dRG-II) and
Ac ce p
te
d
M
an
100
(WU
Agrotechnology
&
Food
Sciences,
Wageningen,
Netherlands).
Page 5 of 35
115
arabinogalactan-protein (AGP0 and AGP4) were kindly supplied by Dr. Claire Dufour
116
(INRA-UMR408 Sécurité et Qualité des Produits d’Origine Végétale, Avignon, France).
117 118
2.2.
119
Apple fruits (Malus domestica Borkh.) of the Avrolles and Marie Ménard varieties
120
were harvested at commercial maturity during the 2000 season in the experimental orchard of
121
the Centre Technique des Productions Cidricoles (CTPC, Sées, France). Fruits were
122
mechanically peeled and cored as previously described by Guyot et al. (1997) and cortex
123
tissues were freeze-dried. Apple pomace was from Val de Vire Bioactives (Condé-sur-Vire,
124
France). Fibrex 615 (Sugar beet pulp) was from Nordic Sugar (Copenhagen, Denmark).
an
us
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Plant material
M
125 126
2.3.
127
Procyanidins were extracted from “Marie Ménard” and “Avrolles” freeze-dried pulps
128
by successive solvent extractions and purified as described by Watrelot et al. (2013). The
129
purified procyanidin fractions were designated DP9 (from “Marie Ménard”) and DP30 (from
130
“Avrolles”). In the two fractions, (−)‐epicatechin was the predominant constitutive unit,
131
accounting for more than 95% and 88% in DP30 and DP9 respectively. For details, see
132
Watrelot et al. (2013)
d
te
Ac ce p
133
Procyanidin extraction and purification
2.4.
Rhamnogalacturonan preparation
134
Rhamnogalacturonans were prepared as described by Renard & Jarvis (1999) using 15
135
g/L of apple pectins saponified at 4 °C, pH > 12 and left overnight. The solution was then
136
brought to pH 6 and precipitated with three volumes of an ethanol/water/hydrochloric acid
137
mixture (96: 3: 1, v/v/v) with stirring at 4 °C and left overnight. This suspension was filtered
138
on a G0 sintered glass filter. After filtration the pectic acid precipitate was washed thoroughly
Page 6 of 35
with an ethanol/water mixture (70:30, v/v) until all remaining chloride ions were eliminated
140
(as verified by silver nitrate precipitation). The resulting powder was dried by solvent
141
exchange (ethanol 96% and acetone), followed by drying at 40 °C in an oven overnight. The
142
pectic acid was dissolved (6 g/L) and hydrolyzed with hydrochloric acid (0.25 N) for 72 h at
143
80 °C (Thibault et al., 1993).The supernatant was concentrated on a rotary evaporator before
144
dialyzing against 0.1 M NaCl followed by water to eliminate monomers and neutral oligomers
145
(galactose and arabinose). The rhamnogalacturonan oligomers (RG) were finally freeze-dried.
us
cr
ip t
139
Apple hairy regions were obtained by a modified alkaline peroxide treatment as
147
described by Renard et al. (1997). Apple pomace was suspended overnight at room
148
temperature with stirring at 50 g/L in water, and pH was stabilised at >13. Polyphenols were
149
then eliminated by adding 200 mL of hydrogen peroxide (30%). The medium was left to react
150
with stirring for 2 h. The pH was adjusted to 5 with acetic acid, and enzymatic cocktail
151
Endozym polifruit liq+ (1mL) was added and left to act overnight with stirring at room
152
temperature. The suspension was filtered on a nylon membrane (250 µm pore size) under
153
vacuum. The filtrate was dialyzed against 0.1 M NaCl, and then water at 4 °C. Finally, the
154
filtrate was freeze-dried to give the apple hairy region fraction (AHR).
156 157
M
d
te
Ac ce p
155
an
146
Acid-treated hairy regions (HR-H) were obtained from sugar beet pulp as described by
Ralet et al. (2010).
Modified hairy regions (MHR1) from apple were prepared by the method of Schols,
158
Posthumus & Voragen, (1990) and were a kind gift from Dr. M.-C. Ralet. Modified hairy
159
regions (MHR2 and MHR3) from pear and onion were prepared by the method of Schols &
160
Voragen, (1994). MHR4 from soybean was prepared by Coenen (2007). MHR2, MH3 and
161
MHR4 were a kind gift from Dr. Henk Schols. All MHRs resulting from drastic enzymic
162
hydrolysis of plant cell walls were isolated as high molar mass polymers with a
163
rhamnogalacturonan backbone. However, the structure of the side chain depends on the
Page 7 of 35
botanical origin (Table 1). MHR1 from apple was mostly composed of β-(1→4) linked
165
galactans and arabinans (Schols & Voragen, 1994; Renard et al., 1991). They were
166
characterized by a high xylose content with a molar ratio Xyl: GalA of 0.4, which
167
corresponds to xylogalacturonan chains (Schols et al., 1995). MHR2 from pear was rich in
168
arabinose present as (1→5)-linked chains (with proportion of 71 %). Its galactans were
169
predominantly highly ramified β-(1→3) and β-(1→6) type II galactan and galactose residues
170
were terminally linked (with proportion of 36 %) (Schols & Voragen,1994).MHR3 from
171
onion was rich in galactose with high proportion of terminal galactose residues (58 %) and in
172
β-(1→4) linked type I galactan(19 %) (Schols & Voragen, 1994), as was MHR4 from
173
soybean with the additionnal presence of β-D-Xyl side chains substituted to (1,4)-linked
174
galacturonic acid, i.e. also xylogalacturonans (Coenen et al., 2007; Nakamura et al., 2001).
M
an
us
cr
ip t
164
Polysaccharides isolated from red wine were kindly donated by Dr. C. Dufour (INRA-
176
UMR408 Sécurité et Qualité des Produits d’Origine Végétale, Avignon, France).
177
Rhamnogalacturonans, RG-IIs, were purified as described in Pellerin et al. (1996). Fractions
178
mRG-II and dRG-II were predominantly monomeric (90% pure) and dimeric (87% pure).
179
Monomer and dimer (mRG-II and dRG-II) were rich in arabinose and composed of rare
180
neutral sugars. The dimer was distinguished by the presence of boron.
te
Ac ce p
181
d
175
Type II arabinogalactan-protein fractions, AGP0 and AGP4, were isolated by anion
182
exchange chromatography according to Pellerin et al. (1995). Fractions were similar in terms
183
of neutral sugar composition except for arabinose and galactose with higher content in AGP0
184
(35.2 % and 57.3 % respectively) than AGP4 (27.1 % and 38.3 %, respectively), while uronic
185
acid content was higher in AGP4 than AGP0. Also, non-reducing arabinose and rhamnose
186
were observed in both AGPs, but rhamnose was 2,4-linked in AGP4. Galactose was 3,6-
187
linked more in AGP4, but 3,4,6-linked more in AGP0 (Pellerin et al., 1995).
188
Page 8 of 35
189
2.5.
Phase diagram
To identify aggregate formation during pectin-tannin interactions, a spectrophotometric
191
method was used at 25 °C. Systematic variation of concentrations and relative amounts was
192
carried out on a 96-well “microplate”. This was done by varying the concentrations of
193
procyanidins (0, 0.015, 0.03, 0.06, 0.117, 0.23, 0.47, 0.94, 1.875, 3.75, 7.5, 15 mmol/L (−)-
194
epicatechin equivalent) along the rows, and varying theconcentrations of hairy regions (0,
195
0.12, 0.23, 0.47, 0.94, 1.875, 3.75, 7.5 mmol/Lgalacturonic acid equivalent) along the
196
columns. Solutions were prepared in citrate/phosphate buffer at pH 3.8 and ionic strength
197
0.1 mol/L. Equal amounts (50 µL) of procyanidin and hairy region fraction solutions were
198
mixed before each spectrum measurement for 20 s. Absorbance spectra (200–650 nm) were
199
recorded, and absorbance at 650 nm was analyzed for each solution using a SAFAS flx-
200
Xenius XM spectrofluorimeter (SAFAS, Monaco). Control spectra were obtained using wells
201
containing hairy regions alone in buffer and procyanidins alone in buffer. After spectra
202
recording, microplates were centrifuged for 10 min at 2100g, and supernatants of control
203
wells (hairy regions at 7.5 mmol/L in buffer (S1A) and procyanidins at 15 mmol/L in buffer
204
(S1B)) and supernatants of wells containing procyanidins at a concentration of 15 mmol/L
205
with hairy regions at a concentration of 7.5 mmol/L (S2) were analyzed by high pressure size
206
exclusion chromatography (HPSEC) and high-performance liquid chromatography – diode
207
array detection (HPLC-DAD) to define changes in partition coefficient (ΔKav= S2-S1A) of
208
hairy region fractions and in average degree of polymerization of flavan-3-ols (ΔDPn = S2-
209
S1B).
Ac ce p
te
d
M
an
us
cr
ip t
190
210 211
2.6.
212
To measure enthalpy changes associated with hairy region-tannin interactions at
213
25 °C, a VP-ITC microcalorimeter (Microcal®, GE Healthcare, Northampton, MA, USA)
Isothermal Titration Microcalorimetry
Page 9 of 35
was used. To allow comparison between the different pectic substrates, and given the
215
polydispersity in molecular weight for both pectic fractions and procyanidins, all
216
concentrations are expressed relative to the main monomers, i.e. galacturonic acid for pectins
217
and (–)-epicatechin for procyanidins. Procyanidins and hairy region fractions were dissolved
218
in the same citrate/phosphate buffer pH 3.8, ionic strength 0.1 mol/L, and filtered on 0.45 µm
219
membrane. All the solutions were degassed before measurements. The reference cell was
220
filled with water. To obtain a hyperbolic curve as recommended for low affinity
221
systems(Turnbull & Daranas, 2003),different concentrations of compounds were tested. The
222
pectic fraction solution was placed in the 1.448 mL sample cell of the calorimeter, and the
223
procyanidin solution was loaded into the injection syringe and titrated into the sample cell by
224
30 injections of 10 µL aliquots. The duration of each injection was 20 s, with a separating
225
delay of 5 min. The contents of the sample cell were stirred throughout the experiment at 307
226
rpm to ensure mixing.
d
M
an
us
cr
ip t
214
Raw data obtained as a plot of heat flow (microcalories per second) against time
228
(minutes) were then integrated peak-by-peak and normalized to obtain a plot of observed
229
enthalpy change per mole of injectant (ΔH, kJ.mol−1) against the molar ratio
230
((−)‐epicatechin/galacturonic acid). Peak integration was performed using Microcal Origin 7.0
231
(Microcal Software, GE Healthcare). Control experiments included the titration of
232
procyanidin fractions into buffer, and were subtracted from titration experiments. The
233
experimental data were fitted to a theoretical titration curve using Microcal Origin 7.0, with
234
ΔH (enthalpy change), Ka (association constant), and n (number of binding sites per molecule)
235
as adjustable parameters, from the relationship
Ac ce p
te
227
∆ 2 236
0
1
1
1
1
2
4
, Eq. 1
Page 10 of 35
where Pt is the total galacturonic acid concentration, At is the total concentration of the ligand,
238
V0 is the volume of the cell, and Qi is the total heat released for injection i.
239
The other thermodynamic parameters (ΔG and ΔS) were calculated from the van’t Hoff
240
equation:
241
ip t
237
ΔG=-RTlnKa= ΔH-TΔS, Eq. 2
where ∆G is free enthalpy, Ka is the association constant, ∆H is the enthalpy and ∆S is the
243
entropy of interaction.
us
cr
242
244 245
2.7.
246
Polyphenols
were
measured
by
an
Analysis methods
high-performance
liquid
chromatography
(HPLC)/diode array detection (DAD) after thioacidolysis as described by Guyot et al. (2001)
248
and Le Bourvellec et al. (2011). Analyses were performed using an ultra fast liquid
249
chromatography Shimadzu Prominence system (Kyoto, Japan) comprising two LC-20AD
250
pumps, UFLCProminence liquid chromatograph, a DGU-20A5 Prominence degasser, a SIL-
251
20ACHT Prominence autosampler, a CTO-20AC Prominence column oven, a SPD-M20A
252
Prominence diode array detector, and a CBM-20A Prominence communication bus module,
253
and was controlled by LC Solution software (Shimadzu, Kyoto, Japan). Separations were
254
achieved in the conditions of Le Bourvellec et al. (2011). The average degree of
255
polymerization (DPn) of procyanidins was measured by calculating the molar ratio of all the
256
flavan-3-ol units (thioether adducts plus terminal units) to (−)-epicatechin and (+)-catechin
257
corresponding to terminal units. Neutral sugars, galacturonic acid and methanol were
258
analyzed as described by Renard & Ginies (2009).
Ac ce p
te
d
M
247
259
The molecular weight distribution of polysaccharides was determined using a high
260
pressure size exclusion chromatography (HPSEC) system comprising a Jasco LC-NET
261
II/ADC interface, a Jasco PU-2080 plus intelligent HPLC pump, a Jasco RI-2031 plus
Page 11 of 35
intelligent RI detector, and a degasser, and was controlled by ChromNav solftware (Jasco,
263
Tokyo, Japan). Separations were achieved using two columns in series: a 8.0 mm × 300 mm
264
i.d. OH-pack SB-802 HQ column (Showa Denko Europe, Munich, Germany) and a 300 ×
265
7.8 mm i.d. TSK-Gel PWXL column (Tosohaas, Stuttgart, Germany) at 35 °C and a 40 ×
266
6.0 mm i.d. guard column TSK-Gel PWXL (Tosohaas, Stuttgart, Germany). Solutions
267
(20 μL) of the extracts (10 g/L) were injected and eluted with 0.4 mol/Lsodium acetate buffer
268
pH 3.5 at 0.8 mL/min. Dextrans T500, T70, T40 and T10 (Pharmacia BioProcess Technology,
269
Uppsala, Sweden) and glucose (Sigma-Aldrich, Deisenhofen, Germany) were used to
270
calibrate the column system.
an
us
cr
ip t
262
271 272
2.8.
273
Results are presented as mean values, and the reproducibility of the results is
274
expressed as pooled standard deviation. Pooled standard deviations were calculated for each
275
series of replicates using the sum of individual variances weighted by the sum of individual
276
degrees of freedom (Box, Hunter & Hunter, 1978).
278 279
M
d
te
Ac ce p
277
Statistical analysis
3. Results 3.1.
Composition of the hairy regions
Page 12 of 35
Table 1. Samples (mg/g)
Kav
RG
0.26
49
3
45
AHR
0.28
23
2
251 16
-
30
19
167
HR-H
0.13 143
-
25
-
199
-
437
1.7
0.1 9.6 0.7
236 128 458
0.9
9.5 5.7
4.5
385
MHR1
−0.06
89
9
83 113
-
134
a
MHR2
−0.03
79
-
240 42
-
101
a
MHR3
0.01 132
-
40
MHR4
-
Arabinan Type I galactan Xylogalacturonan
209
Arabinan Type II galactan
nd
41
33 188 52
-
351 27
165
Galactan Xylogalacturonan Type II Arabinogalactan
17
7
208
-
M
b
-
RG with monomeric galactans
an
a
Arabinan
ip t
-
10
cr
-
15
Characteristic structure
us
Pooled SD
Rha Fuc Ara Xyl Man Gal Glc GalA
283
Type I galactan
c
AGP0
0.08
11
1
358
2
18
583 16
28
c
AGP4
0.02
93
2
250 18
23
354
180
d
280
3
RG, rhamnogalacturonans; AHR, apple hairy regions; HR-H, acid-treated hairy regions; MHR,
282
modified hairy regions (1 from apple, 2 from pear, 3 from onion and 4 from soybean); AGPs,
283
arabinogalactan-proteins; Kav, partition coefficient; Rha, rhamnose; Fuc, fucose; Ara, arabinose; Xyl,
284
xylose; Man, mannose; Gal, galactose; Glc, glucose; GalA, galacturonic acid, Pooled SD: pooled
285
standard deviation, n =11.a: Schols and Voragen 1994; b: Coenen 2007; c: Pellerin et al., 1995.
287
Ac ce p
286
te
281
The characterization of hairy regions is shown in Table 1. RG contained a high
288
proportion of galacturonic acid, with a molar ratio GalA: Rha > 7, indicating the
289
predominance of homogalacturonans in the backbone. Its main neutral sugars were galactose
290
and glucose. AHR contained a substantial portion of homogalacturonans (GalA: Rha = 6), but
291
unlike RG, it also contained a high proportion of arabinan (molar ratio Ara: Rha > 10). HR-H
292
was the simplest rhamnogalacturonan fraction, with the lowest neutral sugar content. The
Page 13 of 35
molar ratio GalA: Rha of 2.5 is consistent with the rhamnogalacturonic backbone, while the
294
low (Gal+Ara): Rha ratio (< 2) corresponds to very short side chains of arabinogalactan
295
(Voragen et al., 2009).MHR1 and MHR4 contained more homogalacturonic backbone (GalA:
296
Rha = 3.6 and 3.4 respectively) than MHR2 and MHR3 with a molar ratio GalA: Rha of 2.2
297
and 1.8 respectively.
ip t
293
The partition coefficients of RG and AHR were identical (Kav of 0.26 and 0.28
299
respectively with retention times of 17.2 min and 17.4 min), corresponding to small polymers
300
obtained after acid hydrolysis from commercial pectins or after enzymatic reaction from apple
301
pomace. The partition coefficient of HR-H fraction was lower (Kav of 0.13 with a retention
302
time of 16.3 min), indicating a higher hydrodynamic volume. Partition coefficients were close
303
between RG-IIs (Kav of 0.27 for mRG-II and 0.22 for dRG-II) and were in good agreement
304
with calculated molecular weight (Pellerin et al., 1996). Partition coefficients of AGPs were
305
low (Kav of 0.02 for AGP4) and similar to commercial apple pectins (Watrelot et al., 2013).
d
M
an
us
cr
298
308 309
3.2.
Interactions with procyanidins of DPn 9
Ac ce p
307
te
306
3.2.1. Hairy regions
Thermograms of titration of hairy region fractions (3.75 mmol/L galacturonic acid
310
equivalent) by procyanidins DP9 (7.5 mmol/L (−)-epicatechin equivalent) are shown in
311
Figure 1.
312 313 314
Figure 1. Error! Reference source not found.
315
The blank experiment (injections of procyanidins DP9 in buffer) yielded small endothermic
316
peaks that were subtracted before integration. The thermogram of titration of RG by
Page 14 of 35
procyanidins DP9 (Figure 1a) showed small exothermic peaks for the first injections, and
318
stronger endothermic peaks after saturation. The thermogram of AHR titration by
319
procyanidins DP9 (Figure 1b) showed strong exothermic peaks (−8 µJ/sec) with clear
320
titration.
Ac ce p
te
d
M
an
us
cr
ip t
317
Page 15 of 35
i RG
AHR
HR-H
0.01
0.02
0.02
2530
1955
∆H (kJ.mol )
−6
∆G (kJ.mol-1)
−19
n -1
Ka (M ) -1
-1
-1
45
MHR1
MHR2
MHR3
MHR4
mRG-II
dRG-II
AGP0
AGP4
0.02
0.02
0.02
-
-
-
0.02
0.02
1850
2105
872
2225
-
-
-
44
57
−3
−18
−5
−16
−26
-
-
-
−67
−38
−19
−19
−19
−17
−19
-
-
-
−9
−10
51
1
47
2
−25
-
-
-
−193
−95
0.000
0.006
0.001
−0.002
−0.001
nd
0.002
−0.001
0.001
0.005
−0.001
Δ DP n DP 30
−1.61
−1.06
−1.07
0.17
0.00
−0.55
nd
0.15
−0.16
−0.22
0.15
RG
AHR
HR-H
MHR1
MHR2
MHR3
MHR4
RGII2
RGII3
AGP0
AGP4
0.02
0.02
0.02
0.02
0.02
0.02
-
0.02
0.02
0.02
0.02
2620
574
5390
1835
875
1170
-
72
62
112
165
∆H (kJ.mol )
−3
−17
−4
−7
−24
−41
-
−16
−14
−54
−31
∆G (kJ.mol-1)
−19
−16
−21
−19
−17
−17
-
−11
−10
−12
−13
56
−4
59
38
−23
−78
-
−18
−12
−142
−61
ΔKav
0.004
0.004
0.015
0.011
0.004
−0.006
nd
0.000
−0.004
0.009
0.011
Δ DP n
−11.82
−10.17
−9.49
−4.89
−5.01
−8.44
nd
0.28
0.27
−3.62
0.53
pt
ΔKav
ce
∆S (J.mol .K )
-1
Ka (M )
Ac
n -1
-1
-1
∆S (J.mol .K )
323
us
cr DP 9
M
(30 mmol/L in (−)-epicatechin equivalent).
ed
322
an
Table 2.Thermodynamic binding parameters of interaction between pectic compounds and procyanidin fractions DP9 and DP30
321
Average of duplicates for each measurement. Stoichiometry corresponds to the molar ratio value of (−)-epicatechin/galacturonic acid. nd: not defined
Page 16 of 35
Thermodynamic parameters are shown in Table 2.Stoichiometry (defined as ratio of (−)-
325
epicatechin/galacturonic acid) was fixed at 0.01 for RG (1 molecule of (−)-epicatechin binds
326
with 100 molecules of galacturonic acid) and 0.02 for other fractions (1 molecule of (−)-
327
epicatechin binds with 50 molecules of galacturonic acid) using a one-site model. The
328
association constant (Ka = 2.53 × 103 M-1) observed for RG and DP9 was of the same order of
329
magnitude as that measured for AHR, HR-H, MHR1 and MHR3 (Ka from 1955 M-1 to 2225
330
M-1). The value of Ka was lower for interaction between MHR2 and procyanidins DP9 (Ka =
331
872 M-1). Analysis of the thermodynamic contributions indicated an enthalpy contribution
332
(∆H = −18 kJ.mol-1,−16 kJ.mol-1 and −26 kJ.mol-1 for HR-H, MHR2 and MHR3 respectively)
333
related to the exothermic interaction. The entropy term between HR-H, MHR2 and
334
procyanidins DP9 was almost null (−T∆S = −0.3 to −0.5 kJ.mol-1 respectively), indicating a
335
weak role of hydrophobic interactions. The analysis of the thermodynamic contributions for
336
MHR3 gave a negative entropy term (−TΔS = 7 kJ.mol-1) indicating that the interaction was
337
driven by enthalpy, which may result from hydrogen bond association. A favorable entropy
338
contribution was observed for RG, AHR and MHR1 (−T∆S ~ −13 kJ.mol-1), indicating that
339
the interaction was driven mostly by entropy, which may result from hydrophobic interactions
340
(Poncet-Legrand et al., 2007; Frazier et al., 2010). The titration of MHR from soybean
341
(MHR4) by procyanidins DP9 (data not shown) displayed strong exothermic peaks, but no
342
thermodynamic parameters could be defined.
Ac ce p
te
d
M
an
us
cr
ip t
324
17
Page 17 of 35
Figure 2.
M
an
us
cr
ip t
343
Ac ce p
te
d
344
345
The absence of differences in affinity between hairy regions and procyanidins DP9
346 347
was confirmed by UV-vis absorbance at 650 nm. Figure 2a, c shows the variation of
348
absorbance at 650 nm between hairy regions (RG, AHR, HR-H, MHR1, MHR2 and MHR3)
349
and procyanidins DP9 at 15 mmol/L (−)-epicatechin equivalent with the increase of
350
concentrations of hairy regions, standardized here in galacturonic acid equivalent. Absorbance
351
at 650 nm was chosen as an indicator for the formation of cloud and precipitate, as neither
352
pectins nor procyanidins absorb at this wavelength. Absorbance of all hairy regions with
353
procyanidins DP9 was low (0.04), quite stable and similar between hairy regions. Only the 18
Page 18 of 35
MHR3 fraction showed an increase in absorption up to 0.08 from 1.875 mmol/L galacturonic
355
acid equivalent. Figure 2b, d shows the variation in absorbance between hairy regions (RG,
356
AHR, HR-H, MHR1, MHR2 and MHR3) at 7.5 mmol/L galacturonic acid equivalent and
357
procyanidins DP9 with the increase in concentrations of procyanidins DP9, given in (−)-
358
epicatechin equivalent. Initially, all hairy regions displayed a slight turbidity, which
359
disappeared with addition of a low concentration of procyanidins. With the increase in
360
procyanidin concentrations, absorbance of hairy regions RG, AHR and HR-H increased
361
slightly (up to 0.04) and in a similar fashion. Among MHRs fractions (Figure 2d), MHR3
362
showed an increase in absorption up to 0.08 from 0.94 mmol/L (−)-epicatechin equivalent,
363
while MHR1 and 2 were stable around 0.01.
an
us
cr
ip t
354
The ∆Kav correspond to the difference in partition coefficient in HPSEC between the
365
pectic hairy regions (at 7.5 mmol/L in galacturonic acid equivalent) that had not formed
366
aggregates with procyanidins (at 15 mmol/L (−)-epicatechin equivalent) (S2) and the initial
367
pectic compounds in buffer (S1A). After centrifugation of hairy regions incubated with
368
procyanidins DP9, values of ∆Kav obtained were close to nil irrespective of the pectic
369
fractions (Table 2). There was no difference in hydrodynamic volume for hairy regions
370
before and after association with procyanidins DP9.The ∆DPn corresponds to the difference in
371
degree of polymerization between procyanidins that had not formed aggregates with pectins
372
(S2) and the initial degree of polymerization of procyanidins in buffer (S1B). The ∆DPn was
373
−1.61 for RG and −1.06 for AHR and HR-H. The degree of polymerization of free
374
procyanidins was slightly lower than the initial degree of polymerization of procyanidins, i.e.
375
hairy regions RG, AHR and HR-H precipitated preferentially with highly polymerized
376
procyanidins. The value of ∆DPn of MHR3 was slightly negative, but there was no difference
377
for MHR2 and MHR1. This means that MHR3, MHR1 and MHR2 were not selective with
378
procyanidins DP9.
Ac ce p
te
d
M
364
19
Page 19 of 35
379
3.2.2. Rhamnogalacturonans II and arabinogalactan-proteins During injection of procyanidins in rhamnogalacturonans type II (mRG-II and dRG-II),
381
peaks were endothermic and heat was absorbed. No titration could be observed. Hence no
382
interaction could be detected by ITC between RGIIs and procyanidins DP9.
ip t
380
A thermogram of titration of arabinogalactan-protein by procyanidins DP9 displayed only
384
exothermic peaks (data not shown). Thermodynamic parameters were calculated from raw
385
data after subtraction of blank experiments (procyanidins DP9 in buffer) (Table 2).
386
Stoichiometry was fixed at 0.02 using a one-site model. Association constants obtained for
387
AGP0 and AGP4 were very weak (Ka = 44 M-1 and 57 M-1 respectively) and correspond to
388
the limit of detection by isothermal titration calorimetry.
M
an
us
cr
383
The variation of absorbance at 650 nm between rhamnogalacturonans II and
390
arabinogalactan-proteins and procyanidins DP9 at 15 mmol/L (−)-epicatechin equivalent with
391
the increase in concentrations of rhamnogalacturonans in galacturonic acid equivalent is
392
shown in Figure 2e. Whatever the pectic fraction, the absorption was stable at around 0.04.
393
The variation of absorbance between RG-IIs and AGPs at 7.5 mmol/L galacturonic acid
394
equivalent and procyanidins DP9 with the increase in concentrations of procyanidins DP9 in
395
(−)-epicatechin equivalent (Figure 2f) was low (0.01) and stable between fractions, but the
396
dimer of RGII (dRG-II) showed absorption at 0.03.
Ac ce p
te
d
389
397
The ΔKav obtained was close to nil for all RGIIs and AGPs. With RGIIs and AGPs, the
398
ΔKav and ∆DPn were very low or nil: there was no selective precipitation of either the
399
procyanidins or the polysaccharides.
400 401
3.3.
Interaction with procyanidins of DPn 30 20
Page 20 of 35
402
3.3.1. Hairy regions ITC measurements were made with 3.75 mmol/L of pectic compound (galacturonic
403
acid equivalent) and 7.5 mmol/L ((−)-epicatechin equivalent) of procyanidins DP30.
405
Figure 3.
406
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c
ip t
404
Titration of RG, AHR, HR-H and MHR1, MHR2 and MHR3 by procyanidins DP30
408
showed complex curves characterized by strong exothermic peaks (Figure 3a and 3b); to
409
avoid over-fitting, the stoichiometry was fixed at 0.02 for all fractions to allow fit and
410
determination of association parameters. Thermodynamic parameters are presented in Table
411
2. The association constant Ka ranged from 574 M-1 to 5390 M-1 in the order
412
AHR
