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Isolation from Sugar Beet Cell Walls of Arabinan Oligosaccharides Esterified by Two Ferulic Acid Monomers1 Se´bastien V. Levigne, Marie-Christine J. Ralet*, Bernard C. Que´me´ner, Brigitte M.-L. Pollet, Catherine Lapierre, and Jean-Franc¸ois J. Thibault Unite´ de Recherche sur les Polysaccharides, leurs Organisations et Interactions, Institut National de la Recherche Agronomique (INRA), boîte postale 71627, 44316, Nantes cedex 03, France (S.V.L., M.-C.J.R., B.C.Q., J.-F.J.T.); and Laboratoire de Chimie Biologique, INRA-Institut National Agronomique Paris-Grignon, 78850 Thiverval-Grignon, France (B.M.-L.P., C.L.)
Side chains of sugar beet (Beta vulgaris) pectins, which are mainly composed of arabinose (Ara) and galactose (Gal) residues, are esterified by ferulic acid units. Enzymatic hydrolysis of beet cell walls yielded several feruloylated oligosaccharides, which were separated by hydrophobic interaction chromatography. Two new oligomers were isolated in the fraction eluted by 25:75 (v/v) ethanol:water. An arabinotriose and an arabinotetraose esterified by two ferulic acid residues were obtained, and their structure was elucidated by mass spectrometry. It is shown that feruloyl groups are linked to O-5 of Ara residues, in addition to the known O-2 position. This work establishes for the first time, to our knowledge, that two neighboring Ara units may be esterified by two ferulic acid units. This close proximity may have important biochemical implications.
It is now well established that ferulic acid is one of the major phenolic acids in ester linkages of polysaccharides in the cell walls of several plants (Ishii, 1997). In the family Poaceae, ferulic acid is mostly linked to O-5 of l-Araf residues from arabinoxylans (Ishii, 1997; Saulnier and Thibault, 1999). In dicotyledons, phenolic acids seem to be restricted to the order Caryophyllales (Hartley and Harris, 1981). This order contains the Chenopodiaceae family that has a number of economically important species, including spinach (Spinacia oleracea) and sugar beet (Beta vulgaris). In beet and spinach cell walls, ferulic acid mainly esterifies neutral sugars (Ara and Gal) of pectic side chains (Fry, 1983; Rombouts and Thibault, 1986; Guillon and Thibault, 1989). The exact locations of these ester linkages were determined by treating spinach-leaf walls and sugar beet pulp with Driselase, a commercial enzymatic mixture from the fungus Irpex lacteus that contains several pectolytic enzymes but lacks hydroxycinnamoyl esterase activities. Linkages of ferulic acid to O-2 of l-Araf and O-6 of d-Galp were shown (Ishii and Tobita, 1993; Colquhoun et al., 1994) by the isolation of: a feruloyl arabinobiose [O-(2-O-transferuloyl)-␣-l-Araf-(135)-l-Araf], a feruloyl arabi1
This work was supported by the European Community (Europectin QLK3–1999 – 00089 Program), by the Re´gion Ile de France (SESAME Grant for financial support of the LC-MS equipment in Grignon), and by INRA (financial support of the LC-MS equipment in Grignon). * Corresponding author; e-mail
[email protected]; fax 33– 240675066. Article, publication date, and citation information can be found at http://www.plantphysiol.org/cgi/doi/10.1104/pp.103.035311.
notriose [O-␣-l-Araf-(133)-O-(2-O-trans-feruloyl)-␣l-Araf-(135)-l-Araf], and a feruloyl galactobiose [O-(6-O-trans-feruloyl)--d-Galp-(134)-d-Galp]. The recovered feruloyl groups (approximately 29% and approximately 10% of the ferulic acid initially present in the cell wall for sugar beet and spinach, respectively; Ishii and Tobita, 1993; Ralet et al., 1994) are linked to the Araf residues of the main core of ␣-(1–5)-linked arabinan chains and to the Galp residues of the main core of -(1–4)-linked galactan chains. A feruloyl Ara-Xyl trisaccharide {O-[5-O(trans-feruloyl)-␣-l-arabinofuranosyl-(133)-O--dxylopyranosyl-(134)-d-xylopyranose} was also isolated from cell walls of sugar beet, demonstrating that some ferulic acid residues are linked to arabinoxylans in these dicotyledon cell walls (Ishii, 1994). Feruloylated oligosaccharides also have been isolated from sugar beet by mild acid hydrolysis (Ralet et al., 1994). After acid hydrolysis, Ara residues were mostly removed (approximately 90%) together with 54% of the ferulic acid. Therefore, it was concluded that approximately 55% of the ferulic acid was linked to Ara residues and the remaining 45% to Gal ones. It was calculated that one in 56 Ara residues and one in 16 Gal residues carry a feruloyl group (Ralet et al., 1994). Ferulic esters are of great interest because they may form dehydrodimers through in vivo oxidative coupling reactions (Fry, 1986). Such reactions enable cross-linking of cell wall polysaccharides (Ishii, 1991; Saulnier et al., 1999), which controls cell wall extensibility and mechanical properties (Fry, 1986; Kamisaka et al., 1990; Ralph and Helm, 1993). For many years, the formation of arabinoxylan-linked diferulates has been assumed to be catalyzed exclusively in
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Water-Eluted Oligosaccharides
Figure 1. Fractionation of the soluble material of CWM after enzymatic hydrolysis on Sephadex LH-20. First elution step: water. Œ, A325; ‚, total neutral sugars; F, GalUA.
muro by wall-bound peroxidases. However, recent studies have shown that the formation of arabinoxylan-bound diferulates occurs not only in muro but also intracellularly, presumably in the Golgi apparatus (Fry et al., 2000; Obel et al., 2003). Feruloyl esters may be also involved in the crosslinking of polysaccharides to lignins (Ralph et al., 1995) and potentially to proteins (Fry, 1986). In the present paper, we describe the isolation of novel feruloylated oligosaccharides obtained by enzymatic hydrolysis of sugar beet cell walls and their structural identification by electrospray ionization (ESI)-ion trap (IT)-mass spectrometry (MS). The regiochemical distribution of two distinct ferulic acids on an arabinotriose and an arabinotetraose revealed novel insights on the structure of hydrophobic pectic fragments. Our view of the chemical structure of pectic side chains is complemented, and the role of ferulic acid in dicotyledons cell walls is discussed.
The elution profile (Fig. 1) shows a series of peaks eluting between 0 and 4 column volumes. The unresolved peak eluting at less than 1 column volume contained ferulic acid (16% of total injected ferulic acid), neutral sugars (96% of total injected neutral sugars), and GalUA (100% of total injected GalUA). Another broad peak containing ferulic acid (23% of total injected ferulic acid) and neutral sugars (2.5% of total injected neutral sugars) was eluted between 1 and 1.8 column volumes. Ralet et al. (1994) showed that this peak corresponds to feruloylated oligosaccharides with high degrees of polymerization. Three well-defined peaks eluted at 2.0 (fraction watereluted oligosaccharide 1 [WO1]), 2.7 (fraction WO2), and 3.7 (fraction WO3) column volumes. The structure of these oligomers, previously identified by NMR spectroscopy (Colquhoun et al., 1994), was confirmed by ESI-IT-MS (data not shown). WO1 was identified as feruloylated galactobiose, O-[6-O(trans-feruloyl)--d-Galp]-(134)-d-Galp; WO2 as feruloylated arabinotriose, O-␣-l-Araf-(133)-O-[2-O(trans-feruloyl)-␣-l-Araf]-(135)-l-Araf; and WO3 as feruloylated arabinobiose, O-[2-O-(trans-feruloyl)-␣l-Araf]-(135)-l-Araf. The small peak eluting at 5.6 column volumes was composed of free ferulic acid (⬍3% of total ferulic acid injected). 25% (v/v) Ethanol-Eluted Oligosaccharides
The elution profile (Fig. 2) showed three major peaks eluting at 9 (fraction ethanol-eluted oligosaccharide 1 [EO1]), 9.7 (fraction EO2), and 12 (fraction EO3) column volumes. The fractions of each peak were pooled, concentrated, and freeze dried. Their compositional analyses are presented in Table I. All the 25% (v/v) ethanol-eluted fractions contained minor amounts of Glc, probably arising from column “bleeding.”
RESULTS Isolation of Feruloylated Oligosaccharides
Sugar beet cell wall material (CWM; 213 mg g⫺1 GalUA, 212 mg g⫺1 Ara, 56 mg g⫺1 Gal, 8.2 mg g⫺1 ferulic acid, and 0.65 mg g⫺1 ferulic dehydrodimers) was degraded with Driselase. The solubilized material was loaded onto a column of Sephadex LH-20 eluted successively by water, 25% (v/v) ethanol, 50% (v/v) ethanol, and 100% (v/v) ethanol. Because the 50% (v/v) ethanol and 100% (v/v) ethanol pools were devoid of neutral sugars or ferulic compounds, these fractions were not studied further. 1174
Figure 2. Fractionation of the soluble material of CWM after enzymatic hydrolysis on Sephadex LH-20. Second elution step: 25% (v/v) ethanol. Plant Physiol. Vol. 134, 2004
Feruloylated Oligoarabinans from Sugar Beet
Table I. Chemical composition (mol%) of feruloylated fractions eluted with water (WO) and 25:75 (v/v) EtOH:water (EO) Fractions
WO1 WO2 WO3 EO1 EO2 EO3
Rha
Ara
Xyl
Gal
Glc
Ferulic Acid
Ferulic Dimers
⫺ ⫺ ⫺ 1 ⫺ ⫺
3 74 67 43 48 38
⫺ ⫺ ⫺ 27 ⫺ ⫺
66 ⫺ 1 7 ⫺ ⫺
⫺ 1 ⫺ 9 8 13
31 25 32 11 41 49
⫺ ⫺ ⫺ 3 3 ⬍1
Fraction EO1 (0.6 mg) was mainly composed of Ara, Xyl, and ferulic acid. Rha, Gal, Glc, and ferulic dimers were also detected in lower amounts (Table I). The HPLC-MS (ESI-MS in the negative ion mode) analysis (data not shown) revealed several chromatographic peaks with distinct [M-H]⫺ ions. This fraction EO1, which was constituted of a mixture of components, was not studied further. Fraction EO2 (0.5 mg) was mainly composed of Ara and ferulic acid with minor amounts of ferulic dimers (Table I). The HPLC-ESI-MS analysis (negative ion mode) revealed that it essentially corresponded to a unique chromatographic peak with a deprotonated [M-H]⫺ ion at mass-to-charge ratio (m/z) 897 (data not shown). The corresponding molecule (mass 898 D), therefore, could correspond to
four Ara residues associated with two ferulic acid units. To confirm this tentative assignment and to determine the structure of this feruloylated oligosaccharide, EO2 was directly infused into the electrospray source, and a unique ion at m/z 897 was detected (Fig. 3a). MSn experiments were performed by negative ESI-IT-MS (Fig. 3, b–d). The fragment ions generated were from the A, C, and Z series, according to the nomenclature established by Domon and Costello (1988), as previously reported for neutral disaccharides (Garozzo et al., 1990) and oligogalacturonates (Que´ me´ ner et al., 2003a). Figure 4 shows the proposed structure and the different cleavages observed. After isolation and collision induced dissociation of the [M-H]⫺ ion at m/z 897, a series of fragment ions appeared (Fig. 3b). An ion at m/z 879 corresponding to the loss of one water molecule was detected. The major ions at m/z 837 and 807 corresponding to losses of 60 (C2H4O2) and 90 (C3H6O3) D, respectively, resulted from cross-ring cleavages (Garozzo et al., 1991). By analogy with the fragmentation of other carbohydrates (Harvey, 2000), they were assigned to the 0,2A4 and 0,3A4 fragment ions. Therefore, the reducing Ara residue is linked through O-5. A fragment ion at m/z 765 (loss of 132 D), corresponding to a C3 fragment ion resulting from the loss of one Ara unit, was produced. Therefore, the reduc-
Figure 3. Negative MSn experiment spectra of EO2. a, Full MS spectrum; b, MS2 experiment (m/z 897 ⬎ products); c, MS3 experiment (m/z 897 ⬎ 765 ⬎ products); d, MS4 experiment (m/z 897 ⬎ 765 ⬎ 457 ⬎ products). Plant Physiol. Vol. 134, 2004
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Figure 4. Proposed structure of EO2 feruloylated oligosaccharide and observed cleavages.
ing Ara residue is not substituted by a ferulic acid unit. It is noteworthy that the cross-ring cleavage ions were more abundant that glycosidic bond cleavage ions. Similar results were previously obtained in negative ESI, particularly on MSn mass spectra of Ci ions substituted on O-2 by sugar residues (Li and Her, 1998; Cheng and Her, 2002) or by an acetyl group (Que´ me´ ner et al., 2003b). A series of minor ions were detected at m/z 661, 631, and 589 corresponding to losses of 236 (60 ⫹ 176), 266 (90 ⫹ 176), and 308 (132 ⫹ 176) D, respectively. Because the mass of ferulic acid is 176 D, these fragments were assigned to 0,2A’4, 0,3A’4, and C’3 fragment ions, released from an isomer. This isomer, characterized by its reducing Ara residue substituted by one ferulic acid on position 2, was not studied further. MS3 analysis of the C3 ion (m/z 897 ⬎ 765 ⬎ products) provided several fragment ions (Fig. 3c). Besides fragment ion at m/z 747 (water loss), fragment ions at m/z 529 and 499 were assigned to the cross-ring cleavage 0,2A3 and 0,3A3 ions with losses of 236 and 266 D, respectively. Such losses, derived from a double cleavage across the Ara ring, correspond to the feruloylated fragments (60 ⫹ 176) and (90 ⫹ 176). This diagnostic fragmentation allowed us to conclude that the second Ara residue from the reducing end carries one ferulic ester group on position 2 and is linked through O-5. Fragment ion at m/z 457 (loss 308 D, i.e. one Ara esterified by one ferulic acid) corresponds to a C2 fragment ion and confirms that the second Ara residue from the reducing end carries one ferulic acid. As before, 0,2Ai ions were produced in greater abundance than Ci ions. A minor Z3 fragment ion (m/z 571/765) also was identified. MS4 analysis of the C2 ion (m/z 897 ⬎ 765 ⬎ 457 ⬎ products) revealed several peaks (Fig. 3d). Fragment 1176
ions at m/z 397 and 367 (losses of 60 and 90 D, respectively) were assigned to the cross-ring cleavage 0,2 A2 and 0,3A2 ions and that at m/z 325 to a C1 fragment ion. This demonstrates that the third Ara residue from the reducing end is not esterified by ferulic acid and is again linked through O-5. Thus, the last ferulic group must be located on the fourth Ara residue from the reducing end. Its exact location was determined by the cross-ring cleavage of the last Ara residue. Fragment ions at m/z 265 and 235 (losses of 60 and 90 D) were again assigned to the cross-ring cleavage 0,2A1 and 0,3A1 ions from the C1 ion at m/z 325, providing assumption that ferulic acid is located on position 5. The fragment ion at m/z 307 may be because of water loss from the C1 fragment ion at m/z 325. The fragment ion at m/z 247 could be assigned to the cross-ring cleavage 0,2A1 from the ion at m/z 307. Therefore, the fraction EO2 main structure was assigned to O-[5-O-(feruloyl)-Ara]-(135)-Ara-(135)-[2O-(feruloyl)-Ara]-(135)-Ara (Fig. 4). Fraction EO3 (0.6 mg) was mainly composed of Ara and ferulic acid (Table I). The HPLC-ESI-MS analysis (negative ion mode) revealed that this fraction mainly comprised one major component and two minor ones (not shown). The major oligosaccharide had a nominal mass of 766 D, as evidenced from the deprotonated ion at m/z 765. Such a mass was tentatively assigned to a feruloylated oligosaccharide comprising three arabinoses associated with two ferulic acids. We employed the same MSn strategy as the one used for fraction EO2 to elucidate the structure of this feruloylated oligosaccharide. Figure 5 shows the proposed structure and the different cleavages observed. Full MS analysis of EO3 by direct infusion revealed the presence of a very major peak at m/z 765 (Fig. 6a). Plant Physiol. Vol. 134, 2004
Feruloylated Oligoarabinans from Sugar Beet
Figure 5. Proposed structure of EO3 feruloylated oligosaccharide and observed cleavages.
MS2 analysis of the [M-H]⫺ ion at m/z 765 (Fig. 6b) provided a series of peaks, the major ones resulting from cross-ring cleavages of the Ara at the reducing end. The ion at m/z 747 corresponds to the loss of one water molecule, and the ions at m/z 735, 705, and 675 correspond to losses of 30, 60, and 90 D, respectively, revealing that the reducing Ara is not esterified and is linked through O-5. The C2 fragment ion at m/z 633 (loss of 132 D) confirmed that this first Ara residue is not substituted. As already shown for the EO2 fraction, series of minor ions were detected at m/z 529, 499, and 457, corresponding to losses of 236, 266, and 308 D, respectively. They were assigned to 0,2A’3, 0,3 A’3, and C’2 fragment ions from an isomeric structure bearing one ferulic acid on position 2 of the reducing Ara residue. This isomer was not studied further. MS3 analysis of the C2 ion (m/z 765 ⬎ 633 ⬎ products; Fig. 6c) showed a major peak at m/z 615 corresponding to the loss of water and other peaks at m/z 397 and 367. These ions were allocated to the 0,2 A2 and 0,3A2 fragments with losses of 236 and 266 D. Such a fragmentation pattern reveals that the central Ara residue is esterified by one ferulic acid on position 2 and linked through O-5. A minor Z2 fragment ion (m/z 439/633) was identified together with ions at m/z 247 and 217 that could correspond to cross-ring cleavages. An ion at m/z 497 was detected but not identified. Because of the low abundance of the C2 ion (m/z 633) before its isolation and collisioninduced dissociation, no C1 fragment ion could be detected. Plant Physiol. Vol. 134, 2004
MS3 analysis of the intense 0,2A3 fragment ion (m/z 765 ⬎ 705 ⬎ products) was tentatively performed to determine the substitution location of the nonreducing Ara residue. The C2 fragment ion at m/z 633 was detected together with the ion at m/z 615 corresponding to one water loss. A minor Z2 fragment ion (m/z 439) was also identified. Trace ions at m/z 529 and 457 could be assigned to the loss of one ferulic acid (⫺176) from the 0,2A3 (m/z 705) and C2 (m/z 633) ions, respectively. Fragment ions at m/z 397 and 367 were detected. These ions were again assigned to the 0,2 A2 and 0,3A2 fragment ions with losses of 236 and 266 D, confirming that the second Ara residue from the reducing end is esterified by one ferulic acid on position 2 and linked through O-5. The C1 fragment ion at m/z 325 was detected together with a fragment ion at m/z 235. This last ion was assigned to the cross-ring cleavage 0,3A1 from ion at m/z 325 (C1 fragment ion), providing assumption that the ferulic acid is located on position 5. Furthermore, fragment ions at m/z 247 and 217 could be assigned to the cross-ring cleavage 0,2A1 and 0,2A1 from ion at m/z 307 (C1 fragment ion—water), confirming the ferulic acid location on position 5. The fraction EO3 structure was assigned to O-[5-O-(feruloyl)-Ara]-(135)-[2-O(feruloyl)-Ara]-(135)-Ara (Fig. 5). DISCUSSION
Degradation of CWM of sugar beet by Driselase followed by hydrophobic interaction chromatography led to the recovery of a series of peaks containing 1177
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Figure 6. Negative MSn experiment spectra of EO3. a, Full MS spectrum; b, MS2 experiment (m/z 765 ⬎ products); c, MS3 experiment (m/z 765 ⬎ 633 ⬎ products); d, MS3 experiment (m/z 765 ⬎ 705 ⬎ products).
ferulic acid (total recovery approximately 95%). Beside already known feruloylated oligomers, two new compounds were isolated: a tetramer of 135-linked Ara residues esterified by two ferulic acids, one on position 2 and the other on position 5: O-[5-O-(feruloyl)Ara]-(135)-Ara-(135)-[2-O-(feruloyl)-Ara]-(135)Ara, and a trimer of 135 linked Ara residues esterified by two ferulic acids: O-[5-O-(feruloyl)-Ara](135)-[2-O-(feruloyl)-Ara]-(135)-Ara. To the best of our knowledge, there has been no previous report that dicotyledon pectins contain esterified ferulic acids in such position. Conversely, the feruloylated arabinoxylan-oligosaccharides prepared so far from the family Poaceae have shown good consistency of structure. The Araf residues are attached to O-3 positions of -(134)-linked d-xylan and are esterified at position O-5 with the feruloyl group. A feruloyl Ara-Xyl trisaccharide {O-[5-O-(trans-feruloyl)-␣-l-arabinofuranosyl(133)-O--d-xylopyranosyl-(134)-d-xylopyranose} was also isolated from cell walls of sugar beet, demonstrating that ferulic acid is not only linked to pectic arabinan and galactan chains but also to the minor arabinoxylan components of these dicotyledon cell walls (Ishii, 1994). However, the nature of the linkages between the Ara residues in the oligosaccharides de1178
scribed in the present paper ascertains clearly their pectic origin. Polysaccharides feruloylation is thought to occur intracellularly (Obel et al., 2003). Based on the one linkage-one enzyme assumption (Mohnen, 1999) and on the location of feruloyl groups, there could be at least three feruloyl transferases required in sugar beet cell walls (one for position 2 of Ara in pectin, one for position 6 of Gal in pectin, and one for position 5 of Ara in arabinoxylan). This last enzyme could also catalyze the transfer of ferulic acid to pectic arabinan chains, which could explain the peculiar feruloylation site on O-5 of arabinan chains presented in this paper. The proximity of two ferulic acid monomers on arabinan chains is demonstrated here for the first time, to our knowledge, although the significance of this fact is not known. The structural characteristics of sugar beet arabinan are well documented (Guillon and Thibault, 1989; Guillon et al., 1989; Oosterveld et al., 2000, 2002). Based on the results of these studies, it can be stated that sugar beet arabinans have a backbone of 60 to 70 (135)-linked ␣-l-arabinofuranose units, onehalf of which are substituted—on O-3 (approximately 90%) and on O-2 and O-3 (approximately 10%)—by Plant Physiol. Vol. 134, 2004
Feruloylated Oligoarabinans from Sugar Beet
single-unit arabinofuranose and by rare short (133)linked ␣-l-arabinofuranose chains. According to these structural characteristics, the location of ferulic acid on O-5 of Ara residues is speculated to exist at the nonreducing end of the arabinan chains main core. Oligoarabinans esterified by two ferulic acid residues are minor compounds, and only one in 40 ferulic acid units linked to Ara residues is linked on O-5. However, their significance could be of great consequence. If we assume that only terminal residues of arabinan chains can be esterified in this position, this indicates a peripheral location of some ferulic acids on pectic “hairy” regions. This location could ease the accessibility of ferulic acids to oxidative coupling in muro or before secretion. Some dehydrodiferulates have been identified in fraction EO1 and EO2, but they were present in very low quantity. The isolation of dehydrodiferulates linked to neutral sugars— hence providing direct evidence for these cross-links in the cell wall—has been reported so far only for monocotyledons, namely bamboo (Phyllostahys edulis) shoot and maize (Zea mays) bran (Ishii, 1991; Saulnier et al., 1999). The regiochemistry of dehydrodiferulates in dicotyledons pectins needs to be investigated further. MATERIALS AND METHODS Material The CWM was prepared as previously described (Levigne et al., 2002). Driselase was obtained from Sigma (St. Louis).
Enzymatic Hydrolysis of CWM and Chromatography Hydrolysis of CWM of sugar beet (Beta vulgaris; 10 g in 1 L) was performed at 37°C for 48 h after Driselase (1 g) addition. The hydrolysate was filtered through G4 sintered glass, and the supernatant (91% [w/w] of the CWM) was concentrated to 50 mL by vacuum evaporation at 40°C and loaded onto a column of Sephadex LH-20 (80 ⫻ 2.6 cm) eluted at 34 mL h⫺1 successively by water (8 column volumes), 25% (v/v) ethanol (7 column volumes), 50% (v/v) ethanol (5 column volumes), and 100% (v/v) ethanol (5 column volumes). Fractions (11 mL) were collected, and the absorbance was measured at 325 nm. Water-eluted fractions were also analyzed for GalUA (Thibault, 1979) and total neutral sugars (Tollier and Robin, 1979).
Analytical Methods GalUA content was determined by the automated m-hydroxybiphenyl method (Thibault, 1979) and by the method of Ahmed and Labavitch (1977) in the soluble and insoluble materials, respectively. Neutral sugars (expressed as Ara) were determined by the automated orcinol method (Tollier and Robin, 1979). The individual sugars were analyzed as their alditol acetate derivatives by gas chromatography after hydrolysis by 1 m H2SO4 at 100°C for 2 h (Blakeney et al., 1983). A prehydrolysis step in 72% (v/v) H2SO4 (30 min, 25°C) was used in the case of insoluble materials (Saeman et al., 1954). Elution of ferulic compounds was followed by spectrophotometry at 325 nm (Saulnier et al., 1999). Hydroxycinnamic compounds were quantified by HPLC after saponification and extraction as previously described (Levigne et al., 2002).
MS The EO1, EO2, and EO3 fractions were dissolved in 1:1 (v/v) methanol: water and then ultrafiltered before HPLC-MS analysis by the ESI method
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(negative mode). The liquid chromatography (LC) conditions were as follows: HyPURITY-C18 column (ThermoHypersil, 4.6–150 mm, 5-m particle size) with a flow rate of 1 mL min⫺1 and a linear elution gradient (30 min) from 5% to 60% (v/v) solvent B (CH3CN ⫹ HCOOH, 0.1% [v/v]) in solvent A (water ⫹ HCOOH, 0.1% [v/v]). The mass spectral data were collected with an IT mass spectrometer (Thermofinnigan LCQ Deca Instrument, Finnigan Mat, San Jose, CA) equipped with a heated capillary electrospray interface. The sprayer needle voltage was 4 kV, and the temperature of the heated capillary was set at 350°C. The mass spectral elucidation of the major compounds from fractions EO2 and EO3 was carried out by MSn experiments using an LCQ Deca IT mass spectrometer working in the negative (nano) ESI mode. Samples were dissolved in 1:1 (v/v) methanol:water before their infusion into the electrospray source. The MS electrospray analyses were carried out under automatic gain control conditions, using a typical needle voltage of 4.2 kV and a heated capillary temperature of 150°C. For MSn experiments, the various parameters were adjusted for each sample to optimize signal and get maximal structural information from the ion of interest. Received October 24, 2003; returned for revision November 16, 2003; accepted November 23, 2003.
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