2.1 Extraction and purification of xylan

The pectic polysaccharides were extracted by hot water and chelating solution from the cell wall material (CWM) of seed endosperm according to our previous work [3]. The residue was extracted sequentially by cold alkaline solution and hot alkaline solution. Two major polysaccharidic fractions were obtained. Cold alkaline soluble fraction (CASF) precipitated by ethanol after neutralization and hot alkaline soluble fraction (HASF) recovered after precipitation occurring during neutralization of hot alkaline extract (Fig. 1). The yield and sugar composition of CWM, CASF and HASF are given in Table 1.

The CASF was fractionated by ion exchange chromatography and the buffer eluted fraction (CASF1*) was then purified by size-exclusion chromatography to give a homogenous polysaccharide noted CASF1.

The HASF, which was insoluble in water, was purified by solubilization in 1% NaOH solution and precipitation with Fehling solution to give purified fraction noted HASF1.

2.2 Sugar composition and methylation analysis of xylans

A H₂SO₄ hydrolyzate of CASF1 revealed that xylose was the major component with a small amount of fucose (xylose and fucose molar ratio of 12:1). The uronic acid content was estimated by a colorimetric method to be 22% (w/w) [4], which corresponded to a molar ratio of xylose to uronic acid of 6:1. The water insoluble fraction HASF1 was composed exclusively of xylose. The sugar composition of CASF1 and HASF1 are reported in Table 2.

Based on the hitherto published results [5–7], we know that the xylans of all higher plants possess β-(1 → 4) linked xylopyranose units as the backbone. Depending on their origin, i.e. Gramineae, Gymnosperms or Angiosperms the backbone is usually substituted with α-d-glucuronic acid, 4-O-methyl-α-d-glucuronic acid and some neutral sugar units such as α-l-arabinose, α-d-xylose and α-d-galactose. The unusual occurrence of fucose in polysaccharides from cell walls of higher plants and the absence of previous evidence for this sugar as a constituent of xylan family prompted us to undertake a more detailed examination. While referring to the literature, the only xylan type polysaccharide containing fucose residues was isolated by Aspinall et al. [8] from Hyptis suaveolens.

The results of methylation analysis are reported in Table 3 and indicated that the backbone of all xylan fractions consisted of (1 → 4) linked xylopyranose. In the CASF1, the results showed the presence, in addition to 2,3-di-O-methyl-xylitol, of O-2 and O-3 substituted xylose confirmed by the presence of 3-O-methyl-xylitol and 2-O-methyl-xylitol. The presence of 2,3,4-tri-O-methyl-fucositol in approximately equal amount with the 2-O-methyl-xylitol confirmed that the fucose units are linked to position O-3 of the xylan backbone. Furthermore, one can notice the poor yields of 3-O-methyl-xylitol due to incomplete hydrolysis of the 4-O-Me-GlcpA-(1 → 2)-Xylp linkage.

The detection of only 2,3-di-O-methyl-xylitol in the case of HASF1 indicated that it consisted exclusively of (1 → 4) linked xylopyranose units.

2.3 NMR characterization of xylan

The structures of CASF1 and HASF1 were also investigated by NMR spectroscopy. The ¹H and ¹³C NMR spectra of CASF1 recorded in D₂O are given in Figs. 2 and 3. The ¹³C NMR spectrum showed five main signals at δ 102.37 (C-1), 73.45 (C-2), 74.45 (C-3), 77.15 (C-4) and 63.74 (C-5) ppm, corresponding to (1 → 4) linked β-d-Xyl residues. Other less intense signals observed at δ 177.16, 98.30, 72.95, 77.44, 83.03, 73.15 and 60.23 ppm, are characteristic, respectively, of C-6, C-1, C-2, C-3, C-4, C-5 and the methoxyl group of a 4-O-methyl-α-d-glucuronic acid residue. Signals at δ 101.95, 77.41, 72.12, 75.95, 62.39 ppm are characteristics of C-1, C-2, C-3, C-4, C-5, respectively, of β-d-Xyl units substituted with 4-O-methyl-α-d-GlcA in O-2.

The presence of fucose was also confirmed by the presence in ¹³C NMR spectrum of characteristic signals at 99.67 (C-1), 68.53 (C-2), 76.24 (C-3), 69.01 (C-4), 61.41 (C-5) and 20.85 (C-6) ppm). Also the xylose residues substituted with fucose are characterized by signals at 103.12 (C-1), 72.27 (C-2), 76.88 (C-3), 76.24 (C-4) and 63.50 (C-5) ppm.

The average integration of all signals for the different sugar residues, in ¹³C quantitative spectrum, revealed a molar ratio of xylose, fucose to 4-O-methyl-α-d-glucuronic acid, respectively, of 12:1:2.

Examination of the proton spectrum (Fig. 2) of CASF1 showed the relative simplicity of the structure as exhibited by: (i) major signals at δ 4.36 (H-1), 3.97 (H-5eq), 3.60 (H-4), 3.40 (H-3), 3.23 (H-5ax) and 3.14 ppm (H-2), corresponding to non-substituted β-d-Xyl residues; (ii) minor signals at δ 5.14 (H-1), 4.16 (H-5), 3.58 (H-3), 3.46 (H-2), 3.35 (OCH₃), 3.12 ppm (H-4), corresponding to 4-O-methyl-α-d-GlcA acid residues, and at δ 4.51 (H-1), 4.01 (H-5eq), 3.60 (H-4), 3.53 (H-3), 3.38 (H-2) and 3.22 ppm (H-5ax) assigned to β-d-Xyl units substituted with 4-O-methyl-α-d-GlcA. The presence of fucose units was also confirmed by the presence of a doublet at 4.83 ppm assigned to H-1 with a small coupling J_1,2 = 1.5 Hz characteristic of fucosyl units α-linked and another doublet at 1.19 ppm corresponding to the methyl group.

The proton spectrum showed five doublets in the anomeric region at 4.36 [→ 4)-β-d-Xylp-(1→], 4.51 [→ 1,2)-β-d-Xylp-(→ 4], 4.45 [→ 1,3)-β-d-Xylp-(→ 4], 4.83 [α-Fucp] and 5.14 ppm [4-O-Me-α-d-GlcpA] with an intensity ratio of 9:2:1:2:1, respectively. The proton NMR data confirmed the presence of 12 xylose units per two uronic acid and one fucose residues.

The NMR data for CASF1 are reported in Table 4 and are in good agreement with the structures of (4-O-methyl-α-d-glucurono)-β-d-xylans already described in a number of plants [9–12].

The ¹H and ¹³C NMR spectrum of HASF1 recorded in DMSO-d₆ are given in Figs. 2 and 3. The ¹³C NMR spectrum showed only five signals at δ 102.34 (C-1), 73.32 (C-2), 74.59 (C-3), 76.02 (C-4) and 63.81 (C-5) ppm, corresponding to (1 → 4) linked β-d-Xyl residues. Also the ¹H NMR spectrum showed the simplicity of the structure as exhibited by only six signals at δ 4.26 (H-1), 3.05 (H-2), 3.28 (H-3), 3.53 (H-4), 3.18 (H-5_ax) and 3.88 (H-5_eq). The NMR data reported in Table 4 for HASF1 are in good agreement with the structure of linear (1 → 4)-β-d-xylan already described in guar seed husk [13].

3.1 Materials

Fresh mature prickly pear fruits of OFI were collected in November 2000 from the experimental station plantation located in the vicinity of Marrakech (Morocco). The harvested fruits were washed, carefully hand-peeled and the pulp containing the seeds was mixed for a few minutes in a mixer grinder. The seeds were recovered from the resulting pulp juice by straining through a metallic strainer and cleaned by several washings in distilled water. After drying, they were cracked in an analytical grinder for a few minutes and the endosperm was recovered as a fine powder after sieving on a 60 mesh sieve.

3.2 Analytical methods

Uronic acid content was determined according to Blumenkrantz and Asboe-Hansen's [4] method. Neutral sugars were analyzed, after H₂SO₄ hydrolysis, by GLC as their corresponding alditol acetates, using a Packard and Becker 417 instrument coupled to a Hewlett–Packard 3380 A integrator. Glass columns (3 mm × 2 m) packed with 3% SP 2340 on Chromosorb W-AW DMCS (100–120 mesh), or 3% OV 17 on the same support were used. The carboxyl groups of the d-galactosyluronic acid were reduced according to the method of Taylor and Conrad [14]. The carboxyl-reduced and the neutrals samples were methylated twice by the Hakomori procedure, as described by Jansson et al. [15]. The partially methylated carbohydrates were then converted into their alditol acetates by successive treatments with NaBH₄ and pyridine-Ac₂O and analyzed on a fused-silica widebore column (30 m × 0.53 m) half bonded with SP-2380. Peak identification was based on retention times using partially methylated alditol acetates standard and confirmed by GLC by using a SP 2380 capillary column (0.32 mm) coupled to a Nermag R1010C mass spectrometer. Peak areas were corrected by using the molar response factors according to Sweet et al. [16].

3.3 Preparation of CWM

Fats, waxes and oils (11 wt.% of dry material) were removed from endosperm powder by refluxing in a Soxhlet apparatus during 24 h with 38:62 toluene/EtOH. The CWM was prepared by enzymatic digestion according to Habibi et al. (unpublished results).

3.4 Isolation of xylan

CWM was sequentially treated by water (2 × 2 h at 100 °C) and aqueous solution of calcium chelator agent 0.5% EDTA (2 × 2 h at 100 °C) and finally sodium chlorite treatment was performed to remove residual protein and lignin. The bleached residue was extracted by cold alkaline solution (0.5 M, 2 × 1 h at 20 °C) and hot alkaline solution (0.5 M, 2 × 1 h at 80 °C). All extracts were neutralized (pH ≈ 5–6) and the precipitates were recovered by centrifugation. The supernatant was precipitated with EtOH (4 vol). The extraction scheme is given in Fig. 1.

3.5 Ion exchange chromatography

A sample of CASF (450 mg) was suspended in 100 ml of 0.05 M phosphate buffer (pH 6.3) and loaded onto a column of DEAE–Trisacryl M (phosphate form), which was eluted sequentially with phosphate buffer and then with a NaCl gradient (0.125–1 M) in the same buffer. The buffer eluted fraction was then dialyzed against distilled water and freeze-dried to give a polysaccharide fraction CASF1*.

3.6 Purification of xylan

The buffer eluted fraction CASF1* was purified by size-exclusion chromatography on a Shodex-OHpak B-803 (7.5 × 500 mm) column, eluted at 1 ml/min flow rate with 0.05 M NaNO₃ solution, and at room temperature. The column effluent was monitored using a refractive index detector. The salts were removed by dialysis and the solution freeze-dried, to give the purified fraction CASF1.

The insoluble fraction HASF was purified by solubilization in 1% NaOH solution and precipitation with Fehling solution according to Jones and Stoodley [17].

3.7 NMR spectroscopy

¹H experiments were recorded on a Bruker Avance 400 spectrometer (operating frequency of 400.13 MHz). ¹³C NMR experiments were obtained on the same spectrometer (operating frequency: 100.57 MHz). CASF1 was examined as solution in D₂O at 333°K in 5 mm o.d. tube (internal acetone ¹H (CH₃) at 2.1 ppm and ¹³C (CH₃) at 31.5 ppm relative to Me₄Si). HASF1 was examined recorded as solution in DMSO-d₆ at 333°K in 5 mm o.d. tube (internal acetone ¹H (CH₃) at 2.1 ppm and ¹³C (CH₃) at 31.5 ppm relative to Me₄Si).