A novel (1 → 4)-α-d-glucan isolated from the fruits of Opuntia ficus indica (L.) Miller

Article abstract of DOI:10.1016/j.carbpol.2010.06.006

A water-soluble polysaccharide (PS-1) was isolated from the fruits of Opuntia ficus indica (L.) Miller by hot water extraction, anion-exchange and gel-permeation chromatography (yield 167.5 mg/kg raw fruit; [α] _D^{16 1} + 192° (c 1.0, H₂O); total neutral sugar content 96.60% w/w; weight-average molecular weight (M _w) ～360 kDa). Structural characterisation was performed by monosaccharide analysis and linkage analysis (methylation analysis, periodate oxidation and Smith degradation) on full and partial acid hydrolysates, followed by alditol acetylation, and product quantification by GC/GC-MS. Spectroscopic analysis (FT-IR and ¹H/¹³C NMR) was also performed. Polysaccharide PS-1 was found to be an α-d-glucan with a (1 → 4)-linked α-d-Glcp backbone, with (1 → 6)-linked (1 → 4)-α-d-Glcp side chains, side chains being short in length with no additional branching, and a minimum branching of ～1 in every 9-11 backbone units. The distribution of side chains lengths and corresponding branching density requires further investigation.

Full text of DOI:10.1016/j.carbpol.2010.06.006

Carbohydrate Polymers 82 (2010) 848–853
Contents lists available at ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
A novel (1 → 4)-␣-d-glucan isolated from the fruits of Opuntia ficus indica (L.)
Miller
Omar Ishurda,∗, Faraj Zgheela, Mohamed Elghazouna, Mohamed Elmabruka, Adel Kermagib,
John F. Kennedyc, Charles J. Knillc
a Biotechnology Research Centre, Twisha, Tripoli, P.O. Box 30313, Libya
b Faculty of Pharmacy, Seventh of April University, Zawia, P.O. Box 16418, Libya
c Chembiotech Laboratories, Advanced Science and Technology Ltd, 5, The Croft, Buntsford Drive, Stoke Heath, Bromsgrove, Worcestershire B60 4JE, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
A water-soluble polysaccharide (PS-1) was isolated from the fruits of Opuntia ficus indica (L.) Miller
Received 22 April 2010
Received in revised form 28 May 2010
Accepted 3 June 2010
by hot water extraction, anion-exchange and gel-permeation chromatography (yield 167.5 mg/kg raw
fruit; [˛]D 1 + 192◦ (c 1.0, H2O); total neutral sugar content 96.60% w/w; weight–average molecular
16
weight (Mw) ∼360 kDa). Structural characterisation was performed by monosaccharide analysis and link-
age analysis (methylation analysis, periodate oxidation and Smith degradation) on full and partial acid
hydrolysates, followed by alditol acetylation, and product quantification by GC/GC–MS. Spectroscopic
analysis (FT-IR and 1H/13C NMR) was also performed. Polysaccharide PS-1 was found to be an ␣-d-glucan
with a (1 → 4)-linked ␣-d-Glcp backbone, with (1 → 6)-linked (1 → 4)-␣-d-Glcp side chains, side chains
being short in length with no additional branching, and a minimum branching of ∼1 in every 9–11 back-
bone units. The distribution of side chains lengths and corresponding branching density requires further
investigation.
Available online 11 June 2010
Keywords:
Polysaccharides
Opuntia ficus indica
d-Glucan
© 2010 Elsevier Ltd. All rights reserved.
1. Introduction
prickly pears have become an important crop in the semi-arid lands
of Libya, where they play a strategic role in subsistence agriculture.
Opuntia ficus indica (L.) Miller is a tropical or subtropical plant,
which belongs to the Cactaceae family and is mainly used for
fruit (commonly known as prickly pear and cactus pear, or less
commonly as Barbary fig or Indian fig) and stem (commonly
known as ‘nopal’) production. It grows wild in arid and semi-arid
regions, where production of more succulent food plants is severely
restricted, and is therefore routinely consumed in parts of North
and Central America, southern Mediterranean countries, and parts
of Africa (Gurbachan & Felker, 1998; Meyer & McLaughlin, 1981;
Pimienta-Barrios, 1994; Yasseen, Barringer, & Splittstoesser, 1996).
Under optimal conditions, annual fruit production can be as high as
50 t/ha, and detailed compositional (proximate) analysis has been
performed on the stem, fruit (pulp, skin and seeds) (Dominguez-
Lopez, 1995; El Kossori, Villaume, El Boustani, Sauvaire, & Mejean,
1998; Ramadan & Mörsel, 2003; Stintzing, Schieber, & Carle, 2001).
Due to its high adaptation to the harsh desert environment and its
different applications, the Opuntia ficus indica (L.) Miller fruit is an
important and abundant potential raw material for industries in
many countries (Stintzing & Carle, 2005). Within the last decade,
Efforts are currently being made to develop fruit production and
find new food industry applications.
The fruits/stems/juice of Opuntia ficus indica (L.) Miller have
shown antiulcerogenic (Galati, Monforte, Tripodo, d’Aquino, &
Mondello, 2001; Galati et al., 2003), antioxidant (Galati et al.,
2003; Gentile, Tesoriere, Allegra, Livrea, & D’Alessio, 2004; Kuti,
2004; Tesoriere, Butera, Pintaudi, Allegra, & Livrea, 2004; Tesoriere,
Fazzari, Allegra, & Livrea, 2005; Zourgui, El Golli, Bouaziz, Bacha,
& Hassen, 2008), anti-inflammatory/wound healing (Park & Chun,
2001; Park, Kahng, Lee, & Shin, 2001; Panico et al., 2007; Trombetta
et al., 2006), anticancer (Zou et al., 2005), neuroprotective (Dok-
Go et al., 2003), hepatoprotective (Galati et al., 2005; Hfaiedh et
al., 2008; Ncibi, Othman, Akacha, Krifi, & Zourgui, 2008), antipro-
liferative (Sreekanth et al., 2007), and anti-genotoxicity (Zourgui,
Ayed-Boussema, Ayed, Bacha, & Hassen, 2009) activity/effects,
and may be used for the treatment of gastritis, hyperglycemia,
arteriosclerosis, diabetes, and prostate hypertrophy (Agozzino,
Avellone, Caraulo, Ferrugia, & Filizzola, 2005). Different parts of
Opuntia ficus indica (L.) Miller are used in the traditional medicine of
several countries, e.g. in Chinese medicine the fruit is used against
inflammation, pain, and snake bite (Zou et al., 2005). The cladodes
(branches or portions of the stem that replace leaves as the main
photosynthetic plant organs, in this case nopal) have a high water
∗
Corresponding author. Tel.: +218 912139441; fax: +218 214448122.
E-mail address: omarishurd@yahoo.com (O. Ishurd).
0144-8617/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carbpol.2010.06.006

O. Ishurd et al. / Carbohydrate Polymers 82 (2010) 848–853
849
content (95% w/w). They are rich in dietary fibre, carbohydrates,
minerals, proteins, and vitamins, and are consumed as a vegetable.
A significant amount of scientific investigations have been per-
formed on extraction and analysis of polysaccharides from cladodes
(Amin, Awad, & El-Sayed, 1970; Majdoub et al., 2001; McGarvie
& Parolis, 1981a, 1981b; Paulsen & Lund, 1979; Trachtenberg &
Mayer, 1981), fruit pulp (Matsuhiro, Lillo, Sáenz, Urzúa, & Zárate,
2006), and fruit peel/skin (Forni, Penci, & Polessello, 1994; Habibi,
Heyraud, Mahrouz, & Vignon, 2004a, 2004b) of Opuntia ficus
indica, particularly from mucilage, and their biophysical proper-
ties (Trachtenberg & Mayer, 1982). Such mucilages (extracted by a
variety of methods, e.g. cold water, hot water, ethanol, and acid)
contain a complex mixture of glycoproteins and heteropolysac-
charides, which have been fractionated and characterised (at
least in part). Identified polysaccharides include pectin-type mate-
rial (galacturonan, some with low methoxyl/high acetyl content),
arabinogalactans, rhamnogalactans, arabinoxylans, and rhamno-
galacturonans (with arabinan, galactan and/or arabinogalactan
side-chains).
crude polysaccharide fraction (26.0 g) was obtained through pre-
cipitation with 3 vol. of ethanol and desiccation in vacuo. The crude
polysaccharide precipitate was redissolved in distilled water and
applied to a DEAE-Sephadex A-25 column (90× 5 cm). The column
was first eluted with distilled water followed by 0.1, 0.3 and 0.5 M
sodium chloride, respectively, and fractions collected accordingly,
and their neutral carbohydrate and uronic acid contents confirmed
by phenol–sulfuric (DuBois et al., 1956) and m-hydroxydiphenyl
(Filisetti-Cozzi & Carpita, 1991) methods, respectively. Based on
the results of this assay, the neutral carbohydrate-positive frac-
tion eluted with water was further purified/fractionated on a
Sephadex G-200 column (100× 5 cm) eluted with 0.1 M sodium
chloride, yielding three further fractions. The neutral carbohy-
drate and uronic acid contents of these fractions were confirmed
by phenol–sulfuric (DuBois et al., 1956) and m-hydroxydiphenyl
(Filisetti-Cozzi & Carpita, 1991) methods, respectively, and the
uronic acid-free neutral carbohydrate-positive fraction was col-
lected, dialysed and lyophilised to obtain a purified polysaccharide
(PS-1, 670 mg, 2.58% w/w of the crude polysaccharide).
The only evidence for the presence of a neutral glucan was
observed by Paulsen and Lund (1979), who identified one neu-
tral and two acidic polysaccharide-containing fractions from the
cladodes of Opuntia ficus indica cv “Burbank’s Spineless”. From the
neutral fraction a very small quantity of glucan with a molecu-
lar weight of ∼1 × 106 Da was isolated, but in insufficient quantity
to perform more detailed structural characterisation. This article
presents the isolation and structural characterisation of a novel
(1 → 4)-␣-d-glucan from the fruits of Opuntia ficus indica (L.) Miller.
2.4. Purified neutral polysaccharide characterisation
2.4.1. Homogeneity and molecular weight profile
PS-1 homogeneity and molecular weight profile was deter-
mined using a Waters AMLC system (717 plus autosampler and
600 delta AMLC pump) equipped with a TSKgel 4000 PWXL col-
umn (7.8× 300 mm) and a Waters 2414 Refractive Index Detector
(RID). PS-1 solution (0.5% w/v, 20 ␮L sample injections) was anal-
ysed using a 0.05 M sodium chloride mobile phase with a flow
rate of 0.8 mL/min. The AMLC system was calibrated using T-series
Dextran standards (T-10, T-40, T-70 and T-500 kDa standards).
2. Materials and methods
2.1. Materials
2.4.2. Monosaccharide analysis
PS-1 was hydrolysed to its constituent monosaccharides using
trifluoroacetic acid (TFA), and the released monosaccharides
derivatised (alditol acetates), and analysed by gas chromatogra-
phy (GC) (Li et al., 2003). Xylose, glucose, rhamnose, mannose,
and galactose were also separately derivatised as standards, and
acetyl inositol was utilised as an internal standard. Derivatised
monosaccharides (from hydrolysed polysaccharide and monosac-
charide standards) were analysed by GC using a GC-9A (Shimadzu,
Japan), a capillary column (OV-225, China) and flame ionisation
detection (FID). Nitrogen was used as the carrier gas (40 mL/min).
The injector temperature was kept at 250 ◦C (split injection 70:1),
and the detector temperature was maintained at 235 ◦C. GC station
software was Zhida N2000 (Zhida, China).
Natural fruits from Opuntia ficus indica (L.) Miller were utilised
in this study (∼5 kg). The peeled dried (hot air, 40–60 ◦C) fruits
were uniform in shape, size, and colour and were obtained from
the Ejfarah region of Libya at weekly intervals during the period
of mid July to mid August. The peeled dried fruits were cut into
small ellipsoidal pieces (∼7 cm length). The Opuntia ficus indica
(L.) Miller fruits selected for this study were at the full stage of
ripeness/maturity, since these have been previously shown to con-
tain greater amounts of polysaccharides (Ishurd, Sun, Xiao, Ashour,
& Pan, 2002).
2.2. General methods
Specific rotation measurements were performed at 20 1 ◦C
using an automatic polarimeter (Model WZZ-2B, China). UV–vis
absorption spectra were recorded using a Shimadzu MPS-2000
spectrophotometer. FT-IR spectra (KBr discs) were recorded using a
Nicolet 360 FT-IR spectrometer. Elemental analysis (C, H and N) was
conducted on an Elementar Vario EL III instrument. Total neutral
carbohydrate content was determined by the phenol–sulfuric acid
method as d-glucose equivalents (DuBois, Gilles, Hamilton, Rebers,
& Smith, 1956). Uronic acid content was determined according to
the m-hydroxydiphenyl colorimetric method, in which neutral sug-
ars do not interfere (Filisetti-Cozzi & Carpita, 1991). Protein content
was determined by the method of Bradford (1976).
2.4.3. Methylation analysis
Methylation analysis of PS-1 (9.0 mg) was performed using
the method of Hakomori (1964). Methylated polysaccharide was
treated with 90% formic acid (4 mL) for 14 h at 100 ◦C in a sealed
tube. After removal of formic acid by rotary evaporation, residues
were heated with 2 M trifluoroacetic acid (TFA, 2 mL) under the
same conditions and the resulting hydrolysate rotary evaporated to
dryness. The methylated sugars were reduced with sodium boro-
hydride, acetylated with acetic anhydride, and analysed as alditol
acetates by GC (as detailed previously). Identification of methylated
sugars was performed by gas chromatography–mass spectrome-
try (GC–MS) and by relative GC retention time. Molar ratios were
estimated from peak area and response factor analysis (Björndal,
Lindberg, & Svensson, 1967).
2.3. Polysaccharide extraction and fractionation
Cut dried fruits (4.0 kg) were firstly refluxed with methanol
to remove lipophilic compounds, and then successively boiled in
distilled water for 6 h at 100 ◦C. After filtration to remove debris
fragments, the filtrate was concentrated by rotary evaporation. Pro-
tein was removed with the Sevag method (Alam & Gupta, 1986). The
2.4.4. Periodate oxidation and Smith degradation
PS-1 (9.0 mg) was dissolved in 0.014 M sodium metaperiodate
(30 mL) and kept in the dark at 4 ◦C, and the absorption at 223 nm
monitored at daily intervals. The reaction was completed after

850
O. Ishurd et al. / Carbohydrate Polymers 82 (2010) 848–853
Table 1
GC/GC–MS data for alditol acetate derivatives from methylated/hydrolysed neutral polysaccharide (PS-1) isolated from Opuntia ficus indica (L.) Miller fruit.
a
Component
TR
Molar ratio
Ms (m/z)
Linkage
2,3,4,6-Tetra-O-methyl-1,5-di-O-acetyl-d-glucitol
2,3,6-Tri-O-methyl-1,4,5,-tri-O-acetyl-d-glucitol
2,3-Di-O-methyl-1,4,5,6-tetra-O-Acetyl-d-glucitol
1.00
2.76
4.91
1.0
8.9
0.9
43, 45, 71, 87, 101, 117, 129, 145, 161, 205
43, 45, 87, 99, 101, 113, 117, 233
43, 101, 117, 127, 261
Glcp-(1 →
→ 4)-Glcp-(1 →
→ 4,6)-Glcp-(1 →
aRetention time of alditol acetate relative to 2,3,4,6-tetra-O-methyl-1,5-di-O-acetyl-d-glucitol
Fig. 2. 13C NMR (500 MHz) spectrum of neutral polysaccharide (PS-1) isolated from
Opuntia ficus indica (L.) Miller fruit.
2.4.6. Nuclear magnetic resonance (NMR) spectroscopy
NMR spectra were recorded using a Bruker 500 MHz instrument.
For 1H NMR spectroscopy at 70 ◦C, PS-1 (8 mg) was repeatedly dis-
solved in deuterium oxide (D2O, 99.99%, 5× 5 mL), and the solution
repeatedly lyophilised. For 13C NMR spectroscopy at 50◦C, PS-1 was
dissolved in deuterium oxide (D2O, 99.99%, 1 mL, 64 mg/mL).
Fig. 1. 1H NMR (500 MHz) spectrum of neutral polysaccharide (PS-1) isolated from
Opuntia ficus indica (L.) Miller fruit.
120 h and ethylene glycol (0.2 mL) was added to the solution with
stirring for 30 min to decompose excess reagent. Consumption
of sodium metaperiodate was measured spectrophotometrically
(Dixon & Lipkin, 1954; Pazur, 1994) and formic acid production
was determined by titration with 0.01 M sodium hydroxide. The
reaction mixture was dialysed against distilled water for 2 days to
remove small molecules, and the retained material (higher molecu-
lar weight material) was reduced with sodium borohydride (25 mg,
12 h). The pH was adjusted to 5.0, the solution was dialysed, and the
retained material was lyophilised, and then hydrolysed with 2 M
trifluoroacetic acid (TFA, 2 h at 110 ◦C). The resulting hydrolysate
was analysed by GC (as detailed previously).
3. Results and discussion
3.1. Extraction, separation, isolation and basic analysis of purified
neutral polysaccharide
The yield of the crude water-soluble polysaccharide (26.0 g)
from the dried cut fruits (4.0 kg) of the Opuntia ficus indica (L.)
Miller was 0.65% w/w. The crude polysaccharide was separated and
sequentially purified through DEAE–Sephadex A-25 and Sephadex
G-200, and a purified neutral polysaccharide (PS-1, 670 mg, ∼2.6%
w/w of crude polysaccharide) was chromatographically separated
and isolated (based upon spectrophotometric assessment of frac-
tions for their total neutral carbohydrate and uronic acid contents).
PS-1 appeared as an off-white powder, [˛]D 1 + 192◦ (c 1.0,
16
2.4.5. Partial hydrolysis
H2O). The relatively high positive value of optical rotation sug-
gested the dominating presence of ␣-d-form glycosidic linkages
(Zhao, Kan, Li, & Chen, 2005). PS-1 had a negative response to the
Bradford test and no absorption at 280 or 260 nm in its UV spec-
trum, indicating the absence of protein and nucleic acid material.
Elemental analysis found it to be free of nitrogen, confirming it to be
a neutral polysaccharide (i.e. confirming the absence of aminosugar
residues, uronic acids having already been eliminated). The GPC
profile showed a single and symmetrically sharp peak, indicating
PS-1 was partially hydrolysed with aqueous trifluoroacetic acid
solution (adjusted to pH 2.0, 20 mL, 18 h at 100 ◦C). After neutralisa-
tion with sodium hydroxide, a polymeric product was obtained by
precipitation with excess ethanol from a small volume of water,
and then retained on dialysis using a 2 kDa molecular weight
cut-off membrane. The isolated (by lyophilisation) residue was sub-
jected to periodate oxidation and Smith degradation as detailed
above.
Table 2
Potential monosaccharide residues present in the neutral polysaccharide (PS-1) isolated from Opuntia ficus indica (L.) Miller fruit.
Residue code
[A]
Monosaccharide residue
Description of monosaccharide residue
␣-d-Glcp-(1 → 4)-
Main chain or side chain non-reducing
end unit
[B]
[C]
[D]
␣-d-Glcp-(1 → 6)-
Non-reducing end unit attached
directly to branching point unit
Main chain or side chain unbranched
unit
Side chain unbranched unit attached
directly to branching point unit
Main chain branching unit
Reducing end unit
→ 4)-␣-d-Glcp-(1 → 4)-
→ 4)-␣-d-Glcp-(1 → 6)-
[E]
[F]
→ 4,6)-␣-d-Glcp-(1 →
→ 4)-␣-d-Glca
aAn equilibrium of various forms will be present (␣, ␤, open chain, furanose, and pyranose).

O. Ishurd et al. / Carbohydrate Polymers 82 (2010) 848–853
851
Fig. 3. Proposed structural features of the neutral (1 → 6)-branched, (1 → 4)-linked ␣-d-glucan polysaccharide (PS-1) isolated from Opuntia ficus indica (L.) Miller fruit.
that PS-1 was a homogeneous polysaccharide, with a weight aver-
age molecular weight (Mw) of ∼360 kDa (from dextran T-series
calibration).
3.3. Spectroscopic analysis of polysaccharide PS-1
3.3.1. FT-IR spectroscopic analysis
FT-IR spectroscopic analysis (data not shown) of PS-1 showed
a strong band at 3420.19 cm−1 attributed to hydroxyl stretching
vibrations; a band at 2930.83 cm−1 was due to C–H stretching vibra-
tions; the broad band at 1636.52 cm−1 was due to bound water
(Park, 1971); the band at 850.81 cm−1 was ascribed to ␣-type gly-
cosidic linkages (Barker, Bourne, Stacey, & Whiffen, 1954), and the
bands at 850.81 and 915.56 cm−1 are characteristic of (1 → 4)-␣-
d-glucans. Therefore, the FT-IR spectroscopy results, together with
the high positive specific rotation, indicate the presence of ␣-d-
glycosidic linkages in PS-1 (Bao, Duan, Fang, & Fang, 2001). The
FT-IR absorptions at 1020.85, 1046.68 and 1154.57 cm−1 also indi-
cate the ␣-d-pyranose form of the glucosyl residues.
3.2. Monosaccharide composition and linkage analysis of
polysaccharide PS-1
The total neutral sugar content of PS-1 was determined to be
96.60% w/w, and it was composed of only glucose monomers, as
detected by GC analysis of the alditol acetate derivatives of the
components of the PS-1 hydrolysates. PS-1 did not contain any
uronic acid material (as determined by spectrophotometric assay
and confirmed by GC analysis of its hydrolysates).
The fully methylated product of PS-1 was hydrolysed with
acid, converted into alditol acetates, and analysed by GC and
GC–MS, yielding three types of glucitol derivative with a relative
molar ratio of 1.0:8.9:0.9 (according to peak area), correspond-
ing to Glcp-(1 →, → 4)–Glcp–(1 →, and → 4,6)-Glcp-(1 → residues,
respectively (Table 1). Periodate oxidation of PS-1 resulted in perio-
date consumption and formic acid production of 1.04 and 0.10 mol,
respectively, per residue, which is in relatively close agreement
with the theoretical values (1.09 and 0.09 mol, respectively) cal-
culated from the monosaccharide relative molar ratios from the
methylation analysis results above.
Smith degradation of the periodate-oxidised PS–1 produced
glycerol and erythritol with a molar ratio of 1.1:8.5 (determined by
GC after conversion to the corresponding alditol acetates). There-
fore terminal non-reducing Glcp-(1 → residues from the main
backbone (and any side chains) and → 4,6)-Glcp-(1 → residues
(main backbone and possibly side chain, branch points) amounted
to a total of 11.5%, with → 4)–Glcp–(1 → residues (main chain and
side chain unbranched units) amounting to 88.5%, respectively. This
is slightly different from the equivalent values from methylation
analysis (17.6 and 82.4%, respectively). PS-1 was partially hydrol-
ysed with TFA, and after periodate oxidation and Smith degradation
of the partial hydrolysate only erythritol was found, confirming that
PS-1 was a polysaccharide with a (1 → 4)-linked backbone, with
relatively acid labile side chains (i.e. of relatively short length and
without further branching).
3.3.2. NMR spectroscopic analysis
The potential monosaccharide residue environments ([A]–[F])
present in polysaccharide PS-1 (Table 2) have been used to assist
with interpretation of 1H and 13C NMR spectra (Figs. 1 and 2, respec-
tively), as detailed below. The 1H NMR spectrum of PS-1 (Fig. 1)
contains numerous peaks, which were assigned based upon the
results discussed above (identification of residues, linkages, etc.)
and comparison with 1H NMR spectroscopic assignments for ␣-d-
glucans by Papp-Szabó, Kanipes, Guerry, and Monteiro (2005) and
van Leeuwen et al. (2008). The relatively broad unresolved signal
at ∼ı 5.5 ppm corresponds to the anomeric (C-1) protons of [A],
[C] and [E] residues (Table 2), i.e. linked to the C-4 of the next
residue along the chain (main backbone or side chain) forming a
(1 → 4)-linkage between the ␣-d-Glcp residues. The smaller signal
at ∼ı 5.1 ppm corresponds to the anomeric (C-1) protons of [B] and
[D] residues (Table 2), i.e. linked to the C-6 of the next residue,
forming the (1 → 6)-linkage between the ␣-d-Glcp residues (the
branch point linkage). The ratio of these peak areas (1:9) gives an
indication of the relative branching ratio/density. The collection of
broad unresolved peaks in the ∼ı 3.5–4.5 ppm region are assigned
to the remaining (C2–C-6) protons in residues [A]–[E] (Table 2). The
large peak at ∼ı 4.7 ppm corresponds to partially deuterated water
(HOD).
The 13C NMR spectrum of PS-1 (Fig. 2) contains numerous
peaks, which were assigned based upon the results discussed above
(identification of residues, linkages, etc.) and comparison with 13C
NMR spectroscopic assignments for ␣-d-glucans (specifically) by
Seymour, Knapp, and Bishop (1976), Seymour, Knapp, Chen, Jeans,
From these results there are six potential monosaccharide
residue environments ([A]–[F]) present in the neutral polysaccha-
ride PS-1 isolated from Opuntia ficus indica (L.) Miller fruit (Table 2).

852
O. Ishurd et al. / Carbohydrate Polymers 82 (2010) 848–853
and Bishop (1979) and Uzochukwu, Balogh, Loefler, and Ngoddy
(2002). The relatively broad signal at ∼ı 100 ppm corresponds to
the anomeric (C-1) carbons of [A], [C] and [E] residues (Table 2),
i.e. linked to the C-4 of the next residue along the chain (main
backbone or side chain) forming a (1 → 4)-linkage between the ␣-
d-Glcp residues. The smaller signal at ∼ı 98 ppm corresponds to
the anomeric (C-1) carbons of [B] and [D] residues (Table 2), i.e.
linked to the C-6 of the next residue, forming the (1→6)-linkage
between the ␣-d-Glcp residues (the branch point linkage). The sig-
nal at ∼ı 77 ppm corresponds to the C-4 carbons of residues [C]–[E]
(Table 2), i.e. linked to the C-1 of the preceding residue along the
chain (an internal link in the main backbone or side chain) form-
ing a (1 → 4)-linkage between the ␣-d-Glcp residues. The relatively
large signal at ∼ı 61 ppm corresponds to unlinked C-6 carbons of
residues [A]–[D] (Table 2), i.e. not involved in branching points. The
collection of poorly resolved peaks in the ∼ı 70–75 ppm region are
assigned to the remaining carbon atoms, namely the C-2, C-3 and
C-5 carbons in residues [A]–[E], the C-4 carbons in residues [A] and
[B], and the C-6 carbons in residue [E] (Table 2).
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4. Conclusions
The neutral polysaccharide (PS-1), isolated from the fruits of
Opuntia ficus indica (L.) Miller is a lightly branched ␣-d-glucan, with
a weight average molecular weight (Mw) in the region of ∼360 kDa.
The potential structural features of PS-1 polysaccharide are pre-
sented in Fig. 3. PS-1 is composed of a (1 → 4)-linked ␣-d-Glcp
backbone, with (1 → 6)-linked ␣-d-Glcp side chains. Linkage anal-
ysis suggests a minimum branching of ∼1 in every 9–11 backbone
units (∼1 in 9 from periodate oxidation/Smith degradation (ratio
∼1:8), and ∼1 in 11 from methylation analysis (ratio ∼1:10) results,
and ∼1 in 10 from 1H NMR analysis). This is if y = 0 (Fig. 3), i.e.
side chains are composed of only terminal non-redusing ␣-d-Glcp
units linked to the backbone via (1 → 6)-linkages. Periodate oxida-
tion/Smith degradation of partial hydrolysates of PS-1 suggests that
any side chains must be relatively acid labile (i.e. short in length,
withnoadditional branching). Thedistributionofside chainlengths
(and corresponding branching density) requires confirmation (by
analysis of hydrolysates prepared using a debranching enzyme,
such as pullulanase or isoamylase).
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Acknowledgement
The authors would like to thank the Chemistry Department,
Zhejiang University, China for access to NMR spectroscopy facilities.
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