A sulfated galactan with antioxidant capacity from the green variant of tetrasporic Gigartina skottsbergii (Gigartinales, Rhodophyta)

Article abstract of DOI:10.1016/j.carres.2011.11.014

The water soluble polysaccharide produced by the green variant of tetrasporic Gigartina skottsbergii was found to be composed of d-galactose and sulfate groups in a molar ratio of 1.0:0.65. ¹H and ¹³C NMR spectroscopy studies of the desulfated polysaccharide showed a major backbone structure of alternating 3-linked β-d-galactopyranosyl and 4-linked α-d-galactopyranosyl units, and minor signals ascribed to 3-O-methyl-substitution on the latter unit. Ethylation analysis of the polysaccharide indicated that the sulfate groups are mainly located at position O-2 of 4-linked α-d-galactopyranosyl residue and partially located at positions O-6 of the same unit and at position O-2 of 3-linked β-d-galactopyranosyl residue, and confirmed the presence of 3-O-methyl-galactose in minor amounts (4.4%). The sulfated d-galactan presents a similar structure to λ carrageenan but with much lower sulfation at position O-6 of the α-residue and at position O-2 of β-residue. The antioxidant capacity of the sulfated d-galactan was evaluated by the peroxyl radicals (ORAC method), hydroxyl radicals, chelating activity, and ABTS ⁺ assays. Kinetic results obtained in these assays were compared with those obtained for the commercial λ carrageenan. The antioxidant activity toward peroxyl radicals was higher for commercial λ carrageenan, this agrees with its higher content of sulfate group. The kinetics of the reaction of both polysaccharides with hydroxyl and ABTS⁺ radicals showed a complex mechanism, but the antioxidant activity was higher for the polysaccharide from the green variant of tetrasporic Gigartina skottsbergii.

Full text of DOI:10.1016/j.carres.2011.11.014

Carbohydrate Research 347 (2012) 114–120
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
A sulfated galactan with antioxidant capacity from the green variant
of tetrasporic Gigartina skottsbergii (Gigartinales, Rhodophyta)
Tamara Barahona a, María V. Encinas a, Andrés Mansilla b,c, Betty Matsuhiro a, , Elisa A. Zúñiga d
⇑
a Departamento de Ciencias del Ambiente, Facultad de Química y Biología, Universidad de Santiago de Chile, Av. B. O’Higgins 3363, Santiago, Chile
b Departamento de Ciencias y Recursos Naturales, Facultad de Ciencias, Universidad de Magallanes, Casilla 113-D, Punta Arenas, Chile
c Instituto de Ecología y Biodiversidad, Casilla 653, Santiago, Chile
d Departamento de Química, Facultad de Ciencias Básicas, Universidad Metropolitana de Ciencias de la Educación, Av. José Pedro Alessandri 774, Santiago, Chile
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 October 2011
Received in revised form 9 November 2011
Accepted 12 November 2011
Available online 19 November 2011
The water soluble polysaccharide produced by the green variant of tetrasporic Gigartina skottsbergii was
found to be composed of
spectroscopy studies of the desulfated polysaccharide showed a major backbone structure of alternating
3-linked b- -galactopyranosyl and 4-linked -galactopyranosyl units, and minor signals ascribed to
3-O-methyl-substitution on the latter unit. Ethylation analysis of the polysaccharide indicated that the
sulfate groups are mainly located at position O-2 of 4-linked -galactopyranosyl residue and partially
located at positions O-6 of the same unit and at position O-2 of 3-linked b- -galactopyranosyl residue,
and confirmed the presence of 3-O-methyl-galactose in minor amounts (4.4%). The sulfated -galactan
presents a similar structure to k carrageenan but with much lower sulfation at position O-6 of the -res-
idue and at position O-2 of b-residue. The antioxidant capacity of the sulfated -galactan was evaluated
D
-galactose and sulfate groups in a molar ratio of 1.0:0.65. 1H and 13C NMR
D
a-D
a
-D
Keywords:
Green variant red seaweed
k Carrageenan
D
D
a
Antioxidant capacity
D
by the peroxyl radicals (ORAC method), hydroxyl radicals, chelating activity, and ABTSꢀ+ assays. Kinetic
results obtained in these assays were compared with those obtained for the commercial k carrageenan.
The antioxidant activity toward peroxyl radicals was higher for commercial k carrageenan, this agrees
with its higher content of sulfate group. The kinetics of the reaction of both polysaccharides with hydro-
xyl and ABTSꢀ+ radicals showed a complex mechanism, but the antioxidant activity was higher for the
polysaccharide from the green variant of tetrasporic Gigartina skottsbergii.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
based on electron transfer mechanism was studied. These results
were compared with those obtained for the commercial k-carra-
Recently, a green variant of Gigartina skottsbergii (Gigartinales,
Rhodophyta) was found in a natural population, growing next to
red individuals on the south coast of Chile.1 Color mutants are
not uncommon, they have been reported in Mazzaella laminarioides
and species of the genus Gracilaria, most of them are green.2–6
The sulfated polysaccharides produced by the red algae
G. skottsbergii (Gigartinales, Rhodophyta), have been thoroughly
geen, which is widely used as an ingredient in the food industry.
2. Results and discussion
Extraction of tetrasporic plants of the green variant of
G. skottsbergii afforded 50 dry weight percent of a polysaccharide,
hereinafter named as GVT polysaccharide, that presents 64% of
reducing sugars, 25.9% of –SO3 groups and 2.5% of protein. It
showed a molecular weight of 1 ꢀ 106 1.2 ꢀ 105 determined
spectrophotometrically by the reducing end method. Its IR
spectrum presented bands at 1254 cmꢁ1, attributed to the S@O
vibration of the sulfate groups, and a broad band in the region of
850–800 cmꢁ1. The second-derivative spectrum showed bands at
studied. Cystocarpic individuals produce
their precursos carrageenans, and tetrasporophytes synthesize
k type carrageenans.7–12 Carrageenans are sulfated galactans of
alternating 3-linked b- -galactopyranose and 4-linked -galacto-
j/i carrageenans and
l/m
D
a-D
pyranose or 4-linked 3,6-anhydrogalactopyranose.13 The aim of
this work was the structural determination of the soluble polysac-
charide from tetrasporic plants of the green variant of
G. skottsbergii. Also, the relevance of the antioxidant activity
in vitro of the polysaccharide toward oxygen-centered radicals
(peroxyl and hydroxyl), chelating activity, and the ABTSꢀ+ assay
942 and 896 cmꢁ1 indicative of the presence of
a- and b-linked
galactopyranosyl residues, respectively; and bands at 830 cmꢁ1
assigned to absorption of equatorial secondary 2-sulfate group
and at 819 cmꢁ1, due to 6-sulfate group on galactopyranosyl resi-
dues.14 Reductive hydrolysis of the GVT polysaccharide followed
by acetylation and GC analysis only indicated the presence of
hexa-O-acetyl-galactitol. 3,6-Anhydrogalactitol acetate was not
⇑
Corresponding author. Tel.: +56 2 7181159; fax: +56 2 681 2108.
E-mail address: betty.matsuhiro@usach.cl (B. Matsuhiro).
0008-6215/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2011.11.014

T. Barahona et al. / Carbohydrate Research 347 (2012) 114–120
115
detected, which is in agreement with the absence in the IR spec-
trum of the polysaccharide of a band at 930 cmꢁ1 due to 3,6-
anhydrogalactosyl residues.14 Configuration of galactose was
determined by GC analysis of diastereoisomeric amines prepared
by reductive amination and acetylation of the total hydrolysis
product of GVT polysaccharide. Comparison of the single signal
pyranosyl residues. It can be deduced that 40.4% of 2,4,6-tri-O-
ethyl-galactitol acetate comes from 3-linked -galactopyranosyl
residues. About 3.8% of the latter residue is sulfated at position
D
O-2. Therefore, unlike the normal tetrasporic plants of
G. skottsbergii, the green variant produces a sulfated
with a backbone similar to k carrageenan but with much lower sul-
fation at position O-6 of the -residue and at position O-2 of the
D-galactan
with standards compounds derived from
cated that the polysaccharide is only composed of
D
- and
L
-galactose indi-
-galactose. Ta-
a
D
b-residue. A simple relation of the structure of the polysaccharide
with the pigmentation could not be proposed; it can be mentioned
that the green variant of cystocarpic G. skottsbergii mainly
ken together, these results indicate the presence of a sulfated
galactan of carrageenan family with a galactose/sulfate molar ratio
of 1:0.65.
produced
j
carrageenan,20 the expected polysaccharide for
The GVT polysaccharide was submitted to partial acid hydroly-
sis due to its high molecular weight for the analysis by NMR spec-
troscopy. The 1H NMR spectrum in D2O of the partially hydrolyzed
carrageenophytes in the cystocarpic phase.
The peroxyl radical (ROOꢀ) scavenging capacity toward GVT
polysaccharide was measured by the ORAC method using fluores-
cein and C-phycocyanin as fluorescent probes.21,22 Figure 2 shows
the decrease of the fluorescein (Fl) emission mediated by peroxyl
polysaccharide (figure not shown) presented in the
a-anomeric re-
gion, four signals that probably are due to sulfated and unsulfated
a
-
D
-galactopyranosyl residues. Assignments of these signals were
radicals in the absence (Io) and presence (IF) of increased GVT poly-
F
possible using 13C NMR spectrum and 2D homonuclear (COSY
and NOESY) and heteronuclear (HSQC and HMBC) experiments,
and literature data (Table 1).15,16 Noteworthy is the presence of
saccharide concentrations, expressed as mg/mL. These data show
that in the absence of the polysaccharide the fluorescence probe
decreases with negligible inhibition time, and the polysaccharide
addition decreases the probe consumption rate. Kinetic profiles
with C-phycocyanin as fluorescent probe were similar. In both
cases, the area under the curve of the kinetic plots was linearly re-
lated to the amount of polysaccharide addition (Fig. 2 insert). The
ORAC values, taking ascorbic acid as reference, were 3.1 0.4 ꢀ
10ꢁ3 and 0.72 0.05 ꢀ 10ꢁ3 for fluorescein and C-phycocyanin,
respectively, and they are lower than that measured for the com-
mercial k-carrageenan (5.6 0.3 ꢀ 10ꢁ3 and 1.1 0.1 ꢀ 10ꢁ3). In
both cases, the ORAC value is five times higher for fluorescein than
C-phycocyanin. The lower reactivity of the protein toward peroxyl
radicals can be explained considering that the first step of the reac-
tion of ROOꢀ with the probe is the abstraction of a hydrogen atom.23
The complex structure of the protein24 retards the access of per-
oxyl radicals toward the reactive center in the protein. On the other
hand, ORAC values are higher for k carrageenan. A sulfate content
of 33–38% has been reported for this polysaccharide,25,26 which is
quite higher than that found for GVT polysaccharide (25.9%). More-
over, considering that the hydrogen abstraction occurs from the
anomeric hydrogen atom, the presence of the sulfate group on
C-2 should increase the hydrogen abstraction rate. Indeed, k carra-
signals ascribed to 3-O-methyl-
4.66 and 4.61 ppm in the 1H NMR spectrum may be attributed to
the anomeric protons of b- -galactopyranosyl residues, although
a-D-galactopyranose. Signals at
D
the latter is a very small signal and could not be properly assigned.
Better results were obtained in the NMR spectra of the desulfated
polysaccharide. 2D NMR spectra allowed the complete assign-
ments of major 1H NMR signals assigned to alternating ?3)-b-
D-
galactopyranosyl- and ?4)-a-D
-galactopyranosyl units. The 13C
NMR spectrum (figure not shown) presented 12 major signals cor-
responding to desulfated k carrageenan which are in very good
agreement with those reported by Van de Velde et al.16 Smaller
signals were also visible in the 13C NMR spectrum, which were
assigned with the aid of 2D NMR spectra and literature data to
3-O-methyl-a-D
-galactopyranosyl residue.17–19 Figure 1 illustrates
the 1H/13C connectivities in the HSQC spectrum and Table 1 shows
the assignments of the signals.
Confirmation of the presence, in low proportion of a residue
corresponding to 3-O-methyl-galactose in the polysaccharide was
obtained by ethylation analysis of the native polysaccharide. The
polysaccharide was submitted to three rounds of ethylation,
followed by total hydrolysis and the resulting ethylated derivatives
were analyzed by GC–MS as alditol acetates (Table 2).
3-O-Methyl-2,6-di-O-ethyl-galactitol acetate (4.4%) was identified;
furthermore, ethylation analysis gave information about glycosidic
linkages and sulfate positions on the native polysaccharide. Galact-
ose was found to be involved in (1?3) and (1?4) linkages as ex-
pected for carrageenan-type polysaccharides. However, only 9.6%
geenan contains sulfate groups on C-2 of
osyl residues, whereas GVT polysaccharide is mainly sulfated at the
C-2 position of -galactopyranosyl residue. These facts suggest
a- and b-D-galactopyran-
a
-D
that the number as well as the position of sulfate groups play a role
in the antioxidant activity of these polysaccharides toward the per-
oxyl radicals.
The scavenging activity toward hydroxyl radicals produced by
the Fenton reaction has been assessed by the competitive reaction
of these radicals with Brilliant Green (BG). Kinetics of the reaction
of GVT polysaccharide with hydroxyl radicals showed complex
behavior. Figure 3 shows that the BG absorbance decay becomes
of ethylated alditol showed sulfation at position O-6; for ?4)-D-
galactopyranosyl residues in k carrageenan total sulfation was ex-
pected at this position. The presence of 3,6-di-O-ethyl-galactitol
acetate indicates sulfation at position O-2 in ?4)-linked galacto-
Table 1
Assignments of chemical shifts (ppm) in the 1H and 13C NMR spectra of partially hydrolyzed and desulfated GVT polysaccharides
GVT polysaccharidea
H1/C1
H2/C2
H3/C3
4.11
H4/C4
H5/C5
H6/C6
O-CH3
?-4-
?-4-
?-4-
?-4-
a-
a-
a-
a-
D-Galp-2S?
D-Galp-3M?
D-Galp-6S?
D-Galp?
5.45/94.66
5.36/100.13
5.30/95.81
5.19/95.98
4.66/104.73
4.52/75.90
3.63
3.95
3.98
3.74
4.32/68.09
3.77/61.42
?-3-b-
Desulfated GVT
?-3-b- -Galp-1?
D-Galp?
D
4.64/104.80
5.15/96.23
5.35/100.10
3.75/70.58
3.94/69.28
3.75/71.85
4.26/78.97
4.25/70.91
3.62/77.86
4.14/65.66
3.75/78.51
3.62/72.12
3.66/75.36
4.02/70.41
3.96/73.76
3.80/61.21
3.83/61.45
3.61-3.52/63.45
?-4-
?-4-
a
a
-
-
D
-Galp-1?
D-Galp-3M?
3.73/55.85
a
ꢁ
M: –CH3; S: —SO3
.

116
T. Barahona et al. / Carbohydrate Research 347 (2012) 114–120
Figure 1. 13C–1H HSQC NMR spectrum in D2O of desulfated polysaccharide from tetrasporic plants of the green variant of Gigartina skottsbergii. F-1 means C-1 and H-1 of
unit F.
Table 2
fixed end point, for polysaccharides from marine algae show that
Linkage analysis of the constituent sugars of GVT polysaccharide
the antioxidant capacity increases with the sulfate content.28–30
Alditol acetate
Deduced unit and
substitution pattern
Polysaccharidea
Ethylation
The faster reactivity of hydroxyl radicals with GVT polysaccharide
with respect to that of commercial k-carrageenan suggests that
other parameters also contribute to the reactivity of the complex
reaction of polysaccharides with hydroxyl radicals. Kinetic studies
have confirmed that the reaction of HOꢀ radicals with polysaccha-
rides is close to the diffusion control, considering this high reactiv-
ity, it can be expected to have low selectivity.31 Thus, the reaction
of HOꢀ radicals can occur with different groups on the polysaccha-
ride. All of these facts indicate that hydroxyl radical scavenging
determined at a fixed end point is not strictly related to results ob-
tained from kinetic data. Furthermore, it has been proposed that
HOꢀ radicals scavenger capacity can occur by two mechanisms,
the suppression against hydroxyl radical generation and the chem-
ical reaction of radicals with the substrate. In the former, one of the
most important processes is the inhibition of HOꢀ radical genera-
tion by chelation of the metal ion with the macromolecule.
Figure 4 shows the absorbance of the ferrozine–Fe2+ complex32
in the absence and presence of GVT polysaccharide. These data
show that the polysaccharide addition inhibits the formation of
the ferrozine–Fe2+ chelating complex. In the absence of polysac-
charide the complex is formed in a few minutes and then, its absor-
bance remains constant. In the presence of polysaccharide, the
complex formation is slower and reaches an almost constant
absorbance value at longer times. The rate of the ferrozine–Fe2+
complex formation decreases with increasing polysaccharide con-
centration, but at longer times, the absorbance is similar to that in
the absence of the polysaccharide. The same behavior was found
for commercial k-carrageenan. These findings suggest that the fer-
rozine–Fe2+ complex is formed but at slower rate. A plausible
explanation is the electrostatic interactions between the Fe2+ and
the negatively charged sulfate groups in the polysaccharide. This
can contribute, at least in part, to the antioxidant capacity of GVT
polysaccharide and commercial k-carrageenan in the assay with
hydroxyl radicals.
2,4,6-Tri-O-ethyl-Gal
2,3,6-Tri-O-ethyl-Gal
4,6-Di-O-ethyl-Gal
3,6-Di-O-ethyl-Gal
2,3-Di-O-ethyl-Gal
3-O-Ethyl-Gal
?3-Gal
?4-Gal
40.4
18.3
3.8
23.5
9.6
?3-Gal 2 S
?4-Gal 2 S
?4-Gal 6 S
?4-Gal 2,6 S
?4-Gal 3 Mb
—
4.4
3-O-Methyl-2,6-Di-O-ethyl-Gal
S: sulfate.
a
Normalised mol % of monosaccharide having methyl or ethyl groups at the
positions indicated.
b
According to GC–MS analysis.
faster in the presence of the polysaccharide. This behavior is not
expected from the inhibition of the probe reaction by the polysac-
charide, and indicates the generation of free radicals in the reaction
of GVT polysaccharide with hydroxyl radicals, followed by a slower
reaction of these formed reactive species with the probe. According
to these reaction pathways, the absorption spectrum monitored
during the reaction showed a fast consumption of the dye and
the presence of a strong absorption at wavelengths lower than
420 nm, indicating a new product formation (Fig. 3 insert). A de-
crease of the probe consumption with increasing substrate concen-
tration also has been reported for some polysaccharides.26–28
Commercial k-carrageenan also exhibited a descending curve, sug-
gesting that free radical generation and not inhibition occurs.20 On
the other hand, the absorbance decay of the BG in the presence of
polysaccharide is well fitted to an exponential decay, indicating
that the reaction follows pseudo-first order kinetics. Interestingly,
the apparent rate constant (kapp) increases linearly with increasing
the polysaccharide concentration, giving rate constant values of
0.28 0.05 and 0.73 0.03 (mg/mL)ꢁ1 minꢁ1 for commercial k-car-
rageenan and GVT polysaccharide, respectively. The slower reac-
tivity of k-carrageenan does not agree with the higher sulfated
Additionally, the antioxidant capacity of sulfated polysaccha-
rides was measured using the ABTSꢀ+ assay based on electron trans-
fer mechanism, with the cation radical as oxidant.33 Changes of the
content at C-2 of b-
works on the scavenging activity by hydroxyl radicals, measured at
D-galactopyranosyl residues. Recently reported

T. Barahona et al. / Carbohydrate Research 347 (2012) 114–120
117
2.0
1.5
1.0
0.5
0.0
1.0
0.8
0.6
0.4
0.2
0.0
0.7
0.6
0.5
0.4
0.00
0.05
0.10
0.15
0.20
[Polysaccharide] (mg/mL)
0.3
0.2
0.1
a
b
c
d
0
10
30
40
Time2(0min)
0
10
20
Time (min)
Figure 4. Ferrozine complex formation in absence of native polysaccharide from
tetrasporic plants of the green variant of Gigartina skottsbergii (j) and in the
presence of 0.09 mg/mL (}), and 0.55 mg/mL (N); in the presence of 0.55 mg/mL of
commercial k-carrageenan (s).
Figure 2. Fluorescence decay of fluorescein probe in the presence of AAPH at
different concentrations of native polysaccharide from tetrasporic plants of the
green variant of Gigartina skottsbergii at 37 °C, and pH 7.0. (a) In the absence and in
the presence of (b) 0.03 mg/mL, (c) 0.07 mg/mL, and (d) 0.10 mg/mL of polysac-
charide. Insert shows the dependence of the net area under the curve versus
concentration for native polysaccharide from tetrasporic plants of the green variant
of Gigartina skottsbergii with fluorescein probe (N), and C-phycocyanin probe. (j).
1.00
Time (min)
0.75
0.50
0.25
0
0.60
0.56
0.52
0.48
10
45
0.45
0.30
Time (min)
0
4
0.30
5
0.25
0.20
0.15
0.10
0.05
15
24
600
800
400 Wavelength (nm)
0.15
0.00
Wav5e0le0nght (nm)
400
600
700
0
10
20
40
50
Time (min3)0
0
10
20
30
40
Figure 5. Decrease of ABTS radical cation absorbance at 734 nm in the presence of
different concentrations of native polysaccharide from tetrasporic plants of the
green variant of Gigartina skottsbergii. In the presence of 0.47 mg/mL (j), 0.97 mg/
mL (s), 1.41 mg/mL (N), and 1.90 mg/mL (h). Insert shows the absorption spectra
of ABTS radical cation in the presence of 1.90 mg/mL of native polysaccharide from
tetrasporic plants of the green variant of Gigartina skottsbergii, recorded at different
reaction times (from 0 to 45 min).
Time (min)
Figure 3. Decrease of Brilliant Green absorbance at 624 nm in the presence of
different concentrations of native polysaccharide from tetrasporic plants of the
green variant of Gigartina skottsbergii. In the absence of polysaccharide (j) and in
the presence of 0.06 mg/mL (s), 0.07 mg/mL (N), 0.12 mg/mLꢂ(}), and 0.24 mg/mL
(d). Insert shows the absorption spectra of Brilliant Green in the absence of H2O2
(- -), and in the presence of H2O2 and 0.24 mg/mL of native polysaccharide from
tetrasporic plants of the green variant of Gigartina skottsbergii recorded at different
reaction times (from 0 to 24 min).
been reported for some phenolic compounds,34,35 vitamin B deriva-
tives,36 and thiol derivatives37 and it has been related to the forma-
tion of side products. Further information on the side products was
obtained from the absorption spectra of the ABTSꢀ+ monitored at dif-
ferent reaction times (Fig. 5, insert). The absorption bands with max-
imum at 734 nm and 410 nm decrease and the band at 340 nm
increases. Probably, the absorption at 340 nm corresponds to the
second reaction since its enhancement is maintained at long reac-
tion times. These findings indicate that to obtain comparative values
of antioxidant capacity of a series of compounds it is necessary to
know the kinetic pattern involved in the reaction with ABTSꢀ+. Re-
ported data for some marine algae indicate a higher reactivity for
sulfated species.28,38,39 A comparison of results obtained for the
ABTSꢀ+ absorbance radical cation with GVT polysaccharide showed
fast initial kinetics followed by a slow reaction (Fig. 5). The extension
of the fast process was very small; it was completed within five min-
utes. Previously, the same behavior was found for commercial k-car-
rageenan.20 The ABTSꢀ+ absorbance decay could be well fitted to
biexponential kinetics with lifetimes in the range of 0.4–1.8 min
and 20–31 min, depending on the polysaccharide concentration.
This biphasic kinetics suggests a very fast reaction of polysaccharide
with the cation radical to form a product that is followed by a slow
reaction to form other products. Recently, this behavior also has

118
T. Barahona et al. / Carbohydrate Research 347 (2012) 114–120
ABTSꢀ+ bleaching by GVT polysaccharide and commercial k-carra-
geenan shows that, even though the activity is quite moderate, is
it slightly higher than that of the GVT polysaccharide.
4.2. Extraction
The dried algal material (25 g) was extracted with distilled H2O
(1.25 L) at 90 °C during 3 h with stirring and centrifuged (4000ꢀg).
The solid was extracted with 1 L of distilled water, and the extrac-
tion process was repeated once. The aqueous layers were dialyzed
against distilled water using Spectra Por (MWCO 3500) membrane,
water was exchanged four times. The retentate was concentrated
in vacuo, poured over 1 L of ethyl alcohol, and the precipitate
was separated by centrifugation at 8500ꢀg, dissolved in distilled
water and freeze-dried, affording 12.5 g of white cotton-like solid.
3. Conclusions
The polysaccharide synthesized by individuals of the green
variant of tetrasporic G. skottsbergii was different from k carra-
geenan, this polysaccharide shows a novel pattern of sulfation
and methylation units, which is unusual in sulfated galactans from
carrageenophytes. It can be concluded that the ORAC assay is an
adequate method to measure the antioxidant capacity of aqueous
soluble polysaccharides. However, this assay measures the antiox-
idant capacity toward peroxyl radicals but not the total antioxidant
capacity. It was found in the ORAC assay that antioxidant capacity
was dependent on the target molecule employed and on the con-
tent of sulfate groups and their position. k Carrageenan had higher
ORAC value and sulfate group content next to glycosidic bond, than
the native polysaccharide. Kinetic studies indicated that the anti-
oxidant capacity with ABTS+ꢀ and OHꢀ radicals was contrary to
the content of sulfate groups, because in both cases the native
polysaccharide was the most active. The behavior of the native
polysaccharide and commercial k-carrageenan was influenced by
the radical used to measure the antioxidant capacity.
4.3. Chemical analysis
Reducing sugars were determined by the phenol–sulfuric acid
method using
D
-galactose as standard.42 Molecular weight was
determined by the reducing end method as previously de-
scribed.43 Sulfate content was determined by a modification of
Dodgson and Price method;44 a conditioning solution containing
glycerine instead of gelatine was used (6 g NaCl, 20 mL H2O,
0.5 mL HCl, and 25 mL glycerine). The configuration of galactose
was determined through the formation of diastereomeric deriva-
tives with (S)-1-amino-2-propanol and GC analysis of the corre-
sponding 1-deoxy-1-1-(2-hydroxy-propylamino) alditol acetates
on a Ultra 2 column according to Cases et al.45 The corresponding
derivatives of D- and of L-galactose were used as standards. Nitro-
4. Experimental
gen was determined by microanalysis in Facultad de Química,
Pontificia Universidad Católica de Chile, and the protein content
was calculated by multiplying the percentage of nitrogen by the
6.25 factor.46
4.1. Material and general procedures
Tetrasporic plants of the green variant of G. skottsbergii (Gigart-
inales, Rhodophyta) were collected in spring in Fuerte Bulnes (53°
370 S, 70° 550 W), Southern Chile. Specimens were deposited in Her-
bario de Criptógamos, Universidad de Magallanes, Punta Arenas,
Chile. 1H NMR (400.13 MHz) and 13C (100.62 MHz) spectra of the
polysaccharides were recorded in D2O, after isotopic exchange
(3 ꢀ 0.75 mL) at 70 °C on a Bruker Avance DRX 400 spectrometer
using the sodium salt of 3-(trimethylsilyl)-1-propane-d4-sulfonic
acid) as the internal reference. All two-dimensional experiments
were acquired using a pulse field gradient incorporated into NMR
pulse sequence. The two-dimensional homonuclear 1H/1H COSY
experiment was acquired with 128 ꢀ 2048 data points having a
spectral width of 1200 Hz and processed in a 1024 ꢀ 1024 matrix
to give a final resolution close to 2.3 Hz/point in the two-dimen-
sions. The two-dimensional heteronuclear single quantum coher-
ence correlation (HSQC) spectra were acquired with 128 ꢀ 1024
data point and processed in a 1024 ꢀ 1024 matrix to give a final
resolution close to 2.3 Hz/point in 1H and close to 2.4 Hz/point in
13C. The number of scans in each experiment was dependent on
the sample concentration. FT-IR spectra in KBr pellets were regis-
4.4. Reductive hydrolysis
The native polysaccharide (5 mg) was heated with 0.1 M triflu-
oroacetic acid (1 mL) for 3 h at 80 °C. After cooling the trifluoro-
acetic acid (TFA) was evaporated and the residue was washed
with distilled water. One milliliter of aqueous NaBH4 (10 mg/mL)
was added and the solution was stirred for 1 h at room tempera-
ture; then, the reaction was stopped by addition of two drops of
acetic acid, and the resulting syrup was washed with methanol.
The solid was suspended in 2 M TFA (1 mL) and the reaction was
kept for 2 h at 120 °C. The excess acid was removed by co-distilla-
tion with water, and the reduction and washing with methanol
was repeated. The alditols were acetylated in Ac2O–pyridine and
analyzed by GLC.
4.5. Partial hydrolysis
The native polysaccharide (100 mg) was stirred in concen-
trated HCl (1 mL) and concentrated sulfuric acid (0.1 mL) at
25 °C. After 15 min, the solution was poured into acetone, and
the precipitate was washed thrice with portions of acetone
(2 mL) and dissolved in distilled H2O. The resulting solution was
dialyzed against 0.1 M sodium acetate followed by distilled H2O
and freeze-dried.
tered in the 4000–400 cmꢁ1 region using a Bruker IFS 66
v instru-
ment according to Leal et al.40 Absorbance was measured in a
Genesys 5 Thermospectronic spectrophotometer.
Gas–liquid chromatography (GC) was carried out on a Shima-
dzu GC-14B chromatograph equipped with a flame ionization
detector using a SP 2330 column (0.25 mm ꢀ 30 m) and performed
with an initial 5 min hold at 150 °C and then at 5 °C minꢁ1 to
210 °C for 10 min. The helium flow was 20 mL minꢁ1. GC–MS anal-
ysis was performed with an Agilent Technologies 7890A GC fitted
with an Agilent 19091S-433 column (30 m ꢀ 250 um ꢀ 0.25 um)
interfaced to a Agilent Technologies 5975C INERT XL M8D mass
spectrometer. Conversion of GC areas to molar basis was calculated
for the partially ethylated alditol acetates according to the effective
carbon response theory of Sweet et al.41 Unless otherwise stated all
the reagents and chemicals were from Merck (Darmstadt,
Germany).
4.6. Desulfation
The native polysaccharide was desulfated according to Falshaw
and Furneaux.47 Briefly, the polysaccharide (50 mg) was trans-
formed to the pyridinium salt form by dialysis against 0.1 M pyrid-
inium hydrochloride, then it was freeze-dried. The solid was
dissolved in 89:10:1 v/v Me2SO–MeOH–pyridine (0.4 mL/mg),
and heated at 100 °C for 4 h. The mixture was dialyzed against dis-
tilled water for 48 h and freeze-dried.

T. Barahona et al. / Carbohydrate Research 347 (2012) 114–120
119
4.7. Ethylation analysis
500
l
L solution of native polysaccharide and commercial k-carra-
geenan (0.0075–0.377 mg/mL), 500
l
L of ABTSꢀ+ was added. Absor-
Ethylation analysis was conducted according to Ciucanu and
Kerek.48 Briefly, the native polysaccharide (50 mg) in dimethylsulf-
oxide (5 mL) was stirred with finely powdered NaOH (400 mg) for
2 h at rt, then CH3CH2 I (5 mL) was added and the mixture was stir-
red for 1 h. The addition was repeated twice. The reaction was
stopped by addition of 2 mL of water, dialyzed against distilled
water and freeze-dried. The solid obtained was submitted twice
to the same ethylation procedure. The ethylated polysaccharide
was hydrolyzed with 2.0 M TFA for 2 h at 120 °C, and the partially
ethylated monosaccharides were reduced with NaBH4, and acety-
lated with Ac2O–pyridine. The partially ethylated alditol acetates
were analyzed by GC and GC–MS.
bance was registered in order to measure the kinetics of the
reaction.
4.9. Statistical analysis
The data obtained were means S.D. of three determinations,
and followed by the Student’s t-test. Differences were considered
to be statistically significant if P < 0.05. All assays were performed
in triplicate and repeated at least three times on different days.
Acknowledgements
The financial support of Dirección de Investigaciones Científicas
y Tecnológicas, Universidad de Santiago de Chile is gratefully
acknowledged. T. Barahona is grateful for a doctoral fellowship
and Grant AT-24091067 from CONICYT (Chile). A. Mansilla thanks
PFB-23 (Basal-CONICYT).
4.8. Determination of antioxidant capacity in vitro of sulfated
polysaccharides
4.8.1. Oxygen radical absorbance capacity (ORAC) assay
The consumption of fluorescein or C-phycocyanin associated
with its incubation with AAPH [2,20-azo-bis(2-amidinopropane)
dihydrochloride] was estimated from fluorescence measure-
ments.49 A reaction mixture containing 10 mM AAPH with and
without the native polysaccharide or commercial k-carrageenan
with different concentrations (0.001–1.0 mg/mL) in distilled water
was incubated in a phosphate buffer (10 mM, pH 7.0) at 37 °C.
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