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Carbohydrate Research 346 (2011) 19371944

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Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres

Mild acid hydrolysis of fucoidan: characterization by electrophoresis


and FT-Raman spectroscopy
A. Pielesz , W. Binias, J. Paluch
University of Bielsko-Biaa, Faculty of Materials and Environment Sciences, Bielsko-Biaa, Poland

a r t i c l e i n f o a b s t r a c t

Article history: Along with proteins, lipids, water and minerals, polysaccharides are the main chemical compounds of
Received 8 March 2011 which macroalgae are built. Among the chemical compounds now widely examined is fucoidan (fucan,
Received in revised form 12 May 2011 fucosan, sulfate fucan or sulfated fucan), a fucose-containing sulfated polysaccharide. Fucoidans isolated
Accepted 14 May 2011
from different species have been extensively studied because of their varied biological properties, includ-
Available online 30 May 2011
ing anticoagulant and antitumor effects. Methodology based on mild acid hydrolysis can be used as an
efcient tool to study the relationship between molecular weight of the sulfated polysaccharides and
Keywords:
their biological activities. Anticancer activity of fucoidans can be signicantly enhanced by lowering their
Fucoidan hydrolysis
Electrophoresis
molecular weight only when they are depolymerized under mild conditions. In this study, fucoidan was
Cellulose acetate identied during extraction with H2SO4 and HCl; its presence was conrmed by FT-Raman spectroscopy
Raman spectroscopy in aqueous solution. In particular, shifts at 840 cm 1 were analysed, which are due to the presence of sul-
fate at the axial C-4 position, as were the shifts at about 811809 cm 1, for which the sulfated fucoidan is
responsible. Shifts of electrophoretic bands of fucoidan resulting from mild acid hydrolysis in H2SO4 and
HCl were also analysed. The analytical procedure was developed using apparatus for cellulose acetate
membrane electrophoresis and this was supplemented by semi-quantitative analysis.
2011 Elsevier Ltd. All rights reserved.

1. Introduction molecular weights from 20 to 400 kDa with relatively high chain
stiffness and good water solubility, could enhance anticancer
A number of studies have concentrated on the anticancer activ- activity, compared to native and lower molecular weight polysac-
ity of fucoidan polymers. It has been suggested that they activate charides. On the other hand, low molecular weight fucoidans (16
the hosts immune system against tumours.1 For example, Miyeok- and 4 kDa) obtained from Ascophyllum nodosum enhanced the
gui fucoidan from the brown seaweed Undaria pinnatida shows neovascularization of human umbilical vein endothelial cells and
antitumour activity against prostate and cervical cancers. This endothelial progenitor cells in the presence of broblast growth
fucoidan is less cytotoxic to immune cells than the common fuco- factor-2, which resulted in the increase of cell proliferation.6,7
idan from Fucus vesiculosi.2 Recently, several studies have also indicated that sulfate groups
Fucoidan polymers can modulate cell growth in different man- of fucoidan play a major role in the suppression of cancer cell
ners depending on their molecular weight. Koyanagi et al.3 growth by binding with cationic proteins on the cell surface.3,8,9
reported that fucoidan polymers obtained from F. vesiculosus Nishino and Nagumo10 reported that the most potent anticoagu-
(100130 kDa) inhibited the growth of Sarcoma 180, Lewis lung lant activities were found in the range of molecular weights from
carcinoma and B16 melanoma cells by suppressing angiogenesis, 10 to 300 kDa. The mechanism of that appears to be very complex
or the formation of new micro-blood vessel. In a study of other and has not been claried.
polysaccharides, a relationship between the molecular weight In this study, fucoidan polymers were extracted from F. vesicu-
and the anticancer activity of sulfated a-glucans from Poria cocos losus Linnaeus and subsequently hydrolysed by heating in boiling
mycelia was observed.4 However, partially desulfated fucoidans water or in the presence of 0.01 M HCl and 0.1 M H2SO4 for various
with sulfate content below 20% showed a drastic decrease in both times. The objective was to analyse, as reported in the literature,
anticoagulant and anticancer activities.5 Oversulfated fucoidans the anticancer activity of partially hydrolysed fucoidan polymers
had higher anti-angiogenic activity than native fucoidans, and thus and to investigate the effects of molecular weights, also known
more effectively inhibited the growth of tumour cells.3 The authors from the literature, and different hydrolysis conditions on poten-
found that the sulfated a-glucans, having a moderate range of tial inhibition of cancer cell growth. Another objective was to cor-
relate the on-going hydrolysis of fucoidan with the changes of its
Corresponding author. Tel.: +48 33 82 79 114; fax: +48 33 82 79 100. molecular structure, which was examined using Raman spectros-
E-mail address: apielesz@ath.bielsko.pl (A. Pielesz). copy. This study continues earlier analyses of structural changes

0008-6215/$ - see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2011.05.016
1938 A. Pielesz et al. / Carbohydrate Research 346 (2011) 19371944

in fucoidan from F. vesiculosus L., examined by means of cellulose process, and could be due to the band of OH deformation vibra-
acetate membrane electrophoresis and FTIR spectroscopy.11 tion of H2O at about 1640 cm 1.
The FT-IR spectrum of this fraction contains bands at 3400 (OH
2. Results and discussion stretching), 2925 (CH stretching), 1675 and 1420 cm 1 (COO
stretching), characteristic of alginate. Two more bands were ob-
Preliminary electrophoretic examination (Fig. 1) revealed that served,15 at approximately 1100 and 1025 cm 1, which correspond
samples of fucoidan begin to hydrolyse when heated for an hour to mannuronic (M) and guluronic (G) units, respectively. The peak
in 0.1 M H2SO4 at 80 C. This hydrolysis manifests itself by clear at around 1642 cm 1 and the peaks near 10131153 cm 1 suggest
enhancement and shift of Rf for the fth and sixth lanes in the the presence of the CO group.16 In the FT-Raman spectrum, the
electrophoretogram. In water, 0.001 M H2SO4 and in 0.01 M absorption band still occurred at 1632 cm 1 and there was also
H2SO4, the electrophoretogram shows bands of the so-called native an absorption band at 3095 cm 1, indicating the presence of the
fucoidan. Interactions of fucoidan with H2SO4 (mild acid hydroly- unsaturated bond.
sis), leading to changes of band positions in Raman spectra More shifts, as shown in Fig. 2, were observed at 1638 cm 1. The
(Fig. 2), were also traced. IR features at 622 and 583 cm 1, and the corresponding Raman
Generally, in native fucoidan,9,11 the Raman band at 840 cm 1 band at 600 and 553 cm 1, were attributed to the asymmetric
and the corresponding IR band at 836 cm 1 were attributed to and symmetric O@S@O deformation of sulfates.1 In the 950
COS bending vibration of sulfate substituents at the axial C-4 posi- 800 cm 1 and 650540 cm 1 regions of the Raman spectra, the
tion. Comparing the spectra of the native fucoidan (2 F2 in Fig. 2) main bands and their relative width below the main peaks are
with the sulfated fucoidan (5 F2 in Fig. 2) shows clearly that inter- shown in Table 1. These bands are shown in detail in Fig. 3, re-
actions of fucoidan with H2SO4 lead to changes of band positions. solved into Gaussian-shaped bands using GRAMS software.
In particular, the band at 840 cm 1 is markedly shifted to These observations, summarized in Figures 2 and 3 and in Ta-
809 cm 1, a band produced by the sulfated fucoidan. ble 1, have been conrmed by other authors. The effect of oversulf-
Characteristic bands were also identied in other ranges. The ation of fucoidan using chlorosulfonic acidpyridine complex on IR
Raman band of the symmetric vibration of COO groups was found spectra was shown changing a peak position.17 Comparing the
at 1413 cm 1. The IR band at 1380 cm 1 and Raman shoulder at a spectra of native and sulfated fucoidans showed that the sharp
similar position originate from symmetric bending vibration of peak in both compounds at 845 cm 1 was related to the sulfate
methyl groups. The Raman band at 1337 cm 1 was assigned at the axial C-4 position, while only the sulfated fucoidan showed
mainly to HCC, HCO and COH vibrations in the pyranoid ring. A a peak at 811 cm 1 corresponding to C-6 substituted sulfate. Qiu
strong Raman band at 1269 cm 1 was attributed to asymmetric et al.9 found that the sulfation of fucoidan using chlorosulfonic
O@S@O stretching vibration of sulfate esters with some contribu- acidpyridine complex doubled the sulfate content and enhanced
tion of COH, CC and CO vibrations. An intense Raman band at its anticoagulant properties.
1072 cm 1 was assigned mainly to symmetric O@S@O stretching In this study, partially hydrolysed fucoidan was also observed,
vibration of sulfate esters. These bands are characteristic of sul- which was obtained by heating at 80 C, for various times (0, 5,
fated polysaccharides (carrageenans, agar, etc.) and have been used 15, 30, 45, 60 min), in the presence of 0.1 M H2SO4. The results of
as markers of sulfation because their intensity increases with the this examination are shown in Figs. 4 and 6 while band locations
sulfate content.12 in electrophoretograms are shown in Table 2.
An intense IR band at 1223 cm 1 and a very intense Raman For the examination presented in Fig. 6, the method used for
band at 1180 cm 1 were assigned to asymmetric and symmetric sample preparation was slightly modied. After centrifuging, the
stretching vibrations of O@S@O, respectively, which is an evidence supernatant was collected and mixed with 1% CaCl2; the solution
of sulfate esters.13,14 Strong, highly overlapping IR/Raman bands in was kept at 4 C for 3 h to precipitate alginic acid. In this way,
the region of 9001200 cm 1 are characteristic of carbohydrates the precipitated alginic acid and b-D-mannuronic acid, earlier
and correspond to coupled CO (at 1740 cm 1 as reported12) and clearly visible in Figures 1 and 4, were partly eliminated from
CC stretching and COH bending vibrations in polysaccharides. the analysed supernatant. Electrophoretic examination shown in
Apart from these signals, a new band appeared at 1636 cm 1. This Figure 4 was supplemented with an analysis of the Raman band
could be related to the unsaturated bond formed in the sulfation at 840 cm 1 (Fig. 5).

Figure 1. Cellulose acetate membrane electrophoresis (F2) of fucoidan; from left to right: fucoidan obtained by heating at 80 C by 1 h, in the presence of (20 mL: H2O,
0.001 M H2SO4, 0.01 M H2SO4, 0.1 M H2SO4).
A. Pielesz et al. / Carbohydrate Research 346 (2011) 19371944 1939

Figure 2. FT-Raman spectra of fucoidan (F2) samples of F. vesiculosus obtained by heating at 80 C by 1 h, in the presence of 20 mL H2O (native fucoidan 2 F2) and 0.1 M
H2SO4.

Table 1
1
Analysis of bands at 950800 cm in Raman spectra obtained using GRAMS software tained from seaweeds. For example, a branched ionic
Band location (cm 1
) Relative band width
polysaccharide isolated from the sap of the Chinese lac tree (Rhus
vernicifera) was chemically modied by sulfation using sulfur tri-
The native fucoidan (2 F2 in Fig. 2)
949 32.4
oxidepyridine (SO3Py) complex as a reagent.2 The spectrum
922 14.6 showed two characteristic absorption bands, one at 1258 cm 1,
900 32.5 describing an asymmetrical S@O stretching vibration, and the
878 18.8 other at 815 cm 1 with a discernible shoulder at 854 cm 1 indicat-
842 23.5
ing a symmetrical COS vibration. As SO3 has strong dehydration,
629 16.8
601 20.3 under such a reaction condition, it was possible that the polysac-
581 14.7 charide with hydroxy groups was dehydrated. This could contrib-
551 19.0 ute to the decrease of the hydroxy groups in the sulfated
The sulfated fucoidan (5 F2 in Fig. 2) pullulan. On account of dehydrolysis, hydrolytic degradation of
922 26.8 polysaccharides was inevitable during the sulfation. Therefore,
889 21.9 degradation of polysaccharide in the sulfation reaction process
876 13.5
813 39.9
likely involves both loss of water and hydrolytic degradation.
638 14.4 Mild acid hydrolysis in HCl is an apparently nonspecic method
624 23.7 for depolymerizing the sulfated polysaccharides. The anticancer
603 34.4 activity of fucoidan polymers found in partially hydrolysed sample
581 14.7
in the presence of 0.01 M HCl, heated in boiling water for various
times (between 0 and 15 min), changes and the sulfated fucans
During mild acid hydrolysis in sulfuric acid (VI), shifts at identied during the reaction with 0.01 M HCl showed a reduction
840 cm 1 were found. For partially hydrolysed fucoidan obtained in molecular size. The fucoidan polymers having the molecular
by heating at 80 C, for various times (0, 5, 15, 30 and 60 min), in weight of 2200 kDa obtained by 1 min hydrolysis showed a signif-
the presence of 0.1 M H2SO4, the following band shifts in Figure 5 icantly enhanced anticancer activity (71.3%) compared with the
were observed: 1 F5 (0 min) 848, 841, 832, 824, 818, 809 cm 1; 2 native fucoidans (37.6%), which have the Mw of 5100 kDa.
F5 (5 min) 850, a clear minimum at about 840 cm 1, 835, 828, Further increase in the anticancer activity (75.9%) was observed
818, 805 cm 1; 3 F5 (15 min) a clear minimum at about with the fucoidan polymers having the Mw of 490 kDa produced
840 cm 1 and 824 cm 1; 4 F5 (30 min) two bands at 830 and by 5 min hydrolysis.16 When the acid and heat treatment was ap-
812 cm 1; 6 F5 (60 min) bands at 847, 838, 831, 825, 816 and plied for 15 min, the anticancer activity of fucoidan polymers
809 cm 1. (260 kDa) decreased to 61.7%. It seems that the slight decrease
Figure 5 shows that the interaction between fucoidan and sulfu- was probably due to the partial removal of sulfate groups from
ric acid during the 15 min heating apparently results in band the fucoidan polymers during the prolonged acid and heat treat-
broadening, which may be later conrmed by currently prepared ment (15 min). In this study, an examination conducted under
further analyses regarding anticancer and antibacterial activity of the same conditions as in16 did not show any distinct shifts of elec-
fucoidan. By analysing Figure 5, uronic acid was found, identied trophoretic bands (the electrophoretograms are not included).
by CO carbonyl band at 946 cm 1. The band at 848 cm 1 was also Interestingly, desulfation was also observed in fucoidan polymers
found to be best visible for sample 6 F5 (Fig. 5), that is, during the extracted from Strongylocentrotus pallidus when they were heated
60 min hydrolysis. for 20 min at 60 C in the presence of 0.01 M HCl.18
Other authors have also analysed the shifts in S@O band loca- In this study, the interactions of reference crude fucoidan from
tions identied for polysaccharides of various origin, not only ob- F. vesiculosus with 0.01 M HCl were also analysed, which lead to
1940 A. Pielesz et al. / Carbohydrate Research 346 (2011) 19371944

Original Trace + Fitted Trace + Residual +


Original Trace + Fitted Trace + Residual + Peaks + Baseline + 2nd Derivative
Peaks + Baseline + 2nd Derivative .022

.051

.018

.049

.047 .014

Rmn Intensity
.045
Rmn Intensity

.01

.043

.041
.006

.039

.037
C .002
A
.035
File: 5 f2 srednia.SPC -.002
File: f2 2 1-3.SPC
950 930 910 890 870 850 830 810 960 940 920 900 880 860 840
Raman Shift (cm-1) Raman Shift (cm-1)

Original Trace + Fitted Trace + Residual +


Peaks + Baseline + 2nd Derivative
Original Trace + Fitted Trace + Residual +
.09
Peaks + Baseline + 2nd Derivative

.089
.054

.088
.053
D
.087

.052
Rmn Intensity

.086

Rmn Intensity
.051

.085

.05
.084

.049
.083

.048
.082

B
.081 .047
File: f2 2 1-3.SPC File: 5 f2 srednia.SPC
640 630 620 610 600 590 580 570 560 550 540 650 640 630 620 610 600 590 580 570
Raman Shift (cm-1) Raman Shift (cm-1)

Figure 3. The fragment of the FT-Raman spectra from fucoidan samples of F. vesiculosus at 950800 cm 1 and 650540 cm 1 regions: (A and B) native fucoidan (2 F2 in Figure
2) resolved into Gaussian-shaped bands; (C and D) sulfated fucoidan (5 F2 in Figure 2) resolved into Gaussian-shaped bands.

Figure 4. Cellulose acetate membrane electrophoresis (F5) and semi-quantitative analysis of fucoidan; from left to right: partially hydrolysed fucoidan obtained by heating at
80 C, at different times (0, 5, 15, 30, and 60 min), in the presence of 0.1 M H2SO4.

shifts of band positions in the Raman spectra. For reference sam- more active than the puried material, indicating that some highly
ples (Fig. 7) of fucoidan (in 0.01 M HCl), the Raman band at active fraction was discarded in the purication, another indication
847 cm 1 was attributed to COS bending vibration of sulfate sub- that specic structures within these complex mixtures can be
stituents at the axial C-4 position. The Raman band at 890 cm 1 linked to particular biological effects.
may indicate the presence of b-glycosidic linkages between mono- Interactions of fucoidan extracted with 0.01 M HCl (hydrolysis
saccharide units; the corresponding band of a-anomers at in 0.01 M HCl for 1, 5, 10 and 15 min), leading to changes of band
860 cm 1 is not detectable, being overlapped by stronger sulfate positions in Raman spectra, were also examined (Fig. 8). An addi-
band. tional weak sulfate absorption band at 848 cm 1 (COS, secondary
Generally, the IR/Raman band at 899 cm 1 is characteristic of b- axial sulfate) indicates that the sulfate group is located at position 4
polysaccharides. Therefore, according to FT-IR and FT-Raman band of the fucopyranosyl residue.20,21 For bands at 848808 cm 1, dur-
assignments the puried sample is sulfated polysaccharide of ing hydrolysis of samples 3 N5 to 6 N5, band shifts, their disappear-
complex structure containing uronic acids as a minor sugar com- ance or lower intensity are observed. Similar changes can be seen
ponent. As reported previusly,19 crude commercial fucoidan was for the bands at 893 and 948 cm 1 characteristic of b-glycosidic
A. Pielesz et al. / Carbohydrate Research 346 (2011) 19371944 1941

Figure 5. FT-Raman spectra of fucoidan (F5) samples of F. vesiculosus obtained by heating at 80 C, at different times (0, 5, 15, 30 and 60 min), in the presence of 0.1 M H2SO4.

linkages between monosaccharide units and uronic acid. In the


Table 2 850800 cm 1 region of the Raman spectra, the main bands and
Comparison of the calculated parameters, in accordance with the GELSCAN procedures
their relative band width below the main peaks are shown in
(regarding electrophoresis of the samples from Figs. 1, 4, 6 and 10)
Table 3. These bands are shown in detail in Fig. 9, resolved into
Path Band location [pix] Time [min] Gaussian-shaped bands using GRAMS software.
Figure 6 (F8) 1 446 5 in 0.1 M H2SO4 Fig. 10 (N2) and Table 2 show the results of fucoidan hydrolysis
2 488 15 in 0.1 M H2SO4 in 0.01 M HCl for 5, 15, 30, 45 and 60 min. A shift of Rf was found
3 574 30 in 0.1 M H2SO4
for samples hydrolysed for 45 and 60 min.
4 623 45 in 0.1 M H2SO4
Figure 4 (F5) 1 503 0 in 0.1 M H2SO4
The mechanism of why low molecular weight fucoidan poly-
2 506 5 in 0.1 M H2SO4 mers1 from sporophyll of U. pinnatida increase the anticancer
3 545 15 in 0.1 M H2SO4 activity has not been clearly explained. It is suggested16 that low
4 599 30 in 0.1 M H2SO4 molecular weight fucoidans may have an increased molar concen-
Figure 1 (F2) 1 479 60 in H20
tration induced by the depolymerization as well as may allow
2 498 60 in 0.001 M H2SO4
3 503 60 in 0.01 M H2SO4 greater molecular mobility and diffusivity than high molecular
4 605 60 in 0.1 M H2SO4 weight fucoidans. Thus, both effects of increased molar concentra-
Figure 10 (N2) 1 543 5 in 0.01 M HCl tions and improved interactions with cancer cell lines seem to be
2 522 15 in 0.01 M HCl responsible for the enhanced anticancer activity of partially hydro-
3 546 30 in 0.01 M HCl
4 489 45 in 0.01 M HCl
lysed fucoidan polymers.
5 386 60 in 0.01 M HCl According to Haroun-Bouhedja,5 partial removal of sulfate
groups from the fucoidan polymers from S. pallidus drastically
lowered their inhibition activity on the growth of CCL39 cells
from 100% to 12%. This implied that the molecular conformation

Figure 6. Cellulose acetate membrane electrophoresis (F8) and semi-quantitative analysis of fucoidan; from left to right: partially hydrolysed fucoidan obtained by heating at
80 C, at different times (5, 15, 30, and 45 min), in the presence of 0.1 M H2SO4.
1942 A. Pielesz et al. / Carbohydrate Research 346 (2011) 19371944

Figure 7. FT-Raman spectra of reference crude fucoidan from F. vesiculosus in the presence of 0.01 M HCl.

Figure 8. FT-Raman spectra (N2) of fucoidan obtained by heating at boiling water, at different times (3 N5-1, 4 N5-5, 5 N5-10 and 6 N5-15 min), in the presence of 0.01 M
HCl.

of fucoidan, which might inuence the binding properties of sul- Thus, boiling water heating appeared to cause less desulfation
fate groups, could be another factor affecting the inhibition po- from fucoidan polymers during the hydrolysis than the microwave
tency of the cancer cell growth. This is because if fucoidan treatment,16 where the dried sporophyll of brown seaweed U. pin-
polymers were in a compact spherical conformation stabilized natida, which originated from the coast of South Korea was ana-
by intramolecular interactions, the anionic sulfate groups avail- lysed. Because of the acidic pH of the medium, many sulfate
able to bind proteins on the cell surface may be hidden inside groups would be in a protonated form, making possible the occur-
the chains. Consequently, this would cause the reduction of sul- rence of intramolecular hydrogen bonds between two adjacent sul-
fates available to bind the proteins. However, if the fucoidan poly- fate groups. The broad stretching peak around 3384 cm 1 is
mers were in the loosened and entangled conformation, most characteristic of the hydroxyl group, while the weak stretching
sulfate groups existing in the chain would be likely available to band at 2930 cm 1 suggests CH bonds. In contrast, the peak at
bind the proteins, thus effectively enhancing their biological activ- 2069 cm 1 has been shown to indicate the presence of aliphatic
ity. These variations in the anticancer activity between hydrolysed CH bonds.12 Unfortunately, none of the studies quoted includes
fucoidan polymers may be due to their structural differences, Raman spectroscopy examination.
especially their sulfate content rather than their molecular These interactions would facilitate the removal of a sulfate
weight. Therefore, the results suggest that hydrolysis could signif- group in a nucleophilic substitution reaction. As for the increase
icantly inuence the anticancer activity of depolymerized fucoi- in susceptibility to acid cleavage in a desulfated fucose unit com-
dans through the removal of sulfate groups. pared with one of a non-desulfated unit, the sulfate groups can
A. Pielesz et al. / Carbohydrate Research 346 (2011) 19371944 1943

Table 3 promote a steric and/or electrostatic protection of the glycosidic


1
Analysis of bands at 850800 cm in Raman spectra obtained using GRAMS software linkage of the polysaccharide. After its removal, the linkage be-
Band location (cm 1
) Relative band width comes more susceptible to hydrolysis. These data are evidenced
Fucoidan (3 N5 in Fig. 9) by the different degradation of the sulfated fucans from S. palli-
845 5.4 dus/Lytechinus variegatus and Strongylocentrotus franciscanus.22
848 5.9 Interesting ndings are given by Pomin et al.16 who analysed
840 5.7 mild acid hydrolysis of sulfated fucans from S. pallidus and from
832 5.6
824 5.6
L. variegatus over different periods of time: 5, 20, 30 min, 1 and
817 6.2 2 h for the fucan from S. pallidus, and 30 min, 1, 2, 4 and 6 h for
809 6.9 the fucan from L. variegatus. In this study, fucoidan from F. vesicu-
802 5.7 losis was hydrolysed in 0.1 M H2SO4 and 0.01 M HCl over different
Fucoidan (4 N5 in Fig. 9) periods of time: 1, 2, 4 and 6 h. Pomin16 found the molecular
847 3.2 masses of the polysaccharides to be reduced, with no narrow bands
840 4.2
appearing in the PAGE, indicating a heterogeneous mixture of oli-
832 2.8
830 2.2 gosaccharides. Thus, the formation of well-dened molecular-size
825 7.6 oligosaccharides is notably dependent on the particular structure
808 3.9 of the sulfated polysaccharides. Therefore, the specic cleavage of
800 9.4 the polysaccharides by mild acid hydrolysis was inuenced specif-
Fucoidan (5 N5 in Fig. 9) ically by their pattern of sulfation. As part of this study, changes of
839 6.0 Rf for each sample were analysed in Table 4. Whether the band
833 8.4
825 6.6
shifts observed in electrophoretograms and Raman wavenumber
817 6.2 shifts may really result in increased anticancer activity of fucoidan
808 6.7 still remains to be determined in subsequent studies.
802 6.6
794 5.5
3. Conclusions
Fucoidan (6 N5 in Fig. 9)
856 7.5
847 4.8 There is no doubt that methodology based on mild acid hydro-
840 5.7 lysis can be used as an efcient tool for studying the relationship
823 1.0 between molecular weight of sulfated polysaccharides and their
816 5.6 biological activities. The loss of their activity is probably due to
809 8.2
not only the reduction of the molecular size. On comparing the
801 4.6
spectra of the native fucoidan with the sulfated fucoidan or fucoi-
dan after mild acid hydrolysis, the Raman results showed that

Original Trace + Fitted Trace + Residual +


Peaks + Baseline + 2nd Derivative
Original Trace + Fitted Trace + Residual +
.032 Peaks + Baseline + 2nd Derivative
.06

.031 .058

.056
.03

.054
Rmn Intensity

.029
.052
Rmn Intensity

.028 .05

.048
.027

.046

.026
.044

.025
855 845 835 825 815 805
3
File: 3 [n5].SPC .042

845 835 825 815 805


4
File: 4 [n5].SPC

Raman Shift (cm-1) Raman Shift (cm-1)

Original Trace + Fitted Trace + Residual +


Peaks + Baseline + 2nd Derivative
Original Trace + Fitted Trace + Residual +
Peaks + Baseline + 2nd Derivative
.0295
.045

.0285
.043

.0275

.041

.0265
Rmn Intensity

.039
Rmn Intensity

.0255

.037

.0245

.035
.0235

.033
.0225

.031
.0215

845 835 825 815


Raman Shift (cm-1)
805 795 5
File: 5 [n5].SPC
855 845 835 825
Raman Shift (cm-1)
815 805
File: 6 [n5].SPC

6
1
Figure 9. The fragment of the FT-Raman spectra from fucoidan samples of F. vesiculosus at 850800 cm region resolved into Gaussian-shaped bands.
1944 A. Pielesz et al. / Carbohydrate Research 346 (2011) 19371944

Figure 10. Cellulose acetate membrane electrophoresis (N2) of fucoidan; from left to right: partially hydrolysed fucoidan obtained by heating at 80 C, at different times (5,
15, 30, 45, and 60 min), in the presence of 0.01 M HCl.

4.2.3. Raman spectroscopic analysis


Table 4 The Raman spectra of the sample solutions were recorded on an
Comparison of the calculated parameters, in accordance with the GELSCAN procedures FTS 6000 spectrometer equipped with a MAGNA-IR 860, NICOLET,
(regarding electrophoresis of the samples in 0.1 M H2SO4 or 0.01 M HCl at different with FT-Raman accessory. Sample was irradiated with a 1064 nm
periods of time: 1, 2, 4, and 6 h)
line of the T10-8S Nd spectra-physics model: YAG laser and scat-
Path Band location [pix] Time [min] tered radiation were collected at 180, using 4 cm 1 resolution.
Sample N7 1 446 1h in 0.1 M H2SO4
2 488 2h in 0.1 M H2SO4
4.2.4. Vibration spectroscopy
3 574 4h in 0.1 M H2SO4 Synytsya2 FT-IR spectra (spectral region 4000400 cm 1, reso-
4 623 6h in 0.1 M H2SO4 lution, 2 cm 1) of the solid samples in the form of KBr tablets were
Sample N 8 1 503 1h in 0.01 M HCl recorded on Nicolet 6700 spectrophotometer (Thermo Scientic,
2 506 2h in 0.01 M HCl
USA) using OMNIC 7.0 software. Vibrational spectra were 10-point
3 545 4h in 0.1 M HCl
4 599 6h in 0.1 M HCl ltered and baseline corrected using ORIGIN 6.0 (Microcal Origin)
software. The second derivatives of the spectra were used for
wavenumber determination of overlapped bands.
interactions of fucoidan with acids lead to changes of band posi-
tions. Whether any increase in the anticancer activity can be ob- References
served needs further analysis and verication.
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