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Monthly changes in the content and monosaccharide composition of fucoidan

from Undaria pinnatifida (Laminariales, Phaeophyta)

Article  in  Journal of Applied Phycology · February 2010

DOI: 10.1007/s10811-009-9438-5

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J Appl Phycol (2010) 22:79–86
DOI 10.1007/s10811-009-9438-5
Monthly changes in the content and monosaccharide
composition of fucoidan from Undaria pinnatifida
(Laminariales, Phaeophyta)
Anna V. Skriptsova & Nataliya M. Shevchenko &
Tatiana N. Zvyagintseva & Tatiana I. Imbs
Received: 24 November 2008 / Revised and accepted: 3 February 2009 / Published online: 22 April 2009
# Springer Science + Business Media B.V. 2009
Abstract Crude fucoidan was extracted from the brown
alga Undaria pinnatifida collected monthly from April to
last July in Peter the Great Bay (Japan Sea, Russia). The
amount of crude fucoidan rose markedly from April to
June–July (from 3.2 to 16.0% dry weight) as the plant
matures. An analysis of the monosaccharide composition of
the fucoidan extracted showed that the alga synthesized
polysaccharides with various structures which were depen-
dent on the algae age. In juvenile plants collected in April–
May, this was represented by sulfated manno-galactofucan
containing up to 19–28 mol% of mannose and about
20 mol% of galactose, whereas in matured plants collected
in June–July, the polysaccharide was represented by a
sulfated galactofucan containing more than 38 mol%
galactose. It is postulated that the production of sori causes
a subsequent effect on fucoidan synthesis and leads to an
enhanced of crude fucoidan content and an increased molar
concentration of galactose. Crude fucoidan content in
sporophylls increased 5 times, and galactose content in this
polysaccharide rose s1.6 times with sori formation. The
structural characteristics of the fucoidan extracted from
sporophylls of Undaria collected in July were also studied.
The fractionation of crude fucoidan on DEAE-Sephadex A-
25 gave two fractions, F1 and F2 in equal quantities. F1
was characterized as manno-galactofucan sulfate and F2
A. V. Skriptsova (*)
A.V. Zhirmunsky Institute of Marine Biology
Far-East Branch of Russian Academy of Science,
Vladivostok 690041, Russia
e-mail: askriptsova@mail.ru
N. M. Shevchenko : T. N. Zvyagintseva : T. I. Imbs
Pacific Institute of Bioorganic Chemistry
Far-East Branch of Russian Academy of Science,
Vladivostok 690022, Russia
was galactofucan sulfate. The molecular weights of both
fractions were in a range of 30–80 kDa. Analysis of
fucoidan structure using ESI-FTICR mass spectrometry
showed the presence of mixed oligosaccharides consisting
of fucose and galactose. Presumably, the polysaccharide
molecules contain blocks built up of successively linked
residues of fucose and galactose. These blocks are built
from two to five or more residues of monosaccharides.
According to IR-spectroscopy data, the main portion of
sulfates is located at C2; in addition, sulfate esters are also
present at C4 on the fucose and C3 and C6 of the galactose
units.
Keywords Undaria pinnatifida . Fucoidan .
Monosaccharide composition . Monthly changes .
Structural analysis . Sporulation
Introduction
Fucoidans, a unique class of sulfated polysaccharide
(Chevolot et al. 1999) found in marine animals (e.g.
echinoderms) as well as extracellular matrix of brown
seaweeds, have not been isolated from terrestrial organisms.
Fucoidans are members of the family of sulfated homo- and
hetero-polysaccharides mainly built up of α-1,3 and 1,4-
linked L-fucose residues. D-Galactose, D-mannose, D-xy-
lose, L-rhamnose, D-glucuronic acid residues and acetyl
groups were found to be the constituents of fucoidans
(Kloareg and Quatrano 1988). Besides fucan sulfates,
fucoidans can also be designated as fucogalactan sulfates,
xylofucoglycuronan, and glycuronofucoglycan sulfates also
named ascophyllans and sargassans, respectively (Berteau
and Mulloy 2003; Ozawa et al. 2006). Fucoidans are a
heterogenous assemblage with regard to their molecular
80
mass, monosaccharide composition and number of sulfate
and acetyl groups. Different fucoidans types probably
coexist in the thallus of the algae. Although there are clear
links between algal species and fucoidan structure, there is
insufficient evidence so far to establish any systematic
correspondence between structure and algal order. It was
shown that most representatives of the orders Laminariales,
Chordariales and Fucales contain a homofucan; ascophyl-
lans are the common fucoidans in the Fucales and
Dictyotales, while sargassans are prevalent in the Scytosipho-
nales, Dictyosiphonales and the Desmarestiales (Chaubet et
al. 2000). All fucoidans show a wide spectrum of biological
action (Fitton 2005), which seems to be determined by their
degree of sulfatation, fine structure, monosaccharide com-
position, and molecular weight.
In spite of the persistent increase in interest regarding
brown algae as sources of fucoidans, basic knowledge
about factors involved in regulation of these polysaccha-
rides yield and composition is deficient. Several studies
have indicated that variations in the fucoidan content and
their structural characteristics seem to be differentiated
according to the seaweed species, season of collection
(Honya et al. 1999), and age of the plants (Zvyagintseva et
al. 2003). Different parts of a single algal thallus have been
shown to have varying amounts of fucoidans, as in the case
of Alaria fistulosa Postels and Ruprecht (Usov et al. 2005).
Maximal content was found in sporophylls, which was
several times higher than any other part of the thallus.
Moreover, publications establishing a correlation between
fucoidan content and seasonality generally report that the
highest amount of the polysaccharide is obtained during the
reproductive stage (Honya et al. 1999). Reproduction,
however, can be a long process involving several stages
(Lobban et al. 1985). Within this period, the physical and
morphological state of the alga varies, and physiological
and biochemical processes undoubtedly accompany such
changes. Sporulation in the Laminariales is followed by the
degeneration of the blades and loss of biomass. Hence,
within this period, the particular time at which the
maximum amounts of fucoidan with the most suitable
monosaccharide composition occurs needs to be identified
in order to manage the best time for harvest.
Undaria pinnatifida (Harvey) Suringar is native to the
cold temperate seas of the Northern Hemisphere. It is
endemic to Japan, Korea, and China, and has been
introduced in the French Mediterranean, the European
Atlantic, New Zealand, Australia, Argentina, and Italy
(Hay and Luckens 1987; Sanderson 1990; Fletcher and
Manfredi 1995; Casas and Piriz 1996; Wallentinus 1999;
Cecere et al. 2000; Aguilar-Rosas et al. 2004). In Russia, U.
pinnatifida is only found in Peter the Great Bay (Sea of
Japan; see Skriptsova et al. 2004). Sporophylls of this alga
contain up to 8–12% dry wt of sulfated polysaccharide,
J Appl Phycol (2010) 22:79–86
Fitton and Dragar 2006) characterized as galactofucan
sulfate (Mori et al. 1982; Lee et al. 2004). However,
information about fucoidan content and composition in
other parts of the Undaria thallus, as well as variations in
content and composition of fucoidan with season is absent.
No comparison has been made between the fucoidan yields
of different growth stages of U. pinnatifida.
In the present study, we investigated the following
aspects of U. pinnatifida: (1) monthly variations in the
content and composition of fucoidans using entire plants;
(2) the effect of sporulation on polysaccharide composition
in the sporophylls; (3) chemical structure of galactofucan
isolated from mature sporophylls.
Materials and methods
Samples of U. pinnatifida were collected from natural
habitats in Peter the Great Bay (Sea of Japan) in the
southern Primorye region of Russia from April to the end of
July, 2006.
In order to study the population structure, 30–70 plants
were collected monthly by a diver from a depth of 1–1.5 m.
Every month, ten quadrats (0.25 m2) were randomly
positioned along 100 m of coastline. All Undaria thalli
present in each quadrat were completely removed. The
developmental stage of each frond was determined. Plants
were classified as juvenile (large fronds without sporo-
phylls) or adult (thalli with developed sporophylls). Each
adult plant was assessed for development of sori by
histological observation of cross sections of the middle
parts of the sporophylls. The percentage of juvenile and
adult plants with and without sori was determined.
Isolation of polysaccharides from the algal material To
study the monthly changes of content and monosaccharide
composition of fucoidan the entire plants of U. pinnatifida
were used. Rhizoids were removed from all collected
algae and fresh thalli were treated with 96% ethanol
(1:1 w/v) at room temperature for 10 days. The extract
was filtered and the residue air-dried. The effect of sorus
development on the content and monosaccharide composi-
tion of fucoidan was studied using the sporophyll material.
Twenty thalli were collected in May and sporophylls were
severed. Sporophylls with sori and without ones were
separated and treated as described above.
Air-dried algal tissue were ground and extracted at room
temperature for 14 h with 0.1 M HCl (1:5 w/v; Extract 1).
The solution was filtered and algal tissue residues were
subjected to repeated extraction with water (1:5 w/v, 60°C,
5 h) and again filtered (Extract 2). Extracts 1 and 2 were
combined, subjected to ultrafiltration on a Millipore
membrane 1000 NMWL (Sigma, USA), concentrated and
J Appl Phycol (2010) 22:79–86
precipitated with three volumes of ethanol. The precipitate
obtained was washed with ethanol and acetone and dried
under vacuum. Crude fucoidan yield was estimated as a
percentage of the algal dry weight (% dry wt.).
Fractionation of sulfated polysaccharides The crude fucoi-
dan extracted as described above from sporophylls of
Undaria which were collected in July 2006, were fraction-
ated by column chromatography on DEAE-Sephadex A-25
(5.1 × 30.7 cm). The column was successively eluted
stepwise with 1.0 and 2.0 M NaCl solution, each time until
the disappearance in the eluate of a positive reaction for
carbohydrates with phenol and sulfuric acid (Dubois et al.
1956). The eluates were dialyzed by ultrafiltration and
lyophilized to get the polysaccharide fractions F1 and F2.
Analytical procedures Acid hydrolysis of the polysaccharides
was performed with 2 M trifluoroacetic acid (100°C, 7 h). The
monosaccharide composition of the products of the acid
hydrolysis was determined with a Biotronik IC-5000 carbohy-
drate analyzer (Shim-pack ISA-07/S2504; 0.4 × 25 cm; 70°C;
bicinchoninate method (Waffenschmidt and Jaenicke 1987);
Shimadzu C-R2AX detector). Monosaccharides (rhamnose
(Rha), mannnose (Man), fucose (Fuc), galactose (Gal), xylose
(Xyl), and glucose (Glc)), were used as standards for HPLC.
Molecular mass determination was performed by gel-
permeation FPLC on a column (1.5 × 30 cm) with
Superdex 75 HR 10/30 (Amersham Pharmacia Biotech
AB). Elution was carried out with 0.1 M sodium phosphate
buffer, pH 7.4 and 0.15 M NaCl, flow rate 0.4 mL h−1.
Dextrans (10, 20, 40, 80 kDa) were used as standards.
Total sugars were assayed using a phenol–sulfuric method
at an absorbance of 480 nm (Dubois et al. 1956). The sulfate
content was determined turbidimetrically after hydrolysis of
the corresponding fractions in 4 N HCl and an addition of a
gelatin/BaCl2 solution (Dodgson 1961). Content of uronic
acids in each polysaccharide sample was determined with m-
hydroxydiphenyl using D-glucuronic acid as a standard
(Blumenkrantz and Asboe-Hansen 1973).
13
С NMR spectra were recorded on a Bruker Avance
DPX-300 spectrometer (Germany) for solutions in D2O at
60°С (internal methanol, δC 50.15 ppm).
A sample of polysaccharide (Fraction 2) was hydrolyzed
using 0.1 N trifluoroacetic acid, 3 h, 100°C and then
methanol was added to the resulting solution (5:1 v/v) in
order to extract the oligosaccharides. The precipitate was
removed. The dried methanol fraction was dissolved in
1 mL of CH3CN:formic acid (0.1%; 1:1; v/v solution). The
SI-FTICR mass-spectrum of oligosaccharides was recorded
on a Bruker (Germany) Apex Qe electrospray ionization
Fourier-transform ion cyclotron resonance mass-
spectrometer (ESI-FTICR MS) in the positive mode (for
review, see Zaia 2004).
81
IR spectra of polysaccharides (KBr disc) were recorded
with a Vector 22 FT-IR spectrophotometer (Bruker,
Germany).
Statistical analysis ANOVA was performed using Statistica
6.0 (StatSoft). The data were tested for normality (Lillie-
form tests) and homogeneity of variances using the Leven
test. Tukey's Honest Significance Difference (HSD) test
was applied for comparison of the means. Statistical
significance of the fucoidan yield and monosaccharide
composition data among the reproductive and non-
reproductive thalli was determined using the Student's t-
test. p values less than 0.05 were considered statistically
significant.
Results and discussion
Monthly changes in crude fucoidan content
and composition
Fucoidan yield increased from April to July five times
(from 3.21 to 16.00% dry wt; one way ANOVA, F(2.2) =
361.86; p < 0.01; Fig. 1a). A similar observation was
reported by Honya et al. (1999) for Laminaria japonica J.E.
Areschoug in which the fucoidan content increased from
April to September. Also, the monosaccharide composition
of fucoidan was significantly affected by the season of
collection (Fig. 1b). The content of galactose increased
from 20.5 mol% in April to 38–39.5 mol% in June–July
2006 (one way ANOVA, F(6.2) = 66.43; p < 0.01). The
content of mannose showed an opposite tendency (one way
ANOVA, F(6.2) = 12.51; p < 0.05). The fucose content was
constant from April to July (one way ANOVA, F(6.2) =
1.53; p = 0.348). Only traces of other monosaccharide
constituents were found. The molar ratio of fucose and
galactose varied from 1:0.34 in April to 1:0.66–0.69 in the
June–July period.
The observed variations in chemical composition of
algae may be related to the influence of complex exogenous
factors (e.g., temperature, light intensity, light day length,
and concentration of nutrients) as well as endogenous
changes in the organisms (e.g., growth, morphological
change, reproduction). However, identification of possible
individual regulatory factors is particularly impossible due
to close relations between environmental interactions and
the physiological history of the alga. Exogenous factors
influence algal growth rate, morphology of thalli and
trigger reproduction and germination. At the same time, it
had been shown for the red algae that composition of
structural polysaccharides is determined to a large extent by
life cycle stage rather than by effects of environmental
82 J Appl Phycol (2010) 22:79–86

20
a b b Table 1 Percentage population of juveniles and adult thalli of
Undaria pinnatifida in different months
Crude fucoidan yield

15
Month Juveniles Adult
% dry wt

10 Sterile Fertile
a
5
a April 62 38 0
May 37 43 20
0 June 12 28 60
April May June July July 0 5 95
70
b
60
Monosaccharide content

50 Man Fig. 2a). Fucoidan yield increased fivefold with sorus

development (t-test = −8.895; df = 3; p < 0.01). Similarly,
mol%

40
30
20
10
0 occurred when the thalli matured for sporulation
April May June July
Fig. 1 Seasonal variation in the content (a) and monosaccharide
composition (b) of crude fucoidan from entire thalli of Undaria
pinnatifida. Man mannose, Fuc fucose, Gal galactose. Mean ± SD, n =
2. Columns with the same letters are not significantly different at p < 0.05
factors. Sporogenesis in Undaria in Peter the Great Bay
begins at the end of May, when water temperature is higher
than 14°C (Skriptsova et al. 2004). At this time, about 20%
of plants became fertile and by June 2006, up to 60%
individuals in the population have developed sori (Table 1).
Several reports indicated that the physiological and chem-
Fuc
the highest quantities of fucoidans in Laminaria cichor-
Gal
ioides Miyabe are accumulated in mature algae while
unripe algae contain a substantially smaller percentage of
fucoidan (Zvyagintseva et al. 2003).
Variations in the composition of neutral sugars also
(Fig. 2b). The content of mannose decreased 4.7-fold
with development of sori (t-test = 17.223; df = 3; p <
0.001), whereas the galactose content rose from 27.0 to
42.8 mol% (t-test = −7.184; df = 3; p < 0.01). The amount
of fucose was unchanged (t-test = −0.013; df = 3; p =
0.99). The molar proportion of fucose and galactose
changed from 1:0.51 to 1:0.80.
It can therefore be postulated that production of sori
affected metabolism and, as such, fucoidan synthesis.
20 a
Crude fucoidan yield

ical changes occur in seaweeds from vegetative to repro- 15

% dry wt

ductive growth and development of the subsequent

reproductive structures. In this period, growth and photo- 10
synthetic rates decline (Lüning 1988; Van Patten and Yarish
1993; Nimura and Mizuta 2001), synthesis of nucleic acids 5
increases (Nimura and Mizuta 2001), and amount of
alginate decreases (Apoya et al. 2002). In our study, we
0
observed enhanced crude fucoidan content and an increase
in molar proportion of galactose in the polysaccharide 60 b
Monosaccharide content

Man
extracted from the June–July samples which also may be 50 Fuc
the result of possible changes in the metabolism of 40 Gal
Undaria due to formation of sporangia in sporophylls. To
mol%

30
assay this suggestion, the effect of sorus development on
fucoidan content and monosaccharide composition in 20
sporophylls of Undaria was studied in May 2006, when 10
the population consisted of both fertile and vegetative adult 0
plants. without sorus with sorus
Our results indicate that reproductive status in U.
Fig. 2 Content (a) and monosaccharide composition (b) of fucoidan
pinnatifida had a significant effect on the polysaccharide from Undaria pinnatifida sporophylls with and without sori. Man
content (one way ANOVA, F(2.2) = 79.124, p < 0.01; mannose, Fuc fucose, Gal galactose. Mean ± SD, n = 2
J Appl Phycol (2010) 22:79–86
However, understanding of the physiological role of sulfated
polysaccharides in the sporulation of marine algae is
incomplete. It has been suggested that fucoidans, due to
their hydroscopic nature, can aid the discharge of reproduc-
tive cells (Evans et al. 1973). In accordance with the general
view, simultaneous gamete release in fucoids is provided by
instant swelling of the extracellular matrix in the concepta-
cle that enables the extrusion of gametes (Pearson and
Brawley 1998). It has been shown that fucoidans from
laminarian algae and fucoids also have antioxidant activities
(Xue et al. 2001; Hu et al. 2001; Le Tutour et al. 1998) and
therefore, presumably, may protect seaweeds against reac-
tive oxygen species induced by fluctuations of temperature,
light, salinity etc. in the field. Modification of the
monosaccharide composition of fucoidan during sorus
development suggested that the physiological role of the
polysaccharide is concerned with its structure. This suppo-
sition requires confirmation by additional experiments.
Preparation and preliminary characterization
of polysaccharides from sporophylls of U. pinnatifida
The highest amount of the fucoidan was extracted from
mature sporophylls and entire thalli of Undaria collected in
July 2006. Both extracts were a similar in their monosac-
charide composition. Hereafter, the fucoidan extracted from
mature sporophylls of alga collected in July was chosen for
structural analysis in our study.
Two fractions were obtained from chromatography of
the crude sulfated polysaccharides on the DEAE-Sephadex
A-25 column. The yields and composition of these fractions
are given in Table 2. Both fractions contained fucose and
galactose as main sugars and had a high content of sulfate.
However, F1 had the highest proportion of mannose and
lowest percentage galactose. Thus, F1 could be character-
ized as manno-galactofucan sulfate and F2 was galactofu-
can sulfate. These results agree with those observed by a
number of researchers (Mori et al. 1982; Lee et al. 2004;
Hemmingson et al. 2006), which showed that major
sulfated polysaccharides present in U. pinnatfida is
characterized to be a galactofucan sulfate, differed more
or less in the molar ratio of their constituents. Fraction-
ation of crude fucoidan by anion exchange chromatogra-
Table 2 Characteristics of the fractions obtained by anion exchange chromatography on column with DEAE-Sephadex A-25 of crude sulfated
polysaccharides from Undaria pinnatifida
Fraction NaCl, M Yields, % Mr, kDa SO3Na, %
F1 1.0 31.7 30–80 14
F2 2.0 29.7 60–80 29
83
phy gave various fractions which differed in their uronic
acid and sulfate contents and had a varying ratio of
galactose to fucose (Hemmingson et al. 2006). Similar
monosaccharide composition polysaccharides were also
shown earlier in Laminaria gurjanovae A.D. Zinova
(Shevchenko et al. 2007), L. japonica (Honya et al.
1999; Xue et al. 2001; Ozawa et al. 2006), Ecklonia
kurome Okamura (Nishino et al. 1991), Alaria fistulosa
(Usov et al. 2005), Adenocystis utricularis (Bory de Saint-
Vincent) Skottsberg (Ponce et al. 2003). These polysac-
charides were also characterized by a large proportion of
galactose; the molar ratio of fucose to this sugar was in the
range of 1.0:0.2–1.1.
In our study, the molecular weights (Mr) of both
fractions of fucoidan were in the range of 30–80 kDa
(Table 2). A previous study showed that Mr of fucoidan
from Undaria may vary significantly (Fitton and Dragar
2006) and presumably depend on the method of polysac-
charide extraction and purification. Lee et al. (2004) found
the Mr to be about 9 kDa of repeatedly fractionated
fucoidan extracted from sporophylls of U. pinnatifida;
crude polysaccharide extracted from whole fronds have a
Mr of 710 kDa, while Mr of it fractions eluted with 1 M and
2 M NaCl were 150 and 290 kDa, respectively (Hemmingson
et al. 2006). The decreasing of Mr of polysaccharide occurs
presumably due to depolymerization during the fractionation
and recovery process (Hemmingson et al. 2006). Generally,
Mr of crude galactofucan sulfate extracted from U. pinnati-
fida may range from about 30 to 1,200 kDa (Fitton and
Dragar 2006).
The fractions F1 and F2 had very complex 13C
NMR spectra (data not shown). The signals at the 97–
104.8 ppm interval corresponded to C1 of α-L-fucopyr-
anosyl units and β-D-galactopyranosyl residues. The signal
at 17–18 ppm (C6) was indicative of the presence of α-L-
fucopyranosyl units. The signal at 62 ppm showed that a
significant amount of non-6-linked galactose was present in
those fractions.
Despite the fact that the structure of galactofucan from
Undaria has been investigated by a number of researchers
(Mori et al. 1982; Lee et al. 2004; Hemmingson et al.
2006), it is still not clear. There is a suggestion that fucose
and galactose in galactofucan may form separate blocks or
UA, % Monosaccharide composition, mol % Fuc:Gal
Man Fuc Gal Xyl Glc UA
2.0 9.05 58.49 28.68 1.81 1.97 0 1:0.49
tr 1.02 52.38 46.59 0 0 0 1:0.89
84 J Appl Phycol (2010) 22:79–86

Table 3 Data of ESI-FTICR mass-spectrum of products of partial be interspersed in one polymer backbone (see Hemmingson
acid hydrolysis of fraction F2 of fucoidan extracted from sporophylls
et al. 2006). Another possibility is the presence of two
of Undaria pinnatifida collected in July 2006
separate polymers (fucan and galactan). Unfortunately,
m/z Composition previous structural investigations have not clarified these
relationships once the galactose and fucose of galactofucan
333.12 [Fuc2 + Na]+
were analyzed separately.
349.11 [Gal1Fuc1 + Na]+
Fraction F2 was separated for detailed study. The sample
365.11 [Gal2 + Na]+
was given a partial acid hydrolysis and then the methanol-
479.17 [Fuc3 + Na]+
extracted products of hydrolysis were analyzed by ESI-FTICR
495.17 [Gal1Fuc2 + Na]+ mass spectrometry. The peaks of ions corresponding to the
511.16 [Gal2Fuc1 + Na]+ fucose- and hexose-containing components were observed by
527.14 [Gal3 + Na]+ mass spectra (Table 3; Fig. 3). Hexose was shown to be a
625.23 [Fuc4 + Na]+ galactose from the monosaccharide analysis. The peaks
641.23 [Gal1Fuc3 + Na]+ registered as oligosaccharide ions [Fucn + Na]+(n = 2–5)
643.24 [Gal1Fuc3 + 2H + Na]+ and [Galn + Na]+(n = 2–5) under positive ion registration.
657.22 [Gal2Fuc2 + Na]+ Signals differing from the fuco-oligosaccharides peaks by 16,
659.24 [Gal2Fuc2 + 2H + Na]+ 32, 48, and 64 Da indicated the presence of oligomers which
673.25 [Gal3Fuc1 + Na]+ simultaneously comprised the fucose and hexose residues
675.23 [Gal3Fuc1 + 2H + Na]+ (Table 3; Fig. 3). Thus, the spectral analysis showed the
689.25 [Gal4 + Na]+ presence of fuco- and galacto-oligosaccharides with a
771.29 [Fuc5 + Na]+ polymerization degree from two to five and mixed oligo-
787.28 [Gal1Fuc4 + Na]+ saccharides consisting of fucose and galactose in their
789.30 [Gal1Fuc4 + 2H + Na]+ structures (e.g., Gal3Fuc2). This allows us to presume that
803.30 [Gal2Fuc3 + Na]+ the polysaccharide molecules contained blocks built up of the
805.30 [Gal2Fuc3 + 2H + Na]+ successively linked residues of fucose and galactose.
819.31 [Gal3Fuc2 + Na]+ The infrared spectra of the sulfated polysaccharides in
821.29 [Gal3Fuc2 + 2H + Na]+ this study are principally similar to the spectra of fucoidan
835.31 [Gal4Fuc1 + Na]+ from Undaria as published previously (Mori et al. 1982). A
837.29 [Gal4Fuc1 + 2H + Na]+ strong adsorption band of S=O stretching vibration at
851.30 [Gal5 + Na]+ 1,240 cm−1 indicated the presence of ester sulfate. A
moderate band near 820 cm−1 showed an equatorial

Fig. 3 ESI-FTICR mass-spectrum of products of partial acid hydrolysis of fraction F2 of fucoidan extracted from sporophylls of Undaria
pinnatifida collected in July 2006
J Appl Phycol (2010) 22:79–86
position of sulfate groups, while the shoulder at 844 cm−1
demonstrated axial locations of one. Smaller absorption
peaks at around 1,612 to 1,620 cm−1 indicated the presence
of uronic acid. According to IR-spectroscopy data, the main
portion of sulfates is located at C2 remains of fucose and
galactose; in addition, sulfate esters are also available at C4
of fucose and C3 and C6 of galactose.
Thus, the characterization of fucoidan from sporophylls
of U. pinnatifida by different analytical methods showed
that it is a low-molecular weight, highly sulfated hetero-
polysaccharide which comprises manno-galactofucan and
galactofucan. Galactose and fucose residues are successive-
ly linked to one polysaccharide molecule.
Conclusion
Fucoidans of various structures are of doubtless interest for
a comparative study of differential and functional biological
activities. Thus, the investigation of content and structural
variability of fucoidan, synthesized by seaweeds at their
different life-stages, is essential both for seaweed biology
and fucoidan utilization by the medical industry. Applica-
tions of polysaccharide agents in medicine have associated
problems with standardization. One method of standardiza-
tion is a definition of the season characterized by the
maximum quantity of fucoidan with a fixed structure and
the highest biological activity. Our results clearly showed
the highest values of fucoidan in the adult thalli of U.
pinnatifida which were collected in June–July 2006.
However, seaweed harvesting may be conducted during
other periods of the year in order to obtain fucoidans with
different structures and thus, with different pharmacological
properties, e.g., for manno-galactofucan and galactofucan
extraction, U. pinnatifida should be harvested in April and
the summer months, respectively.
Acknowledgments This work was financial supported by Russian
Foundation for Basic Research (projects № 06-04-39010; 09-04-00761),
Far-East Branch of Russian Academy of Science (project № 09-III-A-06-
212) and the program “Molecular and Cellular Biology” for Basic
Research of the Presidium of Russian Academy of Science. The authors
are also grateful to Dr. S.D. Anastyuk for help with mass spectra.
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