Chemical characteristic of an anticoagulant-active sulfated polysaccharide from Enteromorpha clathrata

Article abstract of DOI:10.1016/j.carbpol.2012.07.077

A sulfated polysaccharide FEP from marine green alga Enteromorpha clathrata was extracted with hot water and further purified by ion-exchange and size-exclusion chromatography. Results of chemical and spectroscopic analyses showed that FEP was a high arabinose-containing sulfated polysaccharide with sulfate ester of 31.0%, and its average molecular weight was about 511 kDa. The backbone of FEP was mainly composed of (1 → 4)-linked β-l- arabinopyranose residues with partially sulfate groups at the C-3 position. In vitro anticoagulant assay indicated that FEP effectively prolonged the activated partial thromboplastin time and thrombin time. The investigation demonstrated that FEP was a novel sulfated polysaccharide with different chemical characteristics from other sulfated polysaccharides from marine algae, and could be a potential source of anticoagulant.

Full text of DOI:10.1016/j.carbpol.2012.07.077

Carbohydrate Polymers 90 (2012) 1804–1810
Contents lists available at SciVerse ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Chemical characteristic of an anticoagulant-active sulfated polysaccharide from
Enteromorpha clathrata
Xiaohui Qia, Wenjun Maoa,∗, Yan Gaob, Yin Chena, Yanli Chena, Chunqi Zhaoa, Na Lia,
Chunyan Wanga, Mengxia Yana, Cong Lina, Jimiao Shana
a Key Laboratory of Marine Drugs, Ministry of Education, Institute of Marine Drug and Food, Ocean University of China, 5 Yushan Road, Qingdao 266003, People’s Republic of China
b National Oceanographic Center of Qingdao, 88 Xuzhou Road, Qingdao 266071, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 15 June 2012
Received in revised form 17 July 2012
Accepted 29 July 2012
Available online 4 August 2012
A sulfated polysaccharide FEP from marine green alga Enteromorpha clathrata was extracted with hot
water and further purified by ion-exchange and size-exclusion chromatography. Results of chemical and
spectroscopic analyses showed that FEP was a high arabinose-containing sulfated polysaccharide with
sulfate ester of 31.0%, and its average molecular weight was about 511 kDa. The backbone of FEP was
mainly composed of (1 → 4)-linked ␤-l-arabinopyranose residues with partially sulfate groups at the
C-3 position. In vitro anticoagulant assay indicated that FEP effectively prolonged the activated partial
thromboplastin time and thrombin time. The investigation demonstrated that FEP was a novel sulfated
polysaccharide with different chemical characteristics from other sulfated polysaccharides from marine
algae, and could be a potential source of anticoagulant.
Keywords:
Enteromorpha clathrata
Sulfated polysaccharide
Chemical characteristic
Anticoagulant activity
© 2012 Elsevier Ltd. All rights reserved.
1. Introduction
the sulfated polysaccharides from marine green algae is increas-
ing because of their high anticoagulant activities. Maeda, Uehara,
The leading causes of death are now diseases that involve heart
and blood vessels and as a consequence thrombin thrombosis
(Melo, Pereira, Fogue, & Mourão, 2004). Heparin is one of the agents
more largely used in the therapy and prophylaxis of thrombosis.
However, heparin is associated with various side effects, such as
hemorrhagic effect, development of thrombocytopenia and inca-
pacity to inhibit thrombin bound to fibrin (Liaw, Becker, Stafford,
Fredenburgh, & Weitz, 2001; Warkentin, 1999). In addition, the
usual sources of heparin are pig or bovine tissues. These tissues are
rich in mast cells, presumably resulting from the high foreign para-
site burden in these tissues (Warda, Mao, Toida, & Linhardt, 2003).
The appearance of bovine spongiform encephalopathy, ‘mad cow
disease’ and the incidence of prion-related diseases in mammals
has limited the use of bovine heparin (Schonberger, 1998). There-
fore, the alterative sources of anticoagulant and antithrombotic
compounds are required.
Harada, Sekiguchi, and Hiraoka (1991) revealed that the sulfated
polysaccharide from Monostroma nitidum yielded a 6-fold higher
anticoagulant activity than heparin. A sulfated galactan with high
anticoagulant activity was isolated from marine green alga Codium
cylindricum (Matsubara et al., 2001). Heparinoid-active sulfated
rhamnans were also isolated from Monostroma latissimum (Li et al.,
2011; Zhang et al., 2008). The sulfated polysaccharides from marine
green algae represent a potential source of anticoagulant to be
explored.
Recently, a few structural studies have been done on sul-
fated polysaccharides isolated from marine green algae (Lahaye
& Robic, 2007). The ulvans from Ulva sp. were all based on ␤-d-
glucuronic acid-(1 → 4)-␣-l-rhamnose 3-sulfate and ␣-l-iduronic
acid-(1 → 4)-␣-l-rhamnose 3-sulfate repeating units, as well as
contiguous (1 → 4)-␤-d-glucuronic acids possibly occurring either
in the ulvan or as a separate glucuronan (Lahaye et al., 1999). The
sulfated polysaccharides from M. nitidum and M. latissimum were
mainly composed of (1 → 3)-linked rhamnose and (1 → 2)-linked
rhamnose residues, and the sulfate groups were substituted at
C-2 of (1 → 3)-linked rhamnose and/or C-3 of (1 → 2)-linked rham-
nose residues (Harada & Maeda, 1998; Li et al., 2011). The sulfated
polysaccharides from Codium fragile and Codium vermilara con-
tained linear sulfated arabinans and galactans, (1 → 4)-␣-d-glucans
and (1 → 4)-␤-d-mannans (Ciancia et al., 2007). However, the struc-
tures of sulfated polysaccharides from Enteromorpha species have
One abundant source of new anticoagulant polysaccharides is
marine algae. Various anticoagulant-active polysaccharides, espe-
cially from marine red and brown algae, have been isolated and
characterized (McLellan & Jurd, 1992). In recent years, interest in
∗
Corresponding author. Tel.: +86 532 8203 1560; fax: +86 532 8203 3054.
E-mail address: wenjunmqd@hotmail.com (W. Mao).
0144-8617/$ – see front matter © 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carbpol.2012.07.077

X. Qi et al. / Carbohydrate Polymers 90 (2012) 1804–1810
1805
not yet been fully characterized (Kim, Cho, Karnjanapratum, Shin,
& You, 2011; Ray, 2006).
OHpak SB-804 HQ column (0.8 cm × 30 cm), eluted with 0.2 mol/L
Na2SO4 at a flow rate of 0.5 mL/min. The molecular weight was
estimated by reference to a calibration curve made by a set of
standard dextrans (Mw: 5.9, 9.6, 21.1, 47.1, 107, 200 and 708 kDa)
(Chen et al., 2011). Desulfation of the sulfated polysaccharide was
achieved according to Falshaw and Furneaux (1998). The desulfated
product was recovered by dialysis, freeze-dried and designated as
dsFEP.
Marine green alga Enteromorpha clathrata grows frequently
involved in algal proliferation in eutrophicated coastal and lagoon
water. It has been used as drug in traditional Chinese medicine
for hundreds of years. One particularly interesting feature of the
seaweed is its richness in polysaccharides, which are useful in
clearing away heat, detoxification and eliminating inflammation.
In the present work, an anticoagulant-active sulfated polysaccha-
ride from E. clathrata was isolated and its chemical characteristic
was investigated by a combination of chemical and spectroscopic
methods, including one- and two-dimensional nuclear magnetic
resonance (1D, 2D NMR) spectroscopy.
2.4. Analysis of monosaccharide composition
Monosaccharide composition was measured by reversed-phase
high performance liquid chromatography (HPLC) after pre-column
derivatization (Sun et al., 2009). Briefly, polysaccharide was
hydrolyzed with 2 mol/L trifluoroacetic acid at 100 ◦C for 6 h in
a sealed tube. The completeness of hydrolysis was confirmed
by thin-layer chromatography. Excess acid was removed by co-
distillation with methanol for four times after the hydrolysis was
completed. 1 mg of dry hydrolysate was dissolved in 100 ␮L of
0.3 mol/L NaOH, and then added to 120 ␮L of 0.5 mol/L methanol
solution of 1-phenyl-3-methyl-5-pyrazolone (PMP) at 70 ◦C for 1 h.
Finally, the mixture was added 100 ␮L of 0.3 mol/L HCl solution
and vigorously shaken and centrifuged for 5 min. The supernatant,
containing the labeled carbohydrates, was finally filtered through
0.22 ␮m nylon membranes (Westborough, MA, USA) and 10 ␮L
of the resulting solution was injected into the XDB-C18 column
(4.6 mm × 250 mm) fitted with UV detector (245 nm). The mobile
phase was a mixture of 0.1 mol/L KH2PO4 (pH 6.7)–acetonitrile
(83:17). The flow rate was 1.0 mL/min and column temperature was
30 ◦C. Sugar identification was done by comparison with reference
sugars (d-glucose, l-rhamnose, d-xylose, l-arabinose, d-mannose,
l-fucose, d-galactose, d-glucuronic acid, d-galacturonic acid and
N-acetyl-␤-d-glucosamine). Calculation of the molar ratio of the
monosaccharide was carried out on the basis of the peak area of
the monosaccharide. The complete hydrolysate of FEP was also
analyzed by Whatman 3 mm paper chromatography and develop-
ing with the solvent system of 1-butanol:pyridine:water (6:4:3)
(Harada & Maeda, 1998). The arabinose was obtained by extract-
ing with water and its optical rotation value was measured at 20 ◦C
with a P-1020 digital polarimeter, using a 10-cm light path length
at 589 nm (Mao et al., 2008).
2. Materials and methods
2.1. Materials
E. clathrata was collected on the coast of Fujian, China. The
raw material was thoroughly washed with tap water, air dried,
milled, and then kept in plastic bags at room temperature in a
dry environment. Dialysis membranes (flat width 44 mm, molec-
ular weight cut-off 3500) were from Lvniao (Yantai, China). Q
Sepharose Fast Flow and Sephacryl S-400/HR were from Phar-
macia Bioscience (Uppsala, Sweden). Standard dextrans (Mw: 5.9,
9.6, 21.1, 47.1, 107, 200 and 708 kDa) were from Showa Denko
K.K. (Tokyo, Japan). Standard heparin, d-glucose, l-rhamnose, d-
xylose, l-arabinose, d-mannose, l-fucose, d-galactose, d-glucuronic
acid, d-galacturonic acid and N-acetyl-␤-d-glucosamine were from
Sigma (St. Louis, MO, USA). Activated partial thromboplastin time
(APTT) assay reagent and prothrombin time (PT) assay reagent were
from Shanghai Sun (Shanghai, China). Thrombin time (TT) assay
reagent was from Dade Behring (Deerfield, IL, USA).
2.2. Isolation and purification of the sulfated polysaccharide
The milled alga (50 g) was dipped into 30 volumes of distilled
water and kept at room temperature for 1 h, and then filtrated. The
residue was dipped into 30 volumes of distilled water, homog-
enized and refluxed at 100 ◦C for 2 h. After cooling to the room
temperature, the supernatant was collected by centrifugation, con-
centrated, dialyzed in a cellulose membrane (molecular weight
cut-off 3500) against flowing distilled water at room tempera-
ture for three successive days. The retained fraction was recovered,
concentrated under reduced pressure, precipitated by adding four
volumes of 95% ethanol (v/v) and dried. The protein in the frac-
tion was removed by the method of Sevag (Matthaei, Jone, Martin,
& Nirenberg, 1962). The crude polysaccharide was dissolved in
distilled water and fractionated on a Q Sepharose Fast Flow col-
umn (30 cm × 3 cm) coupled to an AKTA FPLC system, eluted with
a step-wise gradient of 0, 1.0, 3.0 and 4.0 mol/L NaCl. The fractions
were assayed for carbohydrate content by the phenol–sulfuric acid
method (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956). The frac-
tions eluted with 1.0 mol/L NaCl were pooled, dialyzed and further
purified on a Sephacryl S-400/HR column (100 cm × 3 cm) eluted
with 0.2 mol/L NH4 HCO3 at a flow rate of 0.3 mL/min. The major
polysaccharide fraction was pooled, freeze-dried and named as FEP.
2.5. Methylation analysis
Methylation analysis was performed according to Hakomori
(1964) with some modification. Briefly, 2.0 mg of polysaccharide
was dissolved in 1.0 mL of dimethyl sulfoxide, and then 100 mg of
anhydrous sodium hydride was added. The mixture was stirred at
room temperature for 1 h. 1.0 mL of CH3I was added to the mix-
ture and stirred for 2 h. Finally, the reaction was terminated with
addition of distilled water, and the residue was extracted with
CH3Cl. The extract was washed with distilled water and evaporated
to dryness. The methylated polysaccharide recovered after dialy-
sis and freeze-drying was hydrolyzed with 2 mol/L trifluoroacetic
acid at 105 ◦C for 6 h. The methylated sugar residues were con-
verted to partially methylated alditol acetates by reduction with
NaBH4, followed by acetylation with acetic anhydride. The prod-
ucts were analyzed by gas chromatography–mass spectrometric
(GC–MS) on a HP6890II/5973 instrument using a DB 225 fused silica
capillary column (0.25 mm × 30 m) (Agilent Technologies Co. Ltd.,
USA). The temperature was increased from 100 to 220 ◦C at a rate
of 5 ◦C/min then maintained at 220 ◦C for 15 min. Identification of
partially methylated alditol acetates was carried out on the basis of
the retention time and its mass fragmentation patterns.
2.3. General analysis
Total sugar content was determined by the phenol–sulfuric acid
method using arabinose as the standard (Dubois et al., 1956). Sul-
fate ester content was estimated according to Therho and Hartiala
(1971). Protein content was quantified as described by Bradford
(1976). Purity and molecular weight were determined by high per-
formance gel permeation chromatography (HPGPC) on a Shodex

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X. Qi et al. / Carbohydrate Polymers 90 (2012) 1804–1810
2.6. IR spectroscopy
FTIR spectrum of the polysaccharide was measured on a Nicolet
Nexus 470 spectrometer. The polysaccharide was mixed with KBr
powder, ground and pressed into a 1-mm pellet for FTIR measure-
ments in the frequency range of 4000–500 cm−1
.
2.7. NMR spectroscopy
1H NMR and 13C NMR spectra were recorded at 23 ◦C using
a JEOL JNM-ECP 600 MHz spectrometer. 40 mg of polysaccha-
ride was deuterium exchanged by three successive freeze-drying
steps in 99% D2O and then dissolved in 0.5 mL of 99.98% D2O.
Chemical shifts are expressed in ppm using acetone as inter-
nal standard at 2.225 ppm for 1H and 31.07 ppm for 13C. 1H–1H
correlated spectroscopy (COSY), 1H–13C heteronuclear multiple
quantum coherence spectroscopy (HMQC) and 1H–13C heteronu-
clear multiple bond correlation spectroscopy (HMBC) and 1H–1H
nuclear overhauser effect spectroscopy (NOESY) experiments were
also carried out.
2.8. Assay of anticoagulant activity
APTT assay was carried according to Mourâo et al. (1996). In
brief, 90 ␮L of citrated normal human plasma samples and 10 ␮L of
sample solution at concentrations of 0, 10, 20, 50 and 100 ␮g/mL
were incubated at 37 ◦C for 60 s, 100 ␮L of prewarmed APTT assay
reagent was added, and the reaction was allowed to proceed at
37 ◦C for 2 min. Prewarmed 100 ␮L of 0.25 mol/L calcium chloride
was added. APTT was recorded as the time for clot formation. For
TT assay, 90 ␮L of citrated normal human plasma was mixed with
10 ␮L of a solution of polysaccharide and incubated at 37 ◦C for
60 s. Then, 200 ␮L of TT assay reagent prewarmed to 37 ◦C was
added and clotting time was recorded. PT assay was measured as
follows: 90 ␮L of citrated normal human plasma was mixed with
10 ␮L of a solution of polysaccharide and incubated at 37 ◦C for
1 min. Then, 200 ␮L of PT assay reagent pre-incubated at 37 ◦C
for 10 min was added and clotting time was recorded. All clotting
assays were performed in triplicate. The results were expressed as
means standard deviation (SD).
Fig. 1. Isolation and purification of the sulfated polysaccharide from marine green
alga E. clathrata. (A) The crude polysaccharides were applied to a Q Sepharose Fast
Flow column. The fraction eluted with 1.0 mol/L NaCl was pooled and named as FE;
(B) FE was applied to a Sephacryl S-400/HR column and eluted with 0.2 mol/L NH4
HCO3. The peak fractions containing the polysaccharides were pooled and named
as FEP.
3.2. Methylation analysis
In general, methylation analysis is useful to determine the link-
ages and the sulfate substitution position of sugar residues by
comparing the results of native and desulfated polysaccharides. The
yield of desulfated polysaccharide dsFEP from FEP was 62.7%. The
completeness of methylation of FEP and dsFEP was confirmed by
IR spectroscopy as the disappearance of OH bands (Supplementary
Fig. S1). As shown in Table 1, FEP mainly consisted of (1 → 3,4)-
linked arabinose with small amounts of (1 → 4)-linked arabinose,
(1 → 3)-linked rhamnose and (1 → 3,4)-linked rhamnose residues.
Compared with the results for FEP, the amounts of (1 → 4)-linked
arabinose distinctively increased, and the amounts of (1 → 3,4)-
linked arabinose significantly decreased in dsFEP. Therefore, the
sulfate substitution was at C-3 of (1 → 4)-linked arabinose. In addi-
tion, high amounts of (1 → 3)-linked rhamnose residues and low
amounts of (1 → 3,4)-linked rhamnose residues were also detected
3. Results and discussion
3.1. Composition analysis of the sulfated polysaccharide
The sulfated polysaccharide FEP from E. clathrata was extracted
with hot water, and further purified by a combination of ion-
exchange chromatography on a Q Sepharose Fast Flow column and
gel filtration chromatography on a Sephacryl S-400/HR column
(Fig. 1). The yield of FEP from crude polysaccharide (10.3 g/100 g
dry alga) was about 61.9%. FEP appeared as a single and symmet-
rical peak in the HPGPC chromatogram, and its average molecular
weight was about 511 kDa (Fig. 2). Monosaccharide composition
analysis by reversed-phase HPLC demonstrated that FEP consisted
of large amounts of arabinose (80.5%), with small amounts of rham-
nose (10.7%), galactose (4.8%) and glucuronic acid (4.0%) (Fig. 3). The
optical rotation of the arabinose hydrate was +104.8◦, the value is
in agreement with that for l-arabinose hydrate (Uehara, Takeshita,
& Maeda, 1992). FEP contained 31.0% sulfate ester, with a minor
amount of protein (0.4%). The chemical composition of FEP was
markedly different from those of other sulfated polysaccharides
from Enteromorpha species (Kim et al., 2011; Ray, 2006).
Fig. 2. HPGPC chromatogram of the sulfated polysaccharide FEP and the standard
curve of molecular weight. (A) HPGPC chromatogram of FEP on a Shodex OHpak
KB-804 column (0.8 cm × 30 cm); and (B) the standard curve of molecular weight.

X. Qi et al. / Carbohydrate Polymers 90 (2012) 1804–1810
Primary mass fragments (m/z)
1807
Table 1
Results of methylation analyses of FEP and dsFEP.
Retention time (min)
Methylated alditol acetate
Mol (%)
FEP
Deduced linkage
dsFEP
22.45
23.06
24.18
25.62
1,3,5-Tri-O-acetyl-2,4-di-O-methyl-rhamnitol
1,4,5-Tri-O-acetyl-2,3-di-O-methyl -arabinitol
1,3,4,5-Tetra-O-acetyl-2-O-methyl -rhamnitol
1,3,4,5-Tetra-O-acetyl-2-O-methyl-arabinitol
89, 101, 117, 131, 159, 201, 233
87, 101, 117, 129, 161, 189
87, 99, 113, 117, 129, 159, 173, 201, 275
87, 99, 117, 129, 159, 201, 261
2.5
10.5
10.1
76.9
8.4
79.5
4.2
→3)-Rhap-(1→
→4)-Arap-(1→
→3,4)-Rhap-(1→
→3,4)-Arap-(1→
7.9
4). The signal at 828 cm–1 was assigned to the bending vibration of
S of sulfate ester in axial position.
in dsFEP, suggesting that the sulfate groups were substituted at C-4
of (1 → 3)-linked rhamnose residues.
C
O
3.4. NMR spectroscopy
3.3. IR spectroscopy
In the 1H NMR spectrum of FEP (Fig. 5A), a major anomeric
proton signal at 5.18 ppm was assigned to ␤-l-arabinopyranose
residues (Fischer et al., 2004; Glushka et al., 2003; Ishii, Ono,
Ohnishi-Kameyama, & Maeda, 2005). The chemical shifts from 3.87
to 4.71 ppm were attributed to protons of the C-2–C-5 of the ara-
binopyranose residues, and the signal at 1.32 ppm was assigned
to the proton of CH3 group of the rhamnopyranose residues. In
the anomeric region of the 13C NMR spectrum (Fig. 5B), a major
carbon resonance occurred at 98.3 ppm. The signals between 61.4
and 77.1 ppm were assigned to the C-2–C-5 of the arabinopyranose
residues, and the signal at 18.3 ppm was attributed to the C-6 of the
rhamnopyranose residues.
As shown in Fig. 4, the band at 3413 cm−1 was derived from
the stretching vibration of O H, and the signal at 2925 cm−1
was attributed to the stretching vibration of C H. The band at
1647 cm−1 was due to the bending vibration of O H, and the signal
at 1243 cm−1 was assigned to the stretching vibration of S O of sul-
fate group (Mao et al., 2008). The signal at 1080 cm−1 was attributed
to the stretch vibration of C O and change angle vibration of O H.
In addition, the absorption at 868 cm–1 suggests the presence of a
␤-anomeric configuration for arabinopyranose (Zhang, 1999, chap.
1H–1H COSY spectrum (Fig. 5C) of FEP allowed the assignment of
most of the signals of the proton spin systems. The 1H–13C HMQC
spectrum afforded some of the correlations between carbon and
proton signals with the sugar residues, and elaborated the site of
sulfate ester in sugar residues and the linkage patterns between
sugar residues. In the 1H–13C HMQC spectrum (Fig. 5D) of FEP, the
anomeric proton signal at 5.18 ppm was correlated to the anomeric
carbon signal at 98.3 ppm, which was attributed to the (1 → 4)-
linked 3-sulfated l-arabinopyranose residues. In addition, the H-3
signal at 4.71 ppm showed the correlation with the C-3 signal at
77.1 ppm. The H-4 signal at 4.43 ppm was correlated with the C-4
signal at 74.7 ppm. Downfield shifted signals at C-3 and C-4 indi-
cated that the substitution positions were at the C-3 and C-4 of
arabinopyranose residues (Fischer et al., 2004). The results sug-
gested the presence of (1 → 4)-linked arabinopyranose residues
with sulfate groups at the C-3 position.
The 1H–13C HMBC spectrum (Fig. 5E) further confirmed some
of the correlations between carbon and proton signals within
the sugar residues, and the sequence of the sugar residues. The
anomeric proton signal at 5.18 ppm was correlated with the C-4
signal at 74.7 ppm in the 1H–13C HMBC spectrum. The anomeric
proton signal at 5.18 ppm was related to the H-4 signal at 4.43 ppm
in the 1H–1H NOESY spectrum (Fig. 5F). Thus, the (1 → 4)-linked
pattern between arabinopyranose residues was proved.
Fig. 3. HPLC analyses of monosaccharide standards and FEP. (A) Monosaccharide
standards and (B) FEP.
All the results demonstrated that the backbone of FEP was
mainly composed of (1 → 4)-linked ␤-l-arabinopyranose residues,
and the sulfation position was at C-3. The assignments of 1H
and 13C signals of the arabinopyranose residues were shown in
Table 2. Because of the strong signals of (1 → 4)-linked 3-sulfated
l-arabinopyranose residues, other sugar signals were relatively
weak and overlapped. The weak resonance at 5.28 ppm could be
attributed to the anomeric proton of (1 → 3)-linked 4-sulfated ␣-
rhamnopyranose residues (Cassolato et al., 2008; Li et al., 2011).
Other signals of the rhamnopyranose were difficult to identify.
The structural characteristics of FEP were markedly different
from those of the sulfated polysaccharide from Enteromorpha com-
press (Ray, 2006), and the latter was composed of (1 → 4)- and
(1 → 2,4)-linked rhamnopyranosyl, (1 → 4)-linked xylopyranosyl,
(1 → 4)- and terminally linked glucuronosyl residues, and sulfate
Fig. 4. IR spectrum of the sulfated polysaccharide FEP.

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X. Qi et al. / Carbohydrate Polymers 90 (2012) 1804–1810
Fig. 5. NMR spectra of the sulfated polysaccharide FEP. Spectra were performed at 23 ◦C on a JEOL ECP 600 MHz spectrometer using acetone as internal standard. Chemical
shifts are referenced to internal acetone at 2.225 ppm for 1H and 31.07 ppm for 13C. (A) 1H NMR spectrum; (B) 13C NMR spectrum; (C) 1H–1H COSY spectrum; (D) 1H–13
HMQC spectrum; (E) 1H–13C HMBC spectrum; and (F) 1H–1H NOESY spectrum.
C
Table 2
1H and 13C chemical shifts for the sulfated polysaccharide FEP.
Chemical shifts (ppm)c
Residued
H1/C1
H2/C2
H3/C3
H4/C4
H5a, H5b/C5
→4)-␤-l-Arap(3SO4)-1→
5.18/98.3
4.21/67.8
4.71/77.1
4.43/74.7
4.29,3.87/61.4
a
Equatorial bond.
Axial bond.
b
c
The spectra were recorded using a JEOL JNM-ECP 600 MHz spectrometer. Chemical shifts are referenced to internal acetone at 2.225 ppm for 1H and 31.07 ppm for 13C.
d
Arap: arabinopyranose.

X. Qi et al. / Carbohydrate Polymers 90 (2012) 1804–1810
1809
than 120 s at 50 ␮g/mL (Fig. 6B). TT is a measure of conversion of
fibrinogen to fibrin by thrombin, and an increase in the TT sug-
gests either thrombin inhibition or impaired fibrin polymerization.
The anticoagulant activity of FEP was weaker than that of heparin.
The effects of FEP on PT were significantly different from that of
heparin, and lack of prolongation effect of FEP on PT was discov-
ered (Fig. 6C). No prolongation of PT demonstrates no inhibition
of the extrinsic pathway of coagulation. The results suggested that
the sulfated polysaccharide FEP inhibited both the intrinsic and/or
common pathways of coagulation and thrombin activity or conver-
sion of fibrinogen to fibrin, and did not inhibit the extrinsic pathway
of coagulation. Extrinsic pathway activation leads to fibrin clot for-
mation in response to tissue injury while red thrombus or clot in
the absence of tissue injury is the result of the intrinsic pathway.
It was noted that the sulfated polysaccharide FEP had higher
anticoagulant activity than the sulfated polysaccharides from M.
latissimum (Zhang et al., 2008). FEP also exhibited higher antico-
agulant activity than the crude acidic polysaccharide from brown
alga Laminaria cichorioides as evaluated by APTT assay (Yoon, Pyun,
Hwang, & Mourâo, 2007). Pereira et al. (2005) found that the
sulfated galactan from the red alga Gelidium crinale had a lower
anticoagulant activity than a similar polysaccharide from Botryocla-
dia occidentalis with different sulfation patterns. The data suggested
that monosaccharide composition and the sulfate content and
position could influence the interaction of polysaccharide with
coagulation inhibitors and their target proteases. In addition, it
was observed that the sulfated polysaccharide FEP demonstrated
lower anticoagulant activity than the sulfated polysaccharide from
C. latum, possibly due to different linkage patterns of the ara-
binose residues in the polysaccharides (Uehara et al., 1992).
The differences of anticoagulant activities between the sulfated
polysaccharides were directly due to their structural features dis-
crepancy (Pereira, Mulloy, & Mourão, 1999). Further investigation
on the structure–activity relationship of the sulfated polysaccha-
ride is required.
4. Conclusion
The water-soluble polysaccharide FEP was isolated from marine
green alga E. clathrata. On the basis of detailed chemical and
spectroscopic analyses, FEP was characterized to be mainly
composed of (1 → 4)-linked ␤-l-arabinopyranose residues with
partially sulfate groups at the C-3 position. FEP was a novel high
arabinose-containing sulfated polysaccharide with different chem-
ical characteristics from the sulfated polysaccharides isolated from
Enteromorpha species and other sulfated arabinans. FEP had high
anticoagulant activity and specially interfered with the coagulation
cascade at several stages. FEP may be a potential source of anticoag-
ulant polysaccharide with novel structure. Complex relationships
existed between the structure and anticoagulant property of the
sulfated polysaccharides. Further analysis on the structure–activity
relationship of the sulfated polysaccharides will play an important
role in the understanding of anticoagulant property. An in-depth
investigation on the mechanism of anticoagulant activity of the
sulfated polysaccharide is in progress.
Fig. 6. Analysis of the anticoagulant activity by APTT, TT and PT on the sulfated
polysaccharide FEP. Three samples were prepared for assays of every attribute. (A)
APTT; (B) TT; and (C) PT.
groups were mainly located at C-3 of (1 → 4)-linked rhamnose
residues and C-2 of (1 → 4)-linked xylose residues. In addition,
FEP demonstrated different structural characteristics from other
sulfated arabinans from marine green algae (Ciancia et al., 2007;
Uehara et al., 1992). The sulfated polysaccharide isolated from C.
latum was reported to be a (1 → 5)-linked furanosidic arabinan with
20.7% sulfate (Uehara et al., 1992). Ciancia et al. (2007) found that
the sulfated arabinan from C. vermilara was mainly composed of ␤-
l-arabinopyranose residues, and sulfate groups were substituted at
C-2 and C-4. The data suggested that marine green algae could be a
potential source of sulfated polysaccharides with novel structures.
3.5. Assay of anticoagulant activity
Anticoagulant activity of the sulfated polysaccharide FEP was
evaluated by the classical coagulation assays APTT, PT and TT
using heparin as a reference. The anticoagulant activity of FEP was
concentration-dependent. APTT was effectively prolonged by FEP,
and the signal for clotting time was more than 200 s at 50 ␮g/mL
(Fig. 6A). The prolongation of APTT indicates inhibition of the intrin-
sic and/or common pathways of coagulation. Moreover, FEP also
strongly extended the TT, and the signal for clotting time was more
Acknowledgements
This work was supported by National Natural Science Founda-
tion of China (41076086), Doctoral Program of Higher Education of
China (20090132110015), National Oceanographic Center of Qing-
dao, China and the Science and Technology Development Program
of Shandong Province, China (2010GHY10509).

1810
X. Qi et al. / Carbohydrate Polymers 90 (2012) 1804–1810
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Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
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