An anticoagulant fucan sulfate with hexasaccharide repeating units from the sea cucumber Holothuria albiventer

Article abstract of DOI:10.1016/j.carres.2018.05.007

A fucan sulfate was isolated and purified from the sea cucumber Holothuria albiventer by papain enzymolysis, alkaline hydrolysis and ion-exchange chromatography. The water-soluble polysaccharide had high molecular weight and contained fucose and sulfate in a molar ratio of about 1:0.83. Methylation analysis of the native polysaccharide indicated that its glycosidic linkages and sulfate substituents might be at O-3 or O-3, 4 or O-2, 3, or O-2, 3, 4 positions. FT-IR and 2D NMR spectroscopies further revealed that the fucan sulfate is characteristically composed of a regular α (1 → 3) linked hexasaccharide repeating unit which is substituted with sulfate esters in a distinctive pattern. Anticoagulant properties of the fucan sulfate and its depolymerized product were assessed in vitro in comparison with a low-molecular-weight heparin. The fucan sulfate exhibits strong APTT and TT prolonging activities and intrinsic factor Xase inhibitory activity, and its molecular size seemed to be required for these activities.

Full text of DOI:10.1016/j.carres.2018.05.007

CarbohydrateResearch464(2018)12–18
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Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
An anticoagulant fucan sulfate with hexasaccharide repeating units from the
sea cucumber Holothuria albiventer
Ying Caia,b, Wenjiao Yanga,b, Ronghua Yina,b, Lutan Zhoua, Zhongkun Lic,∗, Mingyi Wua,∗
,
Jinhua Zhaoa,∗
a
State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, 650201, China
University of Chinese Academy of Sciences, Beijing, 100049, China
b
c
Department of Pharmacy, Yan'an Hospital Affiliated to Kunming Medical University, Kunming, 650051, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
A fucan sulfate was isolated and purified from the sea cucumber Holothuria albiventer by papain enzymolysis,
alkaline hydrolysis and ion-exchange chromatography. The water-soluble polysaccharide had high molecular
weight and contained fucose and sulfate in a molar ratio of about 1:0.83. Methylation analysis of the native
polysaccharide indicated that its glycosidic linkages and sulfate substituents might be at O-3 or O-3, 4 or O-2, 3,
or O-2, 3, 4 positions. FT-IR and 2D NMR spectroscopies further revealed that the fucan sulfate is character-
istically composed of a regular α (1 → 3) linked hexasaccharide repeating unit which is substituted with sulfate
esters in a distinctive pattern. Anticoagulant properties of the fucan sulfate and its depolymerized product were
assessed in vitro in comparison with a low-molecular-weight heparin. The fucan sulfate exhibits strong APTT and
TT prolonging activities and intrinsic factor Xase inhibitory activity, and its molecular size seemed to be required
for these activities.
Fucan sulfate
Chemical structure
Anticoagulant
Polysaccharide
Sea cucumber
1. Introduction
were found to be species-specific [12,17]. Thus, each new sulfated
polysaccharide purified from a certain sea cucumber or sea urchin
Unfractionated heparin (UFH) and low-molecular-weight heparins
(LMWHs) have been the cornerstones of antithrombotic treatment and
prophylaxis for the last 70 years, which are the only type of sulfated
polysaccharide currently used as anticoagulant drugs [1,2]. The com-
mercial sources of heparins are mainly porcine intestinal mucosa and
bovine lung, where the heparin content is low [3]. The possibility that
concomitants such as prions and viruses may be carried by the biolo-
gical products combined with the increasing demand for antithrombotic
therapies indicates the necessity to research for alternative sources of
anticoagulant agents [4].
Marine echinoderm and brown alga are abundant sources of antic-
oagulant polysaccharides, such as fucoidans and fucan sulfates [4–17].
Brown algal polysaccharides, called fucoidans, usually are mixtures of
several sulfated polymers with complex monosaccharide composition,
thus they are structurally non-regular and heterogeneous [4–10], which
hinders the clarification of their structure-activity relationship [10,11].
Distinguishing from fucoidans, the fucans from echinoderm such as sea
cucumber and sea urchin, often known as fucan sulfates (FSs), are
structurally more regular for they comprise only one kind of mono-
saccharide [12–18]. The chemical structures of these fucan sulfates
would be a new compound with unique structures and, consequently,
with potential novel biological activities.
Recently, when searching for new anticoagulant sulfated poly-
saccharides, we obtained two fucan sulfates from two species of sea
cucumbers Holothuria edulis and Ludwigothurea grisea [16,17]. Both
fucan sulfates have a unique structure composed of a central core of
regular (1 → 2) and (1 → 3)-linked tetrasaccharide repeating units.
Approximately 50% units of the FS from L. grisea (100% for H. edulis FS)
contain side chains that are formed by nonsulfated fucose residues and
linked to the O-4 positions of the central core. Anticoagulant activity
assays indicated that the sea cucumber FSs can strongly inhibit human
blood clotting through the intrinsic pathway of the coagulation cascade.
Their distinctive structure with the tetrasaccharide repeating units
contributes to the anticoagulant action [17].
Further exploration of sulfated polysaccharides from other sea cu-
cumber species would provide a wider insight into the studies on their
chemical structures and functional activities. In the present study, we
discovered a new fucan sulfate from the sea cucumber Holothuria albi-
venter. The chemical structure was analyzed by chemical and instru-
mental methods such as Fourier transform infrared spectroscopy
∗
Corresponding authors.
E-mail addresses: yayylzk@163.com (Z. Li), wumingyi@mail.kib.ac.cn (M. Wu), zhaojinhua@mail.kib.ac.cn (J. Zhao).
https://doi.org/10.1016/j.carres.2018.05.007
Received 31 March 2018; Received in revised form 15 May 2018; Accepted 17 May 2018
Availableonline19May2018
0008-6215/©2018ElsevierLtd.Allrightsreserved.

Y. Cai et al.
CarbohydrateResearch464(2018)12–18
Table 1
GC–MS of alditol acetate derivatives from the methylated product of the fucan sulfate from the sea cucumber H. albiventer.
Methylated Derivatives (as alditol acetates)a
Positions of substitution
Relative retention timeb
Molar ratio
Primary mass fragments (m/z)
2, 4-Me2-Fuc
2-Me-Fuc
4-Me-Fuc
Fuc
3
1.00
1.14
1.22
1.35
1.00
0.51
0.79
5.60
89, 101, 117, 131,159, 173, 233
87, 99, 117, 129, 173, 275
89, 99, 131, 159, 201, 261
3, 4
2, 3
2, 3, 4
115, 128, 145, 170, 217, 231, 289
a
2, 4-Me2-Fuc: 1,3,5-tri-O-acetyl-2,4-di-O-methyl-L-fucitol; 2-Me-Fuc: 1, 3, 4, 5-tetra-O-acetyl-2-O-methyl-L-fucitol; 4-Me-Fuc: 1,2,3,5-tetra-O-acetyl-4-O-methyl-
L-fucitol; Fuc: 1,2,3,4,5-penta-O-acetyl-L-fucitol.
b
Relative retention times of the corresponding alditol acetate derivatives compared to 1,3,5-tri-O-acetyl-2,4-di-O-methyl-L-fucitol.
(FT−IR), high performance liquid chromatography (HPLC), mono-
saccharide composition analysis, and nuclear magnetic resonance
(NMR) spectroscopies (1D 1H, 13C, 2D 1H/1H COSY, TOCSY, ROESY,
2D13C/1H HSQC and HMBC). Moreover, its effect on the clotting time of
human plasma and intrinsic coagulation factor Xase complex were in-
vestigated. Our results may provide valuable information for under-
standing the structure-function relationships of the well-defined poly-
saccharides from invertebrate.
acetyl-2,4-di-O-methyl-L-fucitol (2, 4-Me2-Fuc), 1,3,4,5-tetra-O-acetyl-
2-O-methyl-L-fucitol (2-Me-Fuc), 1,2,3,5-tetra-O-acetyl-4-O-methyl- L-
fucitol (4-Me-Fuc), and 1,2,3,4,5-penta-O-acetyl-L-fucitol (Fuc) with a
molar ratio of 1.00: 0.51: 0.79: 5.60 as calculated by their peak areas,
respectively (Table 1). The result suggested that its glycosidic linkages
and sulfate substituents might be at O-3 or O-3, 4 or O-2, 3, or O-2, 3, 4
positions of fucose residues in the polymer chains. Since the four sub-
stituents were all at O-3 positions, we inferred that the fucan sulfate
might consist of (1 → 3) linked fucose residues sequences. Other tech-
niques such as NMR analysis of its desulfated product would further
confirm the [(1 → 3)-Fuc]n backbone in a structure of the fucan sulfate
(see the following results).
2. Results and discussion
2.1. Purification and physicochemical characterization
The body wall of sea cucumber usually contains two types of sul-
fated polysaccharides, fucosylated chondroitin sulfate and fucan sulfate
[16–19]. The crude polysaccharides, with the yield of about 8.0% by
dry weight, were extracted from the sea cucumber H. albiventer by the
papain enzymolysis and alkaline hydrolysis [16]. The fucosylated
chondroitin sulfate was partially removed by ethanol precipitation and
KOAc salting out [19,20]. Then the crude fucan sulfate was further
purified into different fractions by anion exchange chromatography
using a FPA98 column. The fucan sulfate fraction obtained from the sea
cucumber seemed to display high purity as determined by the high-
performance gel permeation chromatography (HPGPC) (Figure S1).
Ultraviolet absorption at around 260 or 280 nm was not observed as
detected by an UV-detector, indicating the absence of contaminants of
protein or peptides.
Additionally, the HPGPC profile of the fucan sulfate displayed a
single wide peak, indicating that the polysaccharide might be homo-
genous with a wide distribution. And its average molecular weight was
over 2000 kDa as calculated by GPC. The monosaccharide composition
of the sea cucumber polysaccharide was qualitatively identified by re-
verse-phase HPLC after PMP derivatization procedures [21,22]. The
result showed that the fucan sulfate contained the only monosaccharide
fucose, which consists with those from other sea cucumber species
[15,16,18] (Figure S2). The specific rotations of the polysaccharide and
its depolymerized product were −168° and −172°, respectively, si-
milar to those from other sea cucumber species [16,17]. This result is in
conformity with L-configuration of fucose residues [17,23].
Among other diagnostic information, the content of possible
charged groups, such as sulfate groups, is essential to evaluate the
charge distribution along the polyelectrolyte chain. Thus, the charge of
the native fucan sulfate and its depolymerized product was measured
by conductometric titration [24]. Both of conductimetric titration
curves showed only one inflection point (Figure S3), indicating that the
fucan sulfate contains only a negatively charged functional group.
Further calculation indicated that the sulfate (SO4) content of the native
fucan sulfate was 34.4% (37.9% for the depolymerized fucan sulfate),
and the molar ratio of sulfate ester to monosaccharide was 0.83 (0.93
for the depolymerized fucan sulfate).
2.2. IR and NMR analysis
The organic functional groups of the H. albiventer fucan sulfate were
further characterized by IR spectroscopy (Figure S4A). The bands in the
region of 4000–1800 cm−1 showed the characteristic O–H and C–H
stretching vibrations of this polysaccharide at 3442 cm−1 and
2942 cm−1, respectively [26]. Three signal groups were assigned to the
sulfate groups, particularly, those appeared at 1262 and 1231 cm−1
were caused by S=O asymmetric stretching vibration, that at 854 cm−1
were assigned to the symmetric C–O–S stretching vibration and that at
583 cm−1 were caused by S–O stretching vibration [26–28]. These data
confirmed that the sea cucumber polysaccharide was substituted by
sulfate esters. Carbohydrate signals in the 1500–1200 cm−1 region in-
dicated the deformation vibrations of H–C–H, C–O–H, C–H and C–O–C
groups. Bands in this region at 1453 cm−1 and 1384 cm−1 may be as-
signed to the asymmetric and symmetric deformation vibrations of CH3,
respectively. These bands were also observed in the second-derivative
spectrum of fucoidan [29]. In the finger print region, band at 962 cm−1
was assigned to the asymmetric and symmetric deformation vibrations
of the methenyl groups in fucose residues [27]. The FT-IR spectrum of
the depolymerized fucan sulfate (Figure S4B) was similar to that of the
native fucan sulfate, implying their structural group similarity.
The 1D 1H NMR spectrum of the fucan sulfate displayed overlapping
and broad signals with line widths of several Hz (Figure S5), owing to
its highly polymeric nature as high-molecular-weight compound, which
hampered the resolution. It would be useful to study the desulfated
product of the native sulfated polysaccharide to clarify the glycosidic
linkages of its backbone. The sulfate content (Figure S3C) and the molar
rate of sulfate ester to fucose of the desulfated product were 15.6% and
0.28, respectively, and the desulfation rate was about 70% compared to
the native fucan sulfate. Minor signals at about 5.3 ppm in the 1H NMR
spectrum of the soluble desulfated product might indicate incomplete
desulfation (Figure S6). The molecular weight of the desulfated product
was 1638 Da. The result indicated that the fucan sulfate may be ex-
cessively degraded under desulfation conditions. Degradation of other
fucan sulfates was also observed previously in the desulfation proce-
dure [30,31]. Nevertheless, the 1D NMR spectra of the desulfated
product (Figure S6) indeed became simpler than those of the native
sulfated polysaccharide. The chemical shifts of individual residues in
the desulfated product were fully assigned (Table S1) according to 1D
(1H, 13C) and 2D 1H/1H COSY, TOCSY, ROESY, 2D13C/1H HSQC, and
To analyze the positions of its glycosidic linkages and sulfate ester
substituents in the fucan sulfate, methylation and GC-MS analysis was
carried out by the previous method [17,21,25]. Consequently, the four
monosaccharide derivatives identified by MS analysis were 1,3,5-tri-O-
13

Y. Cai et al.
CarbohydrateResearch464(2018)12–18
Table 2
1H/13C NMR chemical shift assignments of the depolymerized fucan sulfate from the sea cucumber H. albiventer.
residues
A
B
C
D
E
F
1H
δ (ppm)
H1
H2
H3
H4
H5
H6
C1
C2
C3
C4
C5
C6
5.379/5.340
4.426a/4.460
4.203b/4.236
4.757/4.741
4.362
1.188
100.47/97.83
75.25
5.346/5.310
4.519
4.344/4.311
4.861/4.851
4.366
1.195
96.63/96.38
77.21
5.012
3.876
3.939/3.873
3.898/4.233
4.386
1.208
97.69/97.65
68.78
78.31/78.46
70.69/77.27
69.55
5.306/5.284
4.499
4.061
4.032
4.261
1.192
97.65/95.84
75.33
76.63
71.75
5.046
3.795
3.885
3.957
4.246
1.188
100.75
69.05
76.85
71.06
68.69
17.79
5.035
3.766
3.943
3.975
4.483
1.216
102.0/101.8
69.01
78.07
13C
δ (ppm)
76.8
82.41/82.21
69.80
76.37/75.66
82.97
69.20
70.85
68.95
18.00
68.69
17.58
17.89
18.14
17.65/17.46
a
Values in bold type indicate the positions of sulfation.
Values in italic type indicate the glycosylated positions.
b
HSQC-TOCSY spectra (Figure S6). The correlation peaks in 2D 1H/1H
ROESY and 13C/1H HSQC-TOCSY spectra (Figure S6E, G) confirmed the
[(1 → 3)-Fuc]n backbone in a structure of the fucan sulfate (Figure S7).
To further elucidate the structures in detail, its low-molecular-
weight depolymerized product was obtained by our previously estab-
lished radical depolymerization method [26]. The chemical shifts of
individual residues in the depolymerized product were fully assigned
(Table 2) according to 1D (1H, 13C) (Fig. 1, Figure S8) and 2D 1H/1H
COSY, TOCSY, ROESY, 2D13C/1H HSQC, and HMBC experiments
(Fig. 2).
generally do not completely agree with our earlier related publication
[17]. The downfield chemical shifts of C-2 of the A, B and D residues
and C-4 of the A, B and C residues further confirmed their respective
sulfated substitution positions, consistent with the proton chemical
shifts analysis (values in boldface indicate the positions bearing sulfate
groups as shown in Table 2).
Furthermore, structural features of the fucan sulfate including the
glycosidic linkages and the residue sequences were confirmed ac-
cording to the correlation peaks in 2D 1H/1H ROESY and 13C/1H HMBC
spectra (Fig. 2C, E). The correlation between H-1 and H-3 of these re-
sidues was identified in the 2D 1H/1H ROESY spectrum (Fig. 2C),
confirming the presence of (1 → 3) linkages in the fucan sulfate. The
sequences of these repeating units were also confirmed by the long
range scalar correlations in the 2D13C/1H HMBC spectrum (Fig. 2E).
In the 1D 1H NMR spectrum of the depolymerized fucan sulfate
(Figure S8A), strong signals observed at around 1.1–1.2 ppm were the
characteristic signals of methyl protons in the L-fucose residues
[17,18]. Notably, six main signals at 5.0–5.5 ppm could be un-
ambiguously observed in the 1D 1H NMR spectrum (Fig. 1A), which
were attributed to the anomeric resonances of α-fucopyranosyl residues
with different sulfation patterns [16–18,32]. Further, 2D 1H/1H COSY
spectrum (Fig. 2A) clearly showed six spin-coupled systems (A, B, C, D,
E, F) and the six types of residues showed roughly equal integral areas
in the 1D 1H NMR spectrum, indicating that the fucan sulfate might be
composed of hexasaccharide repeating units. To assign other proton
signals in the spectra, 2D 1H/1H COSY and TOCSY NMR analyses were
employed to complete these proton assignments (Fig. 2A and B).
Starting from their anomeric protons, some protons from six spin sys-
tems were identified in the 2D 1H/1H COSY spectrum. According to the
correlation peaks in the 2D 1H/1H TOCSY spectrum, the proton che-
mical shifts could further be well assigned as shown in Fig. 1A and
Table 2. The H-2 shift values of A, B, and D residues in the fucan sulfate
at 4.4–4.5 ppm was shifted downfield by about 0.6 ppm compared with
those of non-sulfated fucose residues [17,20,32,33], indicating the
sulfation substitution at O-2 of these residues. Likewise, the H-4 shift
values of A, B, and C residues indicated that the fucan sulfate may
possess 4-O-sulfated fucose residues. The O-4 positions of C residues
were partly sulfated, and the non-sulfated C residues accounted for
about 70% (in mole), which may contribute to the complexity of its
chemical structure. And for the C residues with partially sulfated O-4
positions may have certain effect on the 1H/13C chemical shifts of other
residues such as residues A, B and D, two sets of the NMR signals for the
units A, B and D were thus observed here (Table 2).
Additionally, the chemical shifts values of all carbons in the depo-
lymerized fucan sulfate were assigned according to the 1D13C and
2D13C/1H HSQC spectra (Fig. 1B, Figure S8B, Fig. 2D) and presented in
Table 2. In the 1D13C NMR spectrum of the depolymerized fucan sul-
fate, the signals at 102–96 ppm were produced by the anomeric carbons
(C-1). The strong signals occurred at ∼18 ppm could be assigned to the
methyl groups (C-6) [17,18,20]. Owing to the reference to tri-
methylsilyl-propionic sodium salt, chemical shifts of 13C nuclei
Fig. 1. 1H (A) and 13C (B) NMR spectra of the depolymerized fucan sulfate with
molecular weight of ∼4 kDa. Chemical shifts are relative to the external tri-
methylsilylpropionic acid at 0 ppm in the 1H and 13C spectra, respectively. The
numbers following the letters indicate the positions of 1H and 13C.
14

Y. Cai et al.
CarbohydrateResearch464(2018)12–18
Fig. 2. Expansions of the COSY (A), TOCSY (B), ROESY (C), HSQC (D) and HMBC (E) spectra of the depolymerized fucan sulfate from the sea cucumber H. albiventer.
The order of the six residues was deduced as follow. In the 2D 1H/1H
ROESY spectrum (Fig. 2C), H-1 of residue A showed cross-peak to H-3
of residue B; H-1 of residue B showed cross-peak to H-3 of residue C; H-
1 of residue C showed cross-peak to H-3 of residue D; H-1 of residue D
showed cross-peak to H-3 of residue E. Similarly, the 2D13C/1H HMBC
spectrum (Fig. 2E) also showed the sequence-defining A1–B3, B1–C3,
C1–D3, D1–E3, E1–F3 and F1–A3. These evidences revealed the se-
quence and linkage -3-A-1→3-B-1→3-C-1→3-D-1→3-E-1→3-F-1 as the
linear hexasaccharide unit as shown in Fig. 3.
To further estimate the configurations at the glycosidic linkages, the
direct coupling constant (1JC–H) of C-1 of each monosaccharide residue
was also obtained from the 2D13C/1H HMBC spectrum (Fig. 2E). The
large values of 170–175 Hz for these fucose residues indicated the
protons are equatorial [34]. Taking account of the vicinal coupling
constant (3J1H–2H) of 3 Hz for fucose residues, the configuration at C-1
of these residues was α.
Overall, the combination of chemical analysis, methylation experi-
ments, IR and NMR spectroscopy allowed us to determine the fine
15

Y. Cai et al.
CarbohydrateResearch464(2018)12–18
Fig. 3. Proposed structure of the fucan sulfate from the sea cucumber H. albiventer.
structure of the fucan sulfate from the sea cucumber H. albiventer. The
α-L-fucan sulfate is essentially linear polymer, composed of regular
hexasaccharide repeating units with a distinctive sulfation pattern as
shown in Fig. 3.
For holothurian fucan sulfates, their specific sulfation patterns and
the glycosidic linkages vary from each other, for instance, the fucan
sulfates from the sea cucumbers Isostichopus badionotus, Apostichopus
molpadioides and Thelenota ananas are all linear polysaccharides con-
sisting of α(1 → 3) linked repetitive tetrasaccharide sequences while
differ in sulfation patterns [35–37]. The body wall of sea cucumbers,
Apostichopus japonicus (Stichopus japonicus) and Holothuria edulis
Table 3
Anticoagulant activity and intrinsic FXase inhibitory activity of the fucan sul-
fate.
Sulfated
polysaccharides
Mw (kDa)a APTT
(μg/mL)b
TT (μg/mL)b PT (μg/mL)b Anti-FXase
(ng/mL)c
Native FS
> 2000
25.79
115.47
> 128
2.98
> 128d
> 128
> 128
71.99
> 100000
68.57
depolymerized FS 4.33
> 128
9.50
LMWHe
a
4.5
Mw, weight average molecular weight.
b
c
concentration required to double the APTT or PT or TT of human plasma.
IC50 value, concentration required to inhibit 50% of intrinsic factor Xase.
> 128, this activity was not significant at concentration as high as 128 μg/
d
mL.
e
LMWH,
a low-molecular-weight heparin (Enoxaparin, Sanofi-Aventis,
France).
contains branched polysaccharides consisting of a pentasaccharide re-
peating unit, which is proposed to be the major structural component of
these fucan sulfates [17,32]. In this work, the H. albiventer fucan sulfate
comprises α(1 → 3) linked repetitive hexasaccharide sequences with a
specific sulfation pattern.
2.3. Anticoagulant activity analysis
The anticoagulant activities of the new fucan sulfate and its depo-
lymerized product (dFS) were evaluated by plasma clotting assays
[38,39], including activated partial thromboplastin time (APTT), pro-
thrombin time (PT) and thrombin time (TT) (Fig. 4, Table 3). Such
assays are routinely used to determine the ability of anticoagulants to
inhibit blood clotting through the intrinsic, extrinsic and common
pathways of the coagulation cascade, respectively [38,40]. The con-
centrations of the fucan sulfate required to double the APTT and TT
were 25.79 μg/mL and 115.47 μg/mL, higher than LMWH as a positive
control. The results indicated that the native fucan sulfate exhibited
potent anticoagulant activities by targeting the intrinsic and/or
common pathway. Similar to LMWH, the native polysaccharide showed
no significant PT-prolonging activity at the concentrations tested
(1–128 μg/mL), indicating that this polysaccharide had no or little ef-
fect on the extrinsic coagulation pathway. However, its depolymerized
product (Mw, 4.33 kDa) displayed no APTT, TT, PT-prolonging activ-
ities at the concentrations tested (1–128 μg/mL).
Since the intrinsic coagulation factor Xase complex (FXase) is the last
enzyme complex that can be targeted in the intrinsic coagulation pathway
[40,41], it is becoming recognized as a prime target for developing safer
anticoagulants with potential physiological and therapeutic applications
[42]. Thus, the intrinsic FXase inhibitory activity of the fucan sulfate and
dFS was further tested (Fig. 4B, Table 3). Increasing concentrations of the
sea cucumber fucan sulfate could result in essentially complete inhibition of
FXase (Fig. 4B), indicating that this polysaccharide displayed a strong anti-
FXase
activity
(IC50 = 71.99 ng/mL),
comparable
to
LMWH
Fig. 4. Anticoagulant activities (A) and anti-FXase activities (B) of the H. al-
biventer fucan sulfate, its depolymerized product (dFS) and a low-molecular-
weight heparin (LMWH).
(IC50 = 68.57 ng/mL). The FXase inhibitory activity of dFS was markedly
lower than its native polysaccharide, indicating that anticoagulant activity
of the fucan sulfate might depend on its molecular size.
16

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CarbohydrateResearch464(2018)12–18
3. Conclusions
monosaccharides were respectively hydrolyzed with trifluoroacetic
acid, and evaporated to dryness. The dry samples were dissolved and
reacted with PMP in methanol under the alkaline condition, to prepare
the monosaccharide derivatives of PMP for HPLC analysis. The chro-
matographic conditions were performed according to the previous
method [17,21].
The sulfate contents of the polysaccharides were measured by
conductometric method [24]. The acid form of samples was prepared
using the strong acidic cation exchange resin with Dowex R 50w × 8
50–100H and titrated by a strong base titration solution of 0.2 M NaOH
to record the conductivity value, then the sulfate content and the molar
ratio were calculated by the conductance curve.
In this study, we characterized the chemical structures of the fucan
sulfate from the sea cucumber H. albiventer by chemical analysis, IR and
2D NMR spectroscopy techniques. Our data revealed that the sulfated
polysaccharide is composed of regular α(1 → 3) linked hexasaccharide
repeating units with a distinctive sulfation pattern. Anticoagulant as-
says indicated that the fucan sulfate possessed strong APTT and TT
prolonging activities and intrinsic factor Xase inhibitory activity. The in
vitro data encourage a much more detailed screening of the in vivo
anticoagulant and antithrombotic effects of the well-defined fucan
sulfate.
4. Experimental
4.4. Methylation analysis
4.1. Materials
Methylation experiment of the polysaccharide was performed ac-
cording to the literature [17,21]. The native fucan sulfate was dissolved
in dimethyl sulfoxide and methylated using methyl iodide by ultrasonic
reaction. Then, the methylated polysaccharide was hydrolyzed with
trifluoroacetic acid, reduced by sodium borodeuteride, and the re-
sulting alditols were acetylated by pyridine and acetic anhydride. Fi-
nally, the alditol acetates were detected by GC–MS by a previously
reported method [21].
Dried sea cucumber H. albiventer was purchased in seafood markets
in Guangzhou city of Guangdong province (China). Amberlite FPA98Cl
ion exchange resin was purchased from Rohm and Haas Company
(USA). The monosaccharide standards including D-glucuronic acid
(GlcA), D-glucose (Glc), D-galactose (Gal) and D-mannose (Man) were
purchased from Alfa Aesar. L-rhamnose (Rha) was purchased from TCI.
L-fucose (Fuc) and 1-phenyl-3-methyl-5-pyrazolone (PMP, 99%) were
purchased from Sigma Chemical Co. (Shanghai, China). LMWH
(Enoxaparin, 0.4 mL × 4000 AXaIU) was from Sanofi-Aventis (France).
The activated partial thromboplastin time (APTT), prothrombin time
(PT), thrombin time (TT) reagents, and standard human plasma were
from Teco Medical (Germany). Biophen FVIII: C kit was from Hyphen
Biomed (France). Human factor VIII was from Bayer HealthCare LLC
(Germany). All of other chemicals and reagents were of commercially
available analytical grade.
4.5. Preparation of the desulfated product
Desulfuration of the native polysaccharide were carried out ac-
cording to the method with minor modifications [43]. The acid re-
solution of about 80 mg fucan sulfate was prepared using the strong
acidic cation exchange resin with Dowex R 50w × 8 50–100H, neu-
tralized with pyridine and lyophilized. The pyridinium salt was dis-
solved in 8 mL dimethylsulfoxide/methanol (9: 1, v/v), heated at 80 °C
for 9 h, dialyzed by a dialysis bag with a molecular weight cutoff of
1 kDa and lyophilized to obtain the desulfated product with the yield of
23.9%.
4.2. Extraction and purification of the polysaccharide
The crude polysaccharides were extracted from the milled body wall
of the sea cucumber H. albiventer according to the method in our pre-
vious reports with minor modifications [16,19,20]. The powder of
300 g dried body wall of H. albiventer was mixed up with 3 L of deio-
nized water containing 3 g papain and incubated at 50 °C for 6 h. The
resulting mixture was diluted with 600 mL of 1.5 M sodium hydroxide
and reacted at 50 °C for 2 h. After the reactant solution was adjusted to
pH 7 and centrifuged at 3000 × g for 20 min, the supernatant was
precipitated by adding ethanol to a final concentration of 60% (v/v) to
obtain the crude polysaccharides The crude polysaccharide was dis-
solved with 2 L of distilled water containing 67 mL of 30% H2O2 and
decolorized at pH 10 and 45 °C for 3 h. After adjusted pH to 7 and
centrifuged, the solution was precipitated by adding potassium acetate
to a final concentration of 1 M and ethanol to 40% (v/v). The resulting
precipitate was mainly the crude fucosylated chondroitin sulfate, and
the supernatant was further precipitated with ethanol at 60% (v/v).
After centrifugation, the crude fucan sulfate was obtained with the yield
of 4.17%.
4.6. Preparation of the depolymerized fucan sulfate
In order to further confirm the structure of the native fucan sulfate,
its depolymerized product (4.33 kDa) was prepared according to an
established method with some modifications [26]. The polysaccharide
(209.5 mg) and copper sulfate (1.27 mg) were dissolved in 8 mL of
deionized water, then the mixture was reacted with 1.56 mL of 10%
H2O2 at 35 °C for 7 h. After added 2 mL of saturated sodium chloride
solution, the solution was precipitated by adding ethanol to a final
concentration of 80% (v/v). Then, the precipitate was collected by
centrifugation, dissolved in water, dialyzed by a dialysis bag with a
molecular weight cutoff of 1 kDa, and lyophilized to obtain the depo-
lymerized product (dFS). The yield of dFS was 46.1%. Its physico-
chemical characteristics were analyzed, containing sulfate content,
specific rotation and infrared spectroscopy. The chemical structure was
further deduced by 1D/2D NMR analysis.
The crude fucan sulfate was further purified using strong anion
exchange chromatography on a FPA98 column (5 cm × 50 cm). The
purity and the average molecular weight were analyzed by high-per-
formance gel permeation chromatography (HPGPC) using an Agilent
technologies1200 series (Agilent Co., USA) apparatus with RID de-
4.7. Spectrometry analysis
Infrared Spectroscopy was recorded using a Fourier transform IR
spectrophotometer (Nicolet Is10, Thermo). The mixture of the native
fucan sulfate or the depolymerized product and KBr was ground to
powder, and pressed into pellets for IR spectral measurement in the
frequency range of 400–4000 cm−1 at room temperature.
tector, equipped with
a Shodex OH-pak SB-806 HQ column
(8 mm × 300 mm) and eluted with 0.1 M NaCl (35 °C, 0.5 mL/min).
One-dimensional spectra (1H/13
C NMR) and Two-dimensional
4.3. Monosaccharide composition analysis
spectra (COSY, TOCSY, ROESY, HSQC and HMBC) were acquired at
298 K on a 600/800 MHz Bruker Avance spectrometer with HOD sup-
pression by pre-saturation as previously described [38,40]. All samples
were previously dissolved in deuterium oxide (D2O, 99.9% D) and
lyophilized three times to replace exchangeable protons with D2O. All
The monosaccharide composition of the polysaccharide was ana-
lyzed by reverse-phase HPLC after PMP pre-column derivatization
procedures
[17,21,22].
The
polysaccharide
and
standard
17

Y. Cai et al.
CarbohydrateResearch464(2018)12–18
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Acknowledgments
This work was funded in part by the National Natural Science
Foundation of China (81673330, 81703374, and 81773737), Yunnan
Provincial Science and Technology Department in China (2016FA050
and 2014FB078), a grant from Youth Innovation Promotion Association
(2017435), Kunming Institute of Botany (KIB2017011), Discovery,
Evaluation and Transformation of Active Natural Compounds, Strategic
Biological Resources Service Network Programme (ZSTH-020) of
Chinese Academy of Sciences.
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Appendix A. Supplementary data
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