Comparison of physicochemical characteristics and anticoagulant activities of polysaccharides from three sea cucumbers

Article abstract of DOI:10.3390/md11020399

In order to search for sulfated polysaccharides in different invertebrate connective tissues and to examine their biological activities, we have isolated three types of polysaccharides from the body wall of the three sea cucumbers Holothuria edulis, Apostichopus japonicas and Holothuria nobilis. The physicochemical properties and anticoagulant activities of these polysaccharides were examined and compared. The chemical composition analysis and nuclear magnetic resonance (NMR) analysis indicate that two types of polysaccharides, sulfated fucan and fucosylated chondroitin sulfate (FuCS), were found in all of the three species and in addition a neutral glycan was observed in H. edulis. The neutral a-glucan was firstly obtained from sea cucumber. The same type of polysaccharides from different species of sea cucumbers have similar physicochemical properties and anticoagulant activities, but those of different types of glycans are significantly different, possibly due to their different monosaccharide compositions, electric charges and average molecular weights. The FuCSs have stronger anticoagulant activities than the sulfated fucans, although the molecular sizes of the FuCSs are lower than those of the sulfated fucans, whereas the neutral glucan has no activity, as expected from the absence of sulfate. Thus, anticoagulant activities of the different type of polysaccharides are likely to relate to monosaccharide composition and sulfate content. Preliminary analysis suggests that the sulfation patterns of the FuCSs may result in the difference in anticoagulant activities. Our data could help elucidate the structure-activity relationship of the sea cucumber polysaccharides.

Full text of DOI:10.3390/md11020399

Mar. Drugs 2013, 11, 399-417; doi:10.3390/md11020399
OPEN ACCESS
Marine Drugs
ISSN 1660-3397
www.mdpi.com/journal/marinedrugs
Article
Comparison of Physicochemical Characteristics and
Anticoagulant Activities of Polysaccharides from
Three Sea Cucumbers
1
,2,†
1,†
1
1
1
1
1
Lan Luo
, Mingyi Wu , Li Xu , Wu Lian , Jingying Xiang , Feng Lu , Na Gao ,
1
2
1,
Chuang Xiao , Shengmin Wang and Jinhua Zhao *
1
State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of
Botany, Chinese Academy of Sciences, Kunming 650201, China; E-Mails: luolan_41@163.com (L.L.);
mingyiwu_tju@yahoo.com (M.W.); xulib@mail.kib.ac.cn (L.X.); lw3662410@yahoo.com.cn (W.L.);
wind137@126.com (J.X.); lu.fengai@163.com (F.L.); gn2008.happy@163.com (N.G.);
xchuang@mail2.sysu.edu.cn (C.X.)
2
School of Life Science and Engineering, Southwest Jiaotong University, Chengdu 611756, China;
E-Mail: wangshengmin@sohu.com
†
These authors contributed equally to this work.
*
Author to whom correspondence should be addressed; E-Mail: zhao.jinhua@yahoo.com;
Tel./Fax: +86-871-5226278.
Received: 4 December 2012; in revised form: 24 December 2012 / Accepted: 9 January 2013 /
Published: 5 February 2013
Abstract: In order to search for sulfated polysaccharides in different invertebrate connective
tissues and to examine their biological activities, we have isolated three types of
polysaccharides from the body wall of the three sea cucumbers Holothuria edulis,
Apostichopus japonicas and Holothuria nobilis. The physicochemical properties and
anticoagulant activities of these polysaccharides were examined and compared. The
chemical composition analysis and nuclear magnetic resonance (NMR) analysis indicate that
two types of polysaccharides, sulfated fucan and fucosylated chondroitin sulfate (FuCS),
were found in all of the three species and in addition a neutral glycan was observed in
H. edulis. The neutral α-glucan was firstly obtained from sea cucumber. The same type of
polysaccharides from different species of sea cucumbers have similar physicochemical
properties and anticoagulant activities, but those of different types of glycans are
significantly different, possibly due to their different monosaccharide compositions, electric
charges and average molecular weights. The FuCSs have stronger anticoagulant activities
than the sulfated fucans, although the molecular sizes of the FuCSs are lower than those of

Mar. Drugs 2013, 11
the sulfated fucans, whereas the neutral glucan has no activity, as expected from the absence
400
of sulfate. Thus, anticoagulant activities of the different type of polysaccharides are likely to
relate to monosaccharide composition and sulfate content. Preliminary analysis suggests that
the sulfation patterns of the FuCSs may result in the difference in anticoagulant
activities. Our data could help elucidate the structure-activity relationship of the sea
cucumber polysaccharides.
Keywords: sea cucumber; polysaccharide; fucosylated chondroitin sulfate; sulfated fucan;
glucan; anticoagulant activity
1
. Introduction
Thromboembolic diseases continue to be the leading cause of death throughout the world [1]. Due to
aging and the high incidence of thrombosis, unfractionated heparin (UFH) and low-molecular-weight
heparins (LMWHs) have been the cornerstone in antithrombotic treatment and prophylaxis for the last
6
0 years. However, the heparin source is limited. It is obtained from porcine intestine or bovine lung, and
contamination of samples with pathogens is a serious concern [2]. Therefore, there is a great need for
new compounds from natural sources, and marine organisms are an abundant source of sulfated
polysaccharides with anticoagulant and antithrombotic activities [3–6].
Sea cucumbers are marine invertebrates that have gained popularity among researchers in recent
decades, not only due to their nutritive value, but also due to their potential health benefits and
therapeutic uses [4]. An extensive literature survey reveals that sea cucumber has a long history as a
traditional food and folk medicine [4]. For example, bioactives isolated from sea cucumber, novel
sulfated polysaccharides such as fucosylated chondroitin sulfate (FuCS) and sulfated fucan, have shown
anticoagulant [3,6,7] and antithrombotic activities [8]. They have clear advantages over previously
known anticoagulants such as heparin, since they are not obtained from mammals, occur at high
concentrations in an abundant invertebrate and retain the serpin-independent anticoagulant activity [3].
Thus, there is increasing interest in the anticoagulant activities of these sulfated polysaccharides.
Previous studies have suggested that anticoagulant activity mostly depends on the molecular size
of the polysaccharides and also relates to monosaccharide composition, sulfate content and
position [9–12]. The anticoagulant activity of the FuCS from the sea cucumber Thelenota ananas as
measured by the activated partial thromboplastin time assay varies in proportion to the molecular weight
following a logarithmic-like function [10]. Heparin cofactor II-mediated antithrombin activity of a
sulfated fucan is dependent on both its sulfate content and molecular weight [11]. The inhibitory effects
of fucans on coagulation are dependent on their sulfation degree and molecular weight [12].
Very recently, we have isolated a fucosylated chondroitin sulfate from the body wall of the sea
cucumber T. ananas and elucidated its chemical structure and anticoagulant activity [10,13,14]. In order
to examine the distribution of the sea cucumber polysaccharides and to compare their physicochemical
characteristics and biological activities, we have now expanded our studies to include more species of
sea cucumber, namely Holothuria edulis, Apostichopus japonicas and Holothuria nobilis. The
polysaccharides obtained from these sea cucumbers are three FuCSs, three sulfated fucans and a neutral

Mar. Drugs 2013, 11
401
glucan, respectively. We further found that the polysaccharides from different sea cucumber species
have distinctive physicochemical characteristics. The results where the physicochemical characteristics
have been fully characterized by a systematic analysis of nuclear magnetic resonance (NMR), chemical
composition, electric charge, average molecular weight and intrinsic viscosity, are valuable tools to trace
the relationship between structures versus anticoagulant activities of the sea cucumber polysaccharides.
2
. Results and Discussion
2
.1. Extraction and Purification
The yields of crude polysaccharides isolated from the three species of sea cucumbers,
H. edulis (HE), A. japonicas (AJ) and H. nobilis (HN), were 6.3%, 2.6% and 6.3% by dry weight,
respectively. The high-performance gel permeation chromatography (HPGPC) of the extracted
polysaccharides on a Shodex OH-pak SB-804 HQ column showed three peaks for H. edulis, whereas
two peaks for each of the sea cucumbers A. japonicas and H. nobilis (Figure 1) were found, indicating a
difference in molecular weight.
Figure 1. High-performance gel permeation chromatography (HPGPC) profiles of crude
polysaccharides, sulfated fucans, fucosylated chondroitin sulfates and neutral glucan from
the three species of sea cucumbers H. edulis (Panel A), A. japonicas (Panel B) and
H. nobilis (Panel C).
HE-3
HN-1
A
C
B
AJ-1
HE-2
HN-2
HE-1
HE-1
AJ-2
Crude polysaccharide
Crude polysaccharide
Crude polysaccharide
HN-1
Sulfated fucan
Neutral glucan
AJ-1
Sulfated fucan
Sulfated fucan
HE-3
HN-2
AJ-2
HE-2
FuCS
FuCS
FuCS
V0
V
0
V
t
Vt
V
V
0
t
2
.5
5.0
7.5
Elution volume (mL)
10.0
2.5
5.0
7.5
10.0
2.5
5.0
7.5
Elution volume (mL)
10.0
Elution volume (mL)
Sulfated polysaccharides were purified from the body wall of three species of sea cucumbers.
Purification was achieved by Sephadex G-100 and anion exchange chromatography on a
DEAE-Sepharose FF column. The crude polysaccharides were separated into three or two major fractions,
which were eluted with different NaCl concentrations. Based on their chemical compositions, NMR
analysis data and conductimetric titration curves (Table 1, Figure 2 and see below for discussion), we
found that the three species of sea cucumbers contained sulfated fucans (peaks HE-1, AJ-1 and HN-1 in
the Figure 1) and fucosylated chondroitin sulfates (peaks HE-2, AJ-2 and HN-2 in the Figure 1) as has
also been reported for other sea cucumber species [3,13,15–21], but surprisingly, the sea cucumber
H. edulis contains a neutral glucan (peak HE-3 in the Figure 1, Table 1). In addition, analysis of sulfated

Mar. Drugs 2013, 11
402
fucans and fucosylated chondroitin sulfates by anion exchange chromatography on a DEAE Sepharose
FF column confirms the high negative charge densities of the two polysaccharides (not shown), as
expected from analysis of the sulfate and carboxyl by the conductometric titration (Figure 2).
Figure 2. Conductimetric titration curves of samples of sulfated fucans (Panel A), fucosylated
chondroitin sulfates (Panel B) and neutral glucan (Panel C) from three sea cucumbers.
2
2
1
1
50
00
50
00
250
200
150
100
50
A
B
Fucosylated chondroitin sulfate
Sulfated fucan
Heparin
HE-1
AJ-1
HN-1
5
0
HE-2
AJ-2
HN-2
0
0
0
400
800
1200
1600
0
500
1000
1500
2000
2500
Volume of NaOH (L)
Volume of NaOH (L)
250
200
150
100
C
Neutral glucan
H O
2
50
HE-3
Dextran
0
0
400
800
1200
1600
Volume of NaOH (L)
The purities of these polysaccharides were confirmed by gel filtration chromatography on a Shodex
OH-pak SB-804 HQ column. The results show they each migrate as a single homogeneous peak
(Figure 1) and do not have any ultraviolet absorption near 260 or 280 nm by the measurement of an
UV-detector, indicating no contaminants such as protein and peptide. The contents of the two sulfated
polysaccharides are different: The fucosylated chondroitin sulfates are less abundant than sulfated
fucans in the three species of sea cucumbers by HPGPC profile analysis of the crude
polysaccharides (Figure 1).

Mar. Drugs 2013, 11
Table 1. Chemical composition and physicochemical properties of polysaccharides from the body wall of three sea cucumbers.
403
a
Chemical composition (molar ratios)
Average molecular weight
Specific
[η]
ΚH
ΚK
Samples
−
−
−
−1
−4
−4
GlcA GalNAc Fuc
Glc OSO /COO
SO /Fuc
Mn Mw
M /Mn rotation (mL·g ) (×10 ) (×10 )
3
3
w
Sulfated fucans
HE-1
AJ-1
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
1.00 <0.01
/
/
/
0.53
0.57
0.51
517,900 615,500
225,900 419,900
289,400 475,800
1.19
1.86
1.64
−181.4°
−181.5°
−193.4°
605.6
208.6
428.0
2.12
1.98
2.56
−1.49
−2.37
−1.63
1.00 <0.01
1.00 <0.01
HN-1
Fucosylated chondroitin sulfates and a standard polysaccharide
b
b
b
c
HE-2
AJ-2
1
1
1.28
1.05
0.96
ND
0.82
1.03
0.82
<0.01
<0.01
<0.01
3.50
2.96
2.99
2.04
ND
ND
ND
ND
41,340
46,760
42,460
20,230
51,090
56,820
55,320
26,260
1.24
1.22
1.30
1.30
−37.9°
−56.2°
−45.9°
48.2°
24.6
32.5
34.0
24.5
−8.71
−20.95
−10.48
−11.60
−12.62
−11.50
−14.06
−15.59
HN-2
1
Heparin
ND
ND <0.01
Neutral glucan
197,900 253,300
HE-3
<0.01
<0.01
<0.01 1.00
/
/
1.28
170.2°
0.8
15397
15212
a
GlcA: glucuronic acid; GalNAc: N-acetyl-β-D-galactosamin; Fuc: α-L-Fucose; Glc: D-glucose.
b
The ratios of GlcA and GalNAc were determined by the chemical methods (Experimental Section), and the ratios of GalNAc and Fuc were determined by their –CH3
1
integrals analysis of H-NMR spectra.
c
ND: Not determined.

Mar. Drugs 2013, 11
404
2
.2. Physicochemical Characteristics
2
.2.1. Chemical Composition and Analysis of Sulfate and Carboxyl Groups
These polysaccharide fractions have been characterized by different analytical techniques to compare
their physicochemical properties. The chemical analysis shows that they have various monosaccharide
compositions (Table 1 and Figure S1). Fractions HE-1, AJ-1 and HN-1 contain fucose as the only sugar
with a high content of sulfate ester, whereas the polysaccharide fractions HE-2, AJ-2 and HN-2 are
composed of three monosaccharides, namely glucuronic acid (GlcA), N-acetylgalactosamine (GalNAc)
and fucose (Figure S1). Fractions HE-2, AJ-2 and HN-2 can be assigned to be the chondroitin sulfate as
GlcA and GalNAc are in around 1:0.96–1:1.28 ratios (Table 1), possibly similar to the chondroitin
sulfate backbone structure. The fucose and sulfate contents of these FuCSs obtained from different sea
cucumbers vary from each other, which may account for their structure complexity. Cleavage of the sea
cucumber FuCSs by digestion with specific chondroitin lyases or by deamination with nitrous acid was
followed by HPGPC. These polysaccharides with high molecular mass were not reduced by digestion
with chondroitin AC or ABC lyase or by deamination with nitrous acid (not shown), possibly because
their sulfated fucose branches may be linked to Position 3 of the GlcA residues [13,17–20]. The results
are in agreement with previous reports on the FuCSs from others species of sea cucumbers [17–19].
Chemical analysis of the fraction HE-3 (Table 1) reveals glucose as the only sugar without a content
of sulfate ester. Thus, preliminary analysis indicates that the fraction HE-3 may be a neutral glucan.
Among other information needed, the contents of sulfate and uronic groups are essential to evaluate
the charge distribution along the polyelectrolyte chain [22]. Thus, the sulfate and carboxyl of the sea
cucumber polysaccharide were measured by conductometric titration. The conductance curves obtained
by titrating the acid form of the polysaccharides with a strong base (0.02 M NaOH) are shown in Figure 2.
Clearly, these curves differ in inflexion point. Extrapolation of the three branches of the conductimetric
curve of the FuCS gives two intersections, the first corresponding to the equivalence point of the sulfates
−
−
(
SO ), and the second to that of the carboxyls (COO ) (Figure 2B), which is similar to standard heparin.
3
The curve of the sulfated fucan has only one inflexion point, corresponding to the sulfates, but not
carboxyls (Figure 2A); whereas that of the glucan appears to have no inflection point, similar to a
standard dextran, indicating the absence of charge (Figure 2C).
−
−
Data for the SO3 and COO determinations are given in Table 1. The three sulfated fucans from
different species of sea cucumbers have the same sulfate: sugar molar ratio, close to 0.55. The
molar ratios are different from those of the sulfate fucans from Ludwigothurea grisea [15] and
−
−
Stichopus japonicas [16]. The three fucosylated chondroitin sulfates have different SO to COO molar
3
ratio (3.50, 2.99 and 2.96, respectively), more than the FuCS from L. grisea [17], but less than the FuCS
from T. ananas [10] and S. japonicas [18]. These variations may also reflect other important structural
differences [13].
2
.2.2. Specific Optical Rotation
In certain wavelength and temperature conditions, the optical phenomenon of an optically active
substance reflects the specific structure and specific rotation changes along with the structure changes.
Table 1 shows the specific optical rotation of the purified sea cucumber polysaccharides. The different

Mar. Drugs 2013, 11
405
types of polysaccharides have different specific rotations, but the same type of polysaccharides from
different sea cucumbers have similar specific rotations, as expected from the different monosaccharide
composition (Table 1). The strongly negative specific rotation of the sulfated fucans (Table 1) is
compatible with residues of α-L-fucopyranose [21]. The specific rotations of the FuCSs are levorotatory,
which are different from that of the standard heparin from the mammalian source (48°), possibly due to
the presence of L-fucose branches. The glucan (HE-3) appears to be dextrorotatory (170.2°), similar to
the dextran (182.5°), indicating that the glucan may be a D sugar on the Fischer projection. In addition,
the specific rotations of the FuCSs from −37.9° and −56.2° decrease with the drop in molecular weight
(Table 1). This was also observed in our previous study on the FuCS from T. ananas [10].
2
.2.3. Molecular Weight and Molecular Weight Distribution
As shown in Table 1, molecular weights of the three types of polysaccharides are different from each
other. Molecular weights of the sulfated fucans are around nine-fold of those of the FuCSs, but the
FuCSs from different species of sea cucumbers have similar molecular weights [10,18]. The molecular
weight of the glucan (HE-3) is also very high (>253 kDa). The molecular weight distribution (M /M ) is
w
n
used to measure the width of molecular weight distribution, which represents the homogeneity and
dispersibility of the polymer. The distribution data indicates that the natural polysaccharides are
homogeneous [23].
2
.2.4. Intrinsic Viscosity
Viscosity measurements were made on all sodium salt samples in a 0.1 M aqueous NaCl at 25 °C
using conventional capillary viscometers of the Ubbelohde type. The intrinsic viscosity ([η]) can be
determined by measuring the viscosity of solutions at low concentrations, and then extrapolating to infinite
dilution according to the Huggins or Kraemer relationships [24], respectively:
sp
2

[] k [] c
H
(1)
(2)
c
ln(r )
2

[] k [] c
K
c
where η is the relative viscosity (the ratio of solution viscosity and solvent viscosity η/η ), η is the
r
s
sp
specific viscosity (η − 1), and k and k are the Huggins coefficient and Kraemer coefficient, respectively.
r
H
K
According to Equations 1 and 2, the intrinsic viscosity [η] was determined by Huggins plots and
2
Kraemer plots with good linear regressions (R > 0.96) (Figure 3A,B,C). The intrinsic viscosities [η] of the
three types of polysaccharides were found to be obviously different (Table 1). The intrinsic viscosities of
the sulfate fucans are about eight-fold higher than those of the FuCSs, whereas the value of the glucan
(HE-3) is fairly low in the same solution (0.8 mL/g), although it also has a very high molecular weight
(
>253 kDa). Furthermore, Huggins and Kraemer constants (Κ and Κ ) also reflect the various
H
K
viscosity properties.
In contrast to the sulfated fucans, the three FuCSs from different species of sea cucumbers have
similar intrinsic viscosities, possibly due to their similar molecular size and chemical structures [9].

Mar. Drugs 2013, 11
406
Additionally, comparison analysis of the three types of polysaccharides indicates that the absence of
negative charge could result in the low intrinsic viscosity of the neutral glucan.
Figure 3. Plots of reduced viscosity (η /c) or logarithmic viscosity (ln(η )/c) vs.
sp
r
concentrations (c) of sulfated fucan (Panel A), fucosylated chondroitin sulfate (Panel B) and
neutral glucan (Panel C). Panel D shows relationships between the intrinsic viscosity ([η])
and weight average molecular weight (M ) of the sulfated polysaccharides.
w
1
000
34
Fucosylated chondroitin sulfate
A
Sulfated fucan
B
3
3
2
0
HE-2
AJ-2
HE-1
AJ-1
HN-1
8
6
4
2
00
00
00
00
0
HN-2
Heparin



sp/c c
28

c
sp/c
ln( )/c c
r
2
2
2
6
4
2
ln( )/c c
r
1
.0
1.5
2.0
2.5
3.0
3.5
4.0
1.0
1.5
2.0
2.5
3.0
Concentration of polysaccharides (c, g/L)
Concetration of polysaccharides (c, g/L)
40
35
30
25
20
15
10
5
0
C
Neutral glucan
D
2
.8
.4
Sulfated fucan from H. edulis
Sulfated fucan from H. nobilis
HE-3
Dextran
2


2.0
Sulfated fucan from A. japonicas
sp/c c
ln( )/c c
r
1
.6
.2

Three fucosylated chondroitin sulfates
1
4
.0
4.4
4.8
5.2
5.6
6.0
6.4
1.0
1.5
2.0
2.5
3.0
3.5
4.0
Concentration of polysaccharides (c, g/L)
log Mw
Intrinsic viscosity [η] can be related to the weight average M of the polymer through the
w
Mark-Houwink-Sakurada (MHS) relationship [10]. A good linear relationship (Figure 3D) was observed
between log M and log [η] for six different M of the sulfated polysaccharide solutions, but not the
w
w
neutral glucan, and the mathematical equation, log [η] = 1.16 log M − 4.02, was obtained by a linear
w
2
−5
regression (R = 0.973). Thus, the MHS parameters, K and α are given as K = 9.51 × 10 mL/g and
α = 1.16, respectively. The MHS constants are close to those for the chondroitin sulfate and dermatan
−4
sulfate in 0.2 M aqueous NaCl (K = 5 × 10 mL/g, α = 1.14) [25] and those for the FuCS from the sea
−
5
cucumber T. ananas in 0.1 M aqueous NaCl (K = 2.406×10 mL/g, α = 1.305) [10]. The comparatively
high value of α in the present study indicates that the structures of the sea cucumber sulfated
polysaccharide chains may be intermediate between that of an open coil and a stiff rod; in theory the
exponent for a coil should be 0.8 and for a rod 1.8 [25].

Mar. Drugs 2013, 11
.3. NMR Analysis
407
2
In order to compare differences between the fine structures of the sulfated fucans, FuCSs and neutral
1
glucan, their H NMR spectra are shown in Figure 4A,B,C, respectively. In Figure 4A, the signals at
about 1.20–1.30 ppm can be readily assigned to the methyl protons of fucose residues (CH ). In addition,
3
the chemical shifts of the envelope of anomeric signals at 5.0–5.7 ppm (Figure 4A) are consistent with
the presence of α-L-fucopyranosyl units [15,16]. However, the spectra of different species fucans are
quite different from each other. Anomeric signals in the spectra of the three sea cucumber fucans are
spread from 5.7 to 5.2 ppm, including signals at 5.6 and 5.7 ppm that have no counterpart in any of the
other fucans in this study. A feature at around 5.10 ppm can tentatively be assigned to H1 of nonsulfated
fucose residues [15]. The sulfated fucans give overlapping spectra with broad signals hampering
resolution, since line widths are several Hz, as expected for polysaccharides of high molecular mass.
Attempts to record two-dimensional NMR spectra for these polysaccharides gave no useful result in this
study (not shown).
Although the FuCSs also give complex spectra, their several individual signals can easily be
resolved, based on comparison with relatively much literature data for different species of sea
cucumbers [10,13,17–21]. The signals at about 1.20–1.30 ppm and 2.0–2.10 ppm can be readily
assigned to the methyl protons of fucose (CH ) and N-acetyl-β-D-galactosamin (CH CO) residues,
3
3
respectively (Figure 4B). The shifts from 5.1 to 5.6 ppm may be the anomeric protons of distinctive
sulfated fucose residues [13,17–21] and an obvious difference can be observed in this region. The three
major signals at 5.69, 5.41 and 5.35 ppm in the spectra of the A. japonicas FuCS, similar to those in the
H. edulis FuCS, can be assigned to 2,4-O-disulfated fucose (Fuc2S4S), 4-O-monosulfated fucose
(Fuc4S) and 3,4-O-disulfated fucose (Fuc3S4S), respectively (HE-2 and AJ-2 in Figure 4B). The results
are in agreement with the signals found in the spectra of the fucosylated chondroitin sulfate isolated from
the sea cucumber S. japonicas [18]. According to the integrals of the anomeric protons, the contents of
Fuc2S4S in HE-2 and AJ-2 are about 18% and 45%, respectively (Figure 4B). But the anomeric proton
signals of the H. nobilis FuCS are rather weak and complicated (HN-2 in Figure 4B). Anomeric signals
in the spectra of the H. nobilis FuCS are spread from 5.49 to 5.29 ppm, with significant differences from
all of the other FuCSs in this study. Obviously, the H. nobilis FuCS does not show the signals at
5
.69 ppm (HN-2 in Figure 4B), indicating the absence of Fuc2S4S. The peaks at 5.29 ppm are from
Fuc3S, and those at 5.33 and 5.43 ppm are from Fuc3S4S and Fuc4S, respectively, based on the
published reports of FuCSs obtained from other species of sea cucumbers such as T. ananas,
S. japonicas and L. grisea [13,17,18]. Additionally, different sulfation patterns of the fucose branches
also affect the chemical shifts of methyl protons of fucose (CH ) and N-acetyl-β-D-galactosamin
3
(
CH CO), as has been noted [19].
3
In contrast to those of the sulfated polysaccharides, the sea cucumber neutral glucan (Figure 4C)
1
gives H spectrum, which is simple, and although line widths are several Hz, as expected for
polysaccharides of high molecular mass, its signals can be assigned. The anomeric proton shift is
1
3
5
.39 ppm, possible indicating that the glucan may be an alpha sugar. These C signals 100.36, 77.56
(
69.90), 73.83 (73.45), 72.06, 70.96 and 61.09 ppm of the neutral glucan can be readily assigned to the
1
1
C1, C4, C3, C2, C5 and C6, respectively, based on the interpretations of H– H correlation spectroscopy,
1
13
(COSY) and H– C heteronuclear Multiple-Quantum Correlation (HMQC) spectra (Figure S2) and

Mar. Drugs 2013, 11
408
reported data [26]. In addition, based on the HMQC spectrum, comparison of the chemical shifts for
each carbon and proton with shifts for standard glucose showed strong downfield shifts of some signals,
indicating the existence of glycosidic (1→4)- and (1→6)- linkages [26]. Thus, NMR analysis
preliminarily indicates that this neutral glycan is likely to be an α-(1→4)-D-glucan with a (1→6)-linkage
branch (Figure S2), which is consistent with the monosaccharide composition analysis of the fraction
HE-3 (Table 1). The neutral glucan was firstly obtained from sea cucumbers, although it has been
isolated from mussels [26].
1
Figure 4. H NMR spectra of the polysaccharides, three sulfated fucans (Panel A), three
fucosylated chondroitin sulfates (Panel B) and a neutral glucan (Panel C) from three
sea cucumbers.

Mar. Drugs 2013, 11
.4. Anticoagulant Activity
409
2
Anticoagulant activities of the polysaccharides from the three sea cucumbers were assessed by
measuring the activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time
TT), and compared with the same activities of unfractionated heparin from mammalian source. The
(
APTT and TT assays (summarized in Table 2, Figure 5) indicate that the three FuCSs have significant
anticoagulant activities, lower than heparin, but much higher than the sulfated fucans (TT of the sulfated
fucans is not shown in Figure 5B), although the molecular sizes of the FuCSs are much lower than those
of the sulfated fucans (Table 1). The result is highly consistent with that from previous studies on the
other FuCS and sulfated fucan from the sea cucumber L. grisea (40 U/mg for FuCS vs. 2 U/mg for
sulfated fucan) [17,27]. But the neutral glucan has no anticoagulant activity, as expected from the
absence of sulfate [12]. Thus, our studies have suggested that anticoagulant activities of the different
types of polysaccharides are likely to relate to monosaccharide composition and sulfate content.
Additionally, the PT assays were also carried out but none of these polysaccharides showed any effects
on PT at the concentrations tested (1–1000 μg/mL). The absence of PT activities of the sulfated
polysaccharides is similar to that of other sulfated fucans and FuCSs [14,19,28].
Table 2. Anticoagulant properties of the polysaccharides purified from different sea cucumbers.
a
a
a
APTT
μg/mL
TT
μg/mL
PT
μg/mL
Samples
Sources
U/mg
U/mg
U/mg
Sulfated fucans
HE-1
AJ-1
Holothuria edulis
Apostichopus japonicas
Holothuria nobilis
18.71
13
19
9
>128
>128
>128
<1
<1
<1
>1000
>1000
>1000
<1
<1
<1
13.30
26.59
HN-1
Fucosylated chondroitin sulfates
HE-2
AJ-2
Holothuria edulis
Apostichopus japonicas
Holothuria nobilis
2.86
2.20
4.33
89
116
59
16.03
14.20
22.66
6
7
4
>1000
>1000
>1000
<1
<1
<1
HN-2
Neutral glucan
>1000 <1
HE-3
Holothuria edulis
>1000
<1
>1000
24
<1
Standard glycosaminoglycan
1.20 212 0.45
Heparin
Porcine intestine
212
212
a
The activity of the polysaccharides to prolong APTT, TT or PT is expressed by the concentration of each agent that is
required to double the APTT, TT or PT (doubling APTT, TT or PT, μg/mL), and also is expressed as USP units/mg (U/mg)
using a parallel standard curve based on the Heparin 212 units/mg from Sigma (USA).
Interestingly, as shown in Table 2, the anticoagulant activities of the fucosylated chondroitin sulfates
from different species of sea cucumbers are found to be obviously different. The A. japonica FuCS, with
higher Fuc2S4S residues (45% for AJ-2 vs. 18% for HE-2, Figure 4B), has higher activity than the other
FuCSs (116 U/mg for AJ-2 vs. 89 U/mg for HE-2, Table 2). Their sulfation patterns and contents may
result in the difference in anticoagulant activities of the FuCSs, since they have similar chemical
structures and molecular sizes. In addition, comparison of the NMR data of the FuCSs from three species
of sea cucumbers, together with our previous study [13] and a literature report [19], indicates that this
potent effect may be related to the occurrence of 2,4-O-disulfated fucose units.

Mar. Drugs 2013, 11
Figure 5. Anticoagulant activities, activated partial thromboplastin time (APTT) (Panel A)
410
and thrombin time (TT) (Panel B), of the polysaccharides from different species of
sea cucumbers.
3
3
2
2
1
1
50
standard:
sulfated fucan:
Heparin
A
B
00
50
00
50
00
3
00
HE-1
AJ-1
HN-1
Heparin
HE-2
AJ -2
fucosylated chondroitin sulfate:
HE-2
AJ-2
HN-2
250
HN-2
6
4
2
0
0
0
0
5
0
0
0
5
10
15
20
25
30
35
0
10 20 30 40 50 60 70 80 90 100
Concentration of the polysaccharides (g/mL)
Concentration of the polysaccharides (g/mL)
3
. Experimental Section
3
.1. Extraction and Purification of Polysaccharides
The polysaccharides were extracted from the body wall of sea cucumbers as the previous
studies [10,16,17]. The tissue of the dried body wall was digested by 0.5 M sodium hydroxide, and then
core protein combined with polysaccharides was released by the papain (EC 3.4.22.2). The crude
polysaccharides were purified by gel filtration with a Sephadex G-100 (2 cm × 100 cm, GE Healthcare
Technology, Uppsala, Sweden) and ion-exchange chromatography with a DEAE-sephadex FF column
(
3 cm × 7 cm, Amersham Biosciences, Uppsala, Sweden). The purity of preparation was assayed by
high-performance gel permeation chromatography (HPGPC) using a Agilent technologies1200 series
Agilent Co., USA) apparatus with RID (G1362A) and DAD (G1315D) detectors, equipped with a
(
Shodex OH-pak SB-804 HQ column (8 mm × 300 mm). Chromatographic conditions and procedures
were performed according to the previous method [23].
3
.2. NMR Analysis
1
H spectra were recorded with a Bruker Advance III 600 spectrometer of 600 MHz equipped with
Bruker’s CryoProbes. The NMR experiments were recorded at 21 °C with a spectral width of
2335.5 Hz, an acquisition time of 2.6564 s, a pulse width of 7.8 s, a relaxation time of 1 s and a number
of 8 scans. All lyophilized polysaccharides were dissolved in deuterium oxide (D O, 99.9% D) at a
1
2
3
–5 mg/mL concentration. Expansion of the 5.8 to 5.1 ppm region of the spectra of the fucosylated
chondroitin sulfates can be readily assigned to the anomeric protons of distinctive sulfated fucose
residues [17–21]. The integrals are normalized to a total of 100 = protons. Thus, the content of a sulfated
fucose residue can be calculated as a percentage of the total sulfated fucose residues.

Mar. Drugs 2013, 11
.3. Chemical Composition Analysis
411
3
Hexuronic acid was measured as described previously [29]. The ratio of GalNAc and Fuc was
1
obtained by H NMR analysis integrals of their methyl protons (also see Figure 4B). Acetylaminohexose
was carried out as described previously [17]. After strong acid hydrolysis of the polysaccharide
(4.0 M HCl, 100 °C for 6 h), total hexosamine was estimated by a modified Elson-Morgan reaction [30].
The sulfate/carboxyl groups of the polysaccharides were determined by a conductimetric method [22].
3
.4. Analysis of Molecular Weight
The molecular mass, weight-average molecular mass (M ), number-average molecular mass (M ) and
w
n
molecular weight distribution (M /M ) of polysaccharides were estimated by HPGPC. Chromatographic
w
n
conditions and procedures were performed according to the previous method [23]. For molecular weight
estimation, the columns were calibrated by standard D-series Dextrans (D-0, 2, 3, 4, 5, 6, 7, 8 and 2000),
a FuCS from the sea cucumber T. ananas with known relative molecular weight (M 65,820 Da) for the
w
FuCS and sulfated fucan, a dextran from Leuconostoc spp. with relative molecular weight (M 100,000 Da)
w
for the neutral glucan.
3
.5. Specific Rotation
The specific rotation was determined by the optical rotation in Pharmacopoeia of the People’s
Republic of China [31]. In the method, the concentration of the polysaccharides was about 10 mg/mL
and the detection temperature was 20 °C .
3
.6. Measurement of Intrinsic Viscosity
The Intrinsic viscosity was measured by the method of determination of viscosity in Pharmacopoeia
of the People’s Republic of China [32].
3
.7. Anticoagulant Action Measured by APTT, PT and TT
Activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT) clotting
assays were carried out as described previously [14], using a coagulometer (TECO MC-4000, Germany).
4
. Conclusions
In summary, three types of polysaccharides from different species of sea cucumbers were isolated and
purified, and their physicochemical characteristics, such as chemical composition, electric charge,
average molecular weight and intrinsic viscosity, were characterized by different methods. The
polysaccharides obtained from sea cucumber are three FuCSs, three sulfated fucans and a neutral glucan,
respectively. The three types of polysaccharides have obviously different physicochemical
characteristics. However, more attempts to elucidate further refinement of the structure of the sulfated
polysaccharides are necessary.
Comparison analysis of anticoagulant activities of the polysaccharides indicates that anticoagulant
activities of the different types of polysaccharides are likely to relate to monosaccharide composition

Mar. Drugs 2013, 11
412
and sulfate content, and the sulfation patterns of the FuCSs may result in a difference in anticoagulant
activities. Our data may be of help to elucidate the structure-activity relationship of the sea
cucumber polysaccharides.
Acknowledgements
This work was supported in part by Yunnan Provincial Science and Technology Department in China
(
No. 2010CI116, 2012BC011 and 2012FB177), National Natural Science Foundation of China
No. 81102372) and West Light Foundation of the Chinese Academy of Sciences. Bo Li is
(
acknowledged for performing the NMR experiments.
Supplementary Material
S1. Qualitative Analysis of Monosaccharide Composition of the Sea Cucumber Polysaccharide
Samples by HPLC
S1.1. Materials
The sea cucumber polysaccharide samples were prepared from three sea cucumbers. The
monosaccharides including glucuronic acid (GlcA), glucose (Glc), galactose (Gal) were purchased from
Alfa Aesar. The N-acetylgalactosamine (GalNAc) was purchased from TCI and fucose (Fuc) was from
sigma Chemical Co. (Shanghai, China). The 1-phenyl-3-methyl-5-pyrazolone (PMP, 99%) was
purchased from ACROS organics.
S1.2. Methods
S1.2.1. Hydrolysis and PMP Labeling
The dried polysaccharides (5 mg) were dissolved in 1 mL of 2 M trifluoroacetic acid (TFA) in a
hydrolysis tube [33]. The hydrolysis tube was sealed and incubated at 100 °C for 8 h in a heating block.
After reaction, the reaction mixture was evaporated to dryness. The residue was then dissolved in
methanol and again evaporated to dryness to remove residual TFA. This step was repeated four more
times. The polysaccharide samples prepared as described above, and the standard samples were
reconstituted in deionized water [34]. Then 50 μL of the sample solution and 50 μL of 0.5 M PMP in
methanol and 50 μL of 0.6 M sodium hydroxide were added to the mixture. The reaction mixture was
mixed and incubated at 70 °C for 30 min in a heating block. After adjusting the pH to 7 by adding 50 μL
of 0.3 M HCl, 1 mL of chloroform was added to the mixture and mixed for at least 5 s using a vortex
mixer. The bottom chloroform layer was carefully removed using a pipe and discarded. This step was
repeated three more times, and the top aqueous layer was saved for HPLC analysis.
S1.2.2. HPLC Analysis
Analysis of the PMP-labeled polysaccharides was carried out using an Agilent technologies1200
series (Agilent Co., USA) apparatus with DAD (G1315D) detectors, equipped with an Agilent Eclipse
XDB C18 (150 mm × 4.6 mm). The flow rate was 1 mL/min, and UV absorbance of the effluent was

Mar. Drugs 2013, 11
413
monitored at 250 nm. Buffers A and B were 0.1 M ammonium acetate, pH 5.5, containing 22%
acetonitrile, respectively.
S1.3. Results
Figure S1. HPLC of the sulfated fucans, neutral glucan (Panel A) and the fucosylated
chondroitin sulfates (Panel B).
GalNAc
A
standard
Fuc
GlcA
Glc Gal
substance
HE-1
HN-1
HE-3
10
15
20
25
Retention time (min)
HPLC of the sulfated fucans and neutral glucan
GalNAc
B
standard
substance
Glc
Gal
Fuc
GlcA
HE-2
AJ-2
HN-2
3
0
40
50
60
70
80
Retention time (min)
HPLC of three fucosylated chondroitin sulfates

Mar. Drugs 2013, 11
414
S2. NMR Analysis Results of the Neutral Glucan from the Sea Cucumber H. Edulis
1
3
1
1
1
1
13
Figure S2. Parts of the spectra of the neutral glucan ( C, H, H– H COSY, H– C HMQC).

Mar. Drugs 2013, 11
415
Figure S2. Cont.
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2013 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
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