Isolation, Characterization and Anticoagulant Activity of Sulfated Polysaccharides from Brown Algae Sargasam wightii Greville
K. Kumar Ebenezar,
The aim of the present study is to isolate the sulfated polysaccharides from marine macro algae S. wightii. The crude sulfated polysaccharides were fractionated by anionic resin (Amberlite IRA-900) and the separated active fractions were confirmed by agarose gel electrophoresis. The active fractions were pooled, dialyzed and purified by molecular sieve (Sephadex G-100) chromatography. The molecular weight of fractionated as well as purified sulfated polysaccharides was determined through gradient polyacrylamide gel electrophoresis (PAGE) and the disaccharide profile of purified sulfated polysaccharides was also analyzed. The structure of purified sulfated polysaccharides was dogged by 1H-NMR spectrum. The elements such as Carbon, Hydrogen and Nitrogen (CHN) and Activated Partial Thromboplastin Time (APTT) were observed from the purified sulfated polysaccharides. The hexosamine, uronic acid and the CHN content in the purified sulfated polysaccharides were found to be low (except molecular weight) when compared to standard heparan sulfate. The molecular weight of purified sulfated polysaccharides have 6,500 Da and the concentration of disaccharide profile such as 5.52% of uronic acid, 3.47% hexosamine and 5.4% of sulfate. APTT of fractionated and purified sulfated polysaccharides was found to be 24.6 and 31.1 IU mg-1, respectively. These results suggest that the sulfated polysaccharides from S. wightii could be an alternative source of anticoagulant.
March 12, 2013; Accepted: April 10, 2013;
Published: July 10, 2013
The novel biological resources which are widely available in nature have been
used as a source of therapy for thousands of years. To a large extent of the
worlds biological variety remains unexplored and now-a-days, the searches
for new bioactive agents from natural sources are of much importance. Several
of these unique compounds have shown pharmacological activities for many of
the boring diseases like cancer, AIDS, diabetes, arthritis, etc. (Smit,
2004). The marine milieu is an outstanding basin of bioactive natural products
has led to the innovation of many potentially active agents consider worthy
of clinical applications (Saravanan et al., 2009).
Seaweeds have been used since ancient times as food, fodder and fertilizer and
as natural sources with therapeutic and bioactive molecules for treating multiple
diseases. Today seaweeds are the raw material for industrial production of agar,
carrageenan and alginates, but they continue to be widely consumed as food in
Asian countries (Mishra et al., 1993).
Seaweed resources have been used for isolation of major metabolites such as
polysaccharides, lipids, proteins, carotenoides, vitamins, sterols, enzymes,
antibiotics and many other fine chemicals (Ibtissam et
al., 2009). Sulfated polysaccharides are one of the anticoagulant compounds
present in marine algae and occur in the form of sulfated fructose and sulfated
galactans (Painter, 1983). Some algal species trigger
anticoagulant activity through protein or glycoprotein-like compounds (Yasuda
et al., 2004) which binds to the serine protease, an inhibitor of
antithrombin, producing a complex which accelerates the protelysis of the enzyme
responsible for coagulation. The preparation of heparin and sulfated glycosaminoglycans
from natural sources is expensive and therefore an alternative is desirable
(Cassaro and Dietrich, 1997). Hence, an attempt was made
to isolate sulfated polysaccharides from S. wightii which could be a
potent alternative for anticoagulant substance. The brown algae harvested from
the earths marine are beyond doubt one of the most expensive gifts of
the great deep. S. wightii is one of the marine brown algal species broadly
found in India, with incredible biological applications and are known to be
well-off in sulfated polysaccharides content. Sulfated polysaccharides were
found to clutch large pharmacological applications.
MATERIALS AND METHODS
Sample collection: Seaweeds were collected from Kannyakumari, Tamil
Nadu, India, washed and dried at 50°C under ventilation in an oven and ground
Fractionation of sulfated polysaccharides: Fifty grams of the dried
powder were suspended with 5 volumes of 0.1 M NaCl and the pH of the mixture
was adjusted to 8.0 with NaOH. Ten miligram of protease (Sigma, St. Louis, USA)
was then added to the mixture for proteolytic digestion. After 24 h incubation
at 60°C with agitation and periodical adjustments of pH, the mixture was
filtered through cheese cloth. To the filtrate 10 g of Amberlyte IRA-900 (chloride
form, pH 7.0), ion-exchange resin (Sigma, St. Louis, USA) were added and the
resulting mixture was agitated for 24 h at room temperature under a layer of
toluene. The suspension was then filtered again through the same cheese cloth.
The resin retained in the cloth was washed with 1 L of water at 50°C and
subsequently washed with 1 L of 0.1 M NaCl at room temperature (Leite
et al., 1998).
Purification of sulfated polysaccharides: The active sulfated polysaccharides
(confirmed by agarose electrophoresis) eluted from the ionic resin with NaCl
were pooled and further purified by molecular sieving in Sephadex G-100 (100x1.5
cm). About 50 mg of fractionated sulfated polysaccharides were applied in 2
mL of distilled water and the fractions (3 mL) were eluted from the column with
0.2 M acetic acid. Purified sulfated polysaccharides were dialyzed (molecular
membrane cutoff>10 kDa) against distilled water and freeze-dried (Saravanan
and Shanmugam, 2010).
Agarose and gradient PAGE: Agarose gel electrophoresis of fractionated
sulfated polysaccharide in the discontinuous buffer barium acetate/1, 3-diaminopropane
acetate was performed (Toida et al., 1997). The
average molecular masses of fractionated and purified sulfated polysaccharides
were analyzed by gradient PAGE (Edens et al., 1992).
Determination of disaccharide profile: Uronic acid, hexosamine and sulfate
contents of purified sulfated polysaccharides were estimated by following the
methods of Bitter and Muir (1962), Wagner
(1979) and Terho and Hartiala (1971), respectively.
1H-NMR analysis: 1H-NMR of purified sulfated polysaccharides
(Varian BRUKER-500 MHZ) was performed (Sudo et al.,
2001) and compared with standard heparan sulfate. The thoroughly dried sample
was re-dissolved in 0.5 mL of 2 H2O (99.6%) and transferred to the
NMR tube. All spectra were determined on a Varian BRUKER-500 MHZ spectrometer
equipped with 5 mm triple resonance tunable probe with standard Varian software
at 294.9 K on 700 μL samples at 0.5-1.0 mM. The HOD signal was suppressed
by pre-saturation during 3 sec.
Element analysis: In the CHN operating mode the PE 2400 Series II CHNS/O
Analyzer uses a combustion method to convert the sample is first oxidized in
a pure oxygen environment using classical reagents. Products produced in the
combustion zone include CO2, H2O and N2. Elemental,
such as halogens and sulfur are removed by scrubbing reagents in the combustion
zone. The resulting gases are homogenized gases are allowed to de-pressurize
through a column where they are separated in a stepwise steady-state manner
and detected as a function of their thermal conductivities.
Blood coagulation assay: APTT of fractionated and purified sulfated
polysaccharides were performed using kit obtained from Lexington, USA.
The yield of sulfated polysaccharides from S. wghitii was found to be
3.56 g kg-1 in dried seaweeds. Among the collected fractions, only
3 were offered sulfated polysaccharides (1.0, 1.5 and 2.0 M NaCl). These active
sulfated fractions have fast mobility and showed only single band in agarose
gel electrophoresis which was compared with the standard heparan sulfate (Fig.
1). The molecular weight of fractionated sulfated polysaccharides was observed
as 7,500 Da and purified sulfated polysaccharides has 6,500 Da which were compared
with the standard (Fig. 2). The disaccharide profile of purified
sulfated polysaccharides showed 4.35% of uronic acid, 2.78% of hexosamine and
2.8% of sulfate contents whereas the purified sulfated polysaccharides recorded
5.52% of uronic acid, 3.47% hexosamine and 5.4% of sulfate.
||Agarose gel electrophoresis of sulfated polysaccharides from
S. wightti, F-1: 1.0 M NaCl, F-2: 1.5 M NaCl, F-3: 2.0 M NaCl and
||Gradient PAGE of sulfated polysaccharides from S. wightii
F: Fractionated sulfated polysaccharides, P: Purified sulfated polysaccharides
and S: Standard
Furthermore, the 1H-NMR (Fig. 3) showed presence
of iduronic acid and N-acetyl glucosamine from of purified sulfated polysaccharides
S. wightii with impurities of other hexoses which is compared with standard
(Fig. 4). The elemental analysis such as carbon, hydrogen
and nitrogen of sulfated polysaccharides from S. wightii were represented
in Fig. 5.
||1H-NMR sulfated polysaccharides spectrum purified
of S. wightii
||1H-NMR of standard heparan sulfate labelled peaks
are assigned as (a) H-1 of GlcNpS→GlcAp, (b) H-1 of GlcNpAc→IdoAp,
(c) H-1 of IdoAp, (d) H-1 of GlcAp; (e) H-5 of IdoAp, (f) H-4 of IdoAp,
(g) H-2 of GlcAp and (h) methyl of GlcNAc
||CHN analysis of purified sulfated polysaccharides from S.
wightii, HS: Heparan sulfate, SP: Purified sulfated polysaccharides
of S. wightii
From this figure there is major difference between the contents of carbon (22.80%),
hydrogen (3.08%) and nitrogen (2.08%) found to be in HS from bovine where as
the marine brown algae recorded the carbon (14.33%), hydrogen (2.64%) and nitrogen
(1.11%) correspondingly. Finally the APTT activities of fractionated and purified
sulfated polysaccharides from S. wightii were found to be 24.6 and 31.1
IU mg-1, respectively.
In the present investigation sulfated polysaccharides from S. wightii
was estimated and the yield of sulfated polysaccharides from brown algae was
low when compared to the result of Leite et al.
(1998), who extracted total polysaccharide from Sulfated polysaccharides
Spatoglossum schroederi. The molecular weight of purified sulfated polysaccharides
extracted from S. wightii was about 7,500 Da. This is accordance with
the result of Nardella et al. (1996), who reported
the low molecular weight fucoidan (7.2 kDa) from brown seaweed Ascophyllum
nodosum using ion-exchange chromatography.
The disaccharide profile of purified sulfated polysaccharides from S. wightii
has recorded more when compared to fractionated sulfated polysaccharides
from S. wightii. Larsen et al. (1966)
and Medcalf and Larsen (1977) have reported a fucaidan
from A. nodosum contained a central core of β-D-glucuronic acid
with branches of fucose. In CHN content of purified sulfated polysaccharides
was low as judged against to marine scallop heparan sulfate (Saravanan
and Shanmugam, 2011). The variation of CHN contents may be due to the variation
of disaccharide pattern present in the heparin complex isolated from different
sources (Pugazhendi, 2002).
The APTT activity of S. wightii was found to be high when compared to
the anticoagulant activity of sulfated xylogucogluronan from S. schroederi
(Leite et al., 1998). The increase in the
anticoagulant activity of sulfated polysaccharides from S. wightii may
be due to the high sulfate content, this was supported by the result of Mao
et al. (2006) who stated that difference in the potency of thrombin
inhibition may be due to the sugar residue, sulfate content and their structural
features. The anticoagulant activity of sulfated polysaccharides was tested
in vitro by APTT assay and the values were compared with those of standard
heparan sulfate (~170 IU mg-1) (Saravanan and
The APTT activity of purified sulfated polysaccharides from S. wightii
was low when compared to standard. This result suggest that sulfated polysaccharide
less effective in vitro and which can increased by purify the sulfated
polysaccharides through HPLC and this could be used as an potential anticoagulant
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