Polysaccharides from Sargassum tenerrimum: Structural features, chemical modification and anti-viral activity

Article abstract of DOI:10.1016/j.phytochem.2009.10.014

Herpes simplex viruses (HSVs) display affinity for cell-surface heparan sulfate proteoglycans with biological relevance in virus entry. Here, we exploit an approach to inhibiting HSV infection by using a sulfated fucoidan, and a guluronic acid-rich alginate derived from Sargassum tenerrimum, mimicking the active domain of the entry receptor. These macromolecules have apparent molecular masses of 30 ± 5 and 26 ± 5 kDa, respectively. They and their chemically sulfated derivatives showed activity against herpes simplex virus type 1 (HSV-1). Their inhibitory concentration 50% (IC₅₀) values were in the range 0.5-15 μg/ml and they lacked cytotoxicity at concentrations up to 1000 μg/ml. The anti-HSV activity increased with increasing sulfate ester content. Our results suggest the feasibility of inhibiting HSV infection by blocking viral entry with polysaccharide having specific structure.

Full text of DOI:10.1016/j.phytochem.2009.10.014

Phytochemistry 71 (2010) 235–242
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Polysaccharides from Sargassum tenerrimum: Structural features, chemical
modification and anti-viral activity
Sharmistha Sinha a,1, Akram Astani b,1, Tuhin Ghosh a, Paul Schnitzler b, Bimalendu Ray a,
*
a Natural Products Laboratory, Department of Chemistry, The University of Burdwan, West Bengal 713 104, India
b Department of Infectious Diseases, Virology, University of Heidelberg, Im Neuenheimer Feld 324, 69120 Heidelberg, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 26 August 2009
Received in revised form 16 October 2009
Herpes simplex viruses (HSVs) display affinity for cell-surface heparan sulfate proteoglycans with biolog-
ical relevance in virus entry. Here, we exploit an approach to inhibiting HSV infection by using a sulfated
fucoidan, and a guluronic acid-rich alginate derived from Sargassum tenerrimum, mimicking the active
domain of the entry receptor. These macromolecules have apparent molecular masses of 30 5 and
26 5 kDa, respectively. They and their chemically sulfated derivatives showed activity against herpes
simplex virus type 1 (HSV-1). Their inhibitory concentration 50% (IC50) values were in the range 0.5–
Keywords:
Sargassum tenerrimum
Sargassaceae
Herpes simplex virus
Anti-viral activity
Poly-guluronate
Fucoidan
15 lg/ml and they lacked cytotoxicity at concentrations up to 1000 lg/ml. The anti-HSV activity
increased with increasing sulfate ester content. Our results suggest the feasibility of inhibiting HSV infec-
tion by blocking viral entry with polysaccharide having specific structure.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
mediator (HVEM), nectin-1, and specific sites on heparan sulfate
generated by certain 3-O-sulfotransferases (Copeland et al., 2008;
Herpes simplex viruses (HSVs) cause various forms of disease
such as lesions on the lips, eyes, or genitalia to encephalitis and
even disseminated disease in immunocompromised individuals
(Kleymann, 2005). The prevalence of herpes simplex virus type 1
(HSV-1) infection increases progressively from childhood, the sero-
prevalence being inversely related to socioeconomic background.
Primary HSV-1 infections in children can give rise to mucocutane-
ous vesicular eruptions which might be reactivated frequently in
adults (Fleming et al., 1997). Genital herpes, generally caused by
herpes simplex virus type 2 (HSV-2) is characterized by a high
seroprevalence worldwide (8–40%), with an increasing trend in
the last 20 years in nearly all countries (Gupta et al., 2007). Among
the high-risk population, HSV-2 infection is a major public health
problem in young adults. Herpes viruses are circulated and ampli-
fied by transmission through direct contact with a lesion or the
body fluid of an infected individual. Transmission may also occur
through skin-to-skin contact during periods of asymptomatic
shedding. The adsorption of viruses to the host cell surface is the
initial and critical step for viral infection. HSV interaction with tar-
get cells involves cell-surface heparan sulfate (HS) interacting with
several viral envelope proteins, especially glycoproteins gB, gC, and
gD (Adamiak et al., 2007). gD interacts with herpes virus entry
Geraghty et al., 1998; Montgomery et al., 1996; Shukla et al.,
1999). The uniquely distributed sulfation pattern of HS polysaccha-
ride is believed to regulate its functional specificity (Gama et al.,
2006; Liu and Pedersen, 2007). The anti-viral properties of sulfated
polysaccharides have been known for almost 50 years (Vaheri and
Cantell, 1963; Vaheri, 1964; Vaheri et al., 1964). Accordingly, hep-
arin (heparan sulfate analogue) and several sulfated carbohydrate
polymers including dextran, fucoidan, galactan, and xylomannan
have been found to inhibit HSV because of their structural similar-
ity to HS and therefore are potential anti-HSV drug candidates
(Balzarini and Van Damme, 2007; Ghosh et al., 2009; Pujol et al.,
2007; Witvrouw and De Clercq, 1997). To date, the performance
of these macromolecules in efficacy trials has been disappointing
(Cohen, 2008; Grant et al., 2008), but next-generation concepts
now in or approaching clinical trials offer improved prospects for
efficacy (Klasse et al., 2008). The most plausible approach involves
a combination of several drugs, preferentially targeting different
steps in the viral infection process. Because sulfated polysaccha-
rides are safe and acceptable (Bollen et al., 2008; Kilmarx et al.,
2008), development of several second-generation combination for-
mulation based on first generation lead candidates may be more
effective (Brache et al., 2007; Ghosh et al., 2009; Liu et al., 2005).
The identification of active polysaccharides from marine algae
may identify macromolecules with superior efficacy. Structurally
defined polysaccharides generated by chemical sulfation may also
produce drug candidate with higher potency.
* Corresponding author. Tel.: +91 34 22 55 65 66; fax: +91 34 22 63 42 00.
E-mail address: bimalendu_ray@yahoo.co.uk (B. Ray).
1
These authors equally contributed to this work.
0031-9422/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2009.10.014

236
S. Sinha et al. / Phytochemistry 71 (2010) 235–242
The marine brown alga Sargassum tenerrimum possesses vari-
calibration with standard dextrans the apparent molecular mass
of fraction B would be 26 5 kDa.
ous biological activities (Dar et al., 2007; Kang et al., 2008; Ye
et al., 2008). Recently, there is a report on the anti-viral activity
of a crude extract from S. tenerrimum (Zhu et al., 2006). How-
ever, these authors used only crude extracts and the identity
of the active principle has not yet been reported. The present
study reports isolation and chemical characterization of polysac-
charides present in the marine brown alga S. tenerrimum. Using
chemical and chromatographic methods, and various forms of
spectroscopy we have been able to deduce structural features
of an alginic acid and a fucoidan. The possibility to generate
fucoidan and algin derivatives by chemical sulfation in the O-
positions along the polysaccharide chain has led to the synthesis
of various sulfated derivatives with different charge densities
and distribution. With these tailored modifications a range of
macromolecules have been generated that have potential anti-
HSV-1 activity and low cytotoxicity.
2.1.2. IR analysis
The FT-IR spectrum of this fraction contains bands at 3400 (OH
stretching), 2925 (CH stretching), 1675 and 1420 (COOꢁ stretch-
ing) cmꢁ1 characteristic of alginate (Fig. 1). Alginates show two
IR bands at approximately 1100 and 1025 cmꢁ1 assigned to man-
nuronic (M) and guluronic (G) units, respectively (Pereira et al.,
2003). The presence of strong IR band at 1025 cmꢁ1 and a small
shoulder at 1100 cmꢁ1 in the IR spectra of both native and sulfated
alginate fractions (Fig. 1) suggest the presence of small amounts of
mannuronic acid in the homoguluronan enriched alginate fraction.
This is the first report for the presence of guluronate rich alginic
acid in Sargassaceae.
2.1.3. NMR analysis
To specify the anomeric configuration and to confirm the gly-
cosidic linkage pattern and ring form (pyranose or furanose) of
the sugar residues, the native alginic acid (B) was analyzed by
NMR spectrometry. Complete assignments of all the signals were
made by COSY (Fig. 2) and HSQC (Fig. 3) experiments. The carbon
resonances were assigned using heteronuclear (HSQC) and homo-
nuclear (COSY) correlation NMR experiments. To evaluate the
content of G and M linkage, we have investigated the 1H NMR
spectrum of sodium alginate using procedures as described previ-
ously (Chattopadhyay et al., 2010; Grasdalen, 1983). The 1H NMR
spectrum of B showed signals at 4.95 and 4.39 assigned to H-1
and H-5 of guluronic acid in homopolymeric blocks. The absence
2. Results and discussion
The objectives of this study were to analyze the polysaccharides
generated from the brown alga S. tenerrimum and to study their
anti-HSV activities. Sugar compositional analysis of the depig-
mented algal powder (DAP) shows that it contained 61% (w/w)
polysaccharides of which about 46% (w/w) were uronic acids.
The major neutral sugar was fucose and it also contained other
neutral sugars including xylose, galactose, and glucose (Table 1).
TLC analysis of the acid liberated sugars indicates the presence
of, inter alia, guluronic acid. The DAP which contains, inter alia, fu-
can and alginic acid was then extracted sequentially with various
inorganic solvents as described in Section 4.
100
(I)
2.1. Chemical characterization and sulfation of the alginic acid
80
It is well known that sodium alginates form insoluble precip-
itates at acidic pH and with calcium salts, but they are stable in
solution between pH 6 and 9. Therefore, this macromolecule
was extracted with K2CO3. The fractional product (B) had a nega-
60
%T
tive specific rotation ½a D33
ꢁ95° (c 0.2, H2O) and was soluble in
ꢀ
water.
40
20
2.1.1. Molecular mass
The elution profile of this macromolecule on Superdex 30 prep
grade suggests that this polymer is homogeneous. Based on
4000
3000
2000
1000
400
Wavenumber [cm-1]
(II)
Table 1
Yields and sugar composition (mol %) of fractions obtained from Sargassum
tenerrimum.
20
Fractions
DAP
A
B
C
BS
CS
Yieldsa
TSb
1000
61
46
33
51
7
Nd
68
17
Tr
11
4
128
64
62
Nd
Tr
Tr
Nd
Tr
31
59
9
%T
42
41
2
Tr
Tr
Nd
Tr
Nd
38
5
6
70
16
Tr
9
UAb
Sulfateb
Fucosec
Xylosec
Mannosec
Galactosec
Glucosec
2
20
5
45
7
73
15
Tr
9
0
23
Nd
3
5
4000 3500 3000 2500 2000 1500 1000 500
Wavenumber (cm -1
)
A, impure alginate; B, sodium alginate; BS, sulfated sodium alginate; C, fucoidan;
CS, further-sulfated fucoidan; DAP, depigmented algal powder; Nd, not detected;
TS, total sugar; UA, uronic acid; and Tr, trace.
Fig. 1. FT-IR spectrum of the (I) native (B) and (II) sulfated (BS) sodium alginate
isolated from the brown seaweed Sargassum tenerrimum. Absorption band at
1025 cmꢁ1 and 1100 cmꢁ1 arise from guluronic acid (G) and mannuronic acid (M),
respectively.
a
mg quantities per gram of DAP.
Weight % of fraction dry weight.
b
c
mol % of neutral sugars.

S. Sinha et al. / Phytochemistry 71 (2010) 235–242
237
Fig. 2. COSY spectrum (D2O, 25 °C) of the sodium alginate (B) isolated from Sargassum tenerrimum. The cross-peaks labeled are the correlations between H’s of guluronic acid
residues.
Fig. 3. HSQC spectrum (D2O, 25 °C) of the sodium alginate (B) isolated from Sargassum tenerrimum. The cross-peaks (X1–X5) labeled are the correlations between Hs and Cs of
guluronic acid residues. H-1 to H-5 refer to the signals of the protons of guluronic acid residues.
of cross-peaks for mannuronic acid in the COSY (Fig. 2) and HSQC
(Fig. 3) spectra of the native alginic acid (B) isolated from S. ten-
errimum indicates that this algin is a polyguluronic acid enriched.
This result is in accordance with the data obtained from IR
analysis.
1961). In the IR spectrum of the native polymer (B), this absor-
bance was absent (Fig. 1), demonstrating that it was associated
with the sulfate group.
2.2. Chemical characterization and further-sulfation of the alkali
extracted fucan
2.1.4. Sulfation
The purified alginic acid (B) was chemically modified to yield a
sulfated derivative (BS) whose sugar composition is listed in Table
1. The Fourier transform IR spectrum of BS showed an intense
absorption band in the region, 1250 cmꢁ1, which was related to
>S@O stretching vibration of the sulfate group (Lloyd et al.,
2.2.1. Sugar composition
The potassium carbonate extract after removal of alginic acid
by precipitation with HCl, was dialyzed and lyophilized to give
a fucan rich fraction (C). Sugar compositional analysis revealed
that this fraction C consist mainly of fucose as the major neutral

238
S. Sinha et al. / Phytochemistry 71 (2010) 235–242
sugar together with smaller amount of galactose and xylose units
(Table 1). The uronide content of this fraction is 9% and it con-
tained 2% sulfate. Thin layer chromatographic analysis of the
monosaccharides present in the hydrolysate indicates the pres-
ence of an uronic acid with an Rf value similar to that of glucu-
ronic acid. GLC analysis of the TMS derivatives of the derived
methyl glycosides confirmed this result.
2.3.2. Anti-viral activity
The potential anti-viral effect of each of the anionic polysaccha-
rides listed in Table 2 against HSV-1 was evaluated by standard
plaque reduction assays. HSV-1 was incubated for 1 h at room
temperature with various concentrations of compounds, and after-
wards subjected to plaque assay. The 50% inhibitory concentra-
tions (IC50) of the tested anionic compounds for HSV-1 were
determined in a range between 0.5 and 15 lg/ml and these com-
pounds showed activity in a dose dependent manner (Fig. 4). Their
selectivity indices (SI), which are calculated as the TC50/IC50 ratio,
are also listed in Table 2.
2.2.2. FT-IR
The FT-IR spectrum of
C showed an absorption band at
1252 cmꢁ1 related to a >S@O stretching vibration of the sulfate
group. An additional sulfate absorption band at 848 cmꢁ1 (C–O–
S, secondary axial sulfate) indicated that the sulfate group is lo-
cated at position 4 of the fucopyranosyl residue (Lloyd et al., 1961).
2.3.2.1. Influence of the nature of anionic group, constituent sugars
and molecular mass on anti-viral activity. The sample of alginic acid
(B) extracted with dilute alkali showed low anti-viral activity,
while the fucoidan fraction (C) was more active (Table 2). This algi-
nate (B) is rich in 1,4-linked
a-L-guluronate and contains 1.0 car-
2.2.3. Molecular mass
boxyl group per sugar residue whereas the fucoidan (C), which
possesses less anionic character (0.13 carboxyl groups and 0.05
sulfate groups per sugar residue), contains sulfate groups. There-
fore the anti-viral activity of algal polysaccharides of present study
is not merely a function of high charge density, but has distinct
structural specificities of which nature of anionic groups and con-
stituent sugars are undoubtedly important. Sulfates are required
for activity, whereas polymer containing carboxyl groups shows
less potency. This result is in agreement with data published ear-
lier (Ghosh et al., 2009). Indeed, the important parameter for the
anti-viral activity is the degree of sulfation, i.e. the number of sul-
fate groups per monosaccharide residue. It is well documented that
the higher the degree of sulfation, the better its anti-viral potency
(Witvrouw and De Clercq, 1997). Sample B shows only little anti-
The elution profile of this macromolecule on Superdex 30 prep
grade suggests that this polymer is homogeneous. Based on cali-
bration with standard dextrans, the apparent molecular mass of
C would be 30 5 kDa. Notably, the molecular mass of the fucan
of present study is lower from the fucans of Stoechospermum mar-
ginatum and Cystoseira indica (Adhikari et al., 2006; Mandal et al.,
2007).
2.2.4. Sulfation
Sulfate content affects the anti-viral activity of polysaccharides
(Ghosh et al., 2009; Witvrouw and De Clercq, 1997). In general, for
a particular class of polysaccharide, the higher the charge density,
the higher is its activity. In addition to the well documented DS
dependence, the specific position of the sulfate ester group also ap-
pears to be additionally important for the anti-viral activity of sul-
fated polysaccharide (reviewed by Ghosh et al., 2009). To study the
effect of sulfate groups, we have sulfated the fucose containing
fraction (C) to yield sulfated derivative CS. The sulfate content
and sugar composition of these fractions, listed in Table 1 are
similar.
viral activity with an IC50 of 15 lg/ml, whereas all other samples
show a higher anti-herpetic activity, e.g. the sulfated sample CS
is about 30-fold more active against HSV. Thus sample B with no
detectable sulfate content might be comparable to desulfated hep-
arin, which like sample B contains carboxyl group at uronic acid
and possesses little anti-HSV activity. However, the charge density
of sodium alginate of present study is higher than that of desulfat-
ed heparin (1.0 vs. 0.5 mol carboxyl groups per mole of sugar res-
idues). As seen from a structural point of view, a highly charged
molecule is more likely to interfere efficiently with electrostatic
interactions between the positively charged region of a viral glyco-
protein and the negatively charged HS chains of the cell-surface
glycoprotein receptor. Therefore, it is expected that the anti-viral
activity of homoguluronan enriched sodium alginate of present
study will be higher than that of desulfated heparin. Moreover,
in order to exhibit inhibitory activity the anionic groups must be
exposed to the surface of the macromolecule and this arrangement
2.3. Biological activities of anionic polysaccharides
2.3.1. Cytotoxic assay
The 50% toxic concentration (TC50) of the anionic polysaccha-
rides extracted from S. tenerrimum and their sulfated derivatives
against RC-cells was determined with the neutral red assay on sub-
confluent monolayers. No effect on cell viability was detected for
all tested polysaccharides at concentrations up to 1000 lg/ml (Ta-
ble 2). Thus a very low or no toxicity can be assumed for these
compounds.
120
100
Table 2
Cytotoxic effect, anti-HSV-1 activity, and selectivity index of sodium alginate (B) and
fucoidan (C) isolated from Sargassum tenerrimum and their chemically sulfated
derivatives (BS and CS).
80
BS
B
CS
C
60
Polysaccharides
TC50
g/ml)a
IC50
g/ml)b
SI
c
40
20
0
(l
(l
(TC50/IC50)
B
C
BS
CS
P1000
P1000
P1000
P1000
15
1.4
6
P67
P714
P167
P2000
0.1
1
10
100
1000
0.5
Concentration (µg/ml)
a
50% Toxic concentration (TC50), defined as compound concentration required to
reduce cell viability by 50%, was determined from dose–response curves.
Fig. 4. Anti-viral effect of serial dilutions of anionic polysaccharides against herpes
simplex virus type 1. Results represent the mean of three independent experiments.
B, sodium alginate; C, fucoidan; BS, sulfated sodium alginate; and CS, further-
sulfated fucoidan.
b
50% Inhibitory concentration (IC50
) defined as compound concentration
required to reduce virus plaques by 50%.
c
Selectivity index (SI) is the ratio between TC50 and IC50
.

S. Sinha et al. / Phytochemistry 71 (2010) 235–242
239
depends critically on the conformation and dynamic stereochemis-
try (time and solvent dependent aspect of conformation) of the
polysaccharide (Adhikari et al., 2006). Finally, the relatively higher
average molecular mass of sodium alginate (21–31 kDa) compared
to heparin (5–25 kDa) may also play a role.
may interact with the hydrophobic pockets of viral envelope and
makes it more potent. Finally, the higher molecular mass of fucoi-
dan (30 5 kDa) in comparison with alginic acid (26 5 kDa)
might also play a role in exhibiting higher potency.
The higher activity of fucoidan (C and CS) in comparison to na-
tive sodium alginate (B) may be explained by the presence of sul-
fate groups in the former macromolecules (Table 2). However, the
anti-herpetic properties of anionic polysaccharides may also de-
pend on the characteristics of their uncharged portions, which
may be involved in hydrophobic interaction. It has been reported
that the methyl groups of fucoidan interacts with the hydrophobic
pocket of HSV-1 glycoprotein C (reviewed by Ghosh et al., 2009).
This type of hydrophobic interactions was found to be decisive
for the chondroitin sulfate as well as heparan sulfate binding to vir-
al glycoprotein C (gC) (Mardberg et al., 2001). In addition, the
hydrophobic group might additionally be inserted into the viral li-
pid envelope (Uryu et al., 1992). The C-6 methyl groups of fucose
residues in fucoidan are hydrophobic whereas the carboxylate
group in alginic acid is hydrophilic. Therefore, the former polymer
2.3.2.2. Influence of the sulfate content on inhibitory capacity. The
anti-viral activity of sulfated polysaccharides is reported to in-
crease with the sulfate content (Ghosh et al., 2009; Witvrouw
and De Clercq, 1997). To examine this hypothesis, both sulfated
compounds (BS and CS) obtained by chemical sulfation of the na-
tive alginic acid (B) and fucoidan (C), respectively were tested
against HSV-1. In both cases, the chemically sulfated derivatives
exhibited a more potent anti-viral activity in comparison to the na-
tive polymers (Table 2). These data confirmed the correlation be-
tween sulfate content and activity. Importantly, the further-
sulfated fucoidan (CS) inhibited the replication of HSV-1 at signif-
icantly lower concentration than those of B, BS, and C (Table 2). The
SI value for this sulfated polysaccharide was high (P2000). Thus,
CS was the most potent and selective inhibitor of HSV-1 of the four
macromolecules examined.
100
80
60
40
20
0
(I)
untreated
control
acyclovir
acyclovir
acyclovir
BS
B
CS
C
100
80
60
40
20
0
(II)
untreated
control
BS
B
CS
C
120
100
80
60
40
20
0
(III)
untreated
control
BS
B
CS
C
Fig. 5. Inhibitory effect of anionic polysaccharides and acyclovir against herpes simplex virus during different periods of the viral replication cycle. Viruses and cells were
treated with the maximum non-cytotoxic concentration of compounds. (I) Cells were treated with drugs for 1 h prior to infection with HSV, (II) HSV was preincubated with
drugs for 1 h prior to infection of cells, and (III) HSV-infected cells were drug-treated for 3 days during viral replication in the presence of the drugs. Experiments were
repeated independently and performed in triplicate assays.

240
S. Sinha et al. / Phytochemistry 71 (2010) 235–242
2.3.3. Mechanism of action
plastic beaker and air dried to yield depigmented algal powder
(DAP; 88 g).
A time on addition experiment was performed to investigate
the effect of Sargassum-derived polysaccharides on virus infection.
Cells were pretreated with anionic polysaccharides before viral
infection; viruses were incubated with drugs before cell infection
or after penetration of the virus into the host cells. When the host
cells were pretreated with polysaccharides prior to infection, the
tested drug candidates and acyclovir showed no effect on viral
infection (Fig. 5(I)). On the other hand, pretreatment of HSV-1 with
the drugs for 1 h prior to infection caused a significant reduction in
plaque formation for B, BS, C, and CS. At maximum non-cytotoxic
concentrations of the tested polysaccharides, infectivity was re-
duced by >90% (Fig. 5(II)). Acyclovir showed the highest anti-viral
activity when added during the replication period with inhibition
of the viral replication of 98.6%. This drug inhibits specifically the
viral DNA polymerase during the replication cycle when new viral
DNA is synthesized. In contrast, when the anionic polysaccharides
were added to the overlay medium after penetration of the viruses
into the host cells, plaque formation was not significantly reduced
(Fig. 5(III)). These data suggested that the anti-HSV activity of these
macromolecules was exerted directly by interfering with virion
envelope structures or masking viral structures which are neces-
sary for adsorption or entry into host cells as had been shown pre-
viously for plant derived extracts and isolated compounds
(Schnitzler et al., 2008; Astani et al., 2009).
4.1.2. Extraction with hydrochloric acid
Extraction of DAP (5 g) was done with 0.1 M HCl (w:v :: 1:100)
at 25–30 °C for 6 h under constant stirring. Separation of the resi-
due from the extract was performed by filtration through a glass
filter (G-2). The residue was washed with additional distilled water
and the wash was collected to maximize polysaccharide recovery.
This residue was again extracted twice with 0.1 M HCl in a similar
way and the combined extract was dialyzed, concentrated and
lyophilized to give fraction A (162 mg).
4.1.3. Extraction with potassium carbonate
The residue left after extraction with HCl was extracted with 2%
K2CO3 (w:v :: 1:100) at 40–60 °C for 6 h under constant stirring
(thrice). The combined extract was carefully acidified with HCl to
pH ꢂ1 and the precipitate formed was collected by centrifugation
(10,000g, 15 min), washed with water and then dissolved by care-
ful addition of NaOH. The slightly alkaline solution was dialyzed,
concentrated and diluted with 4.0 M CaCl2 solution to make a final
concentration of 2% CaCl2. The precipitate formed at this stage was
isolated by centrifugation, washed with water and treated with
0.1 M HCl (4 ꢃ 50 ml, stirring at room temperature for ꢂ2 h). Then
it was dissolved in NaOH, the solution was dialyzed and finally
lyophilized to yield sodium alginate B (639 mg). The supernatant
after precipitation with HCl was dialyzed and lyophilized sepa-
rately to give fraction C (152 mg).
3. Conclusions
In conclusion, the marine alga S. tenerrimum contains at least
two different types of polysaccharides. The alkali extracted alginic
acid has a molecular mass of 26 5 kDa and this is the first report
of the presence of a poly-guluronate in any Sargassaceae species.
The presence of fucoidan having a molecular mass of 30 5 kDa
was also indicated. We have shown for the first time that these
macromolecules are responsible for the anti-viral activity of ex-
tracts from S. tenerrimum. Moreover, we found that fucoidan has
a higher potency than alginic acid although the charge density
of latter is higher. Hence, the nature of anionic groups and constit-
uent sugars present in algal polysaccharides are important
parameters for their anti-viral activity. We also found that the fur-
ther-sulfated fucan (CS) is a potent anti-HSV-1 compound. The
inhibition of in vitro HSV replication of this compound was
observed at polymer concentrations which do not have any effect
on the cell viability. The mechanism of anti-viral activity of CS in-
volves inhibition of virus adsorption, although further clarification
is required. Furthermore, as the generation of this polysaccharide
involves a few inexpensive and easy steps it will be an additional
point of interest. Given the interesting chemical characteristics of
the sulfated fucan and the promising in vitro anti-herpetic proper-
ties reported here, this macromolecule represents a good candidate
for further anti-viral research.
4.1.4. Size exclusion chromatography (SEC)
Solutions (ꢂ2 ml) of samples in 500 mM sodium acetate buffer
(pH 5.5), were loaded to a column (60 ꢃ 2.6 cm) of Superdex 30
prep grade (Pharmacia) equilibrated with the same buffer. The col-
umn was eluted ascendingly with the same buffer at 15 ml hꢁ1 and
the temperature was 30–35 °C. Elution of polysaccharide was ex-
pressed as
a
function of the partition coefficient Kav
[Kav = (Ve ꢁ V0)/(Vt ꢁ V0) with Vt and V0 being the total and void
volume of the column determined as the elution volume of glucose
and blue dextran, respectively and Ve is the elution volume of the
sample]. The column was calibrated with standard dextrans within
a molecular-weight range of 1000–30,000. Fractions (5 ml) were
collected and analyzed for total sugar content by phenol–sulfuric
acid method (Dubois et al., 1956).
4.1.5. Sugar analysis
Total sugars and uronic acids were determined by the phenol–
sulfuric acid (Dubois et al., 1956) and m-hydroxydiphenyl (Ahmed
and Labavitch, 1977) assay, respectively. For the determination of
neutral sugar composition, the monosaccharide residues released
by acid hydrolysis (2 M trifluoro acetic acid, 2 h, 100 °C) were con-
verted into their alditol acetates (Blakeney et al., 1983) and ana-
lyzed by gas–liquid chromatography (GLC; Shimadzu GC-17A;
Shimadzu, Kyoto, Japan). Monosaccharides were identified by
thin-layer chromatography as described previously (Mandal
et al., 2008). Alternatively, trimethylsilyl (TMS) derivatives of
methyl glycosides were analyzed by GLC (York et al., 1985).
4. Experimental
4.1. Characterization and sulfation of polysaccharides from S.
tenerrimum
4.1.6. Sulfate analysis
4.1.1. Plant material and preliminary treatments
Sulfate groups were estimated by IR-spectrometric (Chattopad-
hyay et al., 2008; Rochas et al., 1986) and modified turbidometric
barium chloride (Craigie et al., 1984) methods as described
(Chattopadhyay et al., 2007).
Samples of S. tenerrimum (Sargassaceae) were collected from
the Okha coast of Gujarat, India in August 1995. The seaweeds
were washed thoroughly with tap water, dried by forced air circu-
lation and pulverized in a blender (Waring Products Inc., Torring-
ton, CT, USA). Algal powder (130 g) was depigmented using
sequential extraction with petroleum ether and acetone as solvent
in a Soxhlet apparatus. The unextracted material was placed in a
4.1.7. Sulfation
Sulfation of the purified fraction (C) was carried out as de-
scribed (Chattopadhyay et al., 2008). The freeze-dried polysaccha-

S. Sinha et al. / Phytochemistry 71 (2010) 235–242
241
ride (B, 639 mg) was soaked in dry DMF (10 ml) and SO3-pyridine
complex dissolved in 10 ml DMF mixed with the polysaccharide.
For every mol of SO3-pyridine complex, 1 mol of pyridine was
added to the mixture. The mixture was heated in an oil bath at
90 °C under nitrogen atmosphere for 1.0 h; the solution was subse-
quently neutralized with NaOH, dialyzed and lyophilized to give
the sulfated alginic acid BS. Similarly, sulfated fucoidan fraction
CS was generated from fraction C under conditions described
above.
of plaques observed in virus control monolayers (untreated cul-
tures), the concentration of test compound which inhibited plaque
numbers by 50% (IC50) was determined from dose–response
curves.
4.2.4. Time of addition assay
To identify the step at which the viral infection might be inhib-
ited by polysaccharides, cells were infected with HSV and incu-
bated at 37 °C 1 h. The infected cell monolayer was then washed
three times with PBS and overlaid with medium containing 0.5%
methylcellulose and maximum non-cytotoxic concentration of
the sulfated polysaccharides. The cell pretreatment assay was per-
formed with cells being treated with compounds at indicated con-
centrations for 1 h at 37 °C before viral infection. For the virus
pretreatment assay, the virus suspension was incubated with com-
pounds at indicated concentrations for 1 h before adding to the cell
culture for plaque assay. For comparative purposes, acyclovir was
used as anti-viral control.
4.1.8. Spectroscopy
4.1.8.1. Fourier transform infra red (FT-IR). Recording of IR spectra
were carried out as described previously (Mandal et al., 2008).
4.1.8.2. Nuclear magnetic resonance (NMR). The 1H NMR spectra of
samples were acquired at probe temperatures of 80 and 25 °C with
Varian 400MR spectrometer (Bruker Biospin AG, Fallanden, Swit-
zerland) operating at 400 MHz for 1H. Samples were dissolved in
D2O (99.96 atom%), two-dimensional (2D) correlated spectroscopy
(COSY), 2D nuclear overhauser enhancement and exchange spec-
troscopy (NOESY), and heteronuclear single quantum correlation
(HSQC) were recorded using standard pulse sequences. The chem-
ical shifts are reported relative to an internal acetone standard at
2.225 and 31.55 ppm for 1H and 13C NMR spectra, respectively.
Acknowledgements
This work was supported by DST (Grant No. SR/S1/OC-50/2007)
to BR. We thank the Director, CSMCRI, Gujarat, India for his help for
the collection and identification of the alga and Iveta Uhliarikova,
Bratislava, Slovak Republic, for recording NMR spectra. Standard
dextrans were gift from Dr. Tapani Vuorinen, Finland.
4.2. Biological activities of anionic polysaccharides
4.2.1. Cell culture and herpes simplex virus type 1 (HSV-1)
Anti-viral experiments were performed on RC-37 cells (African
green monkey kidney cells). Cells were grown in monolayer cul-
ture with Dulbecco’s modified Eagle’s medium (DMEM; Gibco
Invitrogen Corporation; Karlsruhe, Germany) supplemented with
References
Adamiak, B., Ekblad, M., Bergstrom, T., Ferro, V., Trybala, E., 2007. Herpes simplex
virus type
2 glycoprotein G. Is targeted by the sulfated oligo- and
5% fetal calf serum, 100 U/ml penicillin and 100 lg/ml streptomy-
polysaccharide inhibitors of virus attachment to cells. J. Virol. 81, 13424–13434.
Adhikari, U., Mateu, C.G., Chattopadhyay, K., Pujol, C.A., Damonte, E.B., Ray, B., 2006.
Structure and antiviral activity of sulfated fucans from Stoechospermum
marginatum. Phytochemistry 67, 2474–2482.
Ahmed, A., Labavitch, J.M., 1977. A simplified method for accurate determination of
cell wall uronide content. J. Food Biochem. 1, 361–365.
Astani, A., Reichling, J., Schnitzler, P., 2009. Comparative study on the antiviral
activity of selected monoterpenes derived from essential oils. Phytother. Res.
doi:10.1002/ptr.2955.
cin. Herpes simplex virus type 1 (HSV-1) strain KOS was used for
all experiments (Schnitzler et al., 2008). Acyclovir was purchased
from GlaxoSmithKline (Bad Oldesloe, Germany), dissolved in ster-
ile water and was used as synthetic anti-viral control.
4.2.2. Cytotoxicity assay
For cytotoxicity assays, cells were seeded into 96-well plates
and incubated for 24 h at 37 °C. The medium was removed and
fresh DMEM containing serial dilutions of sulfated polysaccharides
were added onto subconfluent cells in eight replicates for each
concentration of the drugs. After 3 days of incubation, the growth
medium was removed and viability of the drug-treated cells was
determined in a standard neutral red assay. Neutral red dye uptake
was determined by measuring the optical density (OD) of the
eluted neutral red at 540 nm in a spectrophotometer. The mean
OD of the untreated control wells was assigned a value of 100%.
The cytotoxic concentration of the drug which reduced viable cell
number by 50% (TC50) was determined from dose–response curves.
Additionally the maximum non-cytotoxic concentration of each
drug was determined.
Balzarini, J., Van Damme, L., 2007. Microbicide drug candidate to prevent HIV
infection. The Lancet 369, 787–797.
Blakeney, A.B., Harris, P., Henry, R.J., Bruce, A.B., 1983. A simple rapid preparation of
alditol acetates for monosaccharide analysis. Carbohydr. Res. 113, 291–299.
Bollen, L.J.M., Kelly, B., Kilmarx, P.H., Supaporn, C., Cathy, C., Punneporn, W.,
Nucharee, S., Jullapong, A., Jordan, W.T., Janet, M.M., 2008. No increase in
cervicovaginal proinflammatory cytokines after carraguard use in a placebo-
controlled randomized clinical trial. J. Acquir. Immun. Defic. Syndr. 47, 253–
257.
Brache, V., Horacio, C., Régine, S.W., Robin, A.M., Juan, C.M., Kumar, N., Salvatierra,
A.M., Tejada, A.S., Cochon, C., Forcelledo, M.L., 2007. Effect of a single vaginal
administration of levonorgestrel in CarraguardÒ gel on the ovulatory process: a
potential candidate for dual protection emergency contraception.
Contraception 76, 111–116.
Chattopadhyay, K., Mandal, P., Lerouge, P., Driouich, A., Ghosal, P., Ray, B., 2007.
Sulfated polysaccharides from Indian samples of Enteromorpha compressa
(Ulvales, Chlorophyta): isolation and structural features. Food Chem. 104,
928–935.
Chattopadhyay, K., Ghosh, T., Pujol, C.A., Carlucci, M.J., Damonte, E.B., Ray, B., 2008.
Polysaccharides from Gracilaria corticata: sulfation, chemical characterization
and anti-HSV activities. Int. J. Biol. Macromol. 43, 346–351.
4.2.3. Anti-viral activity
Four sulfated polysaccharides were tested for anti-viral activity
against HSV-1 by a plaque reduction assay with monolayer cul-
tures of RC-37. Usually 2 ꢃ 103 plaque forming unites (pfu) were
incubated with different concentrations of drugs for 1 h at room
temperature. Virus was allowed to adsorb to the cells for 1 h at
37 °C. The residual inoculum was then discarded and infected cells
were overlaid with medium containing 0.5% methylcellulose. Each
concentration was performed in three replicates; the synthetic
anti-herpetic drug acyclovir was used as control. Monolayers
were fixed with 10% formalin, stained with 1% crystal violet and
subsequently plaques were counted. By reference to the number
Chattopadhyay, N., Ghosh, T., Sinha, S., Chattopadhyay, K., Karmakar, P., Ray, B.,
2010. Polysaccharides from Turbinaria conoides: structural features and
antioxidant capacity. Food Chem. 118, 823–829.
Cohen, J., 2008. Microbicide fails to prevent against HIV. Science 319, 1026–1027.
Copeland, R., Balasubramaniam, A., Tiwari, V., Zhang, F., Bridges, A., Linhardt, R.J.,
Shukla, D., Liu, J., 2008. Using a 3-O-sulfated heparin octasaccharide to inhibit
the entry of herpes simplex virus type 1. Biochemistry 47, 5774–5783.
Craigie, J.S., Wen, Z.C., vanderMeer, J.P., 1984. Interspecific, intraspecific and
nutritionally-determined variations in the composition of agars from
Gracilaria spp. Bot. Mar. 27, 55–61.
Dar, A., Baig, H.S., Saifullah, S.M., Ahmad, V.U., Yasmeen, S., Nizamuddin, M., 2007.
Effect of seasonal variation on the anti-inflammatory activity of Sargassum
wightii growing on the N Arabian Sea coast of Pakistan. J. Exp. Mar. Biol. Ecol.
351, 1–9.

242
S. Sinha et al. / Phytochemistry 71 (2010) 235–242
Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., Smith, F., 1956. Colorimetric
method for determination of sugars and related substances. Anal. Chem. 28,
350–366.
Mandal, P., Pujol, C.A., Carlucci, M.J., Chattopadhyay, K., Damonte, E.B., Ray, B., 2008.
Sulfated xylomannan of Scinaia hetai: isolation, structural features and antiviral
activity. Phytochemistry 69, 2193–2199.
Fleming, D.T., McQuillan, G.M., Johnson, R.E., Nahmias, A.J., Aral, S.O., Lee, F.K., Louis,
M.E.St., 1997. Herpes simplex virus type 2 in the United States, 1976 to 1994. N.
Engl. J. Med. 337, 1105–1111.
Mardberg, K., Trybala, E., Glorioso, J.C., Bergstrom, T., 2001. Mutational analysis of
the major heparin sulfate-binding domain of herpes simplex virus type 1
glycoprotein C. J. Gen. Virol. 82, 1941–1950.
Gama, C.I., Tully, S.E., Sotogaku, N., Clark, P.M., Rawat, M., Vaidehi, N., Goddard III,
W.A., Nishi, A., Hsieh-Wilson, L.C., 2006. Sulfation patterns of
glycosaminoglycans encode molecular recognition and activity. Nat. Chem.
Biol. 2, 467–473.
Geraghty, R.J., Krummenacher, C., Cohen, G.H., Eisenberg, R.J., Spear, P.G., 1998.
Entry of alpha herpes viruses mediated by poliovirus receptor related protein 1
and poliovirus receptor. Science 280, 1618–1620.
Ghosh, T., Chattopadhyay, K., Marschall, M., Karmakar, P., Mandal, P., Ray, B., 2009.
Focus on antivirally active sulfated polysaccharides: from structure–activity
analysis to clinical evaluation. Glycobiology 19, 2–15.
Grant, R.M., Hamer, D., Hope, T., Johnston, R., Lange, J., Lederman, M.M., Lieberman,
J., Miller, C.J., Moore, J.P., Mosier, D.E., Richman, D.D., Schooley, R.T., Springer,
M.S., Veazey, R.S., Wainberg, M.A., 2008. Whither or wither micobicides?
Science 321, 532–534.
Montgomery, R.I., Warner, M.S., Lum, B.J., Spear, P.G., 1996. Herpes Simplex Virus-
1entry into cells mediated by a novel member of the TNF/NGF receptor family.
Cell 87, 427–436.
Pereira, L., Sousa, A., Coelho, H., Amado, A.M., Ribeiro-Calro, P.J.A., 2003. Use of FTIR,
FT-Raman and 13C-NMR spectroscopy for identification of some seaweed
phycocolloids. Biomol. Eng. 20, 223–228.
Pujol, C.A., Carlucci, M.J., Matulewicz, M.C., Damonte, E.B., 2007. Natural sulfated
polysaccharides for the prevention and control of viral infections. Top. Hetero.
Chem. 11, 259–281.
Rochas, C., Lahaye, M., Yaphe, W., 1986. Sulfate content of carrageenan and agar
determined by infrared spectroscopy. Bot. Mar. 29, 335–340.
Schnitzler, P., Schneider, S., Stintzing, F.C., Carle, R., Reichling, J., 2008. Efficacy of an
aqueous Pelargonium sidoides extract against herpesvirus. Phytomedicine 15,
1108–1116.
Grasdalen, H., 1983. High-field 1H NMR spectroscopy of alginate: sequential
structure and linkage conformations. Carbohydr. Res. 118, 255–260.
Gupta, R., Warren, T., Wald, A., 2007. Genital herpes. The Lancet 370, 2127–2137.
Kang, J.Y., Khan, M.N.A., Park, N.H., Cho, J.Y., Lee, M.C., Fujii, H., Hong, Y.K., 2008.
Antipyretic, analgesic and anti-inflammatory activities of the seaweed
Sargassum fulvellum and Sargassum thunbergii in mice. J. Ethnopharmacol. 116,
187–190.
Shukla, D., Liu, J., Blaiklock, P., Shworak, N.W., Bai, X., Esko, J.D., Cohen, G.H.,
Eisenberg, R.J., Rosenberg, R.D., Spear, P.G., 1999. A novel role for 3-O-sulfated
heparin sulfate in herpes simplex virus 1 entry. Cell 99, 13–22.
Uryu, T., Ikushima, N., Katsuraya, K., Shoji, T., Takahashi, N., Yoshida, T., Kanno, K.,
Murakami, T., Nakashima, H., Yamamoto, N., 1992. Sulfated alkyl
oligosaccharides with potent inhibitory effects on human immunodeficiency
virus infection. Biochem. Pharmacol. 43, 2385–2392.
Kilmarx, P.H., Kelly, B., Supaporn, C., Barbara, A.F., Nucharee, S., Cathy, C., Paisit, W.,
Vaheri, A., 1964. Heparin and related polyionic substances as virus inhibitors. Acta
Pathol. Microbiol. Scand. Suppl. 171, 1–98.
Somsak, S., Philip, A.M., Thanyanan, C., Tappero, J., 2008.
A randomized,
placebo-controlled trial to assess the safety and acceptability of use of
Carraguard vaginal gel by heterosexual couples in Thailand. Sex. Trans. Dis.
35, 226–232.
Vaheri, A., Cantell, K., 1963. The effect of heparin on herpes simplex virus. Virology
21, 661–662.
Vaheri, A., Ikkala, E., Saxen, E., Penttinen, K., 1964. Biological actions of polyanions. A
comparative study of the effect on herpes virus, blood coagulation, red blood
cells and growth behaviour of cells on a glass surface. Acta Pathol. Microbiol.
Scand. 62, 340–348.
Witvrouw, M., De Clercq, E., 1997. Sulfated polysaccharides extracted from sea
algae as potential antiviral drugs. Gen. Pharmacol. 29, 497–511.
Ye, H., Wang, K., Zhou, C., Liu, J., Zeng, X., 2008. Purification, antitumor and
antioxidant activities in vitro of polysaccharides from the brown seaweed
Sargassum pallidum. Food Chem. 111, 428–432.
York, W.S., Darvill, A., O‘Neill, M., Stevenson, T., Albersheim, P., 1985. Isolation and
characterization of plant cell walls and cell wall components. Methods
Enzymol. 118, 3–40.
Klasse, P.J., Shattock, R., Moore, J.P., 2008. Antiretorviral drug-based microbicides to
prevent HIV-1 sexual transmission. Ann. Rev. Med. 59, 455–471.
Kleymann, G., 2005. Agents and strategies in development for improved
management of herpes simplex virus infection and disease. Expert Opin.
Investig. Drugs 14, 135–161.
Liu, J., Pedersen, L.C., 2007. Anticoagulant heparan sulfate: structural specificity and
biosynthesis. Appl. Microbiol. Biot. 74, 263–272.
Liu, S., Lu, H., Neurath, A.R., Jiang, S., 2005. Combination of candidate microbicides
cellulose acetate 1,2-benzenedicarboxylate and UC781 has synergistic and
complementary effects against Human Immunodeficiency Virus type-1
infection. Antimicrob. Agents Chemother. 49, 1830–1836.
Lloyd, A.G., Dodgson, K.S., Price, R.B., Rose, F.A., 1961. Infrared studies on sulphate
esters. I. Polysaccharide sulphates. Biochim. Biophys. Acta 46, 108–115.
Mandal, P., Mateu, C.G., Chattopadhyay, K., Pujol, C.A., Damonte, E.B., Ray, B., 2007.
Structural features and antiviral activity of sulphated fucans from the brown
seaweed Cystoseira indica. Antivir. Chem. Chemother. 18, 153–162.
Zhu, W., Chiu, L.C.M., Ooi, V.E.C., Chan, P.K.S., Ang Jr., P.O., 2006. Antiviral property
and mechanisms of
a sulphated polysaccharide from the brown alga
Sargassum patens against Herpes simplex virus type 1. Phytomedicine 13,
695–701.