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Abstract

Three carrageenan representatives of each structural type: λ- and ι -family (Gigartina acicu- laris), ι-family (Euchema denticulatum) and κ -family (Kappaphycus cottonii) have been tested for their in vitro antiviral activity. The carrageenans proved to be potent inhibitors of herpes human virus type 1 (HHV-1) and Poliovirus. The best results were obtained with carrageenans from Gigartina acicularis and Euchema denticulatum, which are more sulfated than those from Kappaphycus cottonii. The selective index values (CC50/ID50) ranged from more than 22 to more than 545 for HHV-1 and more than 6.6 to more than 32 for Poliovirus. No citotoxic effects were observed. At 0.75 mg/ml, none of the carrageenans tested showed a virucidal activity against HHV-1 or Poliovirus. Carrageenans from Euchema denticulatum (ι- family) and Gigartina acicularis (λ- and ι -family) exerted their antiviral effect via, in part, by a lower in- hibition of the virus attachment and by the interference in a subsequent stage of the virus replicative cycle. The κ-carrageenan from Kappaphycus cottonii exerted its antiviral effect mainly by a lower inhibition of the virus attachment. In cultures treated with carrageenans from Euchema denticulatum (ι-family) and Gi- gartina acicularis (λ- and ι -family), the HHV-1 viral DNA synthesis had a reduction of threefold and twofold with 0.75 mg/ml, respectively.
443
ISSN 0326-2383
KEY WORDS: Antiviral, Carrageenan, Herpes Simplex viruses, Poliovirus, Polysaccharide, Red algae.
* Author to whom correspondence should be addressed. E-mail: jarbas.montanha@farmacia.ufrgs.br
Latin American Journal of Pharmacy
(formerly Acta Farmacéutica Bonaerense)
Lat. Am. J. Pharm. 28 (3): 443-8 (2009)
Original Article
Received: August 26, 2008
Accepted: March 14, 2009
Antiviral Activity of Carrageenans from Marine Red Algae
Jarbas A. MONTANHA 1*, Nathalie BOURGOUGNON 2, Joel BOUSTIE 3& Maryvonne AMOROS 3
1Faculdade de Farmácia da Universidade Federal do Rio Grande do Sul,
Av. Ipiranga 2752 (UFRGS), Porto Alegre, RS CEP 90610-000, Brazil;
2Laboratoire de Biotechnologie et Chimie Marines Université de Bretagne Sud Centre de
Recherche et d’Enseignement Yves Coppens Campus de Tohannic, BP 573- 56017 Vannes, France;
3Laboratoire dês Substances Licheniques et Photoprotection,
Equipe Pharmacognosie et Mycologie, Faculté de Pharmacie, Université de Rennes I,
2 Avenue du Prof. L. Bernard,35043, Rennes Cedex, France.
SUMMARY. Three carrageenan representatives of each structural type: λ- and ι–family (Gigartina acicu-
laris), ι-family (Euchema denticulatum) and κ–family (Kappaphycus cottonii) have been tested for their in
vitro antiviral activity. The carrageenans proved to be potent inhibitors of herpes human virus type 1
(HHV-1) and Poliovirus. The best results were obtained with carrageenans from Gigartina acicularis and
Euchema denticulatum, which are more sulfated than those from Kappaphycus cottonii. The selective index
values (CC50/ID50) ranged from more than 22 to more than 545 for HHV-1 and more than 6.6 to more
than 32 for Poliovirus. No citotoxic effects were observed. At 0.75 mg/ml, none of the carrageenans tested
showed a virucidal activity against HHV-1 or Poliovirus. Carrageenans from Euchema denticulatum (ι-
family) and Gigartina acicularis (λ- and ι–family) exerted their antiviral effect via, in part, by a lower in-
hibition of the virus attachment and by the interference in a subsequent stage of the virus replicative cycle.
The κ-carrageenan from Kappaphycus cottonii exerted its antiviral effect mainly by a lower inhibition of
the virus attachment. In cultures treated with carrageenans from Euchema denticulatum (ι-family) and Gi-
gartina acicularis (λ- and ι–family), the HHV-1 viral DNA synthesis had a reduction of threefold and
twofold with 0.75 mg/ml, respectively.
INTRODUCTION
Carrageenans are sulfated polydigalactosides
(20-50% -OSO3Na) with molecular weight be-
tween 105and 106that can be extracted from
several red seaweeds. They comprise a broad
range of structures and are divided into families:
the κ-family is defined by the presence of a C-4-
sulfate group on the β-D-galactose unit; the ι-
family is characterized by a C-4-sulfate group on
the β-D-galactose unit and a C-2-sulfate group
on the 3-6-anhydro-α-D-galactose; the λ-family
is characterized by a sulfate group on the C-2 of
β-D-galactose unit and 2,6-dissulfate on α-D-
galactose unit 1. Polysaccharides is a complex
group of peculiar molecules exhibiting a wide
range of biological activities such as antiinflam-
matory, anticoagulant, antithrombotic, antitu-
moral, antimetastatic, antifertilizing and antiviral
2. Unlike antimicrobial drugs against bacteria
and fungi, only a few effective antiviral drugs
are available. One of the most important rea-
sons for the lack of success in developing an-
tiviral drugs is due to the nature of the infec-
tious viral agents, which totally depend upon
the cell they infect for multiplication and sur-
vival, so, compounds that may cause the death
of viruses also are very likely to injure the host
cell that harbour them 3.
Efforts have been made to evaluate the an-
tiviral activity of natural products, including
those from algaes, in order to characterize new
compounds which could inhibit virus replication
and/or treat viral infection, or even serve as
models for new molecules. Numerous car-
rageenans and other polysaccarides have been
tested for their antiviral activity 4-7.
444
MONTANHA J.A., BOURGOUGNON N., BOUSTIE J. & AMOROS M.
We report herein our results concerning the
in vitro activity against HHV-1 and Poliovirus of
three carrageenan representatives of each struc-
tural type: λ- and ι–family (Gigartina acicu-
laris), ι-family (Euchema denticulatum) and κ
–family (Kappaphycus cottonii). Secondly, the
mechanism of viral inhibition was determined.
MATERIALS AND METHODS
Carrageenan
The carrageenans tested were extracted from
three marine red algae (Rhodophyceae) collect-
ed in different countries: Euchema denticulatum
(Philippines) Gigartina acicularis (Bretagne,
France) and Kappaphycus cottonii (Philippines).
They were extracted with hot distilled water, fil-
tered on diatomaceous earth and the filtrate was
poured into absolute ethanol, with stirring. The
precipitate was recovered and washed with 95°
ethanol, dehydrated with diethyl ether and dried
overnight at 50 °C 8. The chemical composition
of each extract differs in the content of sulfated
galactans and type of carrageenan. Each algae
seems to have different types of carrageenan:
Euchema denticulatum (ι-family) 9,10, Gigartina
acicularis (λ- and ι–family) 11 and Kappaphy-
cus cottonii (κ–family) 10.
The carrageenan samples were provided by
Prof. J.M. Kornprobst (SMAB/ISOMer – Nantes/
France). Stock solutions (10 mg/ml) were pre-
pared in PBS buffer and stored at -20 °C; and,
for the experiments, aliquots were diluted with
MEM to obtain the indicated concentrations.
Cells and viruses
African green monkey kidney cells (VERO
cell line ATCC CCL81) were grown in Eagle’s
minimum essential medium (MEM) supplement-
ed with 10 % newborn calf serum, 160 Units ml-
1 penicillin and 80 mg.ml–1 gentamicin. Cells
were routinely passed every three days. The
viruses used were the following: Human Hes-
pesvirus 1 (HHV-1 strain H29S) and poliovirus
type 2, a vaccinal strain Sabin II. Virus stocks
were propagated by serial passages on VERO
cells at a low multiplicity, incubated for 2 days,
then frozen and thawed three times. Afterwards
the preparation was cleaned by centrifugation at
low speed in order to remove the cell debris.
Virus titration was performed by the limiting di-
lution method 12. The virus titre was estimated
from cytopathogenicity and expressed as 50 %
tissue culture infectious doses ml–1 (TCID50/ml).
It was 2.0 x 105.5 TCID50/ml for HHV-1 and 2.0 x
106.57 TCID50 /ml for poliovirus.
Evaluation of cytotoxicity
Aiming assess the effect of carrageenan on
uninfected Vero cells, dilutions ranging from 6
mg/ml to 11.7 µg/ml (i.e. final concentrations in
wells from 1.5 mg/ml to 2.9 µg/ml) in the main-
tenance medium were added to VERO monolay-
ers (a 96-well microplate with 4.0 x 104 cells
per well). After incubation for 72 h, cytotoxicity
was determined by a microscopic examination
of the cell morphology. The concentration at
which the cell number was reduced to 50% in
relation to that of the controls was taken as the
50% cytotoxic concentration (CC50). The maxi-
mum tolerated concentration (MTC) was not
possible to determine because the carrageenan
solutions were very viscous. All assays were car-
ried out in triplicate.
Antiviral activity
Antiviral assays were carried out as described
previously 13. Dilutions of the carrageenan ex-
tracts ranging from 0.75 mg/ml to 0.003 µg/ml
were prepared in the maintenance medium and
added to confluent 1-day-old monolayers of
Vero cells. These cells were grown in microtitre
tissue culture plates just before inoculation, at a
multiplicity of infection (MOI) of 0.01 TCID50
per cell. Toxicity, cell, and virus controls were
run simultaneously. Plates were incubated at 37
°C for 72 h for HHV-1 or 32 h for poliovirus
(multiple replicative cycles). Then, the monolay-
ers were observed for cytopathic effect. The
concentration which inhibited 50% the viral cy-
topathic effect (compared to the virus control)
was expressed as the 50% inhibitory dose
(ID50). In order to quantify the antiviral activity,
the same plates were frozen and thawed three
times. The contents of the identical wells were
harvested, mixed and clarified by low-speed
centrifugation; and, virus titrations were per-
formed on the supernatant fluids by the limiting
dilution method 12. The antiviral activity of each
carrageenan extract was determined as the re-
duction factor (log10) of the viral titre by the
comparison with untreated controls. All experi-
ments were carried out three times.
Study of mode of action of the carrageenan
extracts against HHV-1 and Poliovirus
All experiments with carrageenans from Gi-
gartina acicularis (λ- and ι–family), Euchema
denticulatum (ι-family) and Kappaphycus cot-
tonii (κ–family) were performed at non toxic
concentration of 0.75 mg/ml.
445
Latin American Journal of Pharmacy - 28 (3) - 2009
Virus inactivation
Equal volumes of carrageenan extracts and
HHV-1 or Poliovirus stock suspension were
mixed and incubated for 1 h at 37 °C in order to
test possible virucidal activity. Thereafter, each
mixture was diluted 10-fold serially and infec-
tious titres were compared to those of controls 14.
Yield reduction after a single cycle of
replication
Vero cell monolayers cultured in 4-well cul-
ture plates were infected with HHV-1 or Po-
liovirus at a multiplicity of infections of about 1.
After 60 min at 37 °C, the unadsorbed viruses
were removed and the monolayers were
washed twice with MEM; subsequently, car-
rageenan extract dissolved in MEM was added.
After incubation of 18 h for HHV-1 or 8 h for
Poliovirus, the cultures were frozen and thawed
three times the cell debris was removed by low-
speed centrifugation. The supernatant virus
titres were determined by the limiting dilution
method and compared to that of the controls
without carrageenan extracts 14.
Culture pretreatment
Vero cell monolayers were pretreated with
each carrageenan extract for 24 h at 37 °C. After
being washed with MEM, the cells were ex-
posed to HHV-1 or Poliovirus at an Multiplicity
of Infections (MOI) of about 1. Following incu-
bation, the experiment was performed as de-
scribed above but no carrageenan extract was
added to the medium 14.
Study of viral DNA synthesis inhibition by
nucleic acid hybridization
Purification of HHV-1 DNA
Vero cells grown in 25 cm2tissue culture
flasks were infected at a high multiplicity (MOI
= 1), and HHV-1 adsorbed for 1 h at room tem-
perature, followed by washing with MEM to re-
move unadsorbed virus. From then on, only
maintenance medium (control) or carrageenan
extract dissolved in MEM (assays) was added.
The cultures were reincubated at 37 °C for 18 h.
The medium was discarded and the cells were
disrupted with 100 µl of lysis buffer. Subse-
quently, DNA was purified, as previously de-
scribed by Boom et al. 15, using a diatom sus-
pension. Samples were dissolved in 100 µl of
sterile water.
Hybridization probe
The digoxigenin-labelled DNA probe was a
non radioactive probe prepared by polymerase
chain reaction according to the method de-
scribed by Griffais et al. 16. The DNA template
was the U57 gene of HHV-1 obtained by a first
PCR 16. This template was amplified using two
primers: 5’-CTCACAGCCCCGAT-3’ and 5’-GTCC-
CGCGTTGC-3’. They were mixed with Thermus
aquaticus polymerase (Perkin Elmer Cetus, Nor-
walk, USA) and deoxynucleotide triphosphates:
d ATP, d CTP, d GTP and d UTP linked to
digoxigenin, dig-d UTP (Boehringer Mannheim,
Fr.). The solution was subjected to 38 cycles of
amplification in 3 steps: 92 °C, 55 °C and 72 °C.
Amplification was verified using agarose gel
electrophoresis.
Hybridization procedure
A twofold dilution of DNA extracts was per-
formed serially from 1/10 to 1/320 and dilutions
were heated up to 95 °C to denature DNA. The
samples were deposited on a Nylon membrane
HYBOND N (Amersham International Plc, UK)
and fixed to a HYBRI-SLOT manifold (BRL,
Maryland, USA). Afterwards, the filters were pre-
hybridized in thermally sealed plastic bags for 3
h at 42 °C in a prehybridization solution con-
taining 1% of a blocking agent (Boehringer
Mannheim, Fr.). After denaturation, a non-ra-
dioactive probe was added into the bag and hy-
bridization was carried out at 42 °C for 18 h,
with gentle shaking.
Detection of HHV-1 DNA probe binding
The hybrids were detected by enzyme-linked
immuno-assay using an antibody conjugate: an-
tidigoxigenin sheep antibody conjugated to al-
kaline phosphatase. The colour reaction was ini-
tiated at alkaline pH by the addition of 5-bro-
mo-4-chloro-3-indolyl phosphate (X-phosphate)
and nitroblue tetrazolium (NBT). (The detection
kit was purchased from Boehringer Mannheim,
Fr.). A blue precipitate indicated the presence of
HHV-1 DNA.
RESULTS AND DISCUSSION
We have investigated the anti-HHV-1 and the
antipoliovirus activity “in vitro” of three car-
rageenans extracts from three different algae
species collected in different countries.
The antiviral activity was first evaluated by
the inhibition of the cytopathic effect (CPE) in
cultures inoculated at a multiplicity of infection
of 0.01. The ID50 (mg/ml) values required for a
50% CPE inhibition and the selectivity indexes
(CC50/ID50) are indicated in Table 1.
446
MONTANHA J.A., BOURGOUGNON N., BOUSTIE J. & AMOROS M.
As shown in Table 1, the CC50 for the three
carrageenans was higher than 1.5 mg/ml (maxi-
mal concentration tested). Thus, the selectivity
index (ratio CC50/ID50) in the confluent cultures
used in antiviral assays range from 22 to more
than 545 for HHV-1; and from more than 6.6 to
more than 32 for Poliovirus at 0.01 MOI. Human
Herpes virus type I was more susceptible than
poliovirus to all carrageenan extracts. For HHV-
1 the highest selectivity index values were in the
same range as those previously found for
polysaccharides dextran sulfate and heparin
which were tested against HIV replication in
MT-4 and Molt-4 cell cultures 17 and for λ-car-
rageenan and pentosan polysulfate tested
against ASFV “in vitro” 18.
The effect on HHV-1 and Poliovirus replica-
tion was precisely quantified by infectious titre
reduction after several rounds of multiplication,
being the culture inoculated at 0.01 MOI. To be
considered active, a sample should induce at
least a 2 log10 decrease in virus titre in compari-
son with untreated virus control 19. According to
this criterion, all carrageenans tested were active
since the compounds reduced the virus titre be-
tween 2.0 and 3.0 log10 against HHV-1; whereas,
the activity against Poliovirus was lower with
titre reduction between 1.5 and 2.25 log10.
These results are shown in Table 1. The best re-
sults were obtained with carragenans from Gi-
gartina acicularis and Euchema denticulatum,
the more sulfated according to Amat 1.
Virus inactivation
No loss of infectivity was observed when
HHV-1 or Poliovirus was incubated for 1 h at 37
°C with Gigartina acicularis (λ- and ι–family),
Euchema denticulatum (ι-family) and Kappa-
phycus cottonii (κ–family) extracts of 0.75
mg/ml; it can be concluded that none of them
has a virucidal for the two viruses tested.
CPE inhibition (MOI 0.01) Yield reduction a(MOI 0.01)
HHV-1 Poliovirus HHV-1 Poliovirus
ID50 SI ID50 SI log10
Gigartina acicularis (λand τ) 0.00275 >545 0.047 >32 3.00 ± 0.86 2.50 ± 0.50
Euchema denticulatum (τ) 0.026 >71 0.115 >13 3.00 ± 0.25 1.50 ± 0.25
Kappaphycus cottonii (κ) 0.068 >22 0.227 >6.6 2.00 ± 0.25 1.50 ± 0.25
Table 1.In vitro antiviral activity of carrageenans against HHV-1 and Poliovirus. For three carrageenans the
CC50 is > 1.5 mg/ml; ID50 (mg/ml); SI (CC50/ID50). awhen compared with controls HHV-1 virus titre. Results
are presented as the mean ± SD from three independent tests.
Carrageenan
(type)
Effect of cell culture pretreatment
After a single round of replication, the HHV-
1 yield reduced 0.9, 0.9 and 0 log10 with Eu-
chema denticulatum (ι-family), Gigartina acicu-
laris (λ- and ι–family) and Kappaphycus cottonii
(κ–family), respectively. These results suggest
that the sulfated polysaccharides from Euchema
denticulatum and Gigartina acicularis have a
little effect on virus attachment or virus entry.
Effect of carrageenans on the yield of virus
from a single round of replication
In this experiment, Vero cells were infected
at a MOI of about 1; then, the carrageenans
were added to the culture medium 1 h posterior
to inoculation to exclude any effects on adsorp-
tion and penetration. After a single round of
replication, the HHV-1 yield reduced 2.0, 1.9
and 1.1 log10 with Euchema denticulatum (ι-
family), Gigartina acicularis λ- and ι–family)
and Kappaphycus cottonii (κ–family), respective-
ly. These results suggest that carrageenans from
Euchema denticulatum (ι-family) and Gigartina
acicularis exert their antiviral effect, in part, by
the inhibition of the virus attachment and the in-
terference in a subsequent stage of the virus at-
tachment. To determine this step, HHV-1 DNA
synthesis was evaluated by a hybridization tech-
nique in cultures where the most active car-
rageenans (Euchema denticulatum - ι-family
and Gigartina acicularis - λ- and ι–family) were
added. The effect of carrageenans on HHV-1
DNA synthesis was measured by nucleic acid
hybridization with a digoxigenin-labelled HHV-1
DNA probe. The DNA of untreated virus-infect-
ed controls, serially diluted twofold, was de-
tectable up to a 1:320 dilution whereas the DNA
of uninfected untreated control cells could not
be detected even at a 1:10 dilution, which
proved the probe specificity. In cultures treated
with carrageenans from Euchema denticulatum
447
Latin American Journal of Pharmacy - 28 (3) - 2009
and Gigartina acicularis, DNA synthesis was re-
duced threefold and twofold, respectively, with
0.75 mg/ml (Fig. 1). These results indicate that
carrageenans interfere with viral DNA synthesis.
This test did not, however, allow us to conclude
that replication was actually disrupted. Inhibi-
tion could result from another phenomenon oc-
curring upstream, such as DNA polymerase in-
terference.
These results are consistent with the yield re-
duction after a single cycle of replication, al-
though the decrease of DNA synthesis alone
could not explain the reduction of virus titre.
Other events of the replication cycle, such as
synthesis of late proteins or assembly step can
be inhibited by these polysaccharides. These
findings suggest that these sulfated polysaccha-
rides inhibit a subsequent step in virus replica-
tion to viral internalization but prior to the onset
of late viral protein synthesis as described by
Gonzáles et al. 20 for other polysaccharides
(Table 2).
If we consider the structure-activity relation-
Yield reduction log10 MOI = 1
Culture pretreatment After a single cycle of replication
HHV-1 Poliovirus HHV-1 Poliovirus
Gigartina acicularis (λand τ) 0.9 ± 0.25 0.3 ± 0.10 1.9 ± 0.25 0.5± 0.25
Euchema denticulatum (τ) 0.9 ± 0.25 0.5 ± 0.20 2.0 ± 0.50 0.5 ± 0.10
Kappaphycus cottonii (κ) 0 0.2 ± 0.25 1.1 ± 0.50 0.3 ± 0.10
Table 2. Mechanism of action of the carrageenans. Results are presented as the mean ± SD from three indepen-
dent tests.
Carrageenan
(type)
Figure 1. Slot hybridization of DNA extracts from
HHV-1 infected cells untreated (A1 to A6) and treated
with carrageenans (A7 to A12, B1 to B6 and B7 to
B12).A1 to A6: DNA of untreated virus control (HHV-
1). A7 to A12: DNA of cultures treated with 0.37
mg/ml of Euchema denticulatum (τ-family). B1 to
B6: DNA of cultures treated with 0.75 mg/ml of Eu-
chema denticulatum (τ-family). B7 to B12: DNA of
cultures treated with 0.75 mg/ml of Gigartina acicu-
laris (λ- and τ–family).
ships, the more actives carrageenans (Gigartina
acicularis -λ- and ι–family and Euchema den-
ticulatum -ι-family) are more sulfated than the
less active ones (Kappaphycus cottonii, κ–fami-
ly). These data were also observed by Gonzáles
et al. 20. Regarding the spectrum the of action of
the carrageenans tested, they were more active
against HHV-1 (enveloped virus) and less active
against poliovirus (a naked virus). These results
are also in agreement with the results obtained
by Gonzáles et al. 20.
We conclude that these extracts have an im-
portant antiviral activity and are potential candi-
dates for further studies of mechanism of action.
Acknowledgments. The present paper is dedicated
to the memory of Professor Loïc Girre. We are grate-
ful to Professor J. M. Kornprobst (SMAB/ISOMer –
Nantes/France) for providing the carrageenan sam-
ples. We also thank Mrs. M. Garel and Mrs. F. Baril
for technical assistance. A Research Fellowship held
from CAPES, Brazil was received by J.A. Montanha.
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... On the other hand, S. chordalis and S. muticum SPs presented a very low activity. The carrageenan extracted from Stenogramme interrupta [28], G. skottsbergii [34], and Gigartina acicularis [57] interfered efficiently during the early steps of the viral replication. In our study, the antiviral activity at this step could be related to the presence of the small peak with a molecular weight estimated of 4.4 kDa, since low molecular weight compounds (LMWC) have shown activity at the intracellular level. ...
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Herpes simplex virus 1 (HSV-1) remains a prominent health concern widespread all over the world. The increasing genital infections by HSV-1 that might facilitate acquisition and transmission of HIV-1, the cumulative evidence that HSV-1 promotes neurodegenerative disorders, and the emergence of drug resistance signify the need for new antiviral agents. In this study, the in vitro anti-herpetic activity of sulfated polysaccharides (SPs) extracted by enzyme or hot water from seaweeds collected in France and Mexico from stranding events, were evaluated. The anti-herpetic activity evaluation of the semi-refined-polysaccharides (sr-SPs) and different ion exchange purified fractions showed a wide range of antiviral activity. Among them, the sr-SPs from the Rhodophyta Halymenia floresii showed stronger activity EC50 0.68 μg/mL with SI 1470, without cytotoxicity. Further, the antiviral activity of the sr-SPs evaluated at different treatment schemes showed a high EC50 of 0.38 μg/mL during the viral adsorption assays when the polysaccharide and the virus were added simultaneously, whilst the protection on Vero cell during the post-infection assay was effective up to 1 h. The chemical composition, FTIR and 1H NMR spectroscopic, and molecular weights of the sr-SPs from H. floresii were determined and discussed based on the anti-herpetic activity. The potential utilization of seaweed stranding as a source of antiviral compounds is addressed.
... Carrageenans indicated an antiviral impact by the inhibition of virus attachment and interference in a subsequent stage of the virus replicative cycle. HHV-1 viral DNA synthesis was reduced by 3 folds in cultures treated with sulphated polydigalactosides from E. denticulatum (0.75 mg/mL) [44]. ...
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Malaysia has a long coastline surrounded by various islands, including North Borneo, that provide a suitable environment for the growth of diverse species of seaweeds. Some of the important North Bornean seaweed species are Kappaphycus alvarezii, Eucheuma denticulatum, Halymenia durvillaei (Rhodophyta), Caulerpa lentillifera, Caulerpa racemosa (Chlorophyta), Dictyota dichotoma and Sargassum polycystum (Ochrophyta). This review aims to highlight the therapeutic potential of North Bornean seaweeds and their nutraceutical profiling. North Bornean seaweeds have demonstrated anti-inflammatory, antioxidant, antimicrobial, anticancer, cardiovascular protective, neuroprotective, renal protective and hepatic protective potentials. The protective roles of the seaweeds might be due to the presence of a wide variety of nutraceuticals, including phthalic anhydride, 3,4-ethylenedioxythiophene, 2-pentylthiophene, furoic acid (K. alvarezii), eicosapentaenoic acid, palmitoleic acid, fucoxanthin, β-carotene (E. denticulatum), eucalyptol, oleic acid, dodecanal, pentadecane (H. durvillaei), canthaxanthin, oleic acid, pentadecanoic acid, eicosane (C. lentillifera), pseudoephedrine, palmitic acid, monocaprin (C. racemosa), dictyohydroperoxide, squalene, fucosterol, saringosterol (D. dichotoma), and lutein, neophytadiene, cholest-4-en-3-one and cis-vaccenic acid (S. polycystum). Extensive studies on the seaweed isolates are highly recommended to understand their bioactivity and mechanisms of action, while highlighting their commercialization potential.
... Prior work has shown multiple advantages and health benefits of human consumption of red algae (Rhodophyta) such as cardiovascular, cancer, diabetes, anti-bacterial and anti-viral effects (Gamero-Vega et al., 2020) while another study also showed that marine red algae species have crucial anti-viral roles such as in HHV-1 viral DNA synthesis (Montanha et al., 2009). However, there are still lots of red algae with unknown potentials. ...
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The respiratory infection COVID-19 caused by the virus SARS CoV-2 has continued to be a major health problem worldwide and has caused more than a million mortalities. Even if the development of COVID-19 vaccines has shown much progress, efforts to find novel, natural anti-viral drugs should be pursued. Halymenia durvillei is a marine red alga widely distributed around Southeast Asia. This study aimed to develop new anti SARS CoV-2 compounds from ethanolic and ethyl acetate extracts of H. via a computational approach, focusing onthe inhibitory action against the main protease (3CL-Mpro). In this study, 37 compounds were extracted and identified by GC-MS analysis. The potentials of compounds 1-2 tetradecandiol and E,E,Z-1,3,12-nonadecatriene-5,14-diol were identified for therapeutic purposes based on our pharmacophore study, while cholest-5-En-3-Ol (3.Beta.)- had a high fitness score in molecular docking studies both in monomer and dimer state compared to the N3 inhibitor and remdesivir affinity scores. As these compounds show competitive affinity scores against the 3CL-Mpro, these natural compounds may be effective for the treatment of COVID-19 infection. The ADME and pharmacokinetics study also be employed to assess the ability of the natural compounds as oral drugs. These promising results have shown the potentials of H. durvillei as an alternative drug in addressing COVID-19 infection. Accordingly, further studies should explore the effectiveness of these active compounds.
... Se utilizó la metodología descrita por Montanha et al. con modificaciones [12] . Se realizaron diluciones 1:5 y 1:10 de los extractos con medio de mantenimiento con 2% de suero bovino fetal en placas de 24 pozos con monocapa confluente de células Vero-76, inoculando 500 µl/pozo de cada dilución por duplicado e incubando los cultivos por nueve días. ...
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... At 0.75 mg/ml, no virucidal activity against HHV-1 or poliovirus was noticed. Their antiviral effect could be due to lower inhibition of the virus attachment and by the interference in the virus replication cycle [21]. ...
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Chapter
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References of all chapters from the book: Therapeutic and Nutritional Use of Algae
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Ce projet de thèse a été réalisé au sein du Laboratoire de Biotechnologie et Chimie Marines. Ce travail est le fruit d’une collaboration au niveau régional avec deux entreprises morbihannaises Armen Instruments et Newonat et au niveau international avec l’institut de recherche CINVESTAV au Mexique. Solieria chordalis (Rhodophyta, Gigartinales) est une algue proliférante de la côte de Bretagne sud. Cette algue s’échoue chaque année sur la presqu’île de Rhuys dans le Morbihan, et représente une biomasse importante encore sous exploitée. Les objectifs de cette thèse portent sur l’application de différents procédés éco-responsables (CO2 supercritique, Extraction Assistée par Enzyme et Extraction Assistée par Microondes) utilisés pour l’extraction de molécules à partir de S. chordalis. Dans le cadre de la première partie, l’extraction assistée par microondes a permis la production d’extraits polysaccharidiques de faibles poids moléculaires non cytotoxiques dont l’efficacité antivirale est supérieure à celle observée pour les fractions obtenues après extraction aqueuse à haute température. La deuxième partie concerne le couplage de différents procédés : Extraction Assistée par Enzymes (EAE), CO2 supercritique et Extraction Assistée par Microondes (EAM). En fonction des procédés utilisés, différentes familles de molécules ont pu être caractérisées. Le couplage de procédés permet de proposer un schéma de bioraffinage. La dernière partie porte sur l’optimisation d’une méthode de fractionnement par Chromatographie de Partage Centrifuge (CPC). Ce système de purification innovant a conduit à l’isolement de composés naturels dont des Mycosporines-like Amino Acids ayant différentes propriétés valorisables dans le domaine de la cosmétique. Ces travaux enrichissent les connaissances sur l’algue S. chordalis et ouvrent la voie à la valorisation de cette biomasse proliférante sur les côtes bretonnes.
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Seaweeds are valuable sources of biologically active compounds that could be used as ingredients for pharmacological applications. Industrial fermentation using microorganisms provides a wide array of fermented foods and functional compounds with excellent health benefits. These fermentation-derived natural compounds have potential to be applied as nutraceuticals and functional foods. Therefore, this paper presents an overview of fermentation of sustainable seaweeds in producing valuable components including anticoagulant, antioxidant, antimicrobial, anti-inflammatory, and anticancer. Fermentation-derived compounds are expected to receive more attention due to the environmentally friendly processing as well as consumer desire for natural foods.
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Among the numerous compounds that have been reported to exhibit potent antiviral activity in cell cultures and/or in experimental animals are several natural products isolated from higher plants. Some of these plant compounds exhibit an unique antiviral mechanism and are good candidates for further clinical research. In this review, an approach to the isolation of potential antiviral plant agents and lead compounds is outlined. A discussion of plant selection, followed by a description of antiviral testing, both in vitro and in vivo, is also given. The importance of the plant kingdom as a source of potent antiviral lead substances will be illustrated by presenting a survey on plant-derived anti-herpes virus and anti-human immunodeficiency virus agents.
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Carrageenans from the following red algae, Eucheuma spinosum, Eucheuma cottonii (Solieriaceae), Chondrus crispus gametophytes, and Gigartina stellata (Gigartinaceae) have been fractionated in order to obtain enriched fractions of carrageenan precursor(s) (μ- and ν-carrageenans). I.r. and 13C-n.m.r. spectroscopy have been used to identify structures present in these fractions and in total extracts from Ahnfeltia concinna and Ahnfeltia gigartinoides (Phyllophoraceae) and both methods are compared. ν-Carrageenan (precursor of ι-carrageenan) was the only precursor that could be detected in E. spinosum, E. cottonii, and the two Ahnfeltia species. E. cottonii fraction was also enriched in ι-carrageenan; this structure was partially methylated at O-6 of the d-galactose residue (m.s. determination). μ-Carrageenan (precursor of κ-carrageenan) was identified as the only precursor in the gametophyte fraction of C. crispus. Both structures μ and ν were present in G. stellata. 13C-N.m.r. spectroscopy is shown to be a good method of characterizing these structures, giving more accurate results than i.r. spectroscopy.
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Hypericum connatum (Guttiferae) is used in southern Brazil in the treatment of lesions in the mouth, often related to acute herpetic gingivo-stomatitis. The chemical investigation of the plant revealed the presence of phloroglucinol derivatives and flavonoids. From the n-hexane extract of the aerial parts a phloroglucinol derivative, hyperbrasilol B, was isolated, while the methanolic extract afforded four flavonoids: amentoflavone, hyperoside, guaijaverine and luteoforol. The crude methanolic extract and fractions (n-hexane, dichloromethane and methanol) as well as the isolated compounds were tested for antiviral activity against herpes simplex viruses (HSV). Among the tested samples, luteoforol was the most active inhibiting the cytopathic effect (CPE) and reducing the viral titer of HSV-1 DNA viral strains KOS and VR733 (ATCC).
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Schizymenia dubyi (Gigartinales, gymnophlaeaceae) contains an unusual sulfated heteropolysaccharide with uronic acids that is active against several viruses including human immunodeficiency virus type-1 (HIV-1), Herpes simplex hominis type 1 and type 2 (HSV-1 and HSV-2) and vesicular stomatitis virus (VSV).
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The polyanionic substances lambda and kappa carrageenan, pentosan polysulfate, fucoidan, dextran sulfate and heparin were investigated for their inhibitory effect on the replication of African swine fever virus (ASFV) in vitro. Lambda carrageenan was the most efficacious with a selectivity index, as based on the ratio of the 50% cytotoxic concentration to the 50% antiviral effective concentration, of 120, followed by pentosan polysulfate with 30, kappa carrageenan 13.3 and fucoidan 10. Dextran sulfate and heparin were almost inactive. In general, the substances had low toxicity for Vero cells. The studies with radiolabeled ASF virions suggest that the sulfated polysaccharides inhibit virus adsorption. Inhibition of virus replication was found for all the polysaccharides only when the substances were present during virus adsorption, with the exception of lambda and kappa carrageenan, which were also inhibitory when added immediately after the adsorption period.
Article
L'amplification enzymatique de gène a été utilisé pour incorporer, dans l'ADN, de la dTUP marquée par la digoxigénine. Cette technique qui permet la synthèse, de façon très simple, de quantités importantes d'ADN marqué, a été appliquée pour obtenir des sondes froides pour le virus d'Epstein-Barr et pour Chlamydia trachomatis. Les sondes ont une longueur bien définie, sont directement utilisable sans purification aussi bien pour la technique de Southern que pour les hybridations in situ, et elles sont sensibles et spécifiques.
Article
We evaluated, in cell cultures, the action of a series of 19 aporphine alkaloids against Herpes simplex virus type 1 (HSV-1). On the basis of viral titre reduction, six alkaloids were found to be active. The mode of action of the three most potent inhibitors, oliverine HCl, pachystaudine, and oxostephanine, was studied. These compounds did not have any virucidal or prophylactic effect but they were shown to interfere with the viral replicative cycle. Although DNA synthesis was reduced, their exact target remains to be elucidated. In the discussion, some structure-activity relationships are considered.
Article
Carrageenans extracted from cystocarpic and tetrasporic Stenogramme interrupta were analysed by chemical and spectroscopic methods. The carrageenan from cystocarpic plants is composed predominantly of 0.5 M KCl-insoluble and 1 M KCl-soluble fractions. The insoluble fraction contained iota-carrageenan as the major component with alpha-carrageenan and pyruvated carrageenan as minor components. The soluble fraction is highly heterogeneous and did not contain the precursors mu- and nu-carrageenans. The polysaccharide from tetrasporic plants is composed of zeta- and lambda-carrageenans, and low sulfated galactans. It is soluble in KCl and partly cyclized by alkaline treatment. The antiviral and anticoagulant properties of the insoluble polysaccharide fraction from cystocarpic S. interrupta and the polysaccharide from tetrasporic S. interrupta are reported the results of which suggest promising antiherpetic activity.
Article
A sulfated fucan containing fraction (SmWE) was isolated from water extract of the brown seaweed Stoechospermum marginatum collected from the Arabian Sea. Anion exchange chromatography of the crude fraction results in the production of a sulfated fucan (F3) having a molecular mass of 40 kDa and specific rotation [alpha]D(30) - 124 degrees (c 0.5, H2O). NMR spectroscopic studies and methylation analysis suggested that the polymer consists of a backbone of (1-->4)- and (1-->3)-linked-alpha-L-fucopyranosyl residues that are substituted at C-2 and C-3, and that fucosyl residues are sulfated mostly at C-2 and/or C-4. SmWE and F3 were selective inhibitors of herpes simplex virus type 1 (strain F, thymidine kinase-deficient strains field and B2006 and syncytial variants arising after selection with a natural carrageenan syn 13-8 and 14-1) and type 2 (strain MS) in Vero cells, with antiviral effective concentration 50% (EC50) values in the range 0.63-10.0 microg/ml. The compounds were highly selective due to the lack of cytotoxicity. The antiviral activity was dependent on the presence of the sulfated fucans during the adsorption period. No direct inactivating effect on virions was observed in a virucidal assay. The absence of anticoagulant activity at concentrations near EC50 confirmed that there was no correlation between the antiviral and anticoagulant properties.
  • U Adhikari
  • C G Mateu
  • K Chattopadhyay
  • C A Pujol
Adhikari, U., C.G. Mateu, K. Chattopadhyay, C.A. Pujol, E B. Damonte & B. Ray (2006) Phytochemistry 67: 2474–82.