NPC

Natural Product Communications

Antilonomic Effects of Brazilian Brown Seaweed Extracts

2009
Vol. 4
No. 8
1075 - 1078

Thaisa Francielle Souza Domingosa, Carla Carvalhoa, Laura de Andrade Mouraa,

Valria Laneuville Teixeirab, Renato Crespo Pereirab, verson Miguel Biancoc,
Wilton Jos Ferreirac, Carlos Jos Brito Ramosb, Ana Luiza Palhares de Mirandad,
Paulo Assis Meloe, Jorge Almeida Guimaresf and Andr Lopes Fulya
a

Departamento de Biologia Molecular e Celular, Instituto de Biologia,

Universidade Federal Fluminense, Niteri, RJ, Brazil, 24001-970

Departamento de Biologia Marinha, Instituto de Biologia, Universidade Federal Fluminense, Niteri,

RJ, Brazil, 24001-970
c

Programa de Ps-graduao em Qumica Orgnica, Instituto de Qumica,

Universidade Federal Fluminense, Niteri, RJ, Brazil, 24020-9015

Faculdade de Farmcia, Universidade Federal do Rio de Janeiro, RJ, Brazil, 21944-970

Departamento de Farmacologia Bsica e Clnica, Universidade Federal do Rio de Janeiro, RJ,

Brazil, 21941-590

Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS,
Brazil, 91501-970
valerialaneuville@gmail.com and andfuly@vm.uff.br
Received: May 22nd, 2009; Accepted: July 14th, 2009

The aim of this work was to investigate the hemolysis and blood clotting activity of Lomonia obliqua venom and the ability of
some Brazilian marine algal extracts (Canistrocarpus cervicornis, Stypopodium zonale and Dictyota pfaffi) to antagonize such
biological activities. L. obliqua caterpillars are dangerous to human beings and envenomation symptoms are characterized by
hemorrhagic, hemolytic and blood clotting disorders, and acute renal failure, which sometimes lead to the death of the victims.
Through in vitro experiments we have shown that L. obliqua venom is able to clot human plasma and hemolize human
erythrocytes and that the coagulation activity of the venom is inhibited by the extracts of C. cervicornis, S. zonale and D. pfaffi.
In contrast, C. cervicornis and S. zonale extracts did not inhibit the hemolytic activity of L. oblqua, as did the extract of D.
pfaffi. These finding indicate that marine algae may be used as antivenoms or may contribute to the development of compounds
with antilonomic effects.
Keywords: Canistrocarpus cervicornis, Dictyota pfaffi, Stypopodium zonale, Lonomia obliqua venom, antilonomic effect.

Caterpillar species are a potential danger for humans

and accidents regularly occur after contact is made
with the caterpillars bristles, which induce diverse
symptoms that range from mild cutaneous reactions
(pain, dermatitis, epistaxis and burning sensation) to
systemic ones, such as diffuse bleeding, renal failure,
depletion of fibrinogen and factor XIII levels, which
cause blood incoagulability, and of factor V level, all
of which can lead to the death of the victims [1]. Two
species of Lonomia (Lepdoptera, Saturniidae) are
involved in such accidents, L. obliqua Walker and L.
acheolus (Cramer) found in Brazil and Venezuela,
respectively. The accidents caused by both animals

share common features. Lonomic erucism is the term

used to describe accidents with Lonomia caterpillars,
which were considered rare in Brazil until 1989,
when over a 1,000 cases were reported. Most of them
occurred in the southern states, where, erucism
represents an important public health problem [2].
The venomous spicules and spines of the caterpillars
contain both non-protein and protein components,
such as serine proteases, glycoconjugates,
phospholipase A2 and others, from some of which
active principles have been purified and their
mechanism of action investigated [3]. Furthermore,
the presence of different toxins was confirmed

1076 Natural Product Communications Vol. 4 (8) 2009

Therefore, the objective of this work was to

investigate the ability of three Brazilian brown
seaweeds to inhibit hemolysis and clotting induced
by L. obliqua crude venom. The venom causes
hemolysis of human erythrocytes (Figure 1A) and has
a procoagulant activity (Figure 1B) upon human
plasma in a concentration-dependent manner. Both
biological activities presented by the L. obliqua
venom are mainly due to the presence of a
phospholipase A2 and two procoagulant toxins,
previously identified as factor X activators, and a
prothrombin activator named Losac and Lopap,
respectively [8]. Losac is a protease composed of a
single polypeptide chain of about 43 kDa and
activates factor X, while Lopap is a 69 kDa
tetrameric protein that activates prothrombin leading
to formation of thrombin, which is able to clot
purified fibrinogen or plasma. Hemolysis is related to
a purified phospholipase A2, an enzyme with a
molecular mass of 15 kDA and an isoelectric point of
5.9 [3e].
Different concentrations of L. obliqua venom (10-60
g/mL) were analyzed for their hemolytic (Panel A)
and clotting (Panel B) activities. Data are expressed
as mean SD of three individual experiments (n=3).
The effect of three brown marine algal extracts,
C. cervicornis (Ktzing) De Paula & De Clerck,
S. zonale (Lamouroux) Howe and D. pfaffii Schnetter
on the hemolytic and coagulant activities of

700 B

A
Coagulation time (s)

% Hemolysis

80
60
40
20
0

600
500
400
300
200
100
0
10 20 30 40 50 60

0 10 20 30 40 50 60

Lonomia obliqua (g/mL)

Lonomia obliqua (g/mL)

Figure 1: Effect of L. obliqua venom on blood hemolysis and clotting.

100

250

80

200
Clotting time (s)

Marine organisms produce many products with

different pharmacological properties, such as
antimicrobial, antiviral, anticancer, antimalarial and
antituberculosis [6]. In addition, further studies have
demonstrated that a dolabellane diterpene from
Dictyota species has an active chemical defense
against herbivores and is also able to maintain
species equilibrium [7].

100

% hemolysis

through a transcriptome study, which also revealed

several other molecules that may be involved in the
clinical manifestations [4]. Specific antivenom called
antilonomic serum (ALS), produced by the Butantan
Institute in So Paulo, Brazil, and an antifibrinolytic
drug, epsilon-aminocaproic acid (EACA), are used
to counteract the venom toxins. However, only ALS
has been shown to be an effective treatment for
envenomation by L. obliqua [5]. Nevertheless, the
search for new natural or synthetic venom inhibitors
able to complement antilonomic therapy is relevant.

Domingos et al.

60

40

20

150

100

50

3
4
Columns

Columns

Figure 2: Inhibitory effect of seaweed extracts on hemolysis and

coagulant activity of L. obliqua venom.

L. obliqua was evaluated. As shown in Figure 2A,

D. pfaffii extract was able to inhibit hemolysis, in
contrast to those of C. cervicornis and S. zonale
extracts. These results show that, although
C. cervicornis, D. pfaffii and S. zonale belong to
the same family (Dictyotaceae), they present
different inhibitory profiles on hemolysis induced by
L. obliqua venom.
The three algal extracts were also shown to be
capable of delaying coagulation triggered by L.
obliqua venom with different potencies (Figure 2B).
The
literature
registers
that
anticoagulant
polysaccharides from marine algae have been
isolated and characterized [9]. It is the authors
intention to develop these either as antithrombotic
drugs or to help in the development of new ones.
In conclusion, our results showed that extracts of
C. cervicornis, D. pfaffii and S. zonale had antilonomic
properties. The Brazilian coast is a rich source of
marine species that should be investigated for such
properties, as well as the isolated principles of the
seaweeds, in order to neutralize L. obliqua venom and
other venoms.

Antilonomic effects of seaweed extracts

Experimental
The seaweed extracts (120 g/mL) were incubated
with L. obliqua venom (320 g/mL) after hemolytic
(Panel A) and clotting (Panel B) tests had been
performed. The graphics show: column 1, venom
plus saline; column 2, venom plus DMSO; column 3,
venom plus C. cervicornis; column 4, venom plus S.
zonale; column 5, venom plus D. pfaffii. Data are
expressed as mean SD of three individual
experiments (n=3).
Material: Human plasma and erythrocytes were
collected from healthy donors and other reagents
were of the best grade available.
Caterpillars and bristle extract: L. obliqua
caterpillars from different colonies were collected in
the field and kindly provided by CIT (Centro de
Informaes Toxicolgicas), Porto Alegre, Brazil.
Crude bristle extract was obtained according to [10].
Bristles were cut at the base, homogenized in water
and centrifuged (9600 g X for 20 min.). The
supernatant was stored at -20oC until use.
Algal material: C. cervicornis was collected at
Ribeira Bay, Angra dos Reis, Rio de Janeiro State
(23o0034 S, 44o 2610 W). D. pfaffii from Reserva
Biolgica do Atol das Rocas, Rio Grande do Norte
State (03o51' 03" S, 33o40'29"W), and S. zonale from
Maratazes (21o02'040" S, 40o42'783"W). The air-

Natural Product Communications Vol. 4 (8) 2009 1077

dried algae were extracted with CH2Cl2 at room

temperature and the solvent evaporated under
reduced pressure. The extracts were diluted in DMSO
100% and stored at 4oC until use.
Hemolytic and coagulant activities: Hemolysis was
determined by the indirect hemolytic test upon
human erythrocytes [11]. A pool of human citrated
plasma was pre-incubated with CaCl2 (8.3 mM, final
concentration). Then, clotting was triggered by
adding L. obliqua venom. The clot formation was
monitored in a coagulometer at 37oC.
Neutralization of Lonomia activities: L. obliqua
venom was incubated with seaweed extracts for 30
min. at room temperature and then, hemolytic and
clotting tests were conducted.
Statistical analysis: All data shown represent mean
SD. Differences in mean values were analyzed using
ANOVA test. p values 0.05 were considered
significant.
Acknowledgments The authors thank Conselho
Nacional de Desenvolvimento Cientfico e
Tecnolgico (CNPq), Fundao de Amparo
Pesquisa do Estado do Rio de Janeiro (FAPERJ),
UFF/PROPP and International Foundation for
Science (IFS, Grant F/4571-1) for financial support.

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