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Rosmarinic acid, a new snake venom phospholipase A2 inhibitor
from Cordia verbenacea (Boraginaceae): antiserum
action potentiation and molecular interaction
Fabio K. Ticlia, Lorane I.S. Hagea, Rafael S. Cambraiab, Paulo S. Pereirab,
Angelo J. Magroc, Marcos R.M. Fontesc, Rodrigo G. Stabelid, Jose R. Giglioe,
Suzelei C. Francab, Andreimar M. Soaresa,*, Suely V. Sampaioa
aDepartamento de Analises Clınicas, Toxicologicas e Bromatologicas, FCFRP, Universidade de Sao Paulo,
USP, Ribeirao Preto-SP, BrazilbUnidade de Biotecnologia, Universidade de Ribeirao Preto, UNAERP, Ribeirao Preto-SP, Brazil
cDepartamento de Fısica e Biofısica, IB, Universidade Estadual Paulista, UNESP, Botucatu-SP, BrazildLaboratorio de Bioquımica do Instituto de Pesquisas em Patologias Tropicais (IPEPATRO), FioCruz,
UNIR, Porto Velho-RO, BrazileDepartamento de Bioquımica e Imunologia, FMRP, Universidade de Sao Paulo, USP, Ribeirao Preto-SP, Brazil
Received 3 February 2005; revised 27 April 2005; accepted 28 April 2005
Available online 29 June 2005
Abstract
Many plants are used in traditional medicine as active agents against various effects induced by snakebite. The
methanolic extract from Cordia verbenacea (Cv) significantly inhibited paw edema induced by Bothrops jararacussu
snake venom and by its main basic phospholipase A2 homologs, namely bothropstoxins I and II (BthTXs). The active
component was isolated by chromatography on Sephadex LH-20 and by RP-HPLC on a C18 column and identified as
rosmarinic acid (Cv-RA). Rosmarinic acid is an ester of caffeic acid and 3,4-dihydroxyphenyllactic acid [2-O-cafeoil-3-
(3,4-di-hydroxy-phenyl)-R-lactic acid]. This is the first report of RA in the species C. verbenacea (‘baleeira’, ‘whaler’)
and of its anti-inflammatory and antimyotoxic properties against snake venoms and isolated toxins. RA inhibited the
edema and myotoxic activity induced by the basic PLA2s BthTX-I and BthTX-II. It was, however, less efficient to inhibit
the PLA2 activity of BthTX-II and, still less, the PLA2 and edema-inducing activities of the acidic isoform BthA-I-PLA2
from the same venom, showing therefore a higher inhibitory activity upon basic PLA2s. RA also inhibited most of the
myotoxic and partially the edema-inducing effects of both basic PLA2s, thus reinforcing the idea of dissociation between
the catalytic and pharmacological domains. The pure compound potentiated the ability of the commercial equine
polyvalent antivenom in neutralizing lethal and myotoxic effects of the crude venom and of isolated PLA2s in
Toxicon 46 (2005) 318–327
www.elsevier.com/locate/toxicon
0041-0101/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.toxicon.2005.04.023
Abbreviations Cv-ME, Cordia verbenacea methanolic extract; Cv-RA, rosmarinic acid from Cordia verbenacea; PLA2, phospholipase A2;
PLIs, phospholipase A2 inhibitors; BthTX-I, B. jararacussu bothropstoxin-I; BthTX-II, B. jararacussu bothropstoxin-II; BthA-I-PLA2, B.
jararacussu acidic phospholipase A2; COSY, COrrelation SpectroscopY; HMQC, heteronuclear multiple quantum coherence; HMBC,
heteronuclear multiple bond coherence; CD, circular dichroism.* Corresponding author. Tel.: C55 16 602 4714; fax: C55 16 633 1936.
E-mail addresses: [email protected] (A.M. Soares), [email protected] (S.V. Sampaio).
F.K. Ticli et al. / Toxicon 46 (2005) 318–327 319
experimental models. CD data presented here suggest that, after binding, no significant conformation changes occur either
in the Cv-RA or in the target PLA2. A possible model for the interaction of rosmarinic acid with Lys49-PLA2 BthTX-I is
proposed.
q 2005 Elsevier Ltd. All rights reserved.
Keywords: Cordia verbenacea; Rosmarinic acid; Anti-inflammatory; Antimyotoxic; Antiophidian; Phospholipase A2 inhibitor; Bothrops
jararacussu; Snake venom
1. Introduction
Plants have often been used by humans, sometimes
successfully, against numerous diseases caused by different
pathological agents. Pharmacological studies have demon-
strated that the extracts and fractions from some of these
plants used in traditional medicine possess anti-inflamma-
tory, antiviral and antiophidian properties (Phillipson and
Anderson, 1989; Martz, 1992; Mors et al., 2000). The
antiophidian activity of several plant species in general use
in some Brazilian communities has been investigated
scientifically (Mors et al., 2000; Batina et al., 2000; Borges
et al., 2000, 2001; Biondo et al., 2003, 2004; Januario et al.,
2004; Veronese et al., 2005; Esmeraldino and Sampaio, in
press; da Silva et al., in press; Oliveira et al., 2005).
Snake venoms are complex mixtures of proteins
including phospholipases A2, myotoxins, hemorrhagic
metalloproteases and other proteolytic enzymes, cytotoxins,
cardiotoxins and others. The pathophysiology of snake
envenomation involves a complex series of events that
depend on the combined action of these venom components
(Gutierrez, 2002). Phospholipases A2 (PLA2; EC 3.1.1.4)
are abundant in snake venoms. Besides playing a digestive
role in phospholipid hydrolysis, they may also exert a wide
variety of pharmacological activities such as neurotoxicity,
myotoxicity, edema-inducing activity and others (Gutierrez
and Lomonte, 1995; Soares et al., 2004b). Local edema, a
typical manifestation of Bothrops envenomation, usually in
addition to pain, is due to the action of the venom upon
mastocytes, kininogens and phospholipids, culminating with
release of endogenous mediators (Teixeira et al., 2003).
The hydroalcoholic extract from Cordia verbenacea
(‘baleeira’, ‘whaler’) has been used by Brazilian folk as
cicatrizant and anti-inflammatory (Sertie et al., 1988). We
report now, for the first time, the anti-inflammatory and
antimyotoxic activity of the extract from C. verbenacea and its
active principle, rosmarinic acid, against these effects induced
by Bothrops jararacussu snake venom and by its main isolated
phospholipases A2. A possible model for the interaction of
rosmarinic acid with Lys49-PLA2 BthTX-I is proposed.
2. Material and methods
2.1. Materials
The leaves from C. verbenacea were collected during
the blooming period in the Campus of the University of
Ribeirao Preto (UNAERP). A voucher specimen (No. 259)
identified by specialist Prof. Dr Lin Chau Ming (Departa-
mento de Botanica, UNESP, Botucatu, SP, Brazil) has been
preserved in the Unidade de Biotecnologia Herbarium,
UNAERP. B. jararacussu venom was purchased from Sandrin
Bioagents serpentarium, Batatais, SP. B. jararacussu PLA2s
were isolated on Sephadex G-75 followed by cation-exchange
chromatography as previously described (Andriao-Escarso
et al., 2000, 2002). PLA2 homogeneity was assessed by native
and SDS-PAGE and reverse-phase HPLC.
2.2. Preparation of plant extract
After identification, the leaves were dried in a stove with
circulating air at 40 8C. They were then grounded (375 g)
and macerated with chloroform three times during three
days, then with methanol, followed by filtration and
evaporation of the methanol in a rotary evaporator where-
from the dried methanolic extract (Cv-ME) was obtained.
2.3. Purification and identification of rosmarinic acid
A preliminary Sephadex LH-20 column was used for the
first fractionation of Cv-ME, using 300 mL of methanol as
mobile phase for elution. The resulting fractions, after
drying, were analyzed by thin-layer chromatography and
revealed with vanillin sulfuric acid reagent. Fraction 3 was
then applied on a HPLC semipreparative Supelcosil C18
column, using a concentration gradient of methanol:water at
a flow rate of 2 mL/min. Seven new fractions were so
obtained, which were assayed for edema inhibition, from
which rosmarinic acid (Cv-RA) was fraction 6, as identified
by NMR analysis. NMR spectra were recorded with a
Brucker DPX-300 spectrophotometer, operating at 300 mHz
for 1H and 75 mHz for 13C. For that, 15 mg samples were
used, dissolved in dimethyl-d6-sulfoxide (Aldrich).
2.4. Edema-inducing activity
Edema was induced by i.d. injection, in the right foot pad
of male Swiss mice (18–22 g), of B. jararacussu venom
(25 mg) and its purified PLA2s (50 mg). Inhibition studies
were performed by incubating venom or PLA2 with
Cv-ME/Cv-RA. Control groups were injected with 50 mL
of phosphate-buffered saline (PBS, pH 7.2) alone, or
Cv-ME/Cv-RA alone. The progression of edema was
evaluated with a low pressure pachymeter (Mitutoyo,
F.K. Ticli et al. / Toxicon 46 (2005) 318–327320
Japan) at various time intervals after injection (Soares et al.,
2000).
2.5. Enzymatic activities
PLA2 activity was determined in a gel plate containing
egg yolk (6 egg yolks/L), CaCl2 (0.56 g/L) and agar (20 g/L)
, according to Gutierrez et al. (1988). Crude venom (1 mg),
BthTX-II (1 mg) and BthA-I-PLA2 (1 mg) were inoculated
and the diameter (mm) of the resulting halos were measured
after 2 h. Anticoagulant activity (Alvarado and Gutierrez,
1988) was evaluated using human plasma (200 mL), CaCl2(0.25 mM) and BthTX-II (0.5 mg). The plasma was
primarily equilibrated at 37 8C in a water bath with or
without Cv-RA/Cv-ME. BthTX-II was then added and, after
10 min, the CaCl2 solution (25 mL). Plasma which did not
Fig. 1. Purification of Cv-RA from Cordia verbenacea methanolic extrac
from the Sephadex LH-20. (B) TLC of fraction F3-CL6 (rosmarini
(100:11:11:26, v/v). Staining: NP/PEG, under UV. (C) Assay for purit
clot after 45 min was considered incoagulable. The control
tube received PBS replacing BthTX-II, where the plasma
should clot within 3–6 min.
2.6. Myotoxic activity
Swiss male mice (18–22 g) were injected intramuscu-
larly in the right gastrocnemius muscle with solutions
containing doses of 25 mg/50 mL of Bothrops venoms or
toxins. The mixtures of venom or toxin/Cv-RA (1:1 and
1:10, w/w) were then evaluated. Controls received PBS or
inhibitor alone. Mice were bled from the tail 3 h after
injections and blood was collected into heparinized capillary
tubes. Plasma creatine kinase activity was determined using
the Kit 47-UV (Sigma Chemical Co.) (Soares et al., 2000).
Activity was expressed in units/L, one unit corresponding to
the production of one micromole of NADH per min at 30 8C.
t. (A) Fractionation on Supelcosil C18, by HPLC, of fraction F3
c acid). Mobile fase:ethyl acetate:formic acid:acetic acid:water
y of rosmarinic acid by HPLC-C18.
Fig. 2. Rosmarinic acid from Cordia verbenacea methanolic extract. (A) Molecular structure of Cv-RA from resonance studies (NMR 1H and
NMR 13C). (B) 3D-molecular structure of Cv-RA.
Fig. 3. Inhibition of the edema-inducing activity by rosmarinic acid. (A) Effect of Cv-ME (1:10, w/w) on the edema induced by B. jararacussu
crude venom, Lys49-BthTX-I and Asp49-BthTX-II. (B) Effect of Cv-RA (1:3.5, w/w) on the edema induced by the crude venom, BthTX-I and
BthTX-II. Results are expressed by the meanGSD (nZ6). Means are statistically significantly different from the control means.
F.K. Ticli et al. / Toxicon 46 (2005) 318–327 321
F.K. Ticli et al. / Toxicon 46 (2005) 318–327322
2.7. Potentiation of anti-bothropic serum action
Cv-RA was added to polyvalent antivenom (Instituto
Butantan-SP, Brasil) at an ED50 (effective dose to neutralize
50% of myotoxicity as defined by levels of creatine kinase
in plasma after 3 h post-injection), incubated with
B. jararacussu venom or isolated PLA2s in a final volume
of 50 mL for 30 min at 37 8C and injected intramuscularly
in mice as previously described (Lizano et al., 2003).
2.8. Circular dichroism of Cv-RA
Far UV circular dichroism spectra (190–250 nm) were
measured with a JASCO 810 (JASCO, Inc., Tokyo, Japan)
using 1 mm path length cuvettes and protein concentrations
of 150 mg/mL for both the target myotoxic PLA2s and
450 mg/mL for Cv-RA. In the case of mixtures, the total
Fig. 4. Inhibition of the PLA2 activity by rosmarinic acid. Effect of Cv-RA
Asp49-PLA2 BthTX-II (B) and Asp49-PLA2 BthA-I-PLA2 (C) at rations 1
the meanGSD (nZ6). Means are statistically significantly different (*) fr
protein concentration was 150 mg/mL. In all cases, a total of
10 spectra were collected, averaged and corrected by
subtraction of a buffer blank.
2.9. Molecular modeling
The molecular model of the monomeric BthTX-I
(da Silva-Giotto et al., 1998) complexed with rosmarinic
acid was elaborated using the program O (Jones et al.,
1990). The complex was refined and its energy was
minimized using the program CNS (Brunger et al., 1998).
2.10. Statistical analysis
Results are presented as the mean valueGSD obtained
with the indicated number of tested animals. The statistical
significance of differences between groups was evaluated
on the PLA2 activity induced by B. jararacussu crude venom (A),
:5, 1:10 and 1:50 (venom:inhibitor, w/w). Results are expressed by
om the control means.
F.K. Ticli et al. / Toxicon 46 (2005) 318–327 323
using Student’s unpaired t-test. A P-value !0.05 was
considered to indicate significance.
3. Results and discussion
In many countries, plant extracts have been traditionally
used in the treatment of snakebite envenomations (Martz,
1992; Mors et al., 2000; Soares et al., 2004a), although only
in a few cases there has been a scientific validation of such
claims. Snake venoms are complex mixtures of proteins and,
among these, are phospholipases A2, hemorrhagins,
proteases and myotoxins that act by different mechanisms
(Gutierrez, 2002). A number of PLA2s has been character-
ized from Bothrops venoms, some which are devoid of
catalytic activity upon artificial substrates due to the
substitution of Lys at position 49 for Asp (Soares et al.,
2004b).
Fig. 1 shows the purification of Cv-RA from the
C. verbenacea methanolic extract. Cv-ME represented
Fig. 5. Inhibition of the myotoxic activity by rosmarinic acid. Effect of Cv-
(A), Lys49-PLA2 BthTX-I (B) and Asp49-PLA2 BthTX-II (C) at rations
meanGSD (nZ6). Means are statistically significantly different (*) from
2.25% (8.5 g) of the dried leaves. From its fractionation
on the Sephadex LH-20 column, three fractions were
obtained from which fraction 3 was less heterogeneous
and corresponded to 0.19% (0.7 g) of the dried leaves.
Among the seven subfractions resulting from the HPLC
rechromatography of fraction 3, subfraction 6 (CL-6)
showed to be highly purified (HPLC-C18) and represented
0.03% (0.112 g) of the dried leaves. Spectroscopic analysis
of subfraction 6 identified it as rosmarinic acid. Its chemical
and tridimensional structure is shown in Fig. 2A and B,
respectively
Rosmarinic acid was first isolated from Rosmarinus
officinalis, but recently its synthetic preparation was
described. RA is often described as anti-inflammatory. It
is a polyphenolic compound, isolated from several plants of
Boraginaceae and Laminaceae families (Petersen and
Simmonds, 2003). This is the first report of rosmarinic
acid in the species C. verbenacea and explains the efficiency
of this plant regarding anti-inflammatory and antimyotoxic
properties against snake venoms and isolated toxins.
RA on the myotoxic activity induced by B. jararacussu crude venom
1:1 and 1:10 (venom:inhibitor, w/w). Results are expressed by the
the control means.
Fig. 6. Analysis of B. jararacussu venom and isolated PLA2s,
BthTX-I and BthTX-II by SDS-PAGE 12%. Before incubation
with Cv-ME or Cv-RA: Lanes: 1, BthTX-II; 2, BthTX-I; 3,
B. jararacussu venom; 4, Cv-ME or Cv-RA. After incubation with
the Cv-ME or Cv-RA: Lanes: 1, Cv-RACBthTX-II; 2, Cv-RACBthTX-I; and 3, Cv-RACB. jararacussu.
F.K. Ticli et al. / Toxicon 46 (2005) 318–327324
Edema-inducing activity is a multifactorial pharmaco-
logical activity, depending on the combined action of
various toxins, suggesting that enzymatic activity is not
strictly required to induce this effect. Cv-ME inhibited near
20, 60 and 10% the edema induced by B. jararacussu crude
venom, BthTX-I and BthTX-II, respectively (Fig. 3A),
while Cv-RA inhibited 5, 60 and 10% the edema induced by
Fig. 7. Enhancement of the antimyotoxic properties of polyvalent anti-bot
verbenacea rosmarinic acid (Cv-RA). Cv-RA was added to commercial p
ED50 incubated with B. jararacussu venom or isolated PLA2s for 30 min a
by the meanGSD (nZ6). Means are statistically significantly different (*
these same samples (Fig. 3B). Cv-ME was more efficient in
neutralizing the edema induced by the crude venom than
Cv-RA, thus suggesting that other active principles are
presented in Cv-ME other than Cv-RA. An active flavonoid
component, artemetin, has previously been isolated from
this plant and shown to have anti-inflammatory effects
(Sertie et al., 1990).
RA showed to be more efficient in neutralizing the PLA2
activity induced by the basic Asp49 BthTX-II (Fig. 4B) than
that induced by the crude venom (Fig. 4A) and by the acidic
isoform Asp49 BthA-I-PLA2 (Fig. 4C). These data suggest a
more specific binding with basic PLA2s, intermediated by a
probable electrostatic interaction. Biondo et al. (2003) also
showed that the aqueous extract from Mandevilla velutina
showed a wide inhibition spectrum of toxic, enzymatic and
pharmacological activities of snake venoms and isolated
toxins. However, this extract was more specific for Crotalus
venom and the neurotoxic basic PLA2 when compared with
Bothrops acidic PLA2.
A partial dissociation between the catalytic and edema-
inducing domains is also likely to exist in these PLA2s, since
a 60% inhibition of the edema induced by the basic Lys49
BthTX-I, enzymatically inactive, was observed, against
only 10 and 50% inhibition of the edema and PLA2 activity,
respectively, induced by the basic Asp49 BthTX-II. These
data agree with several authors who suggest distinct
domains or partial overlapping between the catalytic and
other pharmacological sites (Soares and Giglio, 2003).
Muscle tissue damage, myonecrosis, is a common
consequence of envenomation by crotaline snakes of the
genus Bothrops (Gutierrez, 2002). Muscle damaging
activity of Bothrops venoms is partially caused by a group
hropic immunoglobulin antivenom by supplementation with Cordia
olyvalent (Crotalinae) antivenom (Instituto Butantan, Brazil) at an
t 37 8C, and injected intramuscularly in mice. Results are expressed
) from the control means.
Fig. 8. Analysis of circular dichroism spectra for RA in association
with BthTX-I. Spectra for the BthTX-I (open squares) alone or
BthTX-I and RA (closed squares), a mixture at a 1:3 molar ratio are
shown. The spectra shown are unsmoothed and corrected only by
subtraction of buffer blanks as described in Section 2.
F.K. Ticli et al. / Toxicon 46 (2005) 318–327 325
of highly basic proteins with PLA2 structure. Rosmarinic
acid inhibits the myotoxic activity of both Asp49 BthTX-II
and Lys49 BthTX-I phospholipases A2 from B. jararacussu
(Fig. 5). Rosmarinic acid did not inhibit the myotoxic
activity of the crude venom as effectively as that of the
purified PLA2s. This could very well be a result of the
contribution to myonecrosis made by the strong hemor-
rhagic toxins in the crude venom which are lacking in the
purified PLA2 preparations. A series of PLA2s inhibitors
Fig. 9. Molecular model of rosmarinic acid and monomeric BthTX-I compl
interacting with the rosmarinic acid and the BthTX-I are shown in ball-st
(PLIs) has been isolated from natural sources, such as
marine organisms, snakes and plants (Lizano et al., 2003).
Wedelolactone and 12-methoxy-4-methylvoachalotine
(MMV), compounds isolated from Eclipta prostata and
Tabernamontana catharinensis, respectively, effectively
inhibits the myotoxic activity of the venoms of Crotalus
durissus terrificus, B. jararacussu, B. jararaca and Lachesis
muta, as well as various isolated myotoxic PLA2s (Mors
et al., 2000; Soares et al., 2004a).
Although the mechanism of action of C. verbenacea
methanolic extract (Cv-ME) and/or rosmarinic acid
(Cv-RA) is still unknown, the finding that no visible change
was detected in the electrophoretic pattern of B. jararacussu
venom, BthTX-I and BthTX-II, after incubation with Cv/RA
(Fig. 6), excludes proteolytic degradation as a potential
mechanism.
Preliminary studies on supplementation of conventional
antivenom against B. jararacussu or isolated myotoxic
PLA2s with the Cv-RA from C. verbenacea show that the
inhibitor enhances the neutralization potential of the
antivenom in mice (Fig. 7), which is often only partially
effective in neutralizing myotoxicity in vivo. As far as
toxicity is concerned, at least mice inoculated with
B. jararacussu venom and subsequently treated by
intravenous injection of the inhibitor or antivenom
immunoglobulins supplemented with Cv-RA show no
detectable signs of toxicity or adverse effects to the addition
of inhibitor. Similarly, adjuvant effects and antiserum action
ex. Drawn with the program RIBBONS (Carson, 1997). The residues
ick representation.
F.K. Ticli et al. / Toxicon 46 (2005) 318–327326
potentiation by a compound Hemidesmus indicus 2-hidroxy-
4-methoxy-benzoic acid against Vipera russelli venom were
described (Alam and Gomes, 1998).
Possible secondary structural changes either in the
Cv-RA or the target PLA2 following binding of the inhibitor
were evaluated by circular dichroism (CD) spectroscopy
(Fig. 8). The result for mixtures of the Cv-RA with
Lys49-BthTX-I was described, which reveal that the CD
spectra for the mixture of the two components is equal the
sum of the two individual spectra of the Cv-RA and the
BthTX-I alone. The experiments in which Asp49-BthTX-II
substituted for the BthTX-I yielded similar results (data not
shown), which suggests that no significant secondary
structure changes occurred on association of the Cv-RA
with the PLA2s tested.
In order to study the possible mechanism of Lys49-PLA2
BthTX-I inhibition by rosmarinic acid, a molecular model of
the complex was made. The rosmarinic acid was modeled
into the hydrophobic channel leading to the active site. After
energy minimization, the rosmarinic acid remained in the
hydrophobic channel with a hydroxyl group of one of the
aromatic rings bound to His48 and the carboxyl group
bound to the Lys69 (Fig. 9). This is a possible way for the
rosmarinic acid to interact with a phospholipase A2 leading
to its inhibition. His48 belongs to the catalytic network for
class II PLA2s being a strictly conserved residue to this class
of proteins. The majority of PLA2-inhibitor complexes have
these molecules bound to His48 (Watanabe et al., 2005),
which is seen to be essential for the inhibition process.
Lys69 is conserved residue in the most part of class II PLA2s
and is sited in a loop between a-helix 2 and b-wing known
as ‘pancreatic loop’. While, for Asp49-PLA2s, this residue is
associated with anticoagulant activities (Carredano et al.,
1998), no activity is associated with this residue for
Lys49-PLA2 until now.
The presence of PLA2 inhibitory proteins and other
compounds in plants opens the possibility to search for
natural inhibitors of snake venom myotoxic effects in plants
for therapeutic purposes. It is likely that other plants may
also serve as sources for PLIs that could be used in the future
as potent antivenom compounds.
4. Conclusions
Cv-ME and Cv-RA inhibit the edema and myotoxicity
induced by B. jararacussu crude venom and its main
phospholipases A2 homologs, thus showing that this plant is
a good tool with potential antiophidian activity. Cv-RA was
much more efficient to inhibit the edema induced by the Lys49
PLA2 BthTX-I than its isoform Asp49 BthTX-II. However, it
neutralized equally the myotoxicity induced by both toxins.
This fact suggests the presence of distinct domains for these
activities. Co-crystallization studies of this inhibitor with
Lys49 PLA2s are in progress for a better insight into the
mechanism of action of these enzyme and/or inhibitor.
Supplementation of antiophydian serum with natural
anti-toxins such as anti-hemorrhagins and anti-PLA2s could
increase the ability of serum to neutralize snake toxins. It is
interesting to speculate that Cv-RA, or a derivative, may
prove useful in the treatment of snakebite victims, or more
importantly in the treatment of the many human diseases in
which PLA2 enzymes have been implicated. In particular,
the use of cell impermeable PLA2 inhibitors could be a
favorable therapeutic approach in the treatment of inflam-
matory processes.
Acknowledgements
The authors gratefully acknowledge the financial support
by Fundacao de Amparo a Pesquisa do Estado de Sao Paulo
(FAPESP) and Conselho Nacional de Desenvolvimento
Cientıfico e Tecnologico (CNPq). Thanks are also due to
Joao J. Franco (FCFRP-USP), Adelia C.O. Cintra (FCFRP-
USP), Eliandra G. Silva (TT-FAPESP) and Vanessa
C. Fernandes (TT-FAPESP) for their helpful technical
collaboration.
References
Alam, M.I., Gomes, A., 1998. Viper venom-induced inflammation
and inhibition of free radical formation by pure compound (2-
hydroxy-4-methoxy benzoic acid) isolated and purified from
anantamul (Hemidesmus indicus R. BR) root extract. Toxicon
36, 207–215.
Alvarado, J., Gutierrez, J.M., 1988. Anticoagulant effect of myotoxic
phospholipase A2 isolated from the venom of the snake Bothrops
asper (Viperidae). Rev. Biol. Trop. 36, 563–565.
Andriao-Escarso, S.H., Soares, A.M., Rodrigues, V.M., Angulo, Y.,
Diaz, C., Lomonte, B., Gutierrez, J.M., Giglio, J.R., 2000.
Myotoxic phospholipases A2 in Bothrops snake venoms: effect
of chemical modifications on the enzymatic and pharmacologi-
cal properties of bothropstoxin from Bothrops jararacussu.
Biochimie 82, 755–763.
Andriao-Escarso, S.H., Soares, A.M., Fontes, M.R., Fuly, A.L.,
Correa, F.M., Rosa, J.C., Greene, L.J., Giglio, J.R., 2002.
Structural and functional characterization of on acidic platelet
aggregation inhibitor and hypotensive phospholipase A2 from
Bothrops jararacussu snake venom. Biochem. Pharmacol. 64,
723–732.
Batina, M.F., Cintra, A.C., Veronese, E.L., Lavrador, M.A.,
Giglio, J.R., Pereira, P.S., Dias, D.A., Franca, S.C.,
Sampaio, S.V., 2000. Inhibition of the lethal and myotoxic
activities of Crotalus durissus terrificus venom by Tabernae-
montana catharinensis: identification of one of the active
components. Planta Med. 66, 424–428.
Biondo, R., Pereira, A.M.S., Marcussi, S., Pereira, P.S.,
Franca, S.C., Soares, A.M., 2003. Inhibition of enzymatic and
pharmacological activities of some snake venoms and toxins by
Mandevilla velutina (Apocynaceae) aqueous extract. Biochimie
85, 1017–1025.
Biondo, R., Soares, A.M., Bertoni, B.W., Franca, S.C.,
Pereira, A.M.S., 2004. Direct organogenesis of Mandevilla
F.K. Ticli et al. / Toxicon 46 (2005) 318–327 327
illustris (Vell) Wodson and effects of its aqueous extract on
the enzymatic and toxic activities of Crotalus durissus terrificus
snake venom. Plant Cell Rep. 22, 549–552.
Borges, M.H., Soares, A.M., Rodrigues, V.M., Andriao-Escarso, S.H.,
Diniz, H., Hamaguchi, A., Quintero, A., Lizano, S.,
Gutierrez, J.M., Giglio, J.R., Homsi-Brandeburgo, M.I., 2000.
Effects of aqueous extract of Casearia sylvestris (Flacourtiaceae)
on actions of snake and bee venoms and on activity of
phopholipases A2. Comp. Biochem. Physiol. 127, 21–30.
Borges, M.H., Soares, A.M., Rodrigues, V.M., Oliveira, F.,
Fransheschi, A.M., Rucavado, A., Giglio, J.R., Homsi-
Brandeburgo, M.I., 2001. Neutralization of proteases from
Bothrops snake venoms by the aqueous extract from Casearia
sylvestris (Flacourtiaceae). Toxicon 39, 1863–1869.
Brunger, A.T., Adams, P.D., Clore, G.M., DeLano, W.L., Gros, P.,
Grosse-Kunstleve, R.W., Jiang, J.S., Kuszewski, J., Nilges, M.,
Pannu, N.S., Read, R.J., Rice, L.M., Simonson, T.,
Warren, G.L., 1998. Crystallography and NMR system (CNS):
a new software system for macromolecular structure determi-
nation. Acta Crystallogr. 54, 905–921.
Carredano, E., Westerlund, B., Persson, B., Saarinen, M.,
Ramaswamy, S., Eaker, D., Eklund, H., 1998. The three-
dimensional structures of two toxins from snake venom throw
light on the anticoagulant and neurotoxic sites of phospholipase
A2. Toxicon 36, 75–92.
Carson, M., 1997. Ribbons. Meth. Enzymol. 277, 493–505.
da Silva, J.O., Coppede, J.S., Fernandes, V.C., Sant’Ana, C.D.,
Ticli, F.K., Mazzi, M.V., Giglio, J.R., Pereira, P.S., Soares,
A.M., Sampaio, S.V., 2005. Antihemorrhagic, antinucleolytic
and other antiophidian properties of the aqueous extract from
Pentaclethra macroloba. J. Ethnopharmacol. (in press).
da Silva-Giotto, M.T., Garrat, R.C., Oliva, G., Mascarenhas, Y.P.,
Giglio, J.R., Cintra, A.C., de Azevedo Jr., W.F., Arni, R.K.,
Ward, R.J., 1998. Crystallographic and spectroscopic charac-
terization of a molecular hinge: conformational changes in
bothropstoxin I, a dimeric Lys49-phospholipase A2 homologue.
Proteins Struct. Funct. Genet. 30, 442–454.
Esmeraldino, L.E., Sampaio, S.V., 2005. Evaluation of the effect of
aqueous of extract of Croton urucurana Baillon (Euphorbia-
ceae) on the hemorrhagic activity induced by the venom of
B. jararaca using new techniques to quantify hemorrhagic
activity in rat skin. Phytomedicine (in press).
Gutierrez, J.M., 2002. Understanding snake venoms: 50 years of
research in Latin America. Rev. Biol. Trop. 50, 377–394.
Gutierrez, J.M., Lomonte, B., 1995. Phospholipase A2 myotoxins
from Bothrops snake venoms. Toxicon 33, 1405–1424.
Gutierrez, J.M., Avila, C., Rojas, E., Cerdas, L., 1988. An alternative
in vitro method for testing the potency of the polyvalent
antivenom produced in Costa Rica. Toxicon 26, 411–413.
Januario, A.H., Santos, S.L., Marcussi, S., Mazzi, M.V.,
Pietro, R.C.L., Sato, D.N., Ellena, J., Sampaio, S.V.,
Franca, S.C., Soares, A.M., 2004. neo-Clerodane Diterpenoid,
a new metalloprotease snake venom inhibitor from Baccharis
trimera (Asteraceae): anti-proteolytic and anti-hemorrhagic
properties. Chem. Biol. Interact. 150, 243–251.
Jones, T.A., Bergdoll, M., Kjeldgaard, M., 1990. O: a macromol-
ecule modeling environment. In: Crystallographic and
Modeling Methods in Molecular Design. Springer, Berlin, pp.
189–195.
Lizano, S., Domont, G., Perales, J., 2003. Narural phospholipase A2
myotoxin inhibitor proteins from snakes, mammals and plants.
Toxicon 42, 963–977.
Martz, W., 1992. Plants with a reputation against snakebite.
Toxicon 30, 1131–1142.
Mors, W.B., Nascimento, M.C., Pereira, B.M., Pereira, N.A., 2000.
Plant natural products active against snake-bite—the molecular
approach. Phytochemical 55, 627–642.
Oliveira, C.Z., Maiorano, V.A., Marcussi, S., Sant’Ana, C.D.,
Januario, C.D., Lourenco, M.V., Sampaio, S.V., Franca, S.C.,
Pereira, P.S., Soares, A.M., 2005. Anticoagulant and antifibrino-
genolytic properties of the aqueous extract from Bauhinia forficata
against snake venoms. J. Ethnopharmacol. 98, 213–216.
Petersen, M., Simmonds, M.S., 2003. Rosmarinic acid. Review.
Phytochemistry 62, 121–125.
Phillipson, J.D., Anderson, L.A., 1989. Ethnopharmacology and
Western medicine. J. Ethnopharmacol. 25, 61–72.
Sertie, J.A.A., Basile, A.C., Panizza, S., Matida, A.K., Zelnik, R.,
1988. Pharmacological assay of Cordia verbenacea; Part 1.
Anti-inflammatory activity and toxicity of the crude extract of
the leaves. Planta Med. 1, 7–10.
Sertie, J.A.A., Basile, A.C., Panizza, S., Matida, A.K., Zelnik, R.,
1990. Antiinflammatory activity and sub-acute toxicity of
artemetin. Planta Med. 2, 36–40.
Soares, A.M., Giglio, J.R., 2003. Chemical modification on
phospholipases A2 from snake venom: effects on catalytic and
pharmacological properties. Toxicon 42, 855–868.
Soares, A.M., Andriao-Escarso, S.H., Angulo, Y., Lomonte, B.,
Gutierrez, J.M., Marangoni, S., Toyama, M.H., Arni, R.K.,
Giglio, J.R., 2000. Structural and functional characterization of
myotoxin I, a Lys49 phospholipase A2 homologue from
Bothrops moojeni (Caissaca) snake venom. Arch. Biochem.
Biophys. 373, 7–15.
Soares, A.M., Januario, A.H., Lourenco, M.V., Pereira, A.M.,
Pereira, P.S., 2004a. Neutralizing effects of Brazilian plants
against snake venoms. Drugs Future 29, 1105–1117.
Soares, A.M., Fontes, M.R., Giglio, J.R., 2004b. Phospholipase A2
myotoxins from Bothrops snake venoms: function–structure
relationship. Curr. Org. Chem. 8, 1677–1690.
Teixeira, C.F., Landucci, E.C., Antunes, E., Chacur, M., Cury, Y.,
2003. Inflammatory effects of snake venom myotoxic phospho-
lipases A2. Toxicon 42, 947–962.
Veronese, E.L., Esmeraldino, L.E., Trombone, A.P., Santana, A.E.,
Andisson, F.A., Campos, M.I., Bechara, G.H., Ketelhut, I.,
Cintra, A.C., Giglio, J.R., Sampaio, S.V., 2005. Inhibition of the
myotoxic activity of Bothrops jararacussu venom and its two
major myotoxins, BthTX-I and BthTX-II, by the aqueous
extract of Tabernaemontana catharinensis A.DC. (Apocyna-
ceae). Phytomedicine 12, 123–130.
Watanabe, L., Soares, A.M., Ward, R.J., Fontes, M.R.M.,
Arni, R.K., 2005. Structural insights for fatty acid binding in a
Lys49-phospholipase A2: crystal structure of myotoxin II from
Bothrops moojeni complexed with stearic acid. Biochimie 87,
161–167.