Neutralization of proteases from Bothrops snake venom by the aqueous extract from Casearia sylvestris (Flacourtiaceae)

Abstract

Aqueous extract from Casearia sylvestris leaves, a typical plant from Brazilian open pastures, was able to neutralize the hemorrhagic activity caused by Bothrops asper, Bothrops jararacussu, Bothrops moojeni, Bothrops neuwiedi and Bothrops pirajai venoms. It also neutralized two hemorrhagic metalloproteinases from Bothrops asper venom. Proteolytic activity on casein induced by bothropic venoms and by isolated proteases, including Bn2 metalloproteinase from B. neuwiedi venom, was also inhibited by the C. sylvestris extract in different levels. The alpha-fibrinogen chain was partially protected against degradation caused by B. jararacussu venom, when this venom was incubated with C. sylvestris extract. We also observed that this extract partially increased the time of plasma coagulation caused by B. jararacussu, B. moojeni and B. neuwiedi venoms. C. sylvestris extract did not induce proteolysis in any substrate assayed.

Full text

Neutralization of proteases from Bothrops snake venoms by the
aqueous extract from Casearia sylvestris (Flacourtiaceae)
M.H. Borges
a,
*, A.M. Soares
b,c
, V.M. Rodrigues
b
, F. Oliveira
a
, A.M. Fransheschi
c
,
A. Rucavado
c
, J.R. Giglio
b
, M.I. Homsi-Brandeburgo
a
a
Departamento de Gene
Âtica e Bioquõ
Âmica, Universidade Federal de Uberla
Ãndia, UFU, 38400-902 Uberla
Ãndia, MG, Brazil
b
Departamento de Bioquõ
Âmica, Faculdade de Medicina, USP, 14049-900 Ribeira
Äo Preto, SP, Brazil
c
Instituto Clodomiro Picado, Facultad de Microbiologõ
Âa, UCR, San Jose
Â, Costa Rica
Received 23 January 2001; accepted 18 May 2001
Abstract
Aqueous extract from Casearia sylvestris leaves, a typical plant from Brazilian open pastures, was able to neutralize the
hemorrhagic activity caused by Bothrops asper,Bothrops jararacussu,Bothrops moojeni,Bothrops neuwiedi and Bothrops
pirajai venoms. It also neutralized two hemorrhagic metalloproteinases from Bothrops asper venom. Proteolytic activity on
casein induced by bothropic venoms and by isolated proteases, including Bn2 metalloproteinase from B. neuwiedi venom, was
also inhibited by the C. sylvestris extract in different levels. The
a
-®brinogen chain was partially protected against degradation
caused by B. jararacussu venom, when this venom was incubated with C. sylvestris extract. We also observed that this extract
partially increased the time of plasma coagulation caused by B. jararacussu,B. moojeni and B. neuwiedi venoms. C. sylvestris
extract did not induce proteolysis in any substrate assayed. q2001 Elsevier Science Ltd. All rights reserved.
Keywords: Snake venoms; Proteases; Antiophidian activity; Casearia sylvestris extracts
Animal venoms, including snake venoms, are complex
mixtures of proteins. Among these are hemorrhagins,
proteases, phospholipases A
2
(PLA
2
) and myotoxins that
act by different mechanisms. Venom composition may
vary according to nutritional, geographic and seasonal
factors (Assakura et al., 1992; Rodrigues et al., 1998).
Envenomation caused by snake venoms of the genus
Bothrops induces many local effects such as myonecrose,
edema and hemorrhage (Rosenfeld, 1971; Gutie
Ârrez et al.,
1995). These venoms contain many proteolytic enzymes
that degrade a variety of natural substrates such as casein,
hemoglobin, collagen, elastin, ®brinogen, ®bronectin and
others (Iwanaga and Suzuki, 1979). Hemorrhagic toxins
are among these enzymes and are responsible for degrada-
tion of proteins from the extracellular matrix or alterations
in blood coagulation (Matrisian, 1992; Markland, 1998).
Hemorrhage induced by snakebite occurs due to hemorra-
gins, zinc-dependent metalloproteinases, capable of disrupt
the basement membrane of capillaries and causing altera-
tions on capillary vessels (Bjarnason and Fox, 1994).
Medicinal plants play a key role in world health, as they
are source of many pharmacologically active compounds
such as ¯avonoids and tannins. Many of these substances
structurally resemble biological compounds and this simi-
larity is the basis of their physiological action (Havsteen,
1983). The use of plant extracts as antidote for animal
venoms is an old option found in many communities that
do not have a prompt access to serum therapy. In addition,
depending on the time between the accident and treatment,
the ability of the antiserum to neutralize local effects of
envenomation is only partial. Vegetal extracts become
then an attractive research material as an alternative substi-
tute for antiserum (Rizzini et al., 1988; Ruppelt et al., 1990;
Pereira et al., 1992; Martz, 1992).
Toxicon 39 (2001) 1863± 1869
0041-0101/01/$ - see front matter q2001 Elsevier Science Ltd. All rights reserved.
PII: S0041-0101(01)00169-6
www.elsevier.com/locate/toxicon
* Corresponding author: Laborato
Ârio de Venenos e Toxinas
Animais, Departmento Bioquõ
Âmica e Imunologia, Instituto de Cie
Ãn-
cias Biolo
Âgicas-UFMG, Av Antonio Carlos, 6627 Cidade Universi-
ta
Âria, Belo Horizonte, Brazil. Fax: 155-31-3441-5963.
E-mail address: mhborges@mono.icb.ufmg.br (M.H. Borges).
Abbreviations: Cs, Casearia sylvestris aqueous extract; BaP1 and
BH4, metalloproteinases from Bothrops asper venom; Bn2, metal-
loproteinase from Bothrops neuwiedi venom; PBS, Phosphate-
buffered saline solution

In Brazil, the popular name of Casearia sylvestris
(Flacourtiaceae) is GuacËatonga and it has a widespread
use in folk medicine as antiseptic, healer, topical anaes-
thetic, antitumor agent, antiulcer and antiophidian (Itokawa
et al., 1988; Basile et al., 1990). The neutralization activity
of C. sylvestris against crude venoms and puri®ed toxins
from several genera (Bothrops,Crotalus,Micrurus and
Heloderma) of snakes and Apis mellifera bee venom was
investigated (Borges et al., 2000). This neutralization may
be due to the presence of enzymatic inhibitors, chemical
inactivators or immunomodulators (Hart et al., 1989). The
mechanism of action is still unknown.
This study shows the ability of the aqueous extract from
C. sylvestris to neutralize the hemorrhagic, coagulant and
proteolytic activity on casein or ®brinogen, induced by ®ve
bothropic venoms (Bothrops asper,Bothrops jararacussu,
Bothrops moojeni,Bothrops neuwiedi and Bothrops pirajai)
and by three metalloproteinases (BaP1 and BH4 from B.
asper and Bn2 from B. neuwiedi).
Snake venoms were collected in the serpentarium of
Ribeira
Äo Preto School of Medicine, SP, Brazil, and in
Clodomiro Picado Institute, UCR, Costa Rica. The proteases
BaP1 and BH4 from B. asper and Bn2 from B. neuwiedi
were previously isolated by Gutie
Ârrez et al., 1995;
Franceschi et al., 2000; Rodrigues et al., 2000, respectively.
The leaves of C. sylvestris, collected in Uberla
Ãndia-MG,
Brazil, were washed, stirred with deionized water in a
waring blender for 15 min at room temperature and then
sieved. The ®ltrate was centrifuged at 30.000 £gfor
20 min and the supernatant was lyophilized and stored at
2208C. Leaf extract was weighed and dissolved in deion-
ized water before use (Borges et al., 2000). The extract
concentration was expressed in terms of dry weight. Adults
Swiss mice were obtained from the vivarium of Ribeira
Äo
Preto School of Medicine, SP, Brazil.
Hemorrhagic activity was assayed according to the
method of Nikai et al. (1984). Mice (20±25 g) received
three minimum hemorrhagic doses (MHD) of crude venoms
or isolated metalloproteinases (5 and 15 mg) in 50 mlof
PBS, intradermally. Three hours later, the animals were
sacri®ced and the inner surface of the skin was examined.
The MHD (de®ned as the amount of venom that results in a
hemorrhagic spot of about 1 cm in diameter) for the B.
jararacussu venom is 50 mg, B. neuwiedi is 8 mg, B.
moojeni and B. asper is 5 mg.
Fibrinogenolytic (Edgar and Prentice, 1973) or caseino-
lytic activities (Franceschi et al., 2000) were evaluated
trough incubation of the whole venom (B. asper,B. moojeni,
B. pirajai,B. neuwiedi and B. jararacussu) or puri®ed
proteases (BaP1, BH4 and Bn2) with ®brinogen or casein
at 378C for 5 min. For caseinolytic activity, 40 mg each
venom or 20 mg of each protease were utilized, while
1mgofB. jararacussu venom was used to verify ®brinogen
proteolysis.
Coagulant activity on bovine plasma of B. jararacussu
(50 mg), B. moojeni (50 mg) and B. neuwiedi (5 mg) venoms
was assayed according to Assakura et al. (1992); 50 mlof
venom solutions were added to 200 ml of bovine plasma at
378C. The time to clot the plasma solution, in s, was
recorded.
Venoms and toxins were dissolved in phosphate-buffered
saline, pH 7.2 (PBS). Proteins were estimated by the
methods of Itzhaki and Gill (1964) and Bradford (1976).
For neutralization assays of the hemorrhagic, proteolytic
and coagulant activities, toxins and venoms were pre-
viously incubated with the extract at different ratios (1:1;
1:3; 1:5 and 1:10 w/w, venom:extract) for 30 min at room
temperature.
The hemorrhagic activity caused by intradermal injection
of 3MDH of B. asper,B. jararacussu,B. neuwiedi and B.
pirajai venoms and by the proteases BaP1 and BH4 isolated
from B. asper were signi®cantly neutralized by the C.
sylvestris extract at a ratio of 1:3 (w/w venom:extract).
Fig. 1(A) shows the inner surface of the skin and Fig.
1(B) represents the diameter of the hemorrhagic spot.
Antihemorrhagic compounds have puri®ed from different
sources. The compounds Ar turmerone has been isolated
from Curcuma longa roots, Zingiberaceae (Ferreira et al.,
1992) and Wedelolactone was isolated from Eclipta pros-
tata leaves, Asteraceae (Melo et al., 1994). Furthermore,
many antihemorrhagic proteins have already been isolated
from the serum of resistant animals (Soares et al., 1997;
Pe
Ârez and Sa
Ânchez, 1999).
Our results clearly indicate that C. sylvestris extract
contains compounds capable of neutralizing the hemorrha-
gic activity induced by crude venoms or by isolated toxins.
PLA
2
activity also was inhibited by this extract. PLA
2
enzymes are related with a wide variety of pharmacological
activities and among these is miotoxicity, which was
neutralized by C. sylvestris too (Borges et al., 2000).
Hemorrhagins and phospholipases are enzymes that need
a divalent metal ion for their activity. So, we can suggest
that the extract has compounds that bind these ions, causing
inhibition. Similar results were obtained when Wedelolac-
tone, isolated from E. prostrata plant, neutralized PLA
2
and
proteolytic enzymes in venoms responsible for myotoxic
and hemorrhagic activities (Melo and Ownby, 1999).
Fig. 2 shows the ®brigenolytic activity exerted by B.
jararacussu venom. Proteolytic enzymes present in this
venom degraded preferentially the A
a
chain of bovine ®bri-
nogen in few minutes. Partial inhibition of the A
a
chain
degradation was observed when venom and extract were
incubated together before being mixed to ®brinogen at a
ratio of 1:10 (w/w, venom:extract).
Caseinolytic activity induced by crude venoms [Fig.
3(A)] or isolated proteases [Fig. 3(B)] showed a signi®cant
inhibition when these venoms or toxins were incubated with
C. sylvestris leaf extract at different ratios. Neither ®brino-
genolytic activity (results not shown) nor caseinolytic
activity were induced by C. sylvestris extract alone (Fig. 3).
Inhibition of caseinolytic and ®brinogenolytic activities
by other compounds has already been shown. ABC
M.H. Borges et al. / Toxicon 39 (2001) 1863 ±18691864

M.H. Borges et al. / Toxicon 39 (2001) 1863± 1869 1865
Fig. 1. Effect of C. sylvestris extract on the hemorrhagic activity of crude venoms or isolated metalloproteinases. 3MDH crude venom or 5 and
15 mg isolated proteases were incubated for 30 min at room temperature, with either PBS or C. sylvestris. Fifty ml this mixture was injected
intradermally in mice. The skins were removed after 3 h and the diameters of the hemorrhagic spot were measured. Ratio venom:extract 1:3(w/
w, venom:extract). (A) Skins show the spot in the inner side: 1, B. neuwiedi (24 mg); 2, B. neuwiedi 1C. sylvestris;3,B. jararacussu (150 mg);
4, B. jararacussu 1C. sylvestris; 5, PBS; 6, C. sylvestris. (B) Diameter of the skin lesion. Each bar represents the mean ^SD (n6). The
difference between Absence C. sylvestris and Presence C. sylvestris is signi®cant (P,0.05 One-way ANOVA).

complex, an antihemorrhagic factor puri®ed from Didelphis
marsupialis serum, inhibited the caseinolytic and ®brino-
genolytic activities of B. jararaca venom. Electrophoretic
evidences have shown a non-covalent complex formation
between the ABC complex and the component(s) of B.
jararaca venom (Neves-Ferreira et al., 1997).
B. jararacussu,B. neuwiedi and B. moojeni venoms
induced bovine plasma coagulation in 10 ±50 s. When
these venoms were preincubated with C. sylvestris at a
ratio of 1:10(w/w, venom:extract), an increase in coagula-
tion time was observed, but total inhibition was not veri®ed
(Table 1).
Hemostatically active components are largely distrib-
uted in snake venoms. These compounds interact with
proteins of the coagulation cascade and ®brinolytic path-
way. Markland (1998) puri®ed many ®brin(ogen)olytic
enzymes from different snake venoms, the majority of
which were metalloproteinases.
This work shows that C. sylvestris extract is more effec-
tive in neutralizing hemorrhagic metalloproteinases from
bothropic venoms than the serine-proteinases that cause
alterations in the coagulation system. This extract also
neutralizes Bn2 protease from B. neuwiedi venom. Bn2 is
considered a weak hemorrhagic protease and has moderate
proteolytic activity (Rodrigues et al., 2000). The antihemor-
rhagic factor from Didelphis marsupialis serum inhibits
snake venom metalloproteinases by non-covalent complex
formation, but no action on venom serine-proteinases was
recorded (Neves-Ferreira et al., 1997). Both C. sylvestris
extract and the antihemorrhagic factor appear to have speci-
®city for metalloproteinases.
To better understand these inhibition mechanisms, it is
necessary to study isolated compounds. Puri®cation of these
compounds from C. sylvestris extract is in progress, but the
isolation of an active substance can be a problem. Puri®ca-
tion may exclude components that act in combination,
resulting in synergism.
Regarding a possible action mechanism, our previous
studies have shown that no alteration occurs in the electro-
foretic pattern of B. moojeni venom and isolated myotoxin,
after incubation with C. sylvestris extract, excluding proteo-
lytic degradation as a potential mechanism (Borges et al.,
2000). Because of the preference for metalloproteinases, we
could suggest that C. sylvestris extracts may be a natural
chelanting agent, interacting with metals. On the other hand,
components of the extract may occupy sites in the venoms/
toxins, preventing binding of the substrate to the enzyme,
and this interaction may be covalent or non-covalent.
Tannins (Santos et al., 2000) and ¯avonoids (Havsteen,
1983) are able to bind metal ions. Lindahl and Tagesson
(1997) reported that ¯avonoids appear to inhibit PLA
2
-
class II but not PLA
2
-class I, but how this occurs is not
M.H. Borges et al. / Toxicon 39 (2001) 1863 ±18691866
Table 1
Coagulant activity on bovine plasma (n3) of crude venoms incubated or not with C. sylvestris extract
Sample Controls Time (s)
Venom: C. sylvestris (1:3 w/w) Venom: C. sylvestris (1:10 w/w)
a
B. jararacussu (53 mg) 50.81 ^2.30 85.3 ^5.03 134.22 ^4.08
B. moojeni (50 mg) 10.89 ^5.40 58.75 ^3.20 127.65 ^3.90
B. neuwiedi (5 mg) 35.60 ^5.10 27.66 ^1.50 100.21 ^5.60
PBS
b
245.00 ^0.04 ± ±
C. sylvestris
b
(540 mg) 245.05 ^0.10 ± ±
a
Inhibition of coagulant activity by C. sylvestris is signi®cant (P,0.05 One Way ANOVA).
b
Incubation was performed for 245 s. No coagulation was observed.
Fig. 2. Inhibition of proteolysis of bovine ®brinogen induced by B.
jararacussu.B. jararacussu venom (1 mg) was pre-incubated with
C. sylvestris extract for 30 min at room temperature and was mixed
with 50 ml of ®brinogen solution (1 mg/ml) at 378C for 5 min. A
SDS-polyacrilamide gel electrophoresis (16%) shows ®brinogen
degradation. Lane 1: molecular mass markers: phosporilase b
(97,000); bovine serum albumin (67,000); ovoalbumin (43,000);
carbonic anhydrase (30,000); soybean trypsin inhibitor (20,000)
and
a
-lactoalbumin (14,400). Lane 2: ®brinogen control (without
venom or extract). Lane 3: ®brinogen incubated with B. jarara-
cussu. Lane 4: ®brinogen incubated with B. jararacussu in presence
of C. sylvestris extract at a ratio 1:10 (w/w, venom:extract).

clear. Furthermore, ¯avonoids have a high chemical reac-
tivity, can bind to biological polymers, and are able to cata-
lyze electron transport, and numerous studies show
inhibition of a variety of enzymes by ¯avonoids (Havsteen,
1983).
Snake venom inhibitors have been puri®ed from many
plants. Ar turmerone was capable of abolishing the hemor-
rhagic activity caused by Bothrops venom and inhibited
about 70% of the lethal effect of Crotalus venom (Ferreira
et al., 1992). Wedelolactone neutralized hemorrhagic and
myotoxic activity induced by Crotalus and Bothrops
venoms and isolated toxins (Mors et al., 1989; Melo et al.,
1994), and also inhibited myotoxic PLA
2
from Crotalus d.
durissus,Crotalus v. viridis and Agkistrodon contortrix
laticinetus venoms (Melo and Ownby, 1999).
The mechanisms of action of these compounds are still
unknown. Interactions between venom and extract and the
involvement in immunological mechanisms cannot be
excluded. C. longa and Ar turmerone extracts inhibited
lymphocyte proliferation. 2-hidroxy-4-methoxy benzoic
acid isolated from a root extract of the Indian plant Hemi-
desmus indicus, directly neutralized viper venom-induced
lethal, hemorrhagic and coagulant activities. This
compound also indirectly neutralized both lethal and hemor-
rhagic activities of these venoms by increasing the antibody
production in hyper immunized rabbits. The mechanism of
action is not clear, but this study indicates that 2-hidroxy-4-
methoxy benzoic acid may act as an adjuvant thus triggering
the high title of antibodies, which effectively neutralizes the
venoms (Alam and Gomes, 1998).
In conclusion, our results show that the C. sylvestris
aqueous extract contains compounds that neutralize
proteases present in snake venoms. We tested many others
plant extracts in the same conditions and we observed that
these extracts were not capable of inhibiting the same
proteases. It is not clear yet if this inhibition occurs due to
speci®c interactions between speci®c groups from vegetal
extract and metalloproteinases hemorrhagic domains, but
this study show that metalloproteinases are neutralized by
smaller amounts of extract. Fibrinogenolytic and coagulant
activities were only partially inhibited even when higher
ratios of the extract were used.
Identi®cation of metalloproteinase inhibitors has medical
importance, because proteases structurally similar to snake
venom metalloproteinases are found in mammalian cells.
Proteases and their inhibitors are in delicate equilibrium
M.H. Borges et al. / Toxicon 39 (2001) 1863± 1869 1867
Fig. 3. Neutralization of proteolytic activity on casein of the crude venoms (A) or isolated proteases (B) by C. sylvestris extract. The venoms
(40 mg) or toxins (20 mg) were previously incubated with C. sylvestris at different ratios for 30 min at room temperature and were mixed with
substrate at 378C for 5 min. Bars represent the mean ^SD (n3). The difference between incubated or not incubated is statistically signi®cant
(P,0.05 One Way ANOVA).

with each other to maintain a steady state in the cell. When
this balance is broken disturbs such as neoplasia and others
diseases can occur, so effective drugs capable of inhibiting
metalloproteinases could be developed using these inhibi-
tors. Furthermore, snake venom inhibitors can be useful
tools for the elucidation of the mechanisms of action of
puri®ed toxins.
Acknowledgements
The authors gratefully acknowledge the ®nancial support
by FAPESP, FAPEMIG and CNPq. We thank Drs M.M.
Santoro and Dr A. Magalhaes by excellent comments and
suggestions. Thanks are also Alessandra Campos and Fla
Âvia
Almeida by assistance in preparing this manuscript.
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