Pharmaceutical Biology

ISSN: 1388-0209 (Print) 1744-5116 (Online) Journal homepage: https://www.tandfonline.com/loi/iphb20

Anti-snake venom activities of extracts and

fractions from callus cultures of Sapindus
saponaria

Marcos L. da Silva, Silvana Marcussi, Renata S. Fernandes, Paulo S. Pereira,

Ana Helena Januário, Suzelei C. França, Saulo L. Da Silva, Andreimar M.
Soares & Miriam V. Lourenço

To cite this article: Marcos L. da Silva, Silvana Marcussi, Renata S. Fernandes, Paulo S.
Pereira, Ana Helena Januário, Suzelei C. França, Saulo L. Da Silva, Andreimar M. Soares
& Miriam V. Lourenço (2012) Anti-snake venom activities of extracts and fractions
from callus cultures of Sapindus saponaria, Pharmaceutical Biology, 50:3, 366-375, DOI:
10.3109/13880209.2011.608072

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Pharmaceutical Biology, 2012; 50(3): 366–375
© 2012 Informa Healthcare USA, Inc.
ISSN 1388-0209 print/ISSN 1744-5116 online
DOI: 10.3109/13880209.2011.608072

Research Article

Anti-snake venom activities of extracts and fractions from

callus cultures of Sapindus saponaria
Marcos L. da Silva1, Silvana Marcussi2, Renata S. Fernandes3, Paulo S. Pereira1, Ana Helena
Januário4, Suzelei C. França1, Saulo L. Da Silva5, Andreimar M. Soares3,6, and Miriam V. Lourenço1
1
Unidade de Biotecnologia, Universidade de Ribeirão Preto, UNAERP, Ribeirão Preto-SP, Brazil, 2Departamento de
Química, Universidade Federal de Lavras, UFLA, Lavras-MG, Brazil, 3Departamento de Análises Clínicas, Toxicológicas
e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, FCFRP, Universidade de São Paulo, USP,
Ribeirão Preto-SP, Brazil, 4Grupo de Pesquisa em Produtos Naturais, Universidade de Franca, UNIFRAN, Franca-SP,
Brazil, 5Universidade Federal de São João Del Rei, UFSJ, Campus Centro Oeste, Divinópolis-MG, Brazil, and
6
Fundação Oswaldo Cruz, FIOCRUZ Rondônia e Centro de Estudos de Biomoléculas Aplicadas à Medicina, CEBio,
Universidade Federal de Rondônia, UNIR, Porto Velho-RO, Brazil

Abstract
Context: Sapindus saponaria L. (Sapindaceae) bark, root, and fruits are used as sedatives and to treat gastric ulcer and
also demonstrate diuretic and expectorant effects.
Objective: The anti-snake venom properties of callus of S. saponaria are investigated here for the first time.
Materials and methods: In vitro cultivated callus of Sapindus saponaria were lyophilized, and the extracts were
prepared with different solvents, before submitting to phytochemical studies and evaluation of the anti-ophidian
activity. Crude extracts were fractionated by liquid–liquid partition and the fractions were monitored by thin layer
chromatography (TLC). Subsequently, anti-ophidian activities were analyzed toward Bothrops jararacussu Lacerda
(Viperidae), B. moojeni Hoge (Viperidae), B. alternates Duméril (Viperidea) and Crotalus durissus terrificus Lineu
(Viperidae) venoms and isolated myotoxins and phospholipase A2 (PLA2).
Results: Fractions A1, A2 and the extract in MeOH:H2O (9:1) significantly inhibited the toxic and pharmacological
activities induced by snake venoms and toxins, when compared to other extracts and fractions. The lethal, clotting,
phospholipase, edema-inducing, hemorrhagic and myotoxic activities were partially inhibited by the different extracts
and fractions. TLC profiles of the crude extracts (B and C) and fractions (A1 and A2) showed β-sitosterol and stigmasterol
as their main compounds. Stigmasterol exhibited inhibitory effects on enzymatic and myotoxic activities of PLA2.
Discussion and conclusion: Sapindus saponaria extracts and fractions presented anti-ophidian activity and could
be used as an adjuvant to serum therapy or for its supplementation, and in addition, as a rich source of potential
inhibitors of enzymes involved in several pathophysiological human and animal diseases.
Keywords:  Medicinal plants, snake venoms, anti-snake venom, anti-toxins, natural inhibitors

Introduction
Animal venoms, including snake venoms, embody a
complex mixture of toxic enzymes. In Brazil, Bothrops
and Crotalus snakes are responsible for most enveno-
mations, which mainly induce local tissue damage and
systemic effects, including alterations in blood clotting,
neurotoxicity, and shock (Ownby, 1990; Braud et  al.,
2000). The victim’s metabolic response to the venom’s
components varies with the species of snake, the volume
of venom injected, and the species of the bite recipi-
ent. The primary purpose of the venom is not to kill, but
rather to immobilize the prey and predigest its tissues.
The venom is derived from modified salivary glands. Pit

Address for Correspondence:  Dr. A.M. Soares, Fundação Oswaldo Cruz, FIOCRUZ Rondônia e Centro de Estudos de Biomoléculas Aplicadas à
Medicina, CEBio, Universidade Federal de Rondônia, UNIR, Porto Velho-RO, Brazil. Fax: 55-69-3219-6000. E-mail: andreims@pq.cnpq.br;
Dr. S.L. Da Silva, Universidade Federal de São João Del Rei, UFSJ, Campus Centro Oeste, Divinópolis-MG, Brazil. Fax: 55-37-3221-1164.
E-mail: biomol2@hotmail.com
(Received 26 November 2010; revised 09 June 2011; accepted 02 July 2011)

366

viper venoms are a complex combination of enzymatic
and non-enzymatic protein; phosphodiesterase, pro-
teolytic enzymes, thrombin–like enzyme, collagenase,
hyaluronidase, phospholipase A2 (PLA2), and l-amino
acid oxidase (Da Silva, et  al., 2008a; Calgarotto et  al.,
2008). The toxins of the Bothrops snake venom affects
mainly the muscle tissues and blood clotting, and symp-
toms include; pain, swelling and bleeding (site of the bite,
and mucous membranes), and in cases where complica-
tions can occur: blisters, gangrene, shock and acute renal
failure. In Crotalus envenomation, the poison affects the
nervous system (neurotoxic), coagulation and muscles,
and in severe cases acute renal failure and death can
occur (Peterson, 2006).
Vegetal extracts constitute an alternative for treatment,
displaying a large diversity of chemical compounds with
several pharmacological activities of medical-scientific
interest. A large number of extracts have been prepared
and showed excellent anti-venom activities (Melo et al.,
1994; Izidoro et  al., 2003; Soares et  al., 2004; Da Silva
et  al., 2005, 2008a, 2009; Oliveira et  al., 2005; Ticli
et al., 2005; Marcussi et al., 2007; Lomonte et al., 2009).
Sapindus saponaria L. (Sapindaceae) bark, root and
fruits are used as sedatives, against gastric ulcer and
demonstrate diuretic and expectorant effects (Meyer
et  al., 2002). Other studies show the presence of lipids
and flavonoids in this plant, characterizing it as a chemi-
cal complex with several scientifically important proper-
ties (Castro et al., 1999). Saponins, present in the fruits,
are related to several activities such as molluscicide,
piscicide, anti-inflammatory, analgesic, expectorant,
antioxidant, spermicide, cholesterol-lowering, antibi-
otic, and antifungal (Murgu, 2002; Tsuzuki et  al., 2007).
Castro et  al. (1999) show that extracts of S. saponaria
inhibit the hemorrhagic activity induced by B. asper
Garman (Viperidae) venom and some compounds such
as catechins, flavones, anthocyanins and condensed
tannins have been identified by chemical analysis,
suggesting their involvement in the inhibitory effect
observed, probably acting by the chelation of the zinc ion,
which is required for the activity of metalloproteases.
Hundreds of plants are reported to have anti-oph-
idian activities, although only a few of these have been
scientifically studied, with the isolation of components
and investigations regarding structure-function rela-
tionships (Soares et al., 2004, 2005). The wedelolactone
isolated from Eclipta prostate Hasskarl (Asteraceae)
and the alkaloid, 12-methoxy-4-methylvoachalotine,
isolated from Tabernaemontana catharinensis Müll.Arg.
(Apocynaceae) (Melo et al., 1994; Melo & Ownby, 1999;
Batina et al., 1999), are examples of isolated and charac-
terized anti-ophidian substances. Januário et  al. (2004)
isolated a neoclerodane diterpenoid from Baccharis
trimera Less (Asteraceae), an inhibitor of snake venom
metalloproteases, with anti-proteolytic and anti-hemor-
rhagic properties.
This study shows the activity of extracts from cal-
lus of S. saponaria, and its major active compound
© 2012 Informa Healthcare USA, Inc.
Sapindus saponaria anti-ophidian plant  367
(stigmasterol), in the inhibition of pharmacological and
toxic effects of snake venoms and isolated proteins from
the Crotalus and Bothrops genera.
Materials and methods
Animals, snake venoms and toxins
Male Swiss mice (18–22 g) were used for biological assays.
Crude lyophilized B. jararacussu (Bjussu), B. moojeni
(Bmooj), B. alternatus (Balter) and C. d. terrificus (Cdt)
venoms were obtained from Bioagents serpentarium,
Batatais-SP and the Instituto Butantan, São Paulo-SP,
Brazil. The isolated toxins, crotoxin and basic PLA2 from
Crotalus durissus terrificus (PLA2Cdt or CB) and myotox-
ins from B. jararacussu (BthTX-I and II) were obtained
as previously described by (Andrião-Escarso et al., 2000;
Soares et al., 2001). Stigmasterol (B9757) was purchased
from Sigma-Aldrich. The experiments described were
approved by the Institutional Committee for Ethics in
Animal Experimentation of the Universidade de São
Paulo (no. 08.1.202.53.1) and were done in accordance
with the guidelines of the Brazilian College for Animal
Experimentation.
Plant in natura and in vitro
Sapindus saponaria plants were collected in Ribeirão
Preto and cultivated in the “Coleção de Plantas Medicinais
da Universidade de Ribeirão Preto (UNAERP), Brazil”.
Voucher specimen no. HPM 603 is deposited in the
“Herbário de Plantas Medicinais (HPM), Unidade de
Biotecnologia, UNAERP”. The species was authenticated
by Professor Hermógenes de Freitas Leitão Filho from
the Instituto de Biologia, UNICAMP, Brazil.
The explants were inoculated in Murashige and
Skoog (MS) culture medium (Murashige & Skoog,
1962), supplemented with different combinations of
1-naphthaleneacetic acid (ANA: 0.5, 1.0 and 2.0 mg·L−1)
and 6-benzylaminopurine (BAP: 0.5, 1.0 and 2.0 mg·L−1),
pH 6.0, and solidified with Phytagel® (2.5 g·L−1) for callus
induction. The cultures grew at 25 ± 2°C with a 16 h pho-
toperiod under a white fluorescent lamp, and were then
lyophilized.
Phytochemical analysis
For phytochemical studies, several extracts from S.
saponaria callus culture were prepared using differ-
ent solvents. Extract B was prepared by maceration in
MeOH:H2O (9:1, v/v; 1 L) for 24 h. Extract C was prepared
by maceration in CH2Cl2:MeOH (85:15, v/v; 0.8 L) for
48 h. After the extractions, the solvents were removed
under vacuum using an evaporator and extracts were
lyophilized.
Lyophilized callus were submitted to extractions with
CH2Cl2:MeOH (85:15, v/v; 100 mL) in ultrasound for
30 min. The extract was transferred to a separation fun-
nel, adding 60 mL of water. The organic phase was evapo-
rated (A1) and the aqueous phase had its pH changed to
8.0 (using NH4OH), before being extracted with 30 mL of
368  M.L. da Silva et al.
CH2Cl2 (3 times). The resulting basic organic phases were
collected and treated with sodium sulfate (B1), and the
neutral aqueous phase (N1) was lyophilized. Extract C
was also resuspended in CH2Cl2:MeOH 85:15 (v/v) and
subjected to a liquid–liquid partition, giving rise to three
fractions denominated as A2 (acid), B2 (basic) and N2
(neutral).
A thin layer chromatography (TLC) procedure was
performed with all extracts and fractions at 20 mg/mL.
The analysis was carried out using 60G silica gel plates
(10 × 10 cm; 0.25 mm; Aldrich®), eluted with Hex:EtOAc
(7:3, v/v), CHCl3:MeOH (8:2, v/v) and 1-butanol: acetic
acid: water (BAW, 4:1:5, v/v, upper phase) and stained
with vanillin-sulfuric acid and ninhydrin reagents.
Neutralization assays of the snake venoms or
isolated toxins
Hemorrhagic, myotoxic and phospholipase A2 activities
were assayed as previously described (Biondo et al., 2003;
Da Silva et al., 2008b, Romero et al., 2010; de Alvarenga
et al., 2011).
PLA2 belong to a superfamily of enzymes that cleave
phospholipids from cell membrane into fatty acids and
lysophospholipids. This reaction is calcium dependent.
In this paper we use an experiment that uses phosphati-
dylcholine from egg yolk in agar plate. It is based on
enzymatic breach of these phospholipids with release
of soluble fatty acids that form a translucent halo in the
agar plate around the point of application of a sample
of the enzyme. So, for the venom inhibition assays,
phosphate-buffered saline (PBS) solutions containing a
fixed amount of venom or isolated enzyme (10–50 μg)
were mixed with different amounts of extracts in order to
achieve several ratios (w/w) of venom or toxin to extract
(or stigmasterol). All mixtures were incubated for 30 min
at 37°C and aliquots were assayed in the different test
systems. The clotting activity was assayed with 200 μL of
citrated human plasma directly incubated with Cdt or
Bjussu snake venoms alone (50 μg) or previously incu-
bated with extracts for 30 min, 37°C, at a ratio of 1:5 (w/w)
venom:extract.
Both the venom, such as PLA2, causes lysis in muscle
cells. The myotoxic activity assay is based on detection of
the enzyme creatine kinase (CK) released into the blood
of the animal when muscle cells are lysed. The enzyme
CK has been widely used as an indirect marker of muscle
damage. In this work, plasma CK activity was determined
using the CK-UV Kit (Bioclin, Brazil).
The presence of hemorrhage, local and systemic, is
common in snake bites. This effect is due principally
by the presence in the venom of zinc-dependent met-
alloproteinases. These enzymes act by damaging the
endothelium of capillaries and plasma extravasation.
After application of the toxins, animals were euthanized
and skinned. The area of hemorrhage observed in the
inner skin was measured with precision paquimeter.
Lethality was evaluated for 48 h after the intraperi-
toneal injection of solutions containing C. d. terrificus
 Pharmaceutical Biology
(20 μg) or crotoxin (10 μg) with extracts (1:10 and 1:50,
w/w) in Swiss mice (18–22 g). Results are presented as
mean values ± S.D. obtained with the indicated number
of tested animals. The statistical significance of differ-
ences between groups was evaluated using Student’s
unpaired t-test. A p-value < 0.05 was considered to
indicate significance. Edema was evaluated after the
subplantar injection of venoms or toxins isolated or
preincubated with extracts or fractions obtained from
S. saponaria in the right paw of male Swiss mice (18–22 g)
using a pachymeter. Inhibition studies were performed
after the preincubation of venom/toxins with extracts
(1:50 and 1:10, w/w, respectively).
Results and discussion
Callus cultures from leaves of S. saponaria were success-
fully established, resulting in material with uniform char-
acteristics. The best induction of callus was obtained in
the culture medium containing 0.5 mg·L−1 ANA plus 0.5
mg·L−1 BAP, resulting in more intensive growth and fri-
ability. The biomass accumulation occurred during the
period from day 27 to 30, and the 30th day was selected
for the collection of material.
Figure 1 (A, C and D) shows the significant inhibi-
tion of the PLA2 activity of C. d. terrificus venom,
PLA2Cdt and crotoxin by S. saponaria extracts and
obtained fractions. The mechanisms of action of the
anti-ophidian substances that have been previously
studied are still unknown, but interactions with cata-
lytic sites of specific venom proteins are suggested
to be involved. In the experiments of inhibition of
PLA2 activity, a greater interaction among the extracts
and their fractions with C. d. terrificus venom was
observed, with a major inhibition of this activity when
compared to the inhibition obtained for B. jararacussu
venom (Figure 1B). The differences observed between
the inhibition of C. d. terrificus and B. jararacussu
venoms can be explained in terms of compositions,
biochemical and pharmacological properties of each
of the venoms. Variations between the venoms of differ-
ent genera should be considered. Among other factors
that may affect the composition of venoms are geo-
graphical location, the animals’ health status, age, type
of prey, etc. (Fry et al., 2001).
The tested extracts and fractions, in the proportions
used, showed no significant inhibition of the edema
induced by C. d. terrificus venom (results not shown), but
some fractions (A1, A2, B2, extract B and extract C) sig-
nificantly inhibited the activity induced by B. jararacussu
and its isolated myotoxins (BthTX-I and II) (Figure 2).
Inflammatory mediators such as histamine, bradyki-
nin, prostaglandins, leukotrienes, thromboxanes and
complement factors may be involved in the formation of
edema (Zamuner & Teixeira, 2002).
In Bothrops snake envenomations, the presence of
both local and systemic hemorrhage is common, mainly
due to the presence of zinc-dependent metalloproteases,
 Sapindus saponaria anti-ophidian plant  369

Figure 1.  Inhibition of the PLA2 activity of snake venoms (A and B) and isolated toxins (C and D) induced by the extracts and fractions of
S. saponaria, preincubated at different ratios (w/w, venoms/proteins: Extracts/fractions) for 30 min at 37°C. Each experiment represents
the mean ± S.D. (n = 6).

which act damaging the endothelium of blood vessels by tissue ischemia. The inhibition of the hemorrhagic
(Gutiérrez & Rucavado, 2000), causing serious injuries effect of Bothrops venoms by extracts and fractions of S.
to the skeletal muscle, such as myonecrosis followed saponaria is shown in Figure 3.

© 2012 Informa Healthcare USA, Inc.

370  M.L. da Silva et al.

Figure 2.  Inhibition of the edema-inducing activity of crude snake venom (A) and isolated toxins (B and C) after incubation for 30 min at
37°C with extracts (B and C) or fractions of S. saponaria (A1, A2, B2) at ratios of 1:50 and 1:10 (w/w) to venom and toxins, respectively. Each
denoted value represents the mean ± S.D. (n = 6).

Results suggest the presence of compounds, both in Studies with tropical plant extracts showed that, of
fractions and extracts, able to inhibit the activity of hem- the 52 hydroalcoholic extracts tested, ten completely
orrhagic proteins present in the venoms. These proteins inhibited the hemorrhagic activity of B. asper venom,
are mostly Zn2+-dependent, and the active compounds of and eight partially inhibited this activity (Castro et  al.,
this plant may be interfering in the binding of the pro- 1999). C. sylvestris SW showed an inhibitory effect on
teins to the cofactor, or may be binding to other active B. moojeni, B. neuwiedi Wagler, and B. asper venoms
sites essential for this activity. (Borges et  al., 2001; Da Silva, 2008). Pithayanukul et  al.
 Pharmaceutical Biology
 Sapindus saponaria anti-ophidian plant  371

Figure 3.  Inhibition of the hemorrhagic activity of crude snake venoms by the extracts and fractions of S. saponaria preincubated at
different ratios for 30 min at 37°C. Each denoted value represents the mean ± S.D. (n = 6).

(2003) showed the inhibitory effects of extracts of Eclipta three of its constituents; wedelolactone, stigmasterol
prostata on the hemorrhagic activity induced by the and sitosterol. Some other studies have shown isolated
venom of Calloselasma rhodostoma Kuhl. Diogo et  al. compounds with inhibitory activity, such as ar-turmer-
(2009) attributed the inhibitory effect of this plant to one, isolated from the roots of Curcuma longa Linnaeus,

© 2012 Informa Healthcare USA, Inc.

372  M.L. da Silva et al.
which completely inhibited the hemorrhagic activity of
B. jararaca snake venom (Ferreira et al., 1992).
The presence of myotoxins in Bothrops snake venoms
is quite common. Various types of anti-venoms studied
were not effective for antagonizing the effects of these
toxins. Biondo et al. (2003) demonstrated that the aque-
ous extract of Mandevilla velutina Mart. inhibited 66.5%
of the activity of B. jararacussu venom, 75% of BthTX-I
and 100% of C. d. terrificus venom and basic phospho-
lipase from crotoxin. Oliveira et  al. (2003) showed the
ability of suramin, a polyanionic compound, to inhibit
the myotoxic activity of BthTX-I. In the present study, the
myotoxicity induced by BthTX-I was significantly inhib-
ited by fractions A1 (79.09%), B2 (79.44%) and A2 (100%),
and for B. jararacussu crude venom, fraction A1 showed
an inhibition of 93.18% of this activity (Figure 4).
Extracts B and C and fractions A1 and A2 significantly
inhibited the coagulation induced by B. jararacussu
venom, while that same activity induced by C. d. terrificus
venom was significantly inhibited only by fractions A2
and B2 (Table 1). The venom:extract/fraction ratio tested
in the clotting activity was 1:5 (w/w), which is consid-
ered to be low when compared with ratios used by other
authors. A study by Maiorano et al. (2005) showed that the
aqueous extract of roots and stems of Mikania glomerata
Spreng. inhibited the clotting activity of Bothrops and C.
d. terrificus venoms. Results show 100% inhibition, pre- Figure 4.  Inhibition of the myotoxic activity of B. jararacussu
venting the formation of blood clots, in the proportion of venom and BthTX-I toxin after incubation for 30 min at 37°C with
1:100 (w/w). This inhibition possibly occurred as a result fractions of S. saponaria at a ratio of 1:50 (w/w, venom or toxin/
fractions). Each denoted value represents the mean ± S.D. (n = 6).
of the interaction of bioactive substances of M. glom-
erata with serine proteases, responsible for the venom
coagulating action. These interactions probably induce Table 1.  Neutralization of the clotting activity induced by B.
jararacussu and C. d. terrificus crude venoms preincubated with
irreversible changes in the catalytic site of these toxins,
extracts and fractions of S. saponaria.
which require conservation of the chemical interactions
Time of coagulation (sec)#
between amino acids to exert their toxic effects (Borges
Without extracts or
et al., 2001; Guiguer et al., 2004). Samples fractions 1:5 (w/w)
Vegetal extracts and natural compounds are proven PBS + Ca2+ 310 ± 0.12 –
to be effective at inhibiting the lethal activity of venoms B. jussu (50 µg) 60 ± 0.07 –
and toxins of several snakes (Núñez et al., 2004). Otero A1 – 188 ± 0.02
et  al. (1999) evaluated the neutralization of the lethal A2 – 155 ± 0.06
effects of B. asper venom using 74 ethanol extracts of B2 – 75 ± 0.12
anti-ophidian plants, showing that 7 species com- Extract B – 120 ± 0.08
pletely inhibited this activity. Similarly, Mahanta and Extract C – 109 ± 0.20
Mukherjee (2001) showed that aqueous extracts of the C. d. terrificus (50 µg) 60 ± 0.10 –
dried roots of Mimosa pudica L. inhibited the lethal A1 – 63 ± 0.07
effect of Naja kaouthia Lesson venom. Soares et  al. A2 – 120 ± 0.17
(2004) demonstrated that compounds such as brede- B2 – 80 ± 0.08
meyeroside D, a triterpenoid saponin, and ar-turmerone, Extract B – 67 ± 0.03
a phenolic compound, are potent inhibitors of the Extract C – 69 ± 0.05
lethality induced by B. jararaca and C. d. terrificus ven- #
Each experiment is represented as mean ± S.D. (n = 6).
oms. Additionally, Adzu et  al. (2005) showed that the
methanol extract of Annona senegalensis Pers. roots It was concluded that extract B of S. saponaria pre-
reduced the lethality caused by Naja nigricollis nigri- sented greater inhibitory effects on the activities of snake
collis Reinhardt venom. In our study, extracts B and C venoms, while extract C and fractions A2 and B2 showed
and fractions A1, A2 and B2 of S. saponaria were able greater inhibitory effects on the PLA2 activity induced by C.
to extend the life of animals envenomed by C. d. ter- d. terrificus venom and the isolated proteins, crotoxin and
rificus venom and crotoxin, with the minor inhibition PLA2Cdt. The myotoxic activity induced by B. jararacussu
observed in the presence of extract C (Table 2). venom and BthTX-I was mostly inhibited by fraction A1.

 Pharmaceutical Biology
 Sapindus saponaria anti-ophidian plant  373

Table 2.  Survival time of mice injected with C. d. terrificus Extract C and fraction A1 presented the greatest inhibi-
venom and crotoxin preincubated with extracts and fractions of tory effects on the hemorrhage induced by B. jararacussu
S. saponaria. venom, while for B. moojeni and B. alternatus venoms,
Survival time (min)* the best results of inhibition were observed with extracts
Without extracts C and B, respectively, and fractions A1, A2 and B2.
Samples or fractions 1:10 (w/w) 1:50 (w/w) With regard to edema, B. jararacussu venom was par-
C. d. terrificus 65 ± 0.02 – – tially inhibited by fractions A1, A2 and B2. The BthTX-I
(20 µg)
protein was mostly inhibited by fractions A2 and B2.
A1 – 195 ± 0.22 201 ± 0.18
Fraction A2 presented the greatest inhibition of the
A2 – 136 ± 0.20 142 ± 0.15
coagulation induced by B. jararacussu and C. d. terrificus
B2 – 158 ± 0.09 158 ± 0.04
venoms. Extract B and fractions A1, A2 and B2, incubated
Extract B – 164 ± 0.14 174 ± 0.11
with C. d. terrificus venom and crotoxin alone, signifi-
Extract C – 74 ± 0.14 85 ± 0.03
cantly extended the life of animals.
Crotoxin (10 µg) 72 ± 0.09 – –
TLC profiles of the crude extracts (B and C) and frac-
A1 – 186 ± 0.18 190 ± 0.13
tions (A1 and A2) showed β-sitosterol and stigmasterol
A2 – 165 ± 0.10 175 ± 0.19
as their main compounds (Figure 5A). β-Sitosterol
B2 – 148 ± 0.05 149 ± 0.07
glucoside was identified in all extracts and fractions
Extract B – 108 ± 0.12 123 ± 0.12
A1, B1, A2 and B2 (Figure 5B). Using ninhydrin reagent,
Extract C – 92 ± 0.05 92 ± 0.10
Extracts and # – –
it was possible to detect nitrogenated compounds in
fractions (500 µg) crude extracts and fractions N1 and N2 (Figure 5C).
#
100% of animal survival (>48 h) Murgu (2002) detected β-sitosterol, stigmasterol,
*Each experiment is represented as mean ± S.D. (n = 8). 3-methoxycinnamyl alcohol and hexadecanoic acid by

Figure 5.  TLC profiles of extracts and fractions from S. saponaria callus culture and standards. Mobile phase: (A): Hex:EtOAc (7:3); (B):
CHCl3:MeOH (8:2); (C): BAW (n-butanol:acetic acid:water, 4:1:5, upper phase). Color reagents: (A) and (B): Vanillin-sulfuric acid; (C):
Ninhydrin. Extracts and fractions are represented by their respective nominations (B, C, A1, A2, B1, B2, N1 and N2). Also shown are the
standards of β-sitosterol (SI), stigmasterol (ST) and β-sitosterol glucoside (SG).

Figure 6.  Effect of stigmasterol (A) on myotoxic (B and C) activity induced by isolated myotoxins, BthTX-I and BthTX-II, respectively. Each
experiment is represented as mean ± S.D. (n = 6).

© 2012 Informa Healthcare USA, Inc.

374  M.L. da Silva et al.
GC/MS in the callus culture from S. saponaria, where
β-sitosterol was found to be the main compound.
From extract B, more polar substances were extracted
by means of concentration than from extract C, thus
this last extract is enriched with β-sitosterol and stig-
masterol, while extract B and fractions N1 and N2 are
enriched with polyamines.
For fractions A1 and A2, a predominance of
β-sitosterol, stigmasterol and β-sitosterol glucoside
were observed, while in fraction B2, these compounds
were found in minor concentrations. Figure 6 shows
the anti-myotoxic activity of commercial stigmasterol
on myotoxins BthTX-I and II. Gomes et  al. (2007)
reported the neutralization of viper and cobra venom
by β-sitosterol and stigmasterol, isolated from the root
extract of Pluchea indica. Thus, β-sitosterol, stigmas-
terol and β-sitosterol glucoside could be responsible
for the inhibition of edema, hemorrhagic, myotoxic and
PLA2 activities induced by snake venoms and toxins.
Moreover, modeling studies on PLA2-inhibitor com-
plexes show favorable interactions with the amino acid
residues at the active site of PLA2 from snake venom and
stigmasterol, which could indicate the mechanism of
action responsible for the inhibition of phospholipase
activity (Nirmal et al., 2008).
In conclusion, Sapindus saponaria L. extracts and
fractions presented significant anti-ophidian activity
and could be used as an alternative treatment to serum
therapy or for its supplementation. Nevertheless, phar-
macological studies should still be performed using the
isolated active principles responsible for the anti-oph-
idian activity, thus providing insights to the elucidation
of the mechanisms of action of the corresponding toxins
and the development of future therapeutic agents for the
treatment of ophidian accidents.
Declaration of interest
The authors gratefully acknowledge the financial support
of Fundação de Amparo à Pesquisa do Estado de São
Paulo (FAPESP), Conselho Nacional de Desenvolvimento
Científico e Tecnológico (CNPq), Universidade de
Ribeirão Preto UNAERP and Faculdade de Ciências
Farmacêuticas de Ribeirão Preto-USP.
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