© 2009 The Authors Doi: 10.1111/j.1742-7843.2008.00350.

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Journal compilation © 2009 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 104, 293–299

Inhibition of Snake Venoms and Phospholipases A2 by Extracts from

Blackwell Publishing Ltd

Native and Genetically Modified Eclipta alba:

Isolation of Active Coumestans
Luciana C. Diogo1, Renata S. Fernandes2, Silvana Marcussi2, Danilo L. Menaldo2, Patrícia G. Roberto1, Paula V. F. Matrangulo1,
Paulo S. Pereira1, Suzelei C. França1, Silvana Giuliatti3, Andreimar M. Soares2 and Miriam V. Lourenço1
1
Department of Biotechnology, University of Ribeirão Preto, Ribeirão Preto, São Paulo, Brazil, 2Department of Clinical, Toxicological
and Bromatological Analysis, Faculty of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, São Paulo,
Brazil, and 3Department of Genetics, Faculty of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, São Paulo, Brazil
(Received 9 May 2008; Accepted 19 June 2008)

Abstract: We genetically modified Eclipta alba using Agrobacterium rhizogenes LBA 9402, with the aim of producing
secondary metabolites with pharmacological properties against phospholipase A 2 and the myotoxic activities of snake
venom. Extracts from in natura aerial parts and roots, both native and genetically modified (in vitro), were prepared and
analysed by high-performance liquid chromatography. In natura materials showed the coumestan wedelolactone at higher
concentration in the aerial parts, while demethylwedelolactone appeared at higher concentration in roots. Among the modified
roots, clone 19 showed higher concentrations of these coumestans. Our results show that the in natura extracts of plants
collected from Botucatu and Ribeirão Preto were efficient in inhibiting snake venom phospholipase A 2 activity. Regarding
in vitro material, the best effect against Crotalus durissus terrificus venom was that of clone 19. Clone 19 and isolated
coumestans (wedelolactone and demethylwedelolactone) inhibited the myotoxic activity induced by basic phospholipases A 2
isolated from the venoms of Crotalus durissus terrificus (CB) and Bothrops jararacussu (BthTX-I and II). The search for
antivenom is justified by the need of finding active principles that are more efficient in neutralizing snake venoms and also
as an attempt to complement serum therapy.

Eclipta alba (L.) Hassk. (syn. Eclipta prostrata), popularly
known as dye weed and belonging to the Asteraceae family,
is an annual herbaceous species [1]. Native to Brazil and other
tropical and subtropical regions, this plant is a source of several
secondary metabolites, such as polypeptides, polyacethylenes
and triterpenes [2], flavonoids, phytosterols and coumestans [3].
Coumestans represent an important class of natural
oxygenated aromatic products, including wedelolactone
and its demethylated form, demethylwedelolactone, both
responsible for the main medicinal effects of Eclipta ,
such as its anti-hepatotoxic, anti-hypertensive, antitumor, anti-
phospholipase A2 and antidote activities against snake
venoms [2,4 –7].
With the development of in vitro plant tissue cultures, it is
possible to produce viable, economically valuable compounds
[8]. Among the procedures for tissue culture, those for genetically
modified plants have developed significantly, and the obtain-
ment of transgenic plants is made by indirect modifications
with a biological vector, such as Agrobacterium [9].
Hundreds of plants have been related with anti-snake venom
action, but only a few have been scientifically investigated
[5–7]. Mors et al. obtained excellent results showing that
Author for correspondence: Andreimar Martins Soares, Department
of Clinical, Toxicological and Bromatological Analysis, Faculty of
Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo,
Av. do Café, s/n°, 14040-903, Ribeirão Preto, São Paulo, Brazil
(fax +55 16 3602 4725, e-mail andreims@fcfrp.usp.br).
wedelolactone from E. alba inhibits the myotoxic and
neurotoxic effects of Crotalus durissus terrificus venom [10].
Melo et al. have shown that E. alba extracts decreased the
myotoxic and haemorrhagic activities of Bothrops jararaca,
Bothrops jararacussu and Lachesis muta venoms [11]. More
recently, the potential antivenom property of wedelolactone
against Calloselasma rhodostoma venom (by neutralizing
isolated phospholipases A2 and venom lethality) has been
observed [12]. In this study, E. alba was genetically modified,
aiming at the production of secondary metabolites of
pharmacological interest, which were assayed against
phospholipase A2 and myotoxic activities of snake venoms
and isolated toxins.
Materials and Methods
Animals, venoms and toxins. Male Swiss mice (20–25 g) were used
for biological assays. Crude lyophilized B. jararacussu and C. d. terrificus
venoms were obtained from Bioagents Serpentarium, Batatais,
São Paulo, and Instituto Butantan, São Paulo, São Paulo, Brazil.
C. d. terrificus CB and B. jararacussu phospholipases A2 were isolated
as previously described [13,14]. The experiments described were
approved by the Institutional Committee for Ethics in Animal
Experimentation of the University of São Paulo and were done in
accordance with the guidelines of the Brazilian College for Animal
Experimentation.
Plant collection. Eclipta alba specimens were collected in the cities
of Botucatu and Ribeirão Preto and were cultivated in hotbeds in

294 LUCIANA C. DIOGO ET AL.
the Campo Experimental, Department of Biotechnology of University
of Ribeirão Preto (UNAERP), São Paulo, Brazil. Voucher specimens
HPMU 848 (Botucatu) and HPMU 58 (Ribeirão Preto) are deposited
in Department of Biotechnology of University of Ribeirão Preto, and
authenticated by Professor H. F. Leitão Filho from the Institute of
Biology of University of Campinas, Campinas, São Paulo, Brazil.
In vitro axenic plantlets, kept in solid culture medium Murashige and
Skoog (MS) [15] under 16:8 hr photoperiod were used for genetic
modification.
Preparation of A. rhizogenes suspension. Agrobacterium rhizogenes
(LBA 9402) suspensions were obtained by inoculation of the bacteria
in yeast mannitol broth (YMB) culture medium containing rifampicin
and streptomycin and left standing for 48 hr under constant stirring
at 10 ×g. Abs550 was then read in a Genesys 2 spectrophotometer.
The suspension was centrifuged at 2500 ×g for 15 min.; the residue
was washed and then resuspended in YMB.
Genetic modification of E. alba. Axenic E. alba plants, cultivated
in vitro, were desiccated and the leaves and roots taken out and dis-
carded. Nodal segments were cut (1.0 cm) and co-cultivated in MS/
2 culture medium (20 ml) containing 200 μg of acetosyringone and
300 μl of A. rhizogenes suspension. The cultures were left standing
under stirring (10 ×g) in the dark for 3 days. Control cultures were
also prepared without A. rhizogenes. After the co-cultivation period,
the explants were transferred to the MS culture medium containing
cefotaxime (500 mg/l) and kept in the dark. The obtained roots
were then excised and cultivated individually in MS culture medium
in the presence of cefotaxime (500 and 250 mg/l) for total elimination
of Agrobacterium. Transformed roots, cultivated in liquid MS culture
medium, were kept in the dark at 25 ± 1°, under stirring (10 ×g) and
subcultivated in intervals of 21 days.
Polymerase chain reaction (PCR) analysis of transformants. Confir-
mation of the genetic modification of roots was obtained through
PCR using specific primers for amplification of the TL region (30
nucleotides) of T-DNA and Vir Dl (22 nucleotides). For the PCR,
2.5 μl of Tp10x, 3 μl of dNTPs (1.25 mM), 0.20 μl Taq (5 U/μl),
1 μl of MgCl2 (50 mM), 2 μl of primer TL(a), TL (b), Vir Dl(a) and
Vir Dl(b) and 5 μl of the clone DNA samples were mixed, using
MilliQ water to complete the final volume to 50 μl [16]. After DNA
extraction and subsequent PCR of the samples, confirmation of the
genetic modification was performed by agarose gel electrophoresis.
Preparation of extracts. In natura aerial parts and roots of E. alba,
in addition to in vitro roots, both control and genetically modified,
were kept in a growing room. The material was desiccated, powdered
and weighed, and the metabolites were extracted with methanol
(20 mg/ml) at room temperature under stirring for 24 hr. The dried
residues were resuspended in 10 ml of MeOH:H2O (2:1, v/v) and
submitted to a partition with hexane followed by ethyl acetate.
The dried residues from this last partition were weighed and
resuspended in spectroscopic grade methanol (J. T. Baker). The
presence of coumestans was analysed by high-performance liquid
chromatography (HPLC) using a Shimadzu liquid chromatography,
with a SupelcosilTM LC18 column (4.6 × 250 mm; Supelco, city of
São Paulo, São Paulo, Brazil) and MeOH:H2O (0.1% acetic acid) as
mobile phase, with a linear gradient from 10:90 to 80:20 (v/v) in
40 min. and flow-rate of 1.0 ml/min. Detection was made at 350 nm.
The quantitative analysis of the coumestans wedelolactone and
demethylwedelolactone was performed and a calibration curve
was outlined with external standard (1.0, 0.5 and 0.1 mg/ml).
Inhibition of the phospholipase A2 and myotoxic activity. Phospho-
lipase A 2 activity was assayed by the indirect radial hemolysis
method, as described by Gutiérrez et al. [17]. The aqueous extracts of
in natura aerial parts and roots from Ribeirão Preto and Botucatu, in
addition to the aqueous extracts of the in vitro material, were assayed
at different ratios (1:100 w/w for crude venoms or 1:50 w/w for isolated
© 2009 The Authors
Journal compilation © 2009 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 104, 293–299
17427843, 2009, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/j.1742-7843.2008.00350.x by Amrita Vishwa Vidyapeetham Kollam Campus, Wiley Online Library on [31/07/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
phospholipases A2) with B. jararacussu venom, C. d. terrificus venom
and isolated phospholipases A 2 (BthTX-II [Bothropstoxin-II
from B. jararacussu], BthA-I-PLA2 [acidic phospholipase A2 from
B. jararacussu] and CB [crotoxin B from C. d. terrificus]). These
samples were incubated for 30 min. at 37° and then applied on
haemolytic plates. After incubation for 12 hr at 37°, the halos were
measured and expressed in mm.
Male Swiss mice (18–22 g) were injected intramuscularly in the
right gastrocnemius muscle with solutions containing 25–50 μg of
snake venoms or toxins in 50 μl of phosphate-buffered saline. The
mixtures of myotoxin/extract (1:10 and 1:30, w/w) were then evaluated
after incubation with the crude venoms or isolated toxins for 30 min.
at 37°, in triplicate. Controls received phosphate-buffered saline or
extract alone. Mice were bled from the tail 3 hr after injections and
blood was collected into heparinized capillary tubes. Plasma creatine
kinase activity was determined using the Kit UV (Bioclin, Belo
Horizonte, Minas Gerais, Brazil) [14] and the myotoxic activity was
measured and expressed in U/l.
Results and Discussion
Snakebites represent a serious problem of public health in
tropical countries due to their frequency and to the mortality
they produce. There are approximately 3000 snake species in
the world, of which 10–14% are venomous. The World Health
Organization estimates that between 1.25 and 1.66 million
accidents occur in the world every year, with about 30,000
to 40,000 deaths. The more affected regions are South and
Southeast Asia and tropical America, and among the South
American countries, Brazil represents the highest number of
accidents per year [18].
Several plants are popularly used against snakebite enveno-
mations and many studies validate their anti-snake venom
properties through the isolation and characterization of
components that are able to inhibit the enzymatic, pharma-
cological and toxic properties of different snake venoms.
The use of plant extracts as antidote for animal venoms is
an old option of many communities, which do not have any
prompt access to serum therapy. In addition, depending on
the time between accident and treatment, the ability of the
antiserum to neutralize the local effects of envenomation is
only partial. Thus, vegetal extracts became an important
research material [5–7,19,20], which could be used as an
alternative for the traditional treatment of human envenom-
ations, the use of plant extracts is also important for the
treatment of animals (as horses and cattle) since there is no
serum therapy for their treatment.
According to Uozumi, Agrobacterium is a common soil
bacterium that is able to infect plants, thus promoting
uncontrolled cell proliferation [21]. Around 15 days after the
co-cultivation period of E. alba with A. rhizogenes (LBA 9402),
emission of roots in the injured points started. Sixty root
clones were obtained, but only 16 were potentially modified,
in addition to the control roots.
The presence of multiple apical meristems in the genetically
modified material induced a higher storage of biomass. Similar
results were obtained by Caro et al., reporting a larger response
in the rooting process of Prosopis chilensis treated with
A. rhizogenes [22]. Mechanisms that integrate the plant genome
modification through Agrobacterium are still unknown [23].

INHIBITION OF SNAKE VENOM AND PHOSPHOLIPASES A2 BY ECLIPTA ALBA
Fig. 1. Agarose gels showing extraction of DNA from potentially
modified roots of Eclipta alba (A) and products of PCR (B). After
PCR (B), clones C1, C2, C4, C10, C12, C13, C14, C17, C19, C20 and
C37 showed a band (800 pb) that evidences the transfer of T-DNA from
A. rhizogenes to the plant DNA, confirming the genetic modification.
The DNA from 16 clones and control roots were extracted
(fig. 1A) and then submitted to PCR to check genetic modifi-
cation. It was noted that the clones C1, C2, C4, C10, C12,
C13, C14, C17, C19, C20 and C37 showed a band on agarose
gel that evidences the transfer of T-DNA from A. rhizogenes
to the plant DNA, thus confirming the genetic modification
(fig. 1B). The genetic modification index of E. alba mediated
by A. rhizogenes was 69%.
Qualitative analyses by HPLC showed the presence of two
coumestans, known as wedelolactone and demethylwedelol-
actone (fig. 2A), in all analysed extracts. According to the
results, the highest wedelolactone concentration was found
in the E. alba aerial parts from Botucatu (16.7 mg/g dry
weight [DW]), followed by aerial parts from Ribeirão Preto
(13.02 mg/g DW) and, in lower proportions, in roots from
Ribeirão Preto (6.45 mg/g DW) and from Botucatu (5.97 mg/g
DW). The highest concentrations of demethylwedelolactone
were found in roots from Botucatu (15.7 mg/g DW) and
Ribeirão Preto (14.4 mg/g DW), and in the clones C1
(8.67 mg/g DW), C12 (7.75 mg/g DW), C13 (10.58 mg/g
DW) and, especially, C19 (19.41 mg/g DW) (fig. 2A).
When the coumestan storage was compared with the
selected and control root clones, we saw that all clones
(except C10 and CR) produce wedelolactone. Regarding
© 2009 The Authors
Journal compilation © 2009 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 104, 293–299
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295
Fig. 2. Analysis of the concentration of coumestans present in
Eclipta alba samples. (A) Isolation of coumestans by reversed-phase
HPLC (RP-HPLC). (B) Peaks of wedelolactone (WL) and deme-
thylwedelolactone (DWL) are indicated by arrows. APB, extracts
from the aerial parts of E. alba from Botucatu; APR, extracts from
the aerial parts of E. alba from Ribeirão Preto; C1–43, E. alba
clones 1– 43; CR, E. alba in vitro control roots; RB, roots of E. alba
from Botucatu; RR, roots of E. alba from Ribeirão Preto. Each
experiment is represented as mean ± standard deviation. Statistical
differences compared to the positive control (P < 0.05).
demethylwedelolactone, all showed higher concentration
than those from CR (1.08 mg/g DW), except C5 and C10.
When we evaluate only demethylwedelolactone production,
we note that clones C1, C12, C13, C14 and C19 were the most
promising and, among them, C19 stood out for displaying
the highest amount of demethylwedelolactone. These
coumestans were isolated by HPLC on a C18 reverse phase
column as shown in fig. 2B.
Although chromatographic studies by França et al. [24]
did not give evidence of the presence of wedelolactone L in
E. alba in vitro plantlets, our results show that all samples
displayed both wedelolactone and demethylwedelolactone,
except CR and C10 (fig. 2A), where only demethylwedelolac-
tone appears. According to Farah, the infected plant may
have a deviation in its metabolism, thus synthesizing
substances not needed to the native plants [25].
Production of secondary metabolites in genetically
modified cultures is a function of the A. rhizogenes cell line,
the transformed clones, the growth kinetics of these cultures,
and of the host plant. Increase of secondary metabolites in
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296 LUCIANA C. DIOGO ET AL.

Fig. 3. Inhibition of phospholipase A2 activity. (A) Effect of Eclipta alba extracts on the phospholipase A2 activity induced by Bothrops jararacussu
and Crotalus durissus terrificus venoms, at 1:100 (w/w, venom:extract) ratio. (B) Effect of E. alba extracts on the enzymatic activity induced by
Asp49-PLA2s isolated from B. jararacussu (BthTX-II and BthA-I-PLA2) and C. d. terrificus (CB) at 1:50 (w/w, PLA2: extract) ratios. APB,
extracts from the aerial parts of E. alba from Botucatu; APR, extracts from the aerial parts of E. alba from Ribeirão Preto; C19, E. alba clone
19; CR, E. alba in vitro control roots; RB, roots of E. alba from Botucatu; RR, roots of E. alba from Ribeirão Preto. Results are expressed as
mean ± standard deviation (n = 03).

hairy roots is probably due to their loss of ability to trans-
port these metabolites towards other parts of the plant, thus
providing a higher accumulation in roots [26]. Giri et al.
provided evidence of these facts with Artemisia annua, where
levels of artemisine varied in clones of hairy roots obtained
from different lines of A. rhizogenes [27]. According to Allan
et al., hairy roots are able to produce higher concentrations
of secondary metabolites when compared with the in vitro
culture and even in the intact plant [28]. In addition, synthesis
of secondary metabolites by hairy roots has the advantage
of reproducibility and stability of production [29].
Anti-snake venom activity has been demonstrated
scientifically for a variety of plants [5,6,19]. In the present
study, all analysed extracts showed inhibitory activity against
B. jararacussu venom. The extracts with higher effects were
that from roots from Botucatu and Ribeirão Preto, with
reduction of the phospholipase A2 activity close to 50%.
Among the assayed extracts against C. d. terrificus venom,
the best result was obtained for C19, which reduced approxi-
mately 70% of the phospholipase A2 activity (fig. 3A). Similar
results were reported by Borges et al. with studies carried
out using Casearia sylvestris extracts [30].

Fig. 4. Inhibition of myotoxic activity. Effect of Eclipta alba clone 19 on myotoxicity induced by Bothrops jararacussu BthTX-I (A) and
BthTX-II (B). Each experiment is represented as mean ± standard deviation (n = 6).

© 2009 The Authors

Journal compilation © 2009 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 104, 293–299
 17427843, 2009, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/j.1742-7843.2008.00350.x by Amrita Vishwa Vidyapeetham Kollam Campus, Wiley Online Library on [31/07/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
INHIBITION OF SNAKE VENOM AND PHOSPHOLIPASES A2 BY ECLIPTA ALBA 297

Fig. 5. Inhibition of myotoxic activity. Effect of Eclipta alba demethylwedelolactone (A and C) and wedelolactone (B and D) on myotoxicity
induced by isolated phospholipases A2, BthTX-I and BthTX-II, respectively. Each experiment is represented as mean ± standard deviation
(n = 6).

Clone 19 showed to be more efficient in neutralizing the
phospholipase A2 activity induced by the basic Asp49 BthTX-
II and CB (fig. 3B) than that induced by the acidic isoform
Asp49 BthA-I-PLA2 (fig. 3B). These data suggest a more
specific binding with basic phospholipases A2, intermediated
by a probable electrostatic interaction. Biondo et al. [31] and
Maiorano et al. [32] also showed that the aqueous extract from
Mandevilla velutina and Mikania glomerata, respectively,
presented a wide inhibition spectrum of toxic, enzymatic and
pharmacological activities.
The muscle-damaging activity of Bothrops venoms is
partially caused by a group of highly basic proteins with
phospholipase A2 structure [33]. Clone 19 inhibited the
myotoxic activity of both Asp49 BthTX-II and Lys49
BthTX-I phospholipases A2 from B. jararacussu (fig. 4).
Figure 5 shows the anti-myotoxic activity of the isolated
coumestans (wedelolactone and demethylwedelolactone) on
BthTX-I and -II.
Several phospholipase A2 inhibitors have been isolated from
natural sources, such as marine organisms, snakes and plants [20].
Wedelolactone and 12-methoxy-4-methylvoachalotine, com-
pounds isolated from E. alba and Tabernaemontana catharinensis
respectively, effectively inhibit the myotoxic activity of the
venoms from C. d. terrificus, B. jararacussu, B. jararaca and
L. muta, as well as various isolated myotoxic phospholipases
A2 [5,6].
During the last years, a great scientific progress has been
initiated involving chemical and pharmacological studies on
medicinal plants, aiming at the identification and obtainment
of new compounds with therapeutic properties. Only 8% of
the vegetal species from the Brazilian flora have been studied
in search of bioactive compounds and 1100 species were
evaluated regarding their medicinal properties [34]. However,
substances showing activity of interest are usually present in
very small amounts, depending on several factors as seasonal
variability and even the day and hour of collection, growth
stage, climate and predominant soil composition in different
regions, and finally the plant parts where these compounds
are produced or stored. Some substances, worthy for the
pharmaceutical industry, are obtained directly from the

© 2009 The Authors

Journal compilation © 2009 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 104, 293–299

298 LUCIANA C. DIOGO ET AL.
plants in great amounts and used in the elaboration of
several medicines. However, chemical synthesis is needed
for other medicines based on natural analogues isolated
from plants in minimal amounts.
Continued extraction of medicinal plants in endemic
regions has endangered perpetuation of natural populations
of native species and extinguished many groups of plants
whose pharmacological activity is already evidenced, many
of them running the risk of extinction, even before ecologic
and agronomic studies have been carried out. Therefore,
development of researches on medicinal plants, native from
the Brazilian open pastures, the Atlantic forest and other
regions, are very important since they offer an additional
stimulus for the study of these species, thus contributing to
the efforts for the preservation of the vegetal diversity. In
addition, in vivo multiplication of medicinal plants has made
possible not only their in natura reintroduction, but also their
genetic modification, thus producing secondary metabolites
of medical-scientific interest in large amounts, drastically
decreasing depredation of these species in the surrounding.
Verification of E. alba susceptibility to A. rhizogenes is a
valuable information that will allow further investigations
aiming at the utilization of this plant as model for studies of
gene insertion of pharmacological interest in vegetal genomes.
Pharmacological studies should still be performed using
new extract fractions, in order to isolate and characterize
the active principle responsible for the anti-snake venom
activity, thus providing insight into the mechanisms of
action of corresponding toxins and development of future
therapeutic agents for treatment of ophidian accidents.
Acknowledgements
The authors gratefully acknowledge the financial support
by the Fundação de Amparo à Pesquisa do Estado de São
Paulo, Coordenação de Aperfeiçoamento de Pessoal de Nível
Superior, and Conselho Nacional de Desenvolvimento
Científico e Tecnológico.
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Journal compilation © 2009 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 104, 293–299