Journal of Ethnopharmacology 155 (2014) 1076–1085

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jep

Research Paper

Antiproliferative activity and new argininyl bufadienolide esters from

the “cururú” toad Rhinella (Bufo) schneideri
Guillermo Schmeda-Hirschmann a,n, Cristina Quispe a, Cristina Theoduloz b,
Paulo Teixeira de Sousa Jr.c,d, Carlos Parizotto c,d
a
Laboratorio de Química de Productos Naturales, Instituto de Química de Recursos Naturales, Universidad de Talca, Casilla 747, 3460000 Talca, Chile
b
Laboratorio de Cultivo Celular, Facultad de Ciencias de la Salud, Universidad de Talca, Casilla 747, 3460000 Talca, Chile
c
Laboratório de Pesquisa em Química de Produtos Naturais, Departamento de Química, Universidade Federal de Mato Grosso, Av. Fernando C. da Costa s/n,
78060-900 Cuiabá, Mato Grosso, Brazil
d
Instituto Nacional de Ciência e Tecnologia em Áreas Úmidas (INAU), Rua Nove, 305, Boa Esperanca, 78068-410 Cuiabá, Mato Grosso, Brazil

art ic l e i nf o a b s t r a c t

Article history: Ethnopharmacological relevance: Toads known as “cururú” (Rhinella schneideri) have been used in the
Received 22 January 2014 Brazilian Pantanal and Paraguayan Chaco wetlands to treat erysipelas and cancer. The aim of the study
Received in revised form was to assess the antiproliferative effect of the venom obtained from Rhinella schneideri and to identify its
2 June 2014
constituents by spectroscopic and spectrometric methods.
Accepted 6 June 2014
Available online 16 June 2014
Materials and methods: The venom was obtained by gentle pressing the parotid glands of the toads. The
dry crude drug was analyzed by HPLC–MS–MS and chromatographed on Sephadex LH-20 to obtain
Keywords: purified compounds and fractions for spectroscopic analysis. The venom and fractions were evaluated for
Rhinella schneideri antiproliferative activity towards normal human lung fibroblasts (MRC-5) and four human cancer cell
Bufo
lines: gastric epithelial adenocarcinoma (AGS), lung cancer (SK–MES-1), bladder carcinoma (J82) and
Argininyl bufadienolides
promyelocytic leukemia (HL-60).
Antiproliferative activity
Brazilian pantanal Results: From the Rhinella schneideri venom, 29 compounds were isolated and/or identified by spectro-
scopic and spectrometric means. Three known alkaloids and five argininyl diacids were identified in the
complex mixture by HPLC–MS–MS. Nine out of fifteen argininyl diacid derivatives of the bufadienolides
bufalin, marinobufagin and telocinobufagin are reported for the first time and four argininyl diacids are
described for the first time as natural products. The venom and the fractions 9–13 showed a remarkable
antiproliferative effect, with IC50 values in the range 0.019–0.022, 0.035–0.040, 0.028–0.064, 0.042–0.056
and 0.044–0.052 mg/mL for MRC-5, AGS, SK-MES-1, J82 and HL-60 cell lines, respectively. Under the same
experimental conditions, IC50 values of the reference compound etoposide were 2.296, 0.277, 1.295, 1.884
and 1.059 mg/mL towards MRC-5, AGS, SK-MES-1, J82 and HL-60 cells, respectively.
Conclusions: The venom showed a strong antiproliferative effect towards human cancer cells and
presented a high chemical diversity in its constituents, supporting its use as anticancer agent. These
findings encourage further work on the chemistry and bioactivity of South American toad venoms.
& 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction powerful hallucinogen Weil, Davis, 1994. The structure of several

Frogs have played a relevant role in South American cultures
since prehistoric times. They were represented in material goods
such as earthen ware, pottery, stone sculpture and gold portrayals
(Wassen, 1934a) as well as in myths (Wassen, 1934b) all over the
South American continent. Dendrobatid frogs were used to poison
arrows and darts in the Amazon basin (Bisset, 1989) and the large
toad Bufo alvarius (now Rhinella alvarius) was described as a
groups of alkaloids discovered in South American amphibians has
been revised and show a remarkable chemical diversity (Daly,
1998; Daly et al., 2008). In the Guarani tradition of Paraguay and
the Brazilian Pantanal, “cururú”, the large frog belonging to genus
Rhinella, is linked to firemyths and to the obtention of fire by the
Guarani culture1.
The large toad Rhinella schneideri (Werner, 1894) (synonims:
Bufo paracnemis A. Lutz, 1925 and Bufo schneideri Werner, 1894) is

n 1
Corresponding author. Tel.: þ 56 71 2200288. (http://www.virtualberks.com/spanishasasecondlanguage/ElPrimerfuego_
E-mail address: schmeda@utalca.cl (G. Schmeda-Hirschmann). esen.html).

http://dx.doi.org/10.1016/j.jep.2014.06.025
0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.
 G. Schmeda-Hirschmann et al. / Journal of Ethnopharmacology 155 (2014) 1076–1085
common in the Paraguayan Chaco wetlands and in the Brazilian
Pantanal2 . The large frogs are poisonous when bitten by dogs or
when the venom from the parotid glands came accidentally into
contact with the mouth of children. In spite of their abundance,
frogs were not eaten but sometimes used in healing practices.
Alves, Alves (2011) reported the use of Rhinella schneideri (Werner,
1894) (“cururu” toad), to heal wounds, erysipelas, acne, cancer, to
treat dental caries and to induce abortion in Brazil. The close
related species Rhinella jimii (Stevaux, 2002) is used to treat
erysipelas in northeastern Brazil (Alves et al., 2012). In the decade
of 1960, a decoction of toad to fight cancer began to be known in
Paraguay. This use seems to be a more recent practice in spite of
some claims that it came from the Guayaki Indians from Eastern
Paraguay. The “cururu” toad is rubbed in varicous wounds and skin
lesions in Paraguayan rural medicine. According to the information
received, the toad is used both to treat cancer, as an aqueous
decoction, taken orally or locally applied to treat erysipelas. For the
use against cancer, the toad is killed, skinned and the skin is placed
in boiling water (about one liter). The decoction is bottled and
stored in a cold place. The usual dose was a glass of about 100–
150 ml of the decoction per day.
The toad venom has been used as a crude drug in Oriental
medicine and traditional Chinese medicine for a very long time. It
is recommended mainly as an anti-inflammatory (Ma et al., 2007)
and anticancer (Meng et al., 2009) agent. According to The State
Pharmacopoeia Commission of People´s Republic of China (2005),
the crude drug and its aqueous extract (cinobufacini) are used as
cardiotonic, antineoplastic, local anesthetic and antimicrobial
agent. In spite of an increasing interest in the toad venoms as
potential sources of bioactive agents, less is known on the use of
frog secretions in South American traditional medicine.
Sciani et al. (2013) described the antiproliferative activity of
crude secretions from Brazilian frogs, obtained by manual compres-
sion of the venom glands. Seven species of Rhinella (formerly Bufo)
and one Phyllomedusa species were investigated. The venom of
Rhinella schneiderii showed an IC50 value of 48 mg/mL towards the
breast cancer cell MCF-7. However, no reference compound for
comparison was included in this study. The extracts of the Brazilian
frogs were investigated for the occurrence of already described
molecules in the Chinese crude drug ChanSu by HPLC-MS. The
alkaloids dehydrobufotenin and serotonin were found in all the
secretions and were more abundant than the steroids (Sciani et al.,
2013). Anjolette et al. (2011) reported two active subfractions from
Rhinella schneiderii venom with effects on the complement system.
In a congress abstract, Baldo et al. (2012a) describe the effect of
different fractions from the Rhinella schneideri venom against
seizures induced by the convulsant agents PTZ and NMDA, suggest-
ing a neuroprotective effect of the toad venom molecules. In the
same congress, Baldo et al. (2012b) report the cytotoxicity of the
Rhinella schneideri poison towards murine melanoma cells, HL-60,
HepG2, PC-12 and PBMC cells. The venom showed different effects
on the cells, but IC50 values were not reported.
In Brazilian Rhinella species, telocinobufagin, hellebregenin and
bufalin were identified as known cytotoxic/antitumoral compounds
(Sciani et al., 2013). The main bufadienolides marinobufagin, bufalin,
telocinobufagin, hellebrigenin, 20 S,21 R-epoxymarinobufagin and
β-sitosterol were reported from Rhinella schneideri captured around
Brasilia in the cerrado phytogeographic region (Cunha-Filho et al.,
2010). The compounds and some semisynthetic derivatives were
assessed for cytotoxicity using several cell lines.
The distribution range of Rhinella schneideri and the phylogenetic
relationship of South American Rhinella species were reported by
Maciel et al. (2010). The results of the study suggest diversification
2
(http://www.iucnredlist.org/details/54755/0).
1077
associated with geographic and environmental changes that could
explain the current distribution of the different Rhinella species. The
formation of the hydrological basins of the Amazon, Paraguay and
Parana rivers in the late Tertiary and Quaternary, could have played a
relevant evolutionary role in the genus speciation. The environmental
conditions of the Chaco and Pantanal wetlands, associated with the
Paraguay River basin, are very different from those of the Brazilian
cerrado, suggesting the occurrence of distinctive bioactive compounds
in the toad venoms. The aim of the present work was to verify the
traditional indication of use of the Pantanal toad as an antiproliferative
agent as well as to identify the toad venom components.
2. Materials and methods
2.1. Collection of the toad venom
The toad venom was collected by gentle pressing the parotid
glands from free living individuals at SESC-Pantanal, Mato Grosso,
Brazil, on April 26, 2013. The toad population surveyed gathers at
the river harbor to feed on insects attracted by the town lights.
About 30 individuals were captured and released after collecting
the venom. The secretion was suspended/dissolved in MeOH for
preservation until work-up at the laboratory. The toad was
identified as Rhinella (Bufo) schneideri by Prof. Domingos de Jesus
Rodrigues, Universidade Federal de Mato Grosso, Núcleo de
Estudos da Biodiversidade da Amazônia Matogrossense (NEBAM),
Instituto de Ciências Naturais, Humanas e Sociais, Cep 78557-267
Sinop, MT, Brazil.
2.2. Isolation
The freeze-dried solid (1.2 g) was treated with 3  20 mL of
DCM:MeOH 1:1 under sonication (5 min each time), filtered and
taken to dryness under reduced pressure to afford 256 mg of a
yellowish oil. The solid remaining after DCM:MeOH extraction was
sonicated with CHCl3 (30 mL, 10 min) and MeOH (30 mL, 10 min)
to obtain additional fractions (97 mg of CHCl3 and 101 mg MeOH-
solubles). After TLC comparison, the three combined extracts
(453 mg) were pooled for chromatography. Some 440 mg of the
combined extracts sample was loaded into a Sephadex LH-20
column (column length 40 cm, i.d. 3 cm, Sephadex load: 20 cm)
equilibrated with DCM:MeOH 1:1 v/v to afford 25 fractions. After
TLC comparison, the fractions were pooled as follows: 1–3
(160 mg), 4 and 5 (46 mg), 6–11 (35 mg), 12 and 13 (23 mg), 14–
21 (10 mg), 22–25 (8 mg).
2.3. HPLC analysis
The HPLC system used for DAD analysis of extracts was a
Shimadzu equipment (Shimadzu Corporation, Kyoto, Japan) con-
sisting of a LC-20AT pump, a SPD-M20A UV diode array detector,
CTO-20AC column oven and a LabSolution software. A MultoHigh
100 RP 18-5m (250  4.6 mm) column (CS-Chromatographie Ser-
vice GmbH- Germany) maintained at 25 1C was used. Approxi-
mately 5 mg of the extract obtained as explained above was
dissolved in 1 mL MeOH, filtered through 0.45 mm PTFE filter
(Waters) and submitted to HPLC-DAD analysis. The compounds
were monitored at 295 nm. The HPLC analysis was performed
using a linear gradient solvent system consisting of 1% formic acid
(A) and acetonitrile (B) as follows: from 92% to 46% A over 45 min.
The flow rate was 1 mL/min. The volume injected was 20 mL. The
HPLC conditions were similar to those reported by Gao et al.
(2010) for a comparison of venoms from different Bufo species.
1078 G. Schmeda-Hirschmann et al. / Journal of Ethnopharmacology 155 (2014) 1076–1085
2.4. QTOF-analysis
ESI–MS–MS analyses were conducted in a Micromass (Man-
chester, UK) Q-TOF micro instrument. The samples were directly
infused at a flow rate of 10.0 mL/min using a syringe pump
(Harvard Apparatus, Holliston, United States of America). ESI mass
and tandem mass spectra were acquired in the positive ion mode.
The following operating conditions were used: 3.0 kV capillary
voltage, 40 V cone voltage and desolvation gas temperature of
100 1C. Tandem ESI–MS–MS spectra were collected by causing
collision-induced dissociation (CID) of the mass-selected proto-
nated molecules using argon as the buffer gas and collision
energies from 5 to 45 eV. Mass-selection was performed by
Quadrupole 1 using a unitary m/z window, and collisions were
performed in the rf-only quadrupole collision cell, followed by
Time of Flight (TOF) mass analysis. ESI–MS were acquired over an
m/z range of 80–1000 amu.
2.5. HPLC–MS–MS analysis
Mass spectra were recorded using an Agilent 1100 (USA) liquid
chromatography system connected through a split to an Esquire
4000 Ion Trap LC/MS system (Bruker Daltoniks, Germany) or a
Merck-Hitachi 6200 Intelligent Pump and L 4000 UV detector
coupled to a EBE trisector VG Autospec Micromass spectrometer
(Micromass-Water Autospec, U.K.) operating at 70 eV. Full scan
mass spectra were measured between m/z 150 and 2000 u in
positive ion mode. For the Ion Trap LC/MS system, nitrogen was
used as nebulizer gas at 27.5 psi, 350 1C and at a flow rate of 8 L/min.
The mass spectrometric conditions for positive and negative ion
mode were: electrospray needle, 4000 V; end plate offset, 500 V;
skimmer 1, 56.0 V; skimmer 2, 6.0 V; capillary exit offset, 84.6 V;
capillary exit, 140.6 V. Collision induced dissociation (CID) spectra
were obtained with a fragmentation amplitude of 1.00 V (MS/MS)
using helium as the collision gas.
2.6. NMR analysis
The NMR spectra were recorded on a Bruker Avance 400
(Bruker, Rheinstetten, Germany) spectrometer at 400 MHz for 1H
and 100 MHz for 13C in CDCl3 or MeOH-d4. Chemical shifts are
given in ppm with residual chloroform or methanol as the internal
standard.
2.7. Antiproliferative assay
The MTT reduction assay was used to determine the viability of
MRC-5 normal human lung fibroblasts (ATCC, CCL-171), AGS
human gastric adenocarcinoma cells (ATCC, CRL-1739), SK-MES-1
human lung cancer cells (ATCC, HTB-58), J82 human bladder
carcinoma cells (ATCC, HTB-1), and HL-60 human promyelocytic
leukemia (ATCC, CCL-240) from the American Type Culture Collec-
tion (Manassas, VA, USA). MRC-5, J82, and SK-MES-1 cells were
grown in Eagle's Minimum Essential Medium, containing 2 mM
L-glutamine, 1 mM sodium pyruvate and 1.5 g/L NaHCO3. AGS cells
were grown in Ham's F-12 medium supplemented with 2 mM L-
glutamine and 1.5 g/L NaHCO3. HL-60 cells were grown in suspen-
sion in RPMI 1640 medium containing 1 mM sodium pyruvate and
2 g/L NaHCO3. All media were additionally supplemented with 10%
heat inactivated fetal bovine serum, 100 IU/mL penicillin G, and
10 mg/mL streptomycin. Cells were sub-cultured once a week and
the medium was changed every two days. For the assay, cells were
plated in 96-well plates (100 mL/well) at a density of 50,000 cells/
mL. One day after seeding, the cells were treated with the medium
containing the compounds at concentrations ranging from 0 to
100 mg/mL, first dissolved in DMSO (final concentration of 1%),
diluted with complete medium, and incubated for 72 h in a
humidified incubator with 5% CO2 in air at 37 1C. Untreated cells
were used as 100% viability controls. Control cells were grown
with complete medium and 1% DMSO. At the end of the incubation
the MTT reduction assay was performed as described previously
(Rodriguez, Haun, 1999). The final concentration of MTT was 1 mg/
mL. Etoposide (E1383), vinblastin (V1377) and camptothecin
(C9911) from Sigma-Aldrich (St. Louis, MO, USA) were used as
reference substances. Each experiment was carried out three times
in quadruplicate. The results were transformed to percentage of
controls and the IC50 value was obtained adjusting the dose-
response curve to a sigmoidal model.
3. Results and discussion
The chemical composition and antiproliferative activity of the
parotid glands venom from the “cururu” toad Rhinella schneideri
was investigated. The crude venom extract was permeated into a
Sephadex LH-20 column and the constituents of the CC fractions
were analyzed by TLC, 1HNMR and QTOF-MS. While the TLC was
helpful in the identification of main known constituents, NMR
spectra allowed identification of the main unknown metabolites
and indicated the occurrence of minor compounds, related to the
main products. The main compounds isolated and fully character-
ized by spectroscopic and spectrometric means were used as
reference both for the analysis of the different fractions by
QTOF-MS as well as for HPLC–MS–MS. The HPLC chromatogram
of the venom is presented in Fig. 1. The structures of the
compounds isolated and/or tentatively identified in the “cururu”
toad venom are shown in Figs. 2 and 3.
3.1. NMR and QTOF–MS analysis
QTOF–MS analysis was performed to get an insight into the
identity of the minor compounds in the fraction pools 1–3, 4 and
5, 6–11, 12 and 13, 14–21 and 22–25. The general MS pattern of the
bufadienolides shows the loss of the terpene moiety leading to the
argininyl diacid, allowing the identification of the steroid and the
diacid linked to the basic aminoacid and the steroid (Hu et al.,
2011; Gao et al., 2010; Lenaerts et al., 2013). The fragmentation
pattern in MS allows a clear differentiation of the bufadienolides
based on bufalin, marinobufagin or telocinobufagin, characterized
by the neutral loss of [M-H2O] of 368, 382 and 384 amu for bufalin,
marinobufagin and telocinobufagin, respectively.
The 1H NMR spectrum of fractions 1–3 presented typical
signals for the α-pyrone ring of bufadienolides, with a main
compound 14 showing a dd at δ 7.92 (1H, J ¼9.6 and 2.4 Hz), a d
at δ 7.48 (1H, J ¼1.6 Hz) and a d at δ 6.30 (1H, J ¼9.6 Hz) and a
minor constituent 7 with a dd at δ 8.01 (1H, J¼ 9.6 and 2.4 Hz), a d
at δ 7.46 (1H, J¼ 1.6 Hz) and a d at δ 6.31 (1H, J¼ 9.6 Hz).
Furthermore, the br s at δ 5.04, dd at δ 4.30 (1H, J ¼6.8, 5.2 Hz),
the s at δ 3.46, the t at δ 2.26 and 2.15 and the multiplets at 1.40–
1.68 and 1.16–1.38, pointed out to argininyl diacid derivatives of
bufadienolides. The methyl singlets at δ 1.01 and 0.81 for the main
and δ 0.98 and 0.74 for the minor constituent, can be assigned to
H-19 and H-18 of the terpene moiety, respectively.
QTOF-MS analysis of the fraction 1–3 showed a main com-
pound with an ion at m/z 713.6476 [Mþ H] þ which in MS/MS
shows loss of water (695.6282) leading to a base peak at 331.2944.
The fragment agrees with a molecular formula of C14H26N4O5
accounting for a C-8 dicarboxylic acid linked to arginine. The
additional fragment at m/z 175.1832 [C6H14N4O2 þH] þ confirms
the identity of the aminoacid. The neutral loss of 382 amu [M–
H2O] agrees with the molecular formula C24H32O5 (400 amu) for
marinobufagin. The compound was identified as 3-(N-suberoyl
 G. Schmeda-Hirschmann et al. / Journal of Ethnopharmacology 155 (2014) 1076–1085
argininyl) marinobufagin, namely marinobufotoxin (compound
14). The spectral data are in agreement with marinobufotoxin,
reported from Bufo ictericus and Bufo marinus by Linde-Tempel
(1970) and synthesized by Pettit, Kamano (1974). A second con-
stituent (compound 7) with m/z 699.5363 [Mþ H] þ shows a neutral
loss of 368 amu pointing out to a terpene of 386 amu, differing from
compound 14 in one oxygen function and an unsaturation equiva-
lent, in agreement with bufalin. The base peak at 331 indicates the
presence of a suberoyl arginine moiety. The compound 7 was
identified as 3-(N-suberoyl argininyl)-3,14-dihydroxybufa-20,22-
dienolide (bufalitoxin), previously reported from Bufo vulgaris for-
mosus by Shimada et al. (1975, 1976, 1977). The compounds 12, 13,
16, A, B and C were identified on the basis of the MS/MS spectra.
The compound 13 with [MþH] þ 699 differs from compound 14 in
one CH2 unit, showing a fragmentation pattern in agreement for 3-
(N-pimeloyl argininyl) marinobufagin, previously reported from
Bufo marinus (Dictionary of Natural Products on DVD, 2013).
The QTOF-MS spectrum of compound 12 with a [MþH] þ ion at
m/z 685.5116 shows the loss of water and a neutral fragment of 382
from the [MþH] þ peak, leading to the base peak at m/z 303.2126
and a fragment at m/z 175, compatible with arginine. The base peak
of compound 12 (303 amu) differs from that of compounds 7 and 14
(331 amu) by 28 amu, and can be explained by a C6 dicarboxylic
acid unit acting as a linker between the terpene and aminoacid part
of the molecule. The fragmentation pattern of the bufadienolide is
in agreement with that of marinobufagin. Therefore, the compound
12 was assigned as 3-(N-adipoyl argininyl) marinobufagin. The
compound 16 with [MþH] þ at 741.3877 shows a base peak at
359.3, differing from the base peak at 331.3 observed for com-
pounds 7 and 14 in 28 amu. Therefore, the diacid linked to the
arginine should be C10 and the neutral loss fragment requires a
molecular formula of C24H32O5 (400 amu) for the terpene moiety, in
agreement with marinobufagin. The compound 16 differs from
compound 14 in two CH2 units in the diacid and was assigned as
3-(N-sebacyl argininyl) marinobufagin. Compounds 12 and 16 are
described for the first time as natural products.
The QTOF-MS of compounds 7, 14, A, B and C shows loss of
water from the [Mþ H] þ peak and neutral loss of a fragment
leading to the base peak at m/z 331 and the arginine fragment at
m/z 175. The diacid in these compounds is a C8 (suberoyl) unit
acting as a linker between the basic aminoacid and the bufadie-
nolide. Related compounds were reported from Bufo bufo gargar-
izans Cantor by Shimada et al. (1985).
The compound A, with a molecular weight of [Mþ H] þ
729.5521 (calc for C38H56N4O10: 728.3996) showed a neutral loss
of 398 amu [terpene – H2O] in agreement with a steroid moiety
with the molecular formula C24H32O6 (416 amu) leading to a base
peak at [M þH] þ of 331 (suberoyl arginine). The ion at m/z 345,
shows the loss of four water molecules from the steroid moiety
[Mþ H  4H2O] þ , suggesting three hydroxy and one epoxy
functions in the bufadienolide. Compound A differs from 14 in
one additional OH group in the steroid moiety and was tentatively
assigned to 3-(N-suberoyl argininyl)-hydroxmarinobufagin. For
compound B, the [MþH] þ at 731.5519 shows the neutral loss of
400 amu, in agreement with a molecular formula C24H34O6
(418 amu) for the terpene moiety, and a base peak at 331,
characteristic for suberoyl arginine. Compound B was identified
as 3-(N-suberoyl argininyl)-hydroxytelocinobufagin. Compound B
might be originated from opening of the 14,15-epoxy ring of A. The
bufadienolide C with a [Mþ H] þ at 741.3877 presents a base peak
at 331.2043, in agreement with suberoyl arginine and a neutral
loss of 414 amu [steroid-H2O], in agreement with dihydroxymar-
inobufagin C24H32O7 (432 amu). The compound was tentatively
assigned as 3-(N-suberoyl argininyl)-dihydroxymarinobufagin.
However, the exact placement of the additional hydroxy function
of compounds A, B and C cannot be given based on the MS–MS
1079
spectra. The compounds A, B and C have not been previously
described as toad venom constituents.
Fractions 4 and 5, showed a 1H NMR spectrum close to that of
fractions 1–3, bearing characteristic signals for argininyl esters of
bufadienolides. The fraction was analyzed by QTOF-MS and then
treated with diazomethane in dietyl ether to afford the methyl
ester derivatives of the natural products. In the 1H NMR spectrum
of the methylated fraction, the OMe singlet at δ 3.37 supports the
presence of the free COOH function in the aminoacid moiety while
the whole spectrum of the bufadienolide remains unaltered.
The fractions 6–11 were further purified by preparative TLC
(EtOAc:2-propanol:conc NH4OH 9:7:4 v/v/v) to afford compounds
10 (5 mg), 24 (2 mg) and 26 (1 mg). The 1H NMR spectrum of
fractions 12 and 13 showed a main compound (10), presenting the
structural features characteristic of a bufadienolide. The dd at δ
7.91 (1H, J ¼9.6 and 2.4 Hz), a d at δ 7.46 (1H, J ¼1.6 Hz) and a d at
δ 6.28 (1H, J ¼9.6 Hz) clearly indicates an α-pyrone ring while an
m at δ 4.14 (1H) and an s at δ 3.61 (1H) were assigned to H-3 and
H-15, respectively. Two methyl singlets at δ 0.99 and 0.80 are in
agreement with H-19 and H-18, respectively. The QTOF-MS of 10
presented a [MþH] þ peak at 401.3618, whose fragmentation
pattern is in full agreement with that of marinobufagin. Therefore,
10 was identified as 14,15-epoxy-3,5-dihydroxybufa-20,22-dieno-
lide (marinobufagin). Marinobufagin was previously reported from
skin secretions of Bufo rubescens (Cunha Filho et al., 2005). A
second constituent, with [MþH] þ at m/z 205.2051 was identified
by its 1H NMR spectrum as well as QTOF-MS as the alkaloid
bufotenin (24) (C12H16N2O: 204.12626). The MS–MS is in accor-
dance with the proposed structure (Maciel et al., 2003). Suberoyl
arginine was identified by QTOF analysis of the fraction, as a minor
constituent. After preparative TLC of fractions 12 and 13 (silica gel,
EtOAc:2-propanol:conc NH4OH 9:7:4 v/v/v), five zones were
scraped off from the plate and analyzed for alkaloids by QTOF-
MS. The fraction 12-13-2 showed the alkaloid bufotenin 24 and a
second constituent (26) with [M þH] þ at m/z 203.1096, in agree-
ment with dehydrobufotenin (C12H14ON2: 202.1106). Fraction 12-
13-4 yielded a mixture of compounds 24 and 26, while fraction 12-
13-5 contained nearly pure compound 26. The MS spectra are in
agreement with those reported by Maciel et al. (2003).
Fractions 14–21 afforded compound 10 while the pooled frac-
tions 22–25 contained the alkaloids 24–26 and compounds 3 and 4.
The QTOF-MS of compounds 3 and 4 shows [MþH] þ at 331.2058
and 345.2282 amu, respectively and fragmentation leading to
arginine, pointing out to diacid derivatives of arginine. Compounds
3 and 4 were assigned to suberoyl arginine and azelayl arginine,
respectively. While compound 3 was previously reported from Bufo
species by Gao et al. (2010), compound 4 was not described as a
natural product but as part of more complex bufadienolides.
3.2. HPLC–MS–MS analysis
The composition of the toad venom was investigated by
positive and negative mode ionization HPLC–MS–MS. The chro-
matograms in the positive and negative ion mode are shown in
Fig. 1. The results are summarized in Table 1 (A and B). The
structures of the compounds 1–26 are shown in Figs. 2 and 3.
3.2.1. Argininyl diacid derivatives
The HPLC–MS–MS chromatograms of the toad venom showed
the occurrence of compound 3 in the positive ion mode and that of
compounds 1–5 in the negative ion mode. All the compounds 1–5
showed the loss of the diacid leading to the arginine moiety and
were assigned as adipyl (C6 diacid), pimeloyl (C7 diacid), suberoyl
(C8 diacid), azelayl (C9 diacid) and sebacyl (C10 diacid) arginine,
respectively. Suberoyl arginine was previously described from Bufo
1080 G. Schmeda-Hirschmann et al. / Journal of Ethnopharmacology 155 (2014) 1076–1085

Fig. 1. HPLC chromatogram of the Rhinella schneideri venom extract. UV chromatogram at 295 nm and total ionic current (TIC). A. Positive ion mode. B. Negative ion mode.
Compounds identification: 1: Adipyl arginine; 2: Pimeloyl arginine; 3: Suberoyl arginine; 4: Azelayl arginine; 5: Sebacyl arginine; 6: Bufalin; 11: 3-(N-glutaryl argininyl)-
marinobufagin; 7: 3-(N-suberoyl argininyl)-bufalin (Bufalitoxin); 8: 3-(N-azelayl argininyl)-bufalin; 9: 3-(N-sebacyl argininyl)-bufalin; 12: 3-(N-adipoyl argininyl)
marinobufagin; 13: 3-(N-pimeloyl argininyl) marinobufagin; 14: 3-(N-suberoyl argininyl) marinobufagin (Marinobufotoxin); 15: 3-(N-azelayl argininyl marinobufagin; 16:
3-(N-sebacyl argininyl) marinobufagin; 17: 3-(N-undecadienoyl argininyl) marinobufagin; 18: 3-(N-dodecadienoyl argininyl) marinobufagin; 19: Telocinobufagin; 20: 3-(N-
pimeloyl argininyl) telocinobufagin; 21: 3-(N-suberoyl argininyl) telocinobufagin (Telocinobufatoxin); 23a: 3-(N-suberoyl argininyl) hellebrigenin; 22: 3-(N-sebacyl
argininyl) telocinobufagin; 24. Bufotenin; 25: Bufotenidin.

venom (Gao et al., 2010). Compounds 1, 2, 4 and 5 were not
previously reported as natural products but as esters of dicar-
boxylic acid arginine conjugates of bufadienolides.
3.2.2. Bufalin argininyl diacids
The compounds 7–9, detected in the positive-ion MS, showed a
common loss of 369 amu corresponding to [Mþ H H2O] þ and
similar fragmentation patterns, leading to the m/z ion at 175 amu,
corresponding to arginine. Compounds 7–9 differ in the number of
CH2 groups in the diacid and were identified as follows: 3-(N-
suberoyl argininyl)-bufalin (bufalitoxin) (7), isolated from Bufo
vulgaris formosus venom (Dictionary of Natural Products on DVD
2013, Shimada et al., 1977) and 3-(N-sebacyl argininyl)-bufalin (9),
found in Venenum Bufonis by Hu et al. (2011). The compound 8,
described here for the first time as a natural product, shows azelaic
acid as the diester and was identified as 3-(N-azelayl argininyl)-
bufalin based on the MS fragmentation pattern.
argininyl) marinobufagin (16), 3-(N-undecadienoyl argininyl) marino-
bufagin (17) and 3-(N-dodecadienoyl argininyl) marinobufagin (18).
3.2.4. Telocinobufagin argininyl diacids
The genine telocinobufagin was isolated from Rhinella jimi by
Tempone et al. (2008). In the positive ion-MS, following the loss of
384 amu corresponding to [M þH H2O] þ , further fragmentation
of compounds 20–22 lead to the arginine ion, allowing to
differentiate the compounds by the number of CH2 units of the
diacid linker. Compounds 20–22 present a diacid with 5, 6 or 8
CH2 units, allowing the assignation of the compounds as pimeloyl,
suberoyl, and sebacyl argininyl derivatives of telocinobufagin. The
compound 21, 3-(N-suberoyl argininyl)-telocinobufagin (Telocino-
bufotoxin) was previously reported from Venenum Bufonis (Hu et
al., 2011) and from Bufo marinus (Dictionary of Natural Products on
DVD, 2013). Compounds 20 and 22 are reported here for the first
time as natural products.

3.2.5. Hellebrigenin argininyl diacids

3.2.3. Marinobufagin argininyl diacids
A second group of argininyl diacids include the compounds
11–18, all of them derivatives of marinobufagin 10, isolated from
Rhinella schneideri venom. The QTOF-MS analysis of marinobufagin
was reported by Cunha Filho et al. (2005) and our data are in
agreement with the literature. The compounds 11–18 were detected
in the positive ion-HPLC-MS, following the common loss of 383 amu
corresponding to [MþH H2O] þ . The compounds showed a similar
fragmentation pattern, leading to the arginine m/z ion at 175 amu, and
differing in the diacid moiety. While compounds 13 and 14 were
already described as 3-(N-pimeloyl argininyl) marinobufagin (13) from
Bufo marinus (Dictionary of Natural Products on DVD, 2013) and 3-(N-
suberoyl argininyl) marinobufagin (Marinobufotoxin) (14) from Bufo
marinus and Bufo ictericus (Dictionary of Natural Products on DVD,
2013), compounds 12, 15–18 are reported here for the first time. The
compounds were identified as 3-(N-adipoyl argininyl) marinobufagin
(12), 3-(N-azelayl argininyl) marinobufagin (15), 3-(N-sebacyl
Hellebrigenin is a genine occurring in several toad venoms (Gao
et al., 2008). In our sample, one of the minor constituents
(compound 23a) was tentatively identified as 3-(N-suberoyl argi-
ninyl) hellebrigenin. The structure follows from the [M-H]– ion at
727.6 and the loss of 398 amu, leading to the argininyl suberoyl
moiety at m/z 329.1 amu.
3.2.6. Alkaloids
The alkaloids 24 and 25 were detected in the positive ion mode
analysis of the venom, in agreement with the NMR studies and
QTOF-MS analysis of the fractions.
3.3. Compounds
3.3.1. Argininyl diacids
Compound 1: Adipyl arginine. Molecular formula: C12H22N4O5;
̄
HPLC–MS–MS [M  H]: 300.9; 300.9 (100), 240.8 (12).
 G. Schmeda-Hirschmann et al. / Journal of Ethnopharmacology 155 (2014) 1076–1085 1081

Table 1
Tentative identification of Rhinella schneideri toad venom constituents by HPLC–MS–MS in the positive (A) and negative ion mode (B).

(A) Positive ion mode.

Rt (min) [MþH] þ MS/MS Tentative identification

7.4 204.8 159.6, 57.4 Bufotenin 24

7.5 218.8 159.6 Bufotenidin 25
9.4 660.8 331.2 Suberoyl arginine 3 [2 M þH] þ
12.0 387.4 351.2 Bufalin 6
21.9 671.7 653.4, 289.1 3-(N-glutaryl argininyl)-marinobufagin 11
22.0 715.7 697.6, 331.3, 278.1 3-(N-suberoyl argininyl) telocinobufagin (Telocinobufatoxin) 21
22.8 701.5 683.6, 317.2 3-(N-pimeloyl argininyl) telocinobufagin 20
23.0 685.6 667.5, 303.2, 250.8 3-(N-adipoyl argininyl) marinobufagin 12
23.9 699.6 681.5, 317.2, 331.3 3-(N-pimeloyl argininyl) marinobufagin 13
24.9 713.7 695.6, 331.3, 278.0 3-(N-suberoyl argininyl) marinobufagin (Marinobufotoxin) 14
25.8 727.7 709.5, 345.3, 343.3 3-(N-azelayl argininyl) marinobufagin 15
27.4 741.7 723.6, 359.3, 278.1 3-(N-sebacyl argininyl) marinobufagin 16
27.5 699.7 681.6, 331.3 3-(N-suberoyl argininyl)-bufalin (Bufalitoxin) 7
27.9 403.2 349.2, 367.2, 350.2, 271.0 Telocinobufagin 19
28.8 755.7 737.6, 373.4 3-(N-undecadienoyl argininyl) marinobufagin 17
28.9 713.7 695.6, 345.4 3-(N-azelayl argininyl)-bufalin 8
30.1 769.7 751.7, 387.4, 306.3 3-(N-dodecadienoyl argininyl) marinobufagin 18
30.2 727.8 709.7, 359.4, 279.0 3-(N-sebacyl argininyl)-bufalin 9

(B) Negative ion mode

̄
Rt (min) [M  H] MS/MS Tentative identification

̄
5.2 603.0 300.9, 240.8 [2M  H] Adipyl arginine 1
̄
6.8 631.0 315.0 [2M  H] Pimeloyl arginine 2
̄
9.2 659.0 329.1 [2M  H] Suberoyl arginine 3
10.3 357.2 312.0, 172.5 Sebacyl arginine 5
12.9 343.2 301.0, 172.6 Azelayl arginine 4
22.5 683.7 641.5, 301.0, 240.8 3-(N-adipoyl argininyl) marinobufagin 12
22.8 699.5 657.4, 315.0, 655.3, 272.8 3-(N-pimeloyl argininyl) telocinobufagin 20
23.4 727.6 685.5, 329.1, 268.9 3-(N-suberoyl argininyl) hellebrigenin 23a
23.6 697.6 655.4, 315.1 3-(N-pimeloyl argininyl) marinobufagin 13
23.8 713.5 671.4, 329.1, 287.0, 268.9 3-(N-suberoyl argininyl) telocinobufagin (Telocinobufatoxin) 21
24.5 711.5 669.4, 329.2, 287.1, 270.1 3-(N-suberoyl argininyl) marinobufagin (Marinobufotoxin) 14
25.8 725.4 683.4, 343.1, 684.3 3-((N-azelayl argininyl) marinobufagin) 15
26.3 741.5 357.1, 699.5, 340.2, 315.1, 297.1 3-(N-sebacyl argininyl) telocinobufagin 22
27.5 739.5 697.6, 357.2, 315.1, 297.0 3-(N-sebacyl argininyl) marinobufagin 16
27.5 697.6 655.6, 329.1 3-(N-suberoyl argininyl)-bufalin (Bufalitoxin) 7

Compound 2: Pimeloyl arginine. Molecular formula:
̄
C13H24N4O5; HPLC–MS–MS [M  H]: 315.0.
Compound 3: Suberoyl arginine. Molecular formula:
C14H26N4O5; QTOF–MS–MS [MþH] þ : 331.2058; 331 (19), 272
̄
(26), 250 (100), 175 (68), 158 (71); HPLC–MS–MS [M  H]: 329.1.
Compound 4: Azelayl arginine. Molecular formula: C15H28N4O5;
̄
HPLC–MS–MS [M  H]: 343.2; 301.0 (100), 172.6 (28). QTOF–MS–
þ
MS [Mþ H] : 345.2282.
Compound 5: Sebacyl arginine. Molecular formula: C16H30N4O5;
̄
HPLC–MS–MS [M  H]: 357.2; 312.0 (100), 172.5 (11).
3.3.2. Bufalin argininyl diacid derivatives
Compound 6: 3,14-dihydroxybufa-20,22-dienolide (Bufalin).
Molecular formula: C24H34O4; HPLC–MS–MS [Mþ H] þ : 387.4;
351.2 (100).
Compound 7: 3-(N-suberoyl argininyl)-bufalin (Bufalitoxin).
Molecular formula: C38H58N4O8; QTOF–MS–MS (40 eV) [MþH] þ :
699.5363 (22), 681.5162 (22), 331.2511 (100), 317.2320 (65),
250.1973 (56), 175.1503 (52). HPLC–MS–MS [MþH] þ : 699.7;
681.6 (100), 331.3 (31); HPLC–MS–MS [M  H]-: 697.5; 655.6
(100), 329.1 (31). 1H NMR: δ 8.01 (dd, J ¼9.6 and 2.4 Hz, H-22), δ
7.46 (d, J ¼1.6 Hz, H-21), δ 6.31 (d, J ¼9.6 Hz, H-23), δ 5.25 m, δ
5.04 (br s, NH), δ 4.30 (dd, J¼ 6.8, 5.2 Hz, H-3), δ 2.61 (d, J ¼10.4,
H-17), δ 2.26 (t, J ¼7.2), δ 2.15 (t, J ¼7.2), δ 1.40-1.68 (m), δ 1.16-1.38
(m), 0.98 s (H-19), 0.74 s (H-18).
Compound 8: 3-(N-azelayl argininyl)-bufalin. Molecular for-
mula: C39H60N4O8; HPLC–MS–MS [Mþ H] þ : 713.7; 695.6 (100),
345.4 (11).
Compound 9: 3-(N-sebacyl argininyl)-bufalin Molecular for-
mula: C40H62N4O8; HPLC–MS–MS [Mþ H] þ : 727.8; 709.7 (100),
359.4 (15), 279.0 (4).
3.3.3. Marinobufagin argininyl diacid derivatives
Compound 10: 14,15-epoxy-3,5-dihydroxybufa-20,22-dienolide
(marinobufagin); Molecular formula: C24H32O5; QTOF–MS–MS
(20 eV) [M þH] þ : 401.3618; 401 (100), 383 (12), 365 (40), 347
(30), 339 (22), 319 (12), 269 (17), 253 (20), 159 (23). Marinobufagin
acetate (compound 10a: QTOF–MS–MS: 907.7390 [2 MþNa] þ ;
465.3669 [M þNa] þ ; 443.3846 [MþH] þ .
Compound 11: 3-(N-glutaryl argininyl)-marinobufagin. Molecu-
lar formula: C36H54N4O8; HPLC–MS–MS [M þH] þ : 671.7; 653.4
(60), 289.1 (100).
Compound 12: 3-(N-adipoyl argininyl) marinobufagin. Molecu-
lar formula: C36H52N4O9; exact mass: 684.3734; QTOF–MS–MS
[MþH] þ : (40 eV): 685.5116 (19), 667.5118 (13), 655.5584 (13),
331.2511 (19), 303.2126 (100), 250.1591 (21), 175.1567 (16); HPLC–
MS–MS [Mþ H] þ : 685.6; 667.5 (67), 303.2 (100), 250.8 (5); HPLC–
MS–MS [M  H]-: 683.7; 641.5 (100), 301.0 (99), 240.8 (34).
Compound 13: 3-(N-pimeloyl argininyl) marinobufagin. Mole-
cular formula: C37H54N4O9; QTOF–MS–MS [M þH] þ : 699.5363;
1082 G. Schmeda-Hirschmann et al. / Journal of Ethnopharmacology 155 (2014) 1076–1085

HPLC–MS–MS [MþH] þ : 699.6; 681.5 (86), 317.2 (100), 331.3 (26);

Fig. 2. Compounds identified in Rhinella (Bufo) schneideri toad venom.
̄
HPLC–MS–MS [M  H]: 697.6; 655.4 (44), 315.1 (100).
Compound 14: 3-(N-suberoyl argininyl) marinobufagin (Mari-
nobufotoxin). Molecular formula: C38H56N4O9; QTOF–MS–MS
(40 eV) [MþH] þ : 713.4387 (100), 695.4443 (4), 331.2131 (56),
278.1512 (10), 175.1186 (5); HPLC–MS–MS [MþH] þ : 713.7; 695.6
(75), 331.3 (100), 278.0 (12); HPLC–MS–MS [M  H]-: 711.5; 669.4
(45), 329.2 (1 0 0), 287.1 (24), 270.1 (9). 1H NMR: δ 7.92 (dd, J ¼10.0
and 2.8 Hz, H-22), δ 7.48 (d, J ¼2.0 Hz, H-21), δ 6.30 (d, J¼ 10.0 Hz,
H-23), δ 5.16 (br s, NH), δ 4.30 (dd, J ¼6.8, 5.2 Hz, H-3), δ 3.64 (s,
H-15), δ 2.61 (d, J¼ 10.4, H-17), Diacid –CH2- δ 2.41 m, 2.34-2.36
(brt, J ¼7.2), δ 2.27 (t, J ¼7., H.4), δ 1.40-1.68 (m), δ 1.16-1.38 (m),
Arginine δ 3.20-3.25 m; 1.01 s (H-19), 0.81 s (H-18).
Compound 15: 3-(N-azelayl argininyl) marinobufagin. Molecu-
lar formula: C39H58N4O9; HPLC–MS–MS [M þH] þ : 727.7; 709.5
̄
(40), 345.3 (100), 343.3 (25); HPLC–MS–MS [M  H]: 725.4 683.4
(55), 343.1 (100), 684.3 (51).
Compound 16: 3-(N-sebacyl argininyl) marinobufagin. Molecu-
lar formula: C40H60N4O9; QTOF MS–MS (40 eV) [Mþ H] þ : 741.3877
(55), 723.4843 (16), 359.2509 (100), 331.1955 (32), 175.1378 (33).
HPLC–MS–MS [Mþ H] þ : 741.7; 723.6 (54), 359.3 (100), 278.1 (11).
̄
HPLC–MS–MS [M  H]: 739.4; 697.6 (90), 357.2 (100), 315.1 (15),
297.0 (30).
Compound 17: 3-(N-undecadienoyl argininyl) marinobufagin.
Molecular formula: C41H62N4O9; HPLC–MS–MS [MþH] þ : 755.7;
737.6 (77), 373.4 (100).
Compound 18: 3-(N-dodecadienoyl argininyl) marinobufagin.
Molecular formula: C42H64N4O9; HPLC–MS–MS [Mþ H] þ : 769.7;
751.7 (54), 387.4 (100), 306.3 (11).

O 3.3.4. Telocinobufagin argininyl diacid derivatives

O Compound 19: Telocinobufagin. HPLC–MS–MS [Mþ H] þ : 403.2;
349.2 (100), 367.2 (42), 350.2 (25), 271.0 (7).
Bufalin argininyl diacids Compound 20: 3-(N-pimeloyl argininyl) telocinobufagin. Mole-
̄
cular formula: C37H56N4O9; HPLC–MS–MS [M  H]: 699.5; 657.4
n Diacid
OH (97), 315.0 (100), 655.3 (44), 272.8 (55). HPLC–MS–MS [M þH] þ :
O 701.5; 683.6 (95), 317.2 (100).
H 7 6 suberic acid
n(H2C) O 8 7 azelaic acid
Compound 21: 3-(N-suberoyl argininyl) telocinobufagin (Telo-
HN NH2 cinobufatoxin). Molecular formula: C38H58N4O9; HPLC–MS–MS
HN O 9 8 sebacic acid ̄
HN [M H]: 713.5; 671.4 (54), 329.1 (100), 287.0 (21), 268.9 (22);
COOH
HPLC–MS–MS [MþH] þ : 715.7; 697.6 (96), 331.3 (100), 278.1 (19).
O Marinobufagin argininyl Compound 22: 3-(N-sebacyl argininyl) telocinobufagin. Mole-
̄
O diacids cular formula: C40H62N4O9; HPLC–MS–MS [M H]: 741.5; 357.1
(100), 699.5 (20), 340.2 (6), 315.1 (27), 297.1 (9).
n Diacid
11 3 glutaric acid
12 4 adipic acid
3.3.5. Hellebrigenin argininyl diacid derivatives
O
13 5 pimelic acid Compound 23a: 3-(N-suberoyl argininyl) hellebrigenin. Mole-
O ̄
OH
14 6 suberic acid cular formula: C39H60N4O9; HPLC–MS–MS [M  H]: 727.6; 685.5
n(H2C) O
(100), 329.1 (31), 268.9 (13).
15 7 azelaic acid
HN NH2
HN O 16 8 sebacic acid
HN 17 9 undecadienoic acid
COOH
18 10 dodecadienoic acid 3.3.6. Alkaloids
Compound 24. Bufotenin. HPLC–MS–MS [M þH] þ : 204.8; 159.6
(100), 57.4 (44); QTOF–MS–MS: [M þH] þ : 205.1263; 205 (100),
O
160 (100).
O
Compound 25: Bufotenidin (BTD). HPLC–MS–MS [Mþ H] þ : 219
Telocinobufagin argininyl
(100), 160 (66).
diacids
Compound 26: Dehydrobufotenin. QTOF–MS–MS: [M þH] þ :
203.1227; 203 (55), 188 (100), 173 (6).
OH
O n Diacid
OH
20 5 pimelic acid
n(H2C) O 3.3.7. Polihydroxy argininyl diacid derivatives
21 6 suberic acid
HN NH2 Compound A. 3-(N-suberoyl argininyl)-hydroxymarinobufagin.
HN O 22 8 sebacic acid
HN Molecular formula: C38H56N4O10, QTOF–MS–MS (40 eV) [M þH] þ :
COOH
729.4277 (51), 711.4509 (18), 345.2379 (33), 331.2043 (100),
Fig. 3. Argininyl diacid derivatives from Rhinella schneideri bufadienolides. 278.1674 (16), 175.1122 (8).
 G. Schmeda-Hirschmann et al. / Journal of Ethnopharmacology 155 (2014) 1076–1085
Compound B. 3-(N-suberoyl argininyl)-hydroxytelocinobufagin.
Molecular formula: C38H58N4O10, QTOF–MS–MS (45 eV) [MþH] þ :
731.4405 (91), 713.4128 (10), 331.2131 (100), 278.1674 (20),
175.1186 (13).
Compound C. 3-(N-suberoyl argininyl)-dihydroxymarinobufa-
gin. Molecular formula: C38H56N4O11, QTOF–MS–MS (45 eV):
745.4355 (44), 727.4439 (14), 379.2471 [MþH  3H2O] (5),
331.2043 (100), 278.1674 (17), 250.1729 (9), 175.1314 (11).
3.4. Bufadienolides and alkaloids in Rhinella schneideri venom
From the parotid gland venom of the Brazilian Pantanal toad
Rhinella schneideri, 29 compounds were identified or tentatively
identified by a combination of spectroscopic and spectrometric
means. The venom showed as main compounds the bufadieno-
lides 7, marinobufagin 10 and marinobufotoxin 14 but also several
minor compounds differing in the homologous diacid side chain
linking arginine and the bufadienolide as well as in the number of
hydroxy groups in the steroidal moiety. Kamano et al. (1968)
described marinobufagin 10 from the crude drug Ch'an Su.
Compound 14 was previously reported by Linde-Tempel (1970)
and Shimada et al. (1976). Compounds 10 and 14 were synthesized
by Pettit, Kamano (1974). A comprehensive 1H and 13C NMR
chemical shift assignment for bufadienolides from the Chinese
drug Ch'an Su was reported (Kamano et al., 2001). Shimada et al.
(1977) pointed out to the possible occurrence of other homologous
diacid side chain linking arginine and marinobufagin in the living
animals. In a careful study of the crude drug, Shimada et al. (1977)
reported the occurrence of several diacid conjugates of arginine,
including the 3-suberoyl arginine ester of bufogenin and the
3-pimeloyl, 3-adipoyl and 3-succinoyl arginine esters of bufalin.
The related compound bufalin 3-succinoylarginine ester was
isolated from the skin of the Japanese toad Bufo vulgaris formosus
Boulenger (Shimada et al., 1975). In a study undertaken with the
dried body of the Korean toad Bufo bufo gargarizans Cantor,
Shimada et al. (1985) described 3-succinyl, 3-adipoyl, 3-pimeloyl
and 3-suberoyl arginine esters of cinobufagin. Compounds 3, 6 and
10 were reported as constituents of the Chinese crude drug
Chansu (Gao et al., 2010). The alkaloids bufotenin (24), bufotenidin
(25) and dehydrobufotenin (26) were previously identified in the
parotid glands secretions of Bufo (Rhinella) schneideri and several
Brazilian Rhinella (Bufo) species by Maciel et al. (2003).
Three additional suberoyl argininyl esters of hydroxymarino-
bufagin, hydroxytelocinobufagin and dihydroxytelocinobufagin,
namely compounds A, B and C, were tentatively identified based
on QTOF–MS–MS analysis. The exact position of the hydroxy group
of the three minor compounds, remains to be established.
Table 2
Antiproliferative activity of Rhinella schneideri crude extract and fractions expressed as IC50 values (mg/mL 7 SEM).
a b
MRC-5 AGS
Crude 0.0417 0.003 0.0767 0.004
Fr. 1-3 0.1267 0.008 0.1837 0.007
Fr. 4-5 0.2357 0.016 0.3127 0.018
Fr. 6-8 0.1007 0.005 0.1677 0.110
Fr. 9-11 0.022 7 0.001 0.0407 0.002
Fr. 12-13 0.0197 0.001 0.0357 0.002
Fr. 14-21 0.026 7 0.001 0.0427 0.002
Etoposide 2.296 7 0.115 0.2777 0.011
Camptothecin 0.0026 7 0.0003 0.00777 0.0006
Vinblastine 0.0025 7 0.0003 0.0025 7 0.0001
a
Normal lung MRC-5 fibroblasts.
b
Human epithelial gastric cells AGS.
c
SK-MES-1 lung cancer cells.
d
J82 bladder carcinoma cells
e
Promyelocytic leukemia HL-60 cells. Each concentration was tested in quadruplicate and repeated three times in separate experiments.
1083
In the Chinese crude drug Ch’an Su, the HPLC pattern of the
venom shows three differential zones, characterized by the elution of
suberoyl arginine in the first third of the chromatogram, followed by
the bufadienolide genines with different oxidation patterns and the
diacid esters of bufadienolides at the end of the chromatographic
run. In the Pantanal toad, the simple alkaloids and argininyl diesters
elutes first, followed by the argininyl diacid derivatives of the
bufadienolides bufalin, marinobufagin and telocinobufagin. Liquid
chromatography/electrospray ionization tandem mass spectrometry
is an efficient method for the analysis of constituents in the Chinese
crude drug Venenum Bufonis (Hu et al., 2011) as well as to compare
the composition of different Bufo (Rhinella) venoms (Gao et al., 2010)
or for dosage methods of marinobufagenin in biofluids (Lenaerts et
al., 2013). In the Chinese (Chan Su) and Japanese (Sen So) crude toad
venom, large variation in the oxidation pattern of the bufadienoides
was observed. In the Chinese crude drug (Gao et al., 2010), sulfate,
acetate and C8 diacids (suberoyl) were the main esters of the
bufadienolides. The composition of the Brazilian Rhinella schneideri
venom show differences with that of the Chinese crude drug Ch'an
Su in the identity of the minor components as well as in the
oxidation patterns of the main compounds.
Looking for novel bufadienolides with relevant biological
activities, Ye et al. (2004, 2005) used the fungi Mucor spinosus
and Mucor polymorphosporus to modify bufalin and resibufogenin,
respectively. The microbial oxidation products were assessed for
cytotoxicity and the authors suggest that 3-OH glycosylation or
hydroxylation at C-1β or C-12β might be relevant to afford more
polar bufadienolides with higher cytotoxicity.
The antimicrobial effect of the bufadienolides telocinobufagin
and marinobufagin was published by Cunha Filho et al. (2005). The
compounds were characterized in the parotid secretion from Bufo
rubescens (Rhinella rubescens) after purification by RP-HPLC and
QTOF and NMR analysis. Both compounds presented antimicrobial
effect with MIC of 128 mg/mL against Staphylococcus aureus ATCC
25213 and MICs of 64 and 16 mg/mL towards Escherichia coli ATCC
25922, respectively. The MICs for the reference compounds amox-
icillin, iminipenem and trimethoprim against Escherichia coli were
o22, 14 and 40 mg/mL, respectively. Trimetoprim, used as refer-
ence against Staphylococcus aureus presented an MIC o160 mg/mL.
Both compounds show an interesting effect against Escherichia coli.
Furthermore, bufadienolides from Rhinella jimi were assessed for
trypanocidal effect against Trypanosoma cruzi trypomastigotes and
Leishmania chagasi promastigotes (Tempone et al., 2008). The
compounds were isolated from the toad venom by HPLC and were
identified by mass spectrometry and NMR spectroscopy analysis.
The bufadienolides telocinobufagin and hellebrigenin did not
induced hemolysis up to 200 mg/mL and reduced growth of
c d e
SK-MES-1 J82 HL-60
0.093 7 0.065 0.086 7 0.004 0.1027 0.005
0.1947 0.009 0.1347 0.008 0.1737 0.011
0.3537 0.021 0.2457 0.017 0.5217 0.026
0.2107 0.015 0.1837 0.011 0.234 7 0.014
0.0647 0.003 0.056 7 0.004 0.054 7 0.002
0.028 7 0.005 0.0427 0.003 0.0447 0.002
0.082 7 0.003 0.0777 0.005 0.052 7 0.003
1.295 7 0.078 1.884 7 0.075 1.059 7 0.002
0.0059 7 0.0004 0.00217 0.0002 0.00457 0.0004
0.00137 0.0001 0.0081 7 0.0006 0.00037 0.00002
1084 G. Schmeda-Hirschmann et al. / Journal of Ethnopharmacology 155 (2014) 1076–1085
Leishmania chagasi with IC50 of 61.21 and 126.2 mg/mL, respec-
tively. The latter activity is not related to the ethnopharmacologi-
cal data on the “cururu” frog.
3.5. Antiproliferative activity
The crude venom and the CC fractions of the venom were
assessed for antiproliferative effect using the MTT reduction assay
in five different human cell lines: normal lung fibroblasts (MRC-5),
human epithelial gastric (AGS), lung cancer (SK-MES-1), bladder
carcinoma (J82) and promyelocytic leukemia (HL-60) cells. Etopo-
side, camptothecin and vinblastine were used as reference anti-
proliferative compounds. The concentrations of the extract/
fractions inhibiting cell growth by 50% (IC50 values, mg/mL) were
obtained adjusting the dose-response curves to a sigmoidal model.
The results are summarized in Table 2.
The crude venom showed a strong antiproliferative effect
towards all the cell lines, with IC50 values ranging from 0.041 to
0.102 mg/mL. After chromatography, the fractions 1–3, 4 and 5 and
6–8 presented less effect than the crude venom. The fractions 9–
13, however, showed a remarkable antiproliferative effect, with
IC50 values in the range 0.019–0.022, 0.035–0.040, 0.028–0.064,
0.042–0.056 and 0.044–0.052 mg/mL for MRC-5, AGS, SK-MES-1,
J82 and HL-60 cell lines, respectively. Under the same experi-
mental conditions, IC50 values (in mg/mL) of etoposide towards the
different cell lines were: 2.296 (MRC-5), 0.277 (AGS), 1.295 (SK-
MES-1), 1.884 (J82) and 1.059 (HL-60). Camptothecin and vinblas-
tine were more active with IC50 values in the range of 0.0021–
0.0077 and 0.0003–0.0081 mg/mL, respectively. In spite of relevant
antiproliferative effect, no selectivity is observed for the toad
venom, fractions or reference compounds. While the less effective
fractions 1–3 and 4 and 5 contained mainly the higher molecular
weight argininyl diesters of bufadienolides, the most active frac-
tions 9–13 and 14–21 yielded marinobufagin 10 as the main
compound with alkaloids 24 and 26 as minor constituents.
The aqueous extract of the skin and parotid glands of Bufo bufo
gargarizans was shown to inhibit cell proliferation of the liver
carcinoma cell lines HepG2 and Bel-7402 (Qi et al., 2010). The
aqueous extract, as used in traditional medicine, induces apoptosis
through a disruption of the mitochondrial membrane potential.
However, in this report, the concentration of solubles is not given.
The total crude drug (20 g) to the final volume after concentration
to 1 ml was referred as 20 g crude drug/mL for the experiments (Qi
et al., 2010). When expressed as mg crude drug/mL, IC50 values for
HepG2 cells at 24, 48 and 72 h were 0.20, 0.06 and 0.02 mg/mL,
respectively while for Bel-7402 cells, the values were 0.15, 0.06
and 0.02 mg/mL, respectively. No reference compound was used in
this study. Further work on the crude drug and the main active
bufadienolides from the crude drug (Qi et al., 2011) showed a
relevant apoptosis-inducing effect of cinobufacini with the follow-
ing IC50 values (in parentheses): human leukemia U937 (1.36 mg/
mL, 24 h), human leukemia HL-60 (0.25 mg/mL, 48 h), human
hepatocellular carcinoma Bel-7402 and HepG2 (80 mg/mL, 48 h).
However, all the structures of bufadienolides shown in the report
of Qi et al. (2011) are wrong.
Giri et al. (2006) reported the cytotoxic, antiproliferative and
apoptosis-inducing effect of extracts from the Indian toad Bufo
melanostictus using human myeloid leukemia U937 and K562
cells. The toad skin was extracted with methanol, with 1.3% w/w
yield. The extract induces cell arrest at the G1 phase of the cell
cycle and showed an IC50 value of 55.0–66.2 mg/mL for U937 and
81.1–92.2 mg/mL for K562 cells, respectively. The antiprolifera-
tive effect of the Brazilian Rhinella marina and Rhaebo guttatus
venom was reported by Ferreira et al. (2013). From Rhinella
marina, the bufadienolides telocinobufagin, marinobufagin,
bufalin and resinobufogenin were identified by HPLC-MS
analysis and assessed for cytotoxicity. According to the authors,
Rhaebo guttatus yielded only marinobufagin. The antiprolifera-
tive effect of five naturally occurring bufadienolides from the
parotid gland secretion of Rhinella schneideri (captured near
Brasilia) was reported by Cunha-Filho et al. (2010). The authors
prepared several semisynthetic derivatives and found that all
natural and synthetic compounds presented moderate to strong
activity towards human HL-60, SF-295, MDA-MB-435 and HCT-8
cancer cells.
4. Conclusions
Our study demonstrated that argininyl diacid esters of bufa-
dienolides are the main constituents of the Rhinella schneideri
venom and that the most active antiproliferative compounds are
bufadienolides. Marinobufagin was associated with higher activity
but the presence of alkaloids as minor constituents in the same
fractions might play a role in the effect. The strong antiprolifera-
tive activity of Rhinella schneideri crude venom extract and frac-
tions, associated with the identification of constituents, supports
the traditional claim of using this toad to treat cancer. However,
further pharmacological studies are required to disclose the
potential of the “cururu” toad venom in antitumor therapy. The
new compounds detected and identified in the venom pointed out
to the relevance of new investigations on South American amphi-
bians, using updated techniques and combining spectroscopic and
spectrometric methods with working hypothesis based on tradi-
tional uses of natural resources.
Acknowledgments
We are grateful to Conselho Nacional de Desenvolvimento Cientí-
fico e Tecnológico (CNPq-Brazil) and FONDECYT No. 1110054 (Chile)
for financial support.
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