Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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Journal of Ethnopharmacology
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Antitumoural effect of Synadenium grantii

Hook f. (Euphorbiaceae) latex
Thais Latansio de Oliveira a, Antônio Carlos Mattar Munhoz a, Bruna Mikulis Lemes a,
Bruno Rodrigo Minozzo a, Angelita Nepel b, Andersson Barison b, Giovani Marino Fávero c,
Eduardo Bauml Campagnoli d, Flávio Luís Beltrame a,n
a
Laboratory of Pharmaceutical Technology, State University of Ponta Grossa, General Carlos Cavalcanti Avenue 4748, Ponta Grossa 84030-900, Paraná, Brazil
b
Nuclear Magnetic Resonance Centre, Federal University of Paraná, Curitiba, Paraná, Brazil
c
Department of General Biology, State University of Ponta Grossa, Ponta Grossa, Paraná, Brazil
d
Laboratory of Histopathology, State University of Ponta Grossa, Ponta Grossa, Paraná, Brazil

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

Article history: Ethnopharmacological relevance: Synadenium grantii Hook f. has traditionally been used to treat various
Received 3 April 2013 neoplastic diseases in southern Brazil.
Received in revised form Aim of study: Evaluation of the antitumoural potential of Synadenium grantii latex against B16F10
4 August 2013
melanoma cell line using in vitro and in vivo models, as well as a phytochemical study of the latex.
Accepted 16 August 2013
Materials and methods: The in vitro antitumoural activity was performed using MTT and trypan blue
assays with different latex concentrations (1.7 mg–7.0 mg/well and 1.22 mg–4.88 mg/well). Flow cytome-
Keywords: try was used to determine the progression of the cell cycle. The in vivo activity was performed by
Synadenium grantii subcutaneously injecting melanoma cells in the dorsum of C57BL6 mice, followed by treating the mice
Euphorbiaceae
with a popular form of use of the latex (garrafada) administered orally. After sacrificing the animals,
Antitumoural
histological analysis of the organs was performed by hematoxylin-eosin staining. The phytochemical
Cytotoxicity
Euphol study of the latex was performed by NMR and chromatographic procedures and the extracts and isolated
Citrostadienol substances were evaluated by IR, 1D and 2D NMR analysis.
Results: The Synadenium grantii latex exhibited decreased cell viability of the melanoma line in a
concentration and time-dependent manner, and also cell cycle arrest in the S-G2/M phase. The latex
caused a 40% reduction in the volume of tumours of the mice with melanomas. Histological examination
of the organs of these animals showed no differences between groups. The phytochemical investigation
resulted in the isolation and identification of triterpene euphol and the steroid citrostadienol, which
were tested against the strain of melanoma. Euphol showed no antitumoural activity, while the steroid
citrostadienol showed reduced cytotoxic activity.
Conclusion: The Synadenium grantii latex presented in vitro and in vivo cytotoxic effects with anti-
tumoural activity against B16F10 melanoma cells.
& 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Bittner et al., 2001; Souza et al., 2005; Rajesh et al., 2006; Rogério
et al., 2007).
The Euphorbiaceae family is complex and heterogeneous and The Synadenium genus, which belongs to this family, has been
contains approximately 300 genera and 7000 species that have linked to some pharmacological properties such as antitumoural
been identified worldwide (Bittner et al., 2001). Previous phyto- (Premaratna et al., 1981), anti-inflammatory (Souza et al., 2005),
cemical investigations of this family, mainly in the search for new fibrinolytic action (Rajesh et al., 2006) and immunoregulation
biologically active compounds, have revealed the presence of (Rogério et al., 2007). Nogueira et al., (2008) demonstrated that
flavonoids, saponins, terpenes, esters, alkaloids, cyanogenic glyco- the ethanol extract of the leaves of Synadenium umbellatum is an
sides, tannins, lectins and glycoproteins (Premaratna et al., 1981; effective antitumoural agent and in vivo it presents an anti-
angiogenic effect. Chloroform extract of the leaves of Synadenium
grantii was recently found (Hassan et al., 2012) to have cytotoxic
activity in human lung fibroblast strain.
n
Corresponding author. Tel.: þ 55 42 3220 3782, þ 55 42 3223 5020. Synadenium grantii Hook f. (the object of the present study) is
E-mail address: flaviobeltra@gmail.com (F.L. Beltrame). widely used by the population of southern Brazil and is popularly

0378-8741/$ - see front matter & 2013 Elsevier Ireland Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jep.2013.08.033

Please cite this article as: de Oliveira, T.L., et al., Antitumoural effect of Synadenium grantii Hook f. (Euphorbiaceae) latex. Journal of
Ethnopharmacology (2013), http://dx.doi.org/10.1016/j.jep.2013.08.033i
2 T.L. de Oliveira et al. / Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

known as tiborna or cola-nota (Oliveira et al., 2005). Its latex is were evaluated and pooled according to TLC analysis based on the
used empirically in the treatment of various diseases such as similarity of the retention factor Rf of the spots that were
allergies, gastric disorders, and especially, cancer (Ortêncio, 1997). observed. The FH sub-fraction 46–59 (0.45 g, 5.6%, w/w) was
In the east of the State of Paraná, people use Synadenium grantii subjected to flash chromatography using elution mode and this
latex as a home preparation named garrafada. They mix 18 drops procedure resulted in substance 1 (citrostadienol—0.150 g, chloro-
of collected latex in 1 L of water, keep it in the refrigerator, and form:ethyl acetate, 5:5, v/v). Similarly, the FH sub-fraction 1–26
take a glass of this solution 3 times a day. Like other species of the (0.96 g, 12%, w/w), was partitioned once more into CC with 35–70
Euphorbiaceae family, it is known that its latex is rich in nonpolar mesh silica, generating 142 sub-fractions. From the sub-fraction
compounds (Costa et al., 2012; Hassan et al., 2012) and that it 118–122 (0.12 g, 12.5%, w/w), after chromatography using the flash
demonstrates activity against tumour cells (Block et al., 2005; elution mode, substance 2 (euphol—0.030 g, hexane: dichloro-
Jassbi, 2006; Aliabadi et al., 2009). methane, 7:3, v/v) was obtained. The structures of the compounds
Therefore, the aim of this work was to evaluate the in vitro and were determined by extensive analysis of IR, 1D and 2D NMR data,
in vivo antitumoural activity of the latex of Synadenium grantii as as well as by comparison with literature data (Zhang et al., 2006;
well as to determine the chemical composition of the latex that Moreira et al., 2010).
could be responsible for this activity.
2.4. Cytotoxicity assays

2. Materials and methods

2.4.1. Tumour lineage
B16F10 melanoma cells were used from cultures maintained in
2.1. Reagents and equipment
the laboratory of the State University of Ponta Grossa, through
continuous expansion in RPMIs 1640 supplemented with 10%
Ethanol (EtOH), methanol (MeOH), ethyl acetate (AcOEt), chloro-
foetal bovine serum (FBS), penicillin and amikacin in an incubator
form (CHCl3), n-hexane (Hex) and xylene were purchased from
at 37 1C with humidified atmosphere containing 5% CO2.
Biotecs (Pinhais, PR, BR). Deuterated chloroform was purchased
from Sigma-Aldrichs (St. Louis, MO, USA.). All reagents used in the
analyses were of analytical grade. Kieselgels 60 M silica, 35–70 mesh 2.4.2. MTT reduction assay
and 230–400 mesh were purchased from Mercks (Darmstadt, HE, In this test, 4  104 cells were seeded in a 96 well plate
DE), RPMI 1640 medium was purchased from Gibco-BRLs (Grand incubated with the popular form of use of latex (garrafada) and
Island, NY, USA), 5-fluorouracil from Sigma-Aldrich (St. Louis, MO, fresh latex at different concentrations (1.7 mg–7.0 mg/well and
USA) and foetal bovine serum from Cripions (Andradina, SP, BR). 1D 1.22 mg–4.88 mg/well, respectively). For the reading, the medium
and 2D NMR analysis were performed on a Brukers Avance III HD was discarded and replaced with 200 mL of MTT solution. The
600 NMR spectrometer, operating at 14.1 T, observing 1H and 13C at plates were incubated at 37 1C for 4 h. The resulting formazan
600.13 and 150.90 MHz, respectively. Flow cytometry was performed crystals were solubilised in 100 mL of dimethylsulphoxide (DMSO).
on a Beckman Coulters FC500 MPL model flow cytometer. IR spectra The optical density was read at 560 nm with ELISA plate reader.
were obtained on a Perkin-Elmer 1420 spectrometer. The extracts Using this methodology, both the isolated substances (euphol and
were concentrated on a Fisatoms 558 evaporator coupled to a citrostadienol) were also tested at concentrations of 2.5, 25, 125
Marconis MA053 vacuum pump, and histological analysis was and 250 μg/mL with incubations at 24 and 48 h (Bagalkotkar et al.,
performed with a Leicas TP1020 tissue processor with Leicas 2011; Yue et al., 2013).
EG1120 support and a Leicas RM2125RT microtome. The histological
slides were analysed using an Olympuss CX31 photomicroscope. 2.4.3. Trypan blue exclusion assay
In this assay, 4  106 cells were distributed into 24 well plates
2.2. Plant material and garrafada preparation and incubated in culture conditions with different aliquots of
garrafada and latex for 24–48 h giving the respective concentra-
Synadenium grantii Hook f. latex was collected in Ponta Grossa, tions (1.7 mg–7.0 mg/well and 1.22 mg–4.88 mg/well). After this
Paraná, Brazil (975 m altitude, 25105'38″ S, 501 09'30″ W), in April time, an aliquot of 10 mL of cell suspension was removed and
2010. A voucher specimen (# 363,509) was identified and depos- homogenised in 10 mL of 0.2% trypan blue. The cells were analysed
ited at the Herbarium of the Municipal Botanical Museum of with aid of a Neubauer chamber.
Curitiba. The garrafada was prepared by adding 18 drops of the
latex in 1 L of distilled water (0.747 g/1 L, w/v). 2.5. Analysis of cellular cycle

2.3. Extraction and isolation After treatment of the B16F10 line with different concentra-
tions of latex (1.7 mg–7.0 mg/well and 1.22 mg–4.88 mg/well) for 24
Fresh latex (48 g, density: 1.22 g/mL) was partitioned in a and 48 h the cell cycle analysis was determined by flow cytometry.
vacuum chromatographic column (VCC—60 silica gel, 35–70 mesh) For this purpose, 2  106 cells were trypsinised, washed three
eluted with Hex, followed by CHCl3, AcOEt and MeOH as solvents times with PBS, fixed in 70% ethanol and stained with propidium
(increasing polarity), obtaining 28.02%, 5.00%, 6.40% and 0.21% iodide (PI). The PI red fluorescence was acquired through a 585/
(w/w) of extraction yield respectively. The resulting respective 42 nm filter and the signals were measured on a linear scale of
fractions were kept under refrigeration (4 1C) prior to use. The 1024 channels. For each sample, 10,000 events were acquired and
fresh latex and the fractions were analysed using NMR to provide a the data were analysed using appropriate software (CellQuests,
fingerprinting profile and to identify the chemical classes of Becton Dickinson, San Jose, CA; WinMDIs 2.8).
compounds present in these materials. So, the hexane fraction
was further subjected to a liquid-liquid partitioning with hexane 2.6. In vivo assays
(FH) and methanol (FM).
Part of the FH (8 g) was subjected to chromatographic column Male, nine-week old, C57BL6 mice, weighing approximately
(CC) containing 35–70 mesh silica (61 g) eluted with Hex, followed 20 g were obtained from the vivarium of the Federal University of
by CHCl3, AcOEt and MeOH to give 207 sub-fractions. The fractions Paraná. Aliquots of 5  104 B16F10 cells were subcutaneously

Please cite this article as: de Oliveira, T.L., et al., Antitumoural effect of Synadenium grantii Hook f. (Euphorbiaceae) latex. Journal of
Ethnopharmacology (2013), http://dx.doi.org/10.1016/j.jep.2013.08.033i
 T.L. de Oliveira et al. / Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎ 3

injected in the dorsum of these animals. On the 10th day after cell The isolated compounds, euphol and citrostadienol, were
injection, the animals were divided into two groups (control and tested against the strain of melanoma (B16F10) by the MTT
treated) and treatment started with 50 mL of the garrafada per method. The readings after 24 and 48 h of incubation of the
animal (corresponding to the popular use of the latex proportional B16F10 cells with the substances demonstrated that the euphol
to the weight of the mouse) administered orally 3 times daily. The did not demonstrate the ability to decrease cell viability, indicating
tumours were measured using a calliper and their size in mm3 was that in the studied concentrations this substance showed no
calculated as follows (Plowman et al., 1997): cytotoxic activity. The steroid, citrostadienol, showed reduced
cytotoxic activity, causing an 8% decrease in cell viability at a
Volume of tumour ¼ longitudinal ðhead tailÞ
concentration of 250 mg/mL.
transverse ðpaw pawÞ  3=4 p:

This study was approved by the Ethics Committee on the use of 3.2. Effects on cellular cycle
animals (no. 19,326/2011 CEUA) and all procedures followed the
rules and conduct of research and animal experimentation. The effect of Synadenium grantii latex on the cell cycle progres-
sion of the B16F10 cell line was determined by flow cytometry. The
2.7. Histological analysis latex induced a dose-dependent cell death (Fig. 1) followed by a
reduction in the number of cells in the S-G2/M phase, as shown in
After the animals were sacrificed, part of the tumour, liver, Table 1.
lungs and mediastinal lymph nodes were removed. The specimens
were fixed in 10% buffered formalin for 24 h and then the 3.3. In vivo antitumoural evaluation
histological processing was performed and the material was
placed in paraffin blocks. Then, histological sections, 5 μm in After treating the mice for seven days, orally 3 times a day, with
thickness, were obtained and they were stained with hematoxylin a solution corresponding to the popular use (garrafada) of Syna-
and eosin (HE). The slides were analysed using light microscopy denium grantii there was a 40% inhibition of tumour growth in
and the histological images were captured with the aid of a light comparison with the untreated control group, as can be seen in
microscope. Fig. 2.

2.8. Statistical analysis 3.4. Histological evaluation

The experimental values were expressed as mean 7standard The histological evaluation of the analysed organs was similar
deviation. The in vitro cytotoxicity assays were analysed by the in both the treated and the control group. The melanoma tumours
difference of experimental statistical significance using analysis of presented areas that contained a great number of cells and other
variance (ANOVA) followed by Tukey's test and the in vivo tests
were analysed using Student's t-test. The data were analysed using Table 1
Graph Pad Prism 4.0 software (GraphPad Software Inc., SanDiego, Values of means of cell populations in the phases of the cycle of B16F10 melanoma
cells after 24 h of treatment with Synadenium grantii latex (garrafada and fresh
CA, USA). The level of P r0.05 was used to determine statistical latex).
significance.
Concentration (mg/well) Sub G1 G0/G1 S–G2/M

Negative control 13.7070.28 34.55 7 1.62 51.75 7 1.34

3. Results Positive control 52.00 7 9.12 25.727 7.40 12.617 5.10
0.0017 28.20 71.75 24.607 1.21 47.20 7 1.08
3.1. In vitro cytotoxicity assays 0.003 27.57 71.40 25.677 4.47 46.777 4.33
0.007 23.20 71.13 20.707 1.95 49.907 9.00
1.22 19.00 70.50 46.83 7 16.37 23.23 7 5.03n
Using the trypan blue method, a 98% cellular inhibition for the
2.44 71.50 712.02n 28.337 18.58 13.337 6.02n
garrafada and 100% for the latex was obtained, after 48 h of 4.88 50.33710.02n 31.007 11.53 18.677 3.05n
incubation, and for the MTT technique, the percentage of cellular
inhibition was up to 40% for the garrafada and up to 64% for the Values represent the mean 7 standard deviation of triplicates.
fresh latex after 48 h of incubation (data not shown). n
p o 0.001. Positive control: 5-fluorouracil 16 uM.

Fig. 1. Histograms of B16F10 cells after 24 h of incubation with Synadenium grantii latex (garrafada and fresh latex) demonstrating increased concentration-dependent
cell death.

Please cite this article as: de Oliveira, T.L., et al., Antitumoural effect of Synadenium grantii Hook f. (Euphorbiaceae) latex. Journal of
Ethnopharmacology (2013), http://dx.doi.org/10.1016/j.jep.2013.08.033i
4 T.L. de Oliveira et al. / Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

and time-dependent cell viability during the studied time periods,

with the best results achieved using the trypan blue assay.
According to Valadares et al. (2007), due to the different principles
of the methods employed in this assessment of cytotoxicity,
differences in the inhibition of cell values can be expected; this
was also observed in the results of the in vitro experiments in the
present study. Other cell lines were also tested by these methods
and similar results were obtained (data not shown).
The cytotoxic activity of the Euphorbiaceae family has been
reported for different species. Aliabadi et al. (2009) demonstrated
that different extracts of Euphorbia macroclada Boiss. latex showed
cytotoxicity against the breast adenocarcinoma cell line, and the
dichloromethane extract was the most efficient, since it caused
50% of cellular death at a concentration of 30 mg/mL; the methanol
Fig. 2. Inhibition in tumour volume in melanoma-bearing mice, induced by the extract showed no activity. Euphorbia tirucalli L. latex also showed
administration of garrafada of Synadenium grantii. nnp ¼0.0029. in vitro toxicity when evaluated in relation to the culture of
melanoma cells, even at high dilutions (homoeopathic doses)
areas of necrosis. The tumour cells exhibited nuclear hyperchro- (Silva et al., 2011). Likewise, the ethanolic extract of the leaves of
maticism, pleomorphism and eosinophilic cytoplasm (Fig. 3). The Synadenium umbelattum Pax. demonstrated the ability to decrease
deposition of both intra- and extracellular melanin was also in vitro cell viability in Ehrlich ascites tumour cell culture and also
observed, as well as atypical mitosis. Metastasis in the lung tissue, acute myeloid leukaemia, presenting the death of 50% of tumour
associated with extensive inflammation, was histologically identi- cells at concentrations of 0.4, 0.1, and 0.08 mg/mL for the crude
fied in both the treated and untreated group (Fig. 3). Metastasis extract, chloroform fraction and hexane fraction, respectively
was macroscopically observed in the mediastinal lymph nodes in (Nogueira et al., 2008).
both groups, the suspicion of which was confirmed histologically. The toxicity of the Synadenium genus has been demonstrated,
mainly based on the evaluation of extracts from the aerial parts of
3.5. NMR fingerprinting and isolation of compounds the plant (Castro and Valadares, 2005; Valadares et al., 2007;
Cunha et al., 2009; Mota et al., 2012) but few studies have reported
The 1H and 13C NMR analysis of the latex and hexane, chloro- the potential effect of the latex, which is popularly used
form and ethyl acetate fractions showed characteristic signals due for therapeutic purposes, as reported in this present study.
to terpenes (Fig. 4). Melo-Reis et al. (2011) showed that Synadenium umbelattum latex
The hexane fraction obtained from the fractionation of the presents cytotoxic and mutagenic potential (concentration-depen-
fresh Synadenium grantii latex was subjected to chromatographic dent), and in the same species, Oliveira et al. (2005) found a
processes, from which the triterpene euphol and the steroid relatively low LD50 value (110–168 mg/Kg), which shows that
citrostadienol were isolated and identified (Fig. 5) (Zhang et al., substantial care should be taken in its empirical use due to its
2006; Moreira et al., 2010). high toxicity.
Even more relevant to the subject of the present research is the
fact that the toxicity of the Synadenium grantii species has also
4. Discussion been reported, evaluated by Artemia salina, presenting an LD50
value of 26.58 μg/mL for latex from the plant (Costa et al., 2012).
The development of new strategies and the search for new The present study demonstrated that Synadenium grantii latex
substances to treat cancer has been the subject of much research. in the tested concentrations caused the death of specific cells in
In this regard, in vitro cytotoxicity assays have been shown to be a the proliferative phase of the cell cycle (S-G2/M). This finding is
consistent and rapid method for screening natural and synthetic supported by other studies that showed that plants of the
products with antitumoural potential (Rahmat et al., 2003; Zhong Euphorbiaceae family present the ability to cause accentuated cell
et al., 2009; Suárez et al., 2009; Momtaz et al., 2010; Silva et al., 2011). death in tumour cell lines after treatment with their extracts
Kauffmann et al. (2004) have observed that the medical (Giridharan et al., 2002; Harikumar et al., 2009).
benefits of medicinal plants commonly result from the combina- To confirm the results obtained in the in vitro study, an in vivo
tion of actions of secondary metabolites (synergic effect) present experiment was conducted to evaluate the diluted form of use of
in extracts of these plants. However, the isolation and identifica- Synadenium grantii. The induction of melanoma in mice followed
tion of active compounds present in these extracts, and the further by the treatment of these animals with the garrafada, 3 times a
study of the therapeutic capacity of these substances and the day, as related for popular use showed a 40% inhibition of tumour
evaluation of potential mechanisms of action, constitute a great growth compared with the control group. According to the
challenge for pharmacology, biochemistry and chemistry National Cancer Institute (NCI), values of T/C (treated/control) less
(Gebhardt, 2000). Therefore, considering the ethnopharmacologi- than 42% are indicative of significant antitumoural activity
cal use of Synadenium grantii, the latex of this plant was initially (Plowman et al., 1997). This result allows us to infer that the latex
assessed against its potential to cause the inhibition of the (garrafada) of Synadenium grantti presents potential antitumoural
proliferation of B16F10 cells by two distinct methodologies. activity. Others studies with different extracts of other plants
Different concentrations of the garrafada and fresh latex were belonging to the Euphorbiaceae family have showed similar
tested. Two concentrations that were more diluted (1.7 mg and results. Bezerra et al. (2009) obtained 31.8% inhibition of growth
3.0 mg/well) than the concentration recommended (7.0 mg/well) in of sarcoma in mice using the essential oil from leaves of Croton
popular use—garrafada—and three other more concentrated pre- regelianus Müll. Arg., while Alonso-Castro et al. (2012) obtained
parations (1.22 mg, 2.44 mg and 4.88 mg/well)—fresh latex—were 59% inhibition of the human epithelial carcinoma cell line using
prepared and tested, since the aim was to study the results of the methanol extract of the leaves of Croton lechleri Müll. Arg.
higher concentrations compared to the models evaluated. It can be However, histological analysis of liver tissue showed no sig-
seen that this strain showed an in vitro decrease in concentration nificant difference between treated animals that receiving the

Please cite this article as: de Oliveira, T.L., et al., Antitumoural effect of Synadenium grantii Hook f. (Euphorbiaceae) latex. Journal of
Ethnopharmacology (2013), http://dx.doi.org/10.1016/j.jep.2013.08.033i
 T.L. de Oliveira et al. / Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎ 5

Fig. 3. Representative photomicrographs of the tumours of treated animals and control. (A) It shows an overview of the tumour of a treated animal, showing high cellularity
(Ce) and large areas of necrosis (Ne). (B) It shows the tumour of an untreated animal showing the same issues previously described. (C) It shows tumorous cells, deposition of
melanin and the presence of eosinophilic material consistent with the inflammatory exudates in the lungs of animals of the treated group. Note the tumorous cells within the
blood vessels (V). (D) shows the presence of a tumorous island with a necrotic centre (arrow), in the lungs of animals of the untreated group (HE, A, B and C: 200X–400X
highlights; D: 400X).

garrafada of Synadenium grantii and those of control group and noted congestion and inflammatory infiltration only in ani-
(untreated). There was no evidence of damage to liver tissue from mals treated with the latex. As well as the process of metastasis
use of the garrafada of Synadenium grantii. This result is in observed in the lungs, this leucocyte migration may be attributed
agreement with results obtained by Costa et al. (2012), who to the presence of lectins, which are potent agglutinins for
evaluated hepatic biochemical parameters in rats treated with erythrocytes that are mainly present in the latex of the plant
garrafada of Synadenium grantii and found no significant difference (Mrinalini et al., 2002; Souza et al., 2005; Rajesh et al., 2006;
between the treated group and the control group; however, these Cunha et al., 2009).
same authors reported that in the highest concentrations of the The significant antitumoural activity observed in the in vitro
latex, significant changes in biochemical parameters were found. and in vivo assays in the present study supports previous findings
On the other hand, the leucocyte infiltration observed in the for species from the Euphorbiaceae family. Block et al. (2005),
lungs of mice in both groups was also reported by Cunha et al. Jassbi (2006) and Aliabadi et al. (2009) attribute this antitumoural
(2009), who evaluated the acute and sub-acute toxicity of latex activity to nonpolar constituents such as terpenoids that are very
and ethanol extract of the leaves of Synadenium umbellatum in rats abundant in the chemical constitution of the Euphorbiaceae

Please cite this article as: de Oliveira, T.L., et al., Antitumoural effect of Synadenium grantii Hook f. (Euphorbiaceae) latex. Journal of
Ethnopharmacology (2013), http://dx.doi.org/10.1016/j.jep.2013.08.033i
6 T.L. de Oliveira et al. / Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

Fig. 4. 1H (top) and 13

C {1H} (bottom) NMR spectra of hexane fraction from the fresh latex of Synadenium grantii.

However, similar compounds have shown to be effective in

treating some types of cancer (Salvador et al., 2013).

5. Conclusion

The in vitro assays demonstrated that both fresh latex and

garrafada from the latex of Synadenium grantii have concentration
and time-dependent cytotoxic effect against B16F10 melanoma
Fig. 5. Molecular structure of compounds isolated from the hexane fraction of fresh cells lines. They also showed deleterious effects on the cellular
Synadenium grantii latex.
replication, inducing cell death and cell cycle arrest in the S-G2/M
phase. Additionally, in vivo assays showed significant reduction in
tumour volume in melanoma-bearing mice.
family. Terpenes that are present in latex as well as other parts of Phytochemical investigation made it possible for the first time
several species of the genus Synadenium are described to present to identify and isolate euphol and citrostadienol in the latex of
cytotoxic activity (Bagavathi et al., 1988; Olivier et al., 1992; Bittner Synadenium grantii. The latter showed reduced cytotoxic activity
et al., 2001; Costa et al., 2012; Hassan et al., 2012). The 1H and 13C when tested in vitro against the strain of melanoma.
NMR spectra of hexane, chloroform and ethyl acetate fractions of
the latex showed characteristic signals due to terpenes. Moreover,
these signals were also evident in both hexane and chloroform Acknowledgements
fractions. The tetracyclic triterpene euphol is widely described
as a major constituent in species of the Euphorbiaceae family The authors are grateful to Fundação Araucária (Research Grant
(Yasukawa et al., 2000; Dutra et al., 2011; Mata et al., 2011; 14/2009) and the Postgraduate Pharmaceutical Sciences Programme
Tsopmo and Kamnaing, 2011; Dutra et al., 2012; Peng et al., and UEPG for financial support and fellowships.
2012). Moreira et al. (2010) found euphol in the methanolic extract
of the latex of S. carinatum Boiss. Hassan et al. (2012) isolated this References
and four other terpenes from chloroform extract of the leaves of
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activities were assigned to these compounds, such as anti- macroclada on MDA-MB-468 breast cancer cell line. Iranian Journal of Pharma-
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nervous system in rats (Dutra et al., 2011; Dutra et al., 2012). Mata ceae). Journal of Ethnopharmacology 140, 438–442.
et al. (2011) showed molluscicidal activity against Biomphalaria Bagavathi, R, Sorg, B., Hecker, E., 1988. Tigliane-type diterpene esters from
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Please cite this article as: de Oliveira, T.L., et al., Antitumoural effect of Synadenium grantii Hook f. (Euphorbiaceae) latex. Journal of
Ethnopharmacology (2013), http://dx.doi.org/10.1016/j.jep.2013.08.033i
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Please cite this article as: de Oliveira, T.L., et al., Antitumoural effect of Synadenium grantii Hook f. (Euphorbiaceae) latex. Journal of
Ethnopharmacology (2013), http://dx.doi.org/10.1016/j.jep.2013.08.033i