Article

International Journal of Toxicology

1-6
Antiproliferative Effect of Synadenium The Author(s) 2016
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grantii Hook f. stems (Euphorbiaceae) DOI: 10.1177/1091581816659660
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and a Rare Phorbol Diterpene Ester

Adriana Campos1, Debora Barbosa Vendramini-Costa2,

Giovanna Barbarini Longato2, Tailyn Zermiani1,
Ana Lucia Tasca Gois Ruiz2, Joao Ernesto de Carvalho2,
Atanasio Pandiella3, and Valdir Cechinel Filho1

Abstract
Synadenium grantii is frequently used for the treatment of various diseases such as allergies, gastric disorders, and especially cancer.
The aim of this study was to evaluate the possible antiproliferative potential of the methanol extract, fractions, and pure com-
pounds from the stems of S grantii. Phytochemical analysis was carried out by conventional chromatographic techniques, and the
antiproliferative activity was analyzed using the sulforhodamine B assay and an MTT-based assay. Nonpolar fraction and its
subfractions from the stems of S grantii exhibited promising cytostatic effect against several human tumor cell lines (glioma, breast,
kidney, and lung), with total grown inhibition values ranging from 0.37 to 2.9 mg/mL. One of the active principles of this plant was
identified as a rare phorbol diterpene ester, denoted as 3,4,12,13-tetraacetylphorbol-20-phenylacetate. This compound
demonstrated antiproliferative activity against glioma, kidney, lung, and triple-negative breast cancer cell lines. These results
demonstrate that S grantii stems produce active principles with relevant antiproliferative potential.

Keywords
Synadenium grantii, antiproliferative action, nonpolar fraction, phorbol ester

Introduction activity of the crude methanol extract and fractions/subfrac-

The Synadenium genus, which belongs to the family Euphor-
biaceae, has been linked to some relevant pharmacological
properties such as anticancer,1 anti-inflammatory,2 fibrinolytic
action,3 and immunoregulation.4
Synadenium grantii Hook is a shrub from Africa that is
commonly found growing as hedges. It is popularly known in
Brazil as Leitosinha or Janauba. The latex of this plant is
widely used in traditional medicine to treat various diseases
such as allergies, gastric disorders, and especially cancer.5-7
Previous experiments have suggested that S grantii latex
may potentially present an antiulcerogenic effect.7 Moreover,
the chloroform extract of the leaves of this plant demonstrated
cytotoxicity and antiparasitic activity.8
In a recent study conducted with the stem bark of S grantii,
in vitro and in vivo assays demonstrated significant antioxidant
and anti-inflammatory activities, which were associated with
the presence of phenolic compounds and terpenes.9 Other phy-
tochemical investigations indicated the presence of terpenes
and phenolic compounds in bark extract of S grantii.7 Some
other compounds have been found in this plant, such as diter-
pene esters, anthocyanins, and unsaponificable substances.6-10
As part of the Network Ribecancer 212RT 0464 (CYTED/
CNPq), the aim of this study was to evaluate the antiproliferative
tions of the stem of S grantii as well as a rare isolated
compound (phorbol diterpene ester) denoted as 3,4,12,13-
tetraacetylphorbol-20-phenylacetate (compound 1).
Materials and Methods
Plant Material and Phytochemical Analysis
Synadenium grantii was collected in the town of Itaja (SC-
Brazil) in March and August 2013 and identified by Dr Oscar
B. Iza (University of Itaja Valley). A voucher specimen was
1
Programa de Pos-Graduacao em Ciencias Farmaceuticas and Nucleo de
Investigacoes Qumico-Farmaceuticas (NIQFAR), Universidade do Vale do
ItajaUNIVALI, Itaja, Santa Catarina, Brazil
2
Centro Pluridisciplinar de Pesquisas Qumicas, Biologicas e Agrcolas
Universidade Estadual de Campinas (UNICAMP), Campinas, Sao Paulo, Brazil
3
Centro de Investigacion del Cancer (CSIC-USAL), Salamanca, Spain
Corresponding Author:
Adriana Campos, Programa de Pos-Graduacao em Ciencias Farmaceuticas,
Universidade do Vale do ItajaUNIVALI, Rua Uruguai, 458 (88302-202),
Itaja, Santa Catarina, Brazil.
Email: adriicampos@hotmail.com

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2 International Journal of Toxicology

deposited at the Barbosa Rodrigues Herbarium (Itaja-SC) Table 1. Antiproliferative Activity of Doxorubicin (Positive Control),
under number VC Filho 108. Acetone (ac), and Methanolic (me) Fractions From the Stems and
Fresh stems (166 g) and leaves (165 g) of S grantii were cut Leaves of Synadenium grantii Against Human Cancer Cell Lines.a
into small pieces and extracted by maceration with methanol at GI50, mg/mLb
room temperature for a period of 1 week, providing the crude
methanol extract of each part after solvent evaporation. These Crude extracts U251 MCF-7 786-0 NCI-H460
extracts were dissolved in acetone and methanol, and after Doxorubicin 0.025 <0.025 <0.025 <0.025
evaporation, 2 new fractions were obtained from each part of S grantii stems 30.3 28.5 26.7 8.1
the plant. An antiproliferative screening was conducted with all S grantii stemsac 2.9 2.9 1.4 0.37
these fractions. Since only the stem fractions exhibited promis- S grantii stemsme >250 >250 >250 >250
ing biological activity, they were selected for further studies. S grantii leaves 128.7 35.6 69.8 >250
Stems (1 kg) from S grantii were exhaustively extracted by S grantii leavesac 25.0 23.7 10.6 1.1
S grantii leavesme 198.3 187.3 39.5 225.2
maceration with methanol at room temperature for 7 days. The
macerate was filtered and concentrated under reduced pressure Abbreviations: AC, acetone fraction; ME, methanolic fraction; SRB, sulforho-
in a rotary evaporator, yielding 31 g of crude methanol extract. damine B assay.
a
Human tumor cell lines: U251 (glioma); MCF-7 (breast); 786-0 (kidney); and
All the extract was suspended in methanol:water (50:50) mix- NCI-H460 (lung). Assessed by the SRB assay.
ture and subjected to liquidliquid partition using solvents of b
GI50 values represent the concentration required to inhibit 50% of cell growth.
increasing polarity such as chloroform (yield of 2.6%) and Values were determined through nonlinear regression analysis using the
ethyl acetate (yield of 5.8%), respectively. ORIGIN 8.0 (OriginLab Corporation, Wellesley Hills, MA). Dose range tested:
0.25 to 250 mg/mL.
Part of chloroform fraction (700 mg) was subjected to col-
umn chromatography (0.063-0.20 mm, 105.4 g, 3.5  50 cm;
Merck, Darmstadt, Germany) over silica gel and eluted with
In Vitro Anticancer Activity Assay
hexane:ethyl acetate (100:0 ! 0:100) in increasing order of Human tumor cell lines of U-251 (glioma), MCF-7 (breast),
polarity to afford 33 fractions that were combined based on the NCI/ADR-RES (ovary expressing multidrug resistance pheno-
thin-layer chromatography (TLC) profiles, using hexane:ethyl type), 786-0 (kidney), NCI-H460 (lung, nonsmall cells), HT-29
acetate (80:20) as the mobile phase and reaction with sulfuric (colon), and K562 (leukemia) were kindly provided by the
anisaldehyde heated at 100 C. The fractions 5 to 13 (118 mg) NCI. Nontumor cell line HaCat (human keratinocytes) was
were rechromatographed as before, using a solvent system donated by Professor Dr Ricardo Della Coletta, FOP/UNI-
hexane:ethyl acetate, yielding new 19 subfractions, which were CAMP. Stock cultures were grown in medium containing
combined according to their TLC profiles. Two of them, which 5 mL RPMI 1640 (GIBCO BRL, Sao Paulo, SP, Brasil) sup-
presented better TLC profiles, 11 to 15 (37 mg) and 16 to 19 plemented with 5% fetal bovine serum. Penicillin:streptomycin
(26 mg) were evaluated in antiproliferative assay. (1,000 microg/L:1,000 U/L, 1 mL/L) was added to the experi-
A part of subfraction 11 to 15 (30 mg) was analyzed by high- mental cultures. Cells in 96-well plates (100 mL cells/well)
performance liquid chromatography (HPLC) and then sub- were exposed to sample concentrations in dimethyl sulfoxide
mitted to a flash chromatography eluted with hexane:acetone (DMSO)/RPMI (0.25, 2.5, 25, and 250 mg/mL) at 37 C, 5% of
8:2, yielding 12 mg of the phorbol diterpene ester 3,4,12,13- CO2 in air for 48 hours. The final DMSO concentration did not
tetraacetylphorbol-20-phenylacetate (compound 1), which was affect cell viability. Afterward, cells were fixed with 50% tri-
identified by spectroscopic data (nuclear magnetic resonance chloroacetic acid, and cell growth was determined by spectro-
[NMR] 1H and 13C) and compared with those recently pub- photometry (540 nm) of cellular protein content using the
lished in the literature.8 sulforhodamine B assay.11 Doxorubicin was used as standard
(positive control). The concentrationresponse curve for each
High-performance liquid chromatography analysis. A Shimadzu cell line and the total growth inhibition (TGI) values were deter-
LC-20AT LC system (Shimadzu, Tokyo, Japan) was used, con- mined through nonlinear regression analysis using the software
sisting of a SPD-M20A photodiode array detector, SIL-20AHT ORIGIN 8.0 (OriginLab Corporation, Wellesley Hills, MA).12
autosampler, and software LC-Solution (Shimadzu). The sub- For the cell proliferation assays, using the triple-negative
fractions 11 to 15 from S grantii stems and compound 1 were breast cancer (TNBC) cell lines MDA-MB231 and HBL100,
diluted in methanol at 1 mg/mL and filtered through a 0.45-mm kindly provided by the ATCC, the cells were cultured in Dul-
cellulose regenerated membrane filter. Samples (20 mL) were becco modified Eagle medium (DMEM) supplemented with
injected on a C18 column (Luna Phenomenex, 250  4.6 mm; 10% fetal bovine serum and antibiotics (penicillin at
0.5-mm film thickness) conditioned at 35 C. The mobile phase 100 mU/mL and streptomycin at 100 mg/mL) at 37 C in a
consisted of acetonitrile (A) and water (pH 2.5, phosphoric humidified atmosphere in the presence of 5% CO2 and 95% air.
acid; B) eluted in a gradient system, starting with 2% A and Cells were plated in 24-well plates at 8,000 cells/well
after 98 minutes 100% A. This was followed by a 10-minute (MDA-MB231) and 15,000 cells/well (HBL100) and cultured
equilibrium period prior to the injection of next sample. The overnight in DMEM 10% fetal bovine serum. The next day,
analyses were monitored at 200 nm. All solvents used were medium was replaced with DMEM containing compound 1 in
HPLC grade and were degassed in an ultrasonic bath. the concentrations 1, 5, 10, 25, 50, and 100 mmol/L.

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Campos et al 3

Figure 1. High-performance liquid chromatography (HPLC) analysis of subfractions 11 to 15 from S grantii stems and compound 1.

Cell proliferation was analyzed by an MTT-based assay as

described previously.13

Results
Crude methanol extracts of both S grantii stems and leaves and
their respective acetone (apolar to medium polar compounds)
and methanol (polar compounds) fractions were evaluated in a
panel of 4 human cancer cell lines (U-251 [glioma], MCF-7
[breast], 7860 [kidney], and NCI-H460 [lung, nonsmall cells])
in order to select the more promisor extract and/fraction for
further evaluations.
This way, stems extract (GI50 between 8.1 and 30.3 mg/mL)
showed better antiproliferative results than leaves extract (GI50
between 35.6 and >250 mg/mL). Moreover, the acetone fraction
of S grantii stems demonstrated higher potency (50% growth
inhibition, GI50, between 0.37 and 2.9 mg/mL) than that observed
for crude stems extract, suggesting that active compound was
concentrated in acetone fraction (Table 1). A similar profile was
observed between acetone and methanol leaf fraction being acet-
one leaf fraction more active than methanol fraction and metha-
nol crude extract (Table 1). Based on these results, S grantii
Figure 2. Molecular structure of 3,4,12,13-tetraacetylphorbol-20-
stems methanol extract was selected for phytochemical analysis. phenylacetate (compound 1) isolated from S grantii stems.
Thus, a bioguided phytochemical study of S grantii stems
methanol extract was performed seeking to determine the pronounced and promising biological results, but 11 to 15
active principles. First, methanol crude extract was partitioned exhibited the best chemical profile. Figure 1 indicates the
by liquidliquid partition into chloroform (nonpolar com- HPLC profile of this fraction (11-15). It furnished a rare phor-
pounds) and ethyl acetate (polar compounds) fractions. bol diterpene ester identified as 3,4,12,13-tetraacetylphorbol-
Based on the initial results (Table 1), in which the nonpolar 20-phenylacetate (compound 1; Figure 2).
fraction showed greater antiproliferative activity, as well as the Phytochemical analysis using TLC, together with standard
recent study conducted by other researcher group,14 part of compounds already isolated from the plant (friedelin, 3b-frie-
chloroform fraction was subjected to column chromatography, delinol, eufol, and citrostadienol)9-14 indicated that these were
yielding 2 subfractions, 11 to 15 and 16 to 19. Both fractions absent, suggesting other active principles that could act as anti-
presented suitable but complex chromatographic profiles and proliferative agents, like compound 1.

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4 International Journal of Toxicology

Table 2. Antiproliferative Activity of Doxorubicin (Positive Control), Subfractions From S grantii Stems and Compound 1 Against Human
Cancer Cell Lines.a

TGI, mg/mLb

U251 MCF-7 NCI-ADR 786-0 NCI-H460 HT29 K562 HaCat

Doxorubicin 0.51 3.7 1.7 0.36 0.57 10.4 >25 0.26
Subfraction 11-15 6.4 9.2 11.7 10.5 9.9 10.5 >250 9.6
Subfraction 16-19 5.6 6.0 8.9 5.3 9.7 8.0 >250 8.7
Compound 1 25.2 >250 >250 24.1 31.1 >250 65.3 >250

Abbreviations: SRB, sulforhodamine B assay; TGI, total growth inhibition.

a
Human tumor cell lines: U251 (glioma); MCF-7 (breast); NCI-ADR/RES (ovary); 786-0 (kidney); NCI-H460 (lung); HT29 (colon); and K562 (leukemia). Nontumor
cell line: HaCat (keratinocyte). Assessed by the SRB assay.
b
The TGI values represent the necessary concentration (mg/mL) for total inhibition of cancer cell proliferation. Values were determined through nonlinear
regression analysis using the ORIGIN 8.0 (OriginLab Corporation). Dose range tested: 0.25 to 250 mg/mL.

compound 1 showed good activity at concentrations of 1 mmol/

L and above. Compound 1 was less potent as an antiproliferative
drug on MDA-MB231 cells, which is a cell line characterized by a
high proliferation rate and resistance to several drugs.

Figure 3. MTT metabolization (percentage control) of compound 1
against 2 triple-negative breast cancer cell lines, MDA-MB231 and
HBL100.
Subfractions 11 to 15 and 16 to 19 and compound 1 were
tested for their antiproliferative activity against different
human cell lines (Table 2). Both subfractions demonstrated
high potency for all human cancer cell lines tested, except
leukemia, with TGI values of between 5.3 and 11.7 mg/mL,
being subfractions 16 to 19 slightly more active, especially
against glioma (U251), breast (MCF-7), and kidney (786-0)
cancer cell lines than subfractions 11 to 15. Compared to the
control doxorubicin, both subfractions were less cytotoxic
against the nontumor cell line (keratinocyte). Compound 1
demonstrated a moderate antiproliferative activity against
glioma (U 251, TGI 25.2 mg/mL), kidney (786-0, TGI
24.1 mg/mL), and lung (NCI-H460, TGI 31.1 mg/mL), sug-
gesting that compound 1 could contribute partly to antiproli-
ferative activity observed for subfractions 11 to 15.
Furthermore, compound 1 did not show activity against the
nontumor cell line (keratinocyte).
The effect of compound 1 was further tested in 2 cellular
models of TNBC: MDA-MB231 and HBL100 cells (Figure 3).
These analyses, using MTT metabolization as a readout of the cell
number, indicated that compound 1 at concentrations between 1
and 100 mmol/L was active against the 2 cell lines, being more
potent on HBL100 than on MDA-MB231. In HBL100 cells,
Discussion
Cancer remains a major public health problem and one of the
leading causes of death in the world, particularly in developing
countries.15,16 The identification of new drugs and treatments
to solve this problem is therefore urgent.
There are a number of distinctly different types of kidney
cancer, each with a different histology and clinical course,
which respond differently to therapy. This cancer is responsible
for over 115,000 deaths annually worldwide.17-19
Malignant gliomas account for 40% to 60% of primary brain
tumors and are responsible for a disproportionate level of mor-
bidity and mortality among patients with cancer. Despite treat-
ments of brain tumors, such as surgery, radiotherapy,
chemotherapy, and immunotherapy, the median survival time
is <15 months. Therefore, the challenges in the treatment of
malignant gliomas remain considerable.20,21
The TNBC accounts for 15% of all breast cancers and tend
to exhibit an aggressive metastatic behavior. These tumors
respond to conventional chemotherapy but have relapse very
frequently and have a worse prognosis.22,23
The toxicity of the Synadenium genus has already been
demonstrated. Valadares et al24 and Mota et al25 found that
Synadenium umbellatum has potential cytotoxic and muta-
genic effects.
In vitro and in vivo assays demonstrated that S grantii latex
possesses cytotoxic effect and antitumoral activity against
B16F10 melanoma cells.14 Citrostadienol, which was isolated
from this plant, showed cytotoxic activity when tested in vitro
against melanoma cells.14 Terpenes that are present in latex as
well as other parts of the S grantii are described as presenting
cytotoxic activity.7,8-10 Interestingly, the majority of the com-
pounds previously described as active principles for this
plant8,9-14 are different from those evidenced in our studies
with subfractions 11 to 15 and 16 to 19, as evaluated by

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Campos et al
spectroscopic (NMR) and chromatographic methods (TLC
and HPLC).
The phorbol diterpene ester 3,4,12,13-tetraacetylphorbol-
20-phenylacetate isolated from the subfractions 11 to 15 is a
rare compound and was isolated only recently from the leaves
of S grantii.8 The antiproliferative action of this compound
was not as good as the one presented by subfraction that was
isolated, suggesting a possible synergy between the com-
pounds, or the presence of other active principles present in
S grantii stems.
Phorbol esters are members of the tigliane family of diter-
penes. These compounds have been isolated from plants
belonging to the family Euphorbiaceae.26-28
Phorbols and their different derivatives are reported to be
tumor promoters. These compounds do not induce tumors but
promote tumor growth following exposure to a subcarcino-
genic dose of a carcinogen. Some phorbol esters isolated from
Jatropha curcas were found to have carcinogenic and muta-
genic properties. However, some naturally occurring phorbols
are tumor inhibitors and possess antileukemic and antimyco-
bacterial activity.28-30
It will be interesting in the future to elucidate the anti-
proliferative mechanism of action of the phorbol isolated in
this study.
In conclusion, these results demonstrate that the stems of S
grantii possess active principles with relevant antiproliferative
potential, distinct from those already described for this plant,
stimulating the progress of studies to discover the compounds
responsible for the indicated effects. Studies are in progress to
confirm these effects in other pharmacological models and to
isolate the other possible active principle of this promising
plant as an anticancer agent.
Acknowledgments
The authors are grateful to the Network Ribecancer 212RT 0464
(CYTED/CNPq), the National Council for Scientific and Technolo-
gical Development (Conselho Nacional de Desenvolvimento Cient-
fico e TecnologicoCNPq), and ProPPEC/UNIVALI for their
financial support
Author Contributions
Adriana Campos substantially contributed to conception or design,
contributed to acquisition, analysis, or interpretation of data, drafted
the manuscript, and critically revised the manuscript for important
intellectual content. Debora Barbosa Vendramini-Costa and Ana
Lucia Tasca Gois Ruiz contributed to acquisition, analysis, or inter-
pretation of data and critically revised the manuscript for important
intellectual content. Giovanna Barbarini Longato and Tailyn Zermiani
contributed to acquisition, analysis, or interpretation of data. Joao
Ernesto de Carvalho substantially contributed to conception or design
and contributed to acquisition, analysis, or interpretation of data. Ata-
nasio Pandiella substantially contributed to conception or design,
acquisition, analysis, or interpretation of data and critically revised
the manuscript for important intellectual content. Valdir Cechinel
Filho substantially contributed to conception or design, contributed
to acquisition, analysis, or interpretation of data, drafted the manu-
script, and critically revised the manuscript for important intellectual
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5
content. All authors gave final approval and agreed to be accountable
for all aspects of the work in ensuring that questions relating to the
accuracy or integrity of any part of the work are appropriately inves-
tigated and resolved.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to
the research, authorship, and/or publication of this article.
Funding
The author(s) received no financial support for the research, author-
ship, and/or publication of this article.
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