fruits. Medicinal uses of this species have not been reported
frequently, although aqueous preparations from stem barks
have been used by Paraguayan communities in the therapy
of ascariasis . Previous bioprospection studies have
demonstrated the presence of guanidine alkaloids, which
exhibited cytotoxic activity against human myeloblastic
leukemia and human glioblastoma cells, and phenolic
compounds with anti-inflammatory, antioxidant, antimuta-
genic, and antidiabetic activities [14–22].
In this study, we demonstrated that two alkaloids isolated
from P. n i t e n s , pterogynine (PGN) and pterogynidine
(PGD; Fig. 1), showed an interesting potential anti-tumor
activity in vitro by inducing programmed cell death. In
addition, we present the results obtained by methods
(Annexin–V, Hoechst and Caspase 3/7) used for the
quantitation of the apoptosis.
Materials and methods
P. nitens leaves were collected at the Botanic Garden of Sao
Paulo, São Paulo State, Brazil, in May 2003. A voucher
specimen (SP204319) has been deposited in the herbarium
of the Botanic Institute (São Paulo State, Brazil).
Extraction, isolation, and identification of alkaloids
The shade-dried leaves (2.8 kg) were ground and defatted
with hexane (2.0 L×5, at room temperature, for 5 weeks)
and exhaustively extracted by maceration with ethanol
(4.0 L×5) at room temperature, for 5 weeks. The ethanol
extract was concentrated under reduced pressure (≤40°C),
to yield 12.7 of a syrup. The concentrate was then diluted
with methanol-water (4:1; 3.5 L) and partitioned succes-
sively with ethyl acetate (5.0 L×3) and n-butanol (5.0 L ×
3). After removal of the solvent, 3.7 and 5.9 g of extract
were afforded, respectively. The n-butanol residue (2.5 g)
was subjected to gel permeation chromatography on a
column of Sephadex LH-20 in methanol to afford nine
fractions, which were combined on the basis of their TLC
visualized with Sakaguchi’s and Dragendorff’s reagents
, to yield an alkaloidal fraction (587 mg). Separation of
this fraction on reversed-phase silica gel column chroma-
tography (RP-18) by elution with increasing amounts of
acetonitrile in water afforded eight fractions (ALK-1–ALK-
8). Fraction ALK-3 (121 mg) was subjected to repeated
column chromatography on silica gel (230-400 mesh),
eluted with chloroform-methanol mixtures (ranging from
0% to 35% of methanol), to furnish the guanidine alkaloids,
pterogynine (PNG, 27 mg) and pterogynidine (PDG,
22 mg). The molecular structures of these compounds were
identified by comparison with literature data [24,25],
C NMR (Fig. 1).
PNG (yield 4.6%, calculated from the alkaloidal frac-
tion): yellow oil.
H NMR δ
position): 7.44-7.64 (br s, H-1 and H-3), 3.72 (d; 6.5, H-1′),
5.16 (t, 6.5, H-2′), 1.62 (s, H-4′), 1.68 (s, H-5′).
(position): 155.7 (C-2), 39.0 (C-1′), 119.2 (C-2′), 135.8
(C-3′), 17.8 (C-4′), 25.2 (C-5′).
PDG (yield 3.8%, calculated from the alkaloidal frac-
tion): yellow oil.
H NMR δ
position): 7.72 (t; 6.5; H-3 and H-4), 7.20 (br s; H-1), 3.70
(t; 6.5, H-1′), 5.16 (t, 6.5, H-2′), 1.63 (s, H-4′), 1.69 (s,
(position): 156.9 (C-2), 38.7 (C-1′),
119.0 (C-2′), 136.0 (C-3′), 17.7 (C-4′), 25.1 (C-5′).
Human infiltrating ductal carcinoma cells (ZR-7531) were
obtained from American Type Culture Collection (ATCC,
USA). Cells were grown at 37°C in Dulbecco’s modified
eagle medium (DMEM) media and Ham’s F10 (Sigma, St.
Louis, MO, USA), supplemented with 10% fetal bovine
serum, 100 units/mL penicillin and 100 μg/mL streptomy-
cin, in an incubator containing 5% CO
To explore the induction of apoptosis (Annexin-V, Hoechst
and Caspase 3/7 assays) and to determine the cytotoxicity
of alkaloids PGN and PGD, cells were grown to 70%
confluence and treated for 24 h with alkaloids (t0) or treated
for 24 h with alkaloids and then for another 24 h with fresh
medium (t24) to observe recuperation. Various concentra-
tions of the alkaloids, from 0.25 to 10 mM, diluted in the
culture media from a 40 mM stock solution in water, were
used in the apoptosis and cytotoxicity tests.
The cytotoxicity of both alkaloids was determined by the
MTT colorimetric assay, which was performed to detect
tumor cell viability after incubation. The cells were cultured
Fig. 1 Molecular structures of guanidine alkaloids, pterogynine
(PNG) and pterogynidine (PDG), isolated from the leaves of
514 Tumor Biol. (2010) 31:513–522