Cytotoxic Orbitide from the Latex of Croton urucurana

Article abstract of DOI:10.1021/acs.jnatprod.5b00724

The bioactive ethyl acetate phase obtained from the latex of Croton urucurana Baillon afforded a novel orbitide (1), [1-9-NαC]-crourorb A1, that proved active against NCI-ADR/RES (ovary, multidrug-resistance phenotype) cells with the same potency as doxor

Full text of DOI:10.1021/acs.jnatprod.5b00724

Article
pubs.acs.org/jnp
Cytotoxic Orbitide from the Latex of Croton urucurana
Priscila de M. Candido-Bacani,†,‡ Patrícia de O. Figueiredo,† Maria de F. C. Matos,‡ Fernanda R. Garcez,†
̂
and Walmir S. Garcez*,†
†Instituto de Química, Universidade Federal de Mato Grosso do Sul, Campo Grande, MS 79074-460, Brazil
‡Centro de Cien
̂
́
́
cias Biologicas e da Saude, Universidade Federal de Mato Grosso do Sul, Campo Grande, MS 79070-900, Brazil
S
* Supporting Information
ABSTRACT: The bioactive ethyl acetate phase obtained from
the latex of Croton urucurana Baillon afforded a novel orbitide
(1), [1−9-NαC]-crourorb A1, that proved active against NCI-
ADR/RES (ovary, multidrug-resistance phenotype) cells with
the same potency as doxorubicin (positive control) and
inactive up to the highest concentration tested against
nontumor NIH/3T3 cells. The structure elucidation was
based on 1D and 2D NMR spectroscopy, further supported by
HRESIMS data and by application of Marfey’s method for
determination of the absolute configuration of its amino acid
residues. This is the first orbitide obtained from C. urucurana.
he genus Croton (Euphorbiaceae) comprises about 1300
against 786-0 (kidney carcinoma), HT-29 (colon carcinoma),
NCI-ADR/RES (ovary adenocarcinoma, multidrug-resistance
phenotype), MCF-7 (breast adenocarcinoma), PC-03 (prostate
carcinoma), and Hep-G2 (hepatocellular carcinoma).
T
species of herbs, shrubs, and trees found in tropical and
subtropical regions.1,2 Some of these species have red-colored
latex, popularly associated with having therapeutic properties,3,4
a feature that may have led to its popular names of “dragon’s
We initially examined the EtOH extract obtained from bark,
which proved inactive (GI50 > 250 μg mL−1), and also the latex,
which proved cytotoxic against all the cancer cell lines
investigated (Table 1). The latex was therefore selected for
further cytotoxicity-guided isolation and phase-partitioned
between MeOH−H2O and hexane and subsequently between
MeOH−H2O and EtOAc. Of the six fractions partitioned from
the bioactive EtOAc phase, fraction 2 (F2) was the most active
against 786-0 (GI50 = 5.42 μg mL−1) and NCI-ADR/RES (GI50
= 3.93 μg mL−1) cells (Table 1). Further fractionation of F2 led
to isolation and structure elucidation of cytotoxic compound 1,
its major constituent. Table 1 depicts the cytotoxicity results
obtained for 1 and the foregoing extracts, phases, and fractions.
HRESIMS of 1 showed an [M + H]+ ion at m/z 817.4199,
compatible with the molecular formula C37H57N10O11 (calcd
for C37H57N10O11, m/z 817.4203). 13C NMR data revealed four
signals for a benzene ring [δC 126.6, 128.3 (×2), 129.4 (×2),
137.2], in addition to signals between δC 60.1 and 15.9 and 10
signals between δC 174.4 and 169.2. These 10 signals,
attributable to amide carbonyl resonances, suggested that 1 is
blood”, “sangre de drago”, and, in Brazil, “sangra-d’ag
́
ua”.5
Phytochemical studies from the latex of some Euphorbiaceae
species have led to the isolation of a large number of
biologically active constituents, namely, caryophyllaceae-type
cyclopeptides.6,7 This class of compounds was recently
redefined as orbitides8,9 and has attracted much attention due
to their important biological activities, such as cytotoxic activity
against several human cancer cell lines,6,7 thus making them a
class of promising therapeutic molecules.
́
Croton urucurana Baillon, one of the “sangra-d’agua”, exhibits
a wide range of pharmacological and biological proper-
ties.5,10−15 This plant is commonly found in Mato Grosso do
Sul State (midwestern Brazil), where its latex is popularly used
to treat several types of cancer. Despite the ethnopharmaco-
logical significance of the species, no phytochemical or
cytotoxic studies of its latex have been published.
As part of an ongoing research program to discover potential
anticancer compounds from plants of midwestern Brazil
(Cerrado and Pantanal biomes), this article reports the
isolation of a novel orbitide (1) from the latex of a specimen
of C. urucurana, as well as the determination of its cytotoxicity
against a panel of human cancer cell lines using the
sulforhodamine B (SRB) assay.
1
a peptide. The H NMR spectrum was consistent with this
possibility, based on resonances between δH 5.05 and 0.82 and
on a group of signals between δH 8.84 and 7.00, characteristic of
hydrogens linked to amide nitrogens (or peptide linkages) and
of aromatic hydrogens (Table 2).
RESULTS AND DISCUSSION
■
The phytochemical investigation of C. urucurana was guided by
the cytotoxic activity (detected using the SRB assay) exhibited
Received: August 13, 2015
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.5b00724
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Table 1. In Vitro Cytotoxicity (GI50) of Latex Extracts, Phases, Fractions, and Compound 1 from Croton urucurana against Six
a
Human Cancer Cell Lines
cell line and GI50 (μg mL−1
HT-29 MCF-7
>250 NT
)
sample
bark
786-0
>250
ADR-RES
NT
Hep-G2
>250
PC-03
>250
EtOH extract
latex
latex
34.92 1.39
30.17 7.42
30.54 4.48
20.55 2.17
>250
29.04 2.26
39.76 4.92
27.64 4.31
29.87 5.56
>250
21.28 1.27
20.83 1.75
19.35 2.41
24.07 3.49
>250
25.82 6.72
33.01 5.30
22.68 7.00
23.68 3.27
>250
26.80 7.57
207.17 6.33
38.19 4.14
28.56 7.71
>250
39.59 3.52
41.92 1.67
28.84 3.10
32.58 6.40
>250
hexane phase
EtOAc phase
MeOH−H2O phase
F1
EtOAc phase
F2
5.42 0.34
38.92 5.59
55.51 3.25
51.65 2.89
59.61 3.34
18.69 0.82
0.15 0.12
42.89 1.76
>250
48.79 0.94
3.93 0.10
41.78 6.68
14.79 1.10
22.29 6.02
38.67 0.65
3.98 0.20
3.00 0.43
82.95 1.40
>250
36.69 5.21
>250
F3
36.545 7.96
18.23 4.20
23.90 3.99
21.34 1.72
35.49 2.59
0.07 0.06
F4
37.18 5.01
27.84 0.11
29.90 3.21
37.28 0.57
0.85 1.01
38.33 4.09
34.98 1.89
39.43 0.52
41.31 2.70
0.25 0.01
35.56 4.87
33.29 0.42
35.47 2.84
29.80 0.34
0.22 0.03
F5
F6
F2
[1−9-NαC]-crourorb A1 (1)
doxorubicin
a
GI50: concentration that inhibits cell growth by 50%. Cancer cell lines: 786-0 (kidney), HT-29 (colon), MCF-7 (breast), NCI-ADR/RES (ovary,
multidrug-resistance phenotype), Hep-G2 (hepatocellular), and PC-03 (prostate). NT: not tested. Doxorubicin was the positive control. Values
represent means obtained from three independent experiments, each run in triplicate.
2D NMR experiments not only corroborated the foregoing
inference but also led to identification of nine amino acid
residues: three glycines (Ga, Gb, Gc), two leucines (La, Lb), one
phenylalanine (F), one asparagine (N), one alanine (A), and
one serine (S) (Table 2).
Reaney,9 compound 1 can be designated as [1−9-NαC]-
crourorb A1.
A range of biological properties have been reported for
orbitides, including immunomodulatory,19,20 antimalar-
ial,21,24,28,29,31 antifungal,23 and antibacterial activities.35 In
recent years, considerable attention has been devoted to the
anticancer activity of natural cyclic peptides,6,27,36−38 which
have been considered a new class of therapeutic molecules, due
to their chemical versatility and specificity for biological
targets.39
The absolute configurations of the A, F, L, and S residues
were identified as L(S) using Marfey’s method.16 On the basis
of NOE data, the configuration of the N residue was
determined to be L(S). This finding is in agreement with
studies showing that all cyclic peptides isolated from Croton
species,17,18 and likewise from other Euphorbiaceae,19−31
consist exclusively of L amino acids.
Evaluated against six human cancer cell lines, compound 1
proved almost as potent against NCI-ADR/RES cells (GI50
=
1
On the basis of correlations observed in the COSY H−1H
4.8 μM) as doxorubicin, the positive control (GI50 = 5.1 μM)
(Table 1), suggesting that 1 is the component responsible for
toxicity against this cell line in the preceding fraction F2. When
tested against 786-0 cells, however, F2 exhibited lower GI50
value than 1, suggesting that the cytotoxic activity of this
fraction against this cell line stems from other active minor
compounds or from synergistic interactions lost during
fractionation. Evaluated against NIH/3T3 nontumor cells, 1
remained inactive up to the highest concentration tested (GI50
> 305.8 μM), thus demonstrating selective inhibition against
NCI-ADR/RES cells. In addition, doxorubicin proved cytotoxic
against NIH/3T3 cells (GI50 = 4.0 μM), emphasizing a possible
advantage in the cytotoxic potential of 1, which would have a
reduced adverse effect in normal tissues. Orbitides have been
reported to have potent cytotoxic activity and may exhibit
differences in responsiveness/sensitivity to different cancer cells
lines.6,7 Nevertheless, further studies are necessary to determine
the cytotoxic effects on nontumor cells and the limitations of
this class of compounds.
Before the present study, the first of its kind reporting the
isolation of a cyclopeptide from C. urucurana latex, this class of
compounds had been identified in other Euphorbiaceae.19−31
Cyclopeptides had also been found in C. gossypifolius17 and C.
sparsiflorus,18 but their cytotoxic activities against cancer cell
lines were not evaluated. These cyclopeptides are characterized
by the presence of nine amino acid residues, of which
phenylalanine, asparagine, alanine, glycine, and leucine occur
in both chains and three glycine residues occupy the same
positions.
and NOESY spectra, the cyclic nature of this peptide and its
amino acid residue sequence were identified (Figure 1).
Noteworthy in the former experiment were the correlations
observed between the amino acid Hα and its NH group, which
permitted all hydrogens of each amino acid residue to be
assigned. Likewise, the NOESY correlations among NH groups
facilitated definition of the other amino acids immediately
preceding or succeeding each residue. A detailed analysis of
these data (Table 2) revealed the amino acid sequence in 1 as
being (cyclo)-GaSLaGbAFGcNLb−.
Characterization of 1 was also confirmed by its HRESIMS/
MS data, which showed fragments consistent with the proposed
sequence of amino acid residues (Table 3). The fragmentations
of cyclic peptides are dominated by the bx−yz pathway, in
which ring opening takes place via oxazolone formation,
resulting in linear peptide ions having a free N-terminus and an
oxazolone ring at the C-terminus.32 These linear ions can give
rise to lower b fragments by the bx−bx−1 pathway, in addition
to undergoing other possible fragmentations, such as CO loss
via the bx−ax pathway, subsequent losses of amino acid residues
by the bx−yz pathway, and common neutral losses (H2O,
NH3).32,33 Although no data are available on the loss of
ammonia from the N-terminal amino group of a linear ion, this
loss does occur from the side chain of asparagine, glutamine,
aspartic acid, and arginine residues.32
On the basis of its skeleton, this compound can be classified
as a caryophyllaceae-type cyclopeptide6 recently redefined as an
orbitide.8,9 According to Shim, Young, Arnison, Gilding, and
B
DOI: 10.1021/acs.jnatprod.5b00724
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a
Table 2. 1D and 2D NMR Data for [1−9-NαC]-Crourorb A1 (1) in DMSO-d6
1
C/H
δC
δH
HMBC H−C
NOE
COSY H−1H
Glycine A (Ga)
NH
α
8.57, br s
8.84 SNH, 3.88 Sα, 3.59 Gaα′, 3.76 Gaα, 7.22 LB2
8.57 GaNH, 7.22 LB NH
3.59 Gaα′, 3.76 Gaα
8.57 GaNH, 3.59 Gaα′
8.57 GaNH, 3.76 Gaα
42.81 3.76 overlapped
169.19 GaCO
α′
3.59 dd, J 17.0,
4.6 Hz
7.22 LBNH
CO
169.19
Serine (S)
NH
8.84, br s
5.05, t J 6.0 Hz
4.55 Laα, 8.57 GaNH, 3.88 Sα, 7.33 LaNH
8.84 SNH
3.88 Sα
OH
3.69 Sβ, Sβ′
5.05 SOH
8.84 SNH
β, β′
α
60.12 3.69 (2H), m
58.11 3.88, t J 6.0 Hz
174.41
174.71 SCO
174.71 SCO
CO
Leucine A (La)
NH
7.33, br s
1.75 Laβ, 1.45 Laβ′, 1.53 Laγ, 4.55 Laα, 3.82 Gbα, 3.43 Gbα 4.55 Laα
′, 7.97 GbNH, 8.84 SNH
δ
23.16 0.90, d, J 6.5 Hz
21.39 0.82, d, J 6.5 Hz
24.10 1.53, m
21.39 Laδ′, 39.80 Laβ
23.16 Laδ, 39.80 Laβ
0.82 Laδ′, 1.53 Laγ
0.90 Laδ, 4.55 Laα
1.53 Laγ
δ′
γ
1.53 Laγ
0.90 Laδ
0.90 Laδ, 0.82 Laδ′
4.55 Laα, 1.45 Laβ′
4.55 Laα, 1.75 Laβ
β
39.80 1.75, br t J 12.0 Hz 170.97 LaCO, 50.35 Laα
7.33 LaNH, 1.45 Laβ′, 4.55 Laα
7.33 LaNH, 1.75 Laβa, 4.55 Laα
β′
1.45, br dd J 12.0,
8.4 Hz
170.97 LaCO, 50.35 Laα
α
50.35 4.55 ddd J 11.4, 8.4, 170.97 LaCO
1.45 Laβ′, 1.75 Laβ, 0,82 Laδ′, 7.33 LaNH, 8.84 SNH
7.33 LaNH, 1.75 Laβ,
1.45 Laβ′
2.8 Hz
CO
170.97
Glycine B (Gb)
NH
7.97, t J 5.9 Hz
3.82 Gbα, 3.43 Gbα′, 7.33 LaNH, 8.52 ANH, 1.22 Aβ
7.97 GbNH, 7.33 LaNH
3.82 Gbα, 3.43 Gbα′
7.97 Gb NH, 3.43
Gbα′
α
43.08 3.82, overlapped
169.22 GbCO
169.22 GbCO
α′
3.43, dd J 17.5,
5.9 Hz
7.97 GbNH, 7.33 LaNH
7.97 GbNH, 3.82 Gbα
CO
169.22
Alanine (A)
NH
β
8.52, br s
15.89 1.22, d J 7.1 Hz
49.60 3.80, overlapped
171.93
4.24 Fα, 3.80 Aα, 7.97 GbNH, 7.58 FNH, 1.22 Aβ
8.52 ANH, 7.97 GbNH, 3.80 Aα
1.22 Aβ
3.80 Aα
49.60 Aα, 171.93 ACO
171.93 ACO
3.80 Aα
α
8.52 ANH, 1.22 Aβ
CO
3.80 2
Phenylalanine
(F)
NH
7.58, d 6.0 Hz
8.31 GcNH, 8.52 ANH, 3.08 Fβ, 3.03 Fβ′, 4.24 Fα, 3.93 4.24 Fα
Gcα, 3.19 Gcα′
4′
3′, 5′
2′, 6′
126.65 7.22 overlapped
128.32 7.28 overlapped
129.39 7.28 overlapped
129.39 F2′,6′
137.24 F1′, 129.39 F2′,6′
126.55 F4′, 128.32 F3′,5′,
137.24 F1′
1′
137.24
β
35.99 3.08, dd J 13.5,
129.39 F2′,6′, 137.25 F1′ 4.24 Fα, 7.58 FNH
129.39 F2′,6′, 137.25 F1′ 4.24 Fα, 7.58 FNH
4.24 Fα
4.24 Fα
7.2 Hz
β′
3.03, dd J 13.5,
8.4 Hz
α
56.03 4.24, br q J 7.2 Hz 173.06 F1
7,58 FNH, 8.52 ANH, 3.08 Fβ, 3.03 Fβ′
7.58 FNH, 3.08 Fβ,
3.03 Fβ′
CO
173.06
Glycine C (Gc)
NH
8.31, m
7.58 FNH, 4.28 Nα, 3.93 Gcα, 3.19 Gcα′
3.19 Gcα′, 8.31 GcNH, 7.58 FNH
3.93 Gcα, 3.19 Gcα′
8.31 GcNH, 3.19 Gcα′
α
42.91 3.93 dd J 16.8,
170.27 GcCO
7.4 Hz
α′
3.19, dd J 16.8,
3.4 Hz
3.93 Gcα, 8.31 GcNH, 7.58 FNH
8.31 GcNH, 3.93 Gcα
CO
170.27
Asparagine (N)
NH
8.22, brd J 6.3 Hz
7.00, br s
4.28 Nα, 4.40 Lbα, 7.22 LbNH
7.52 NNHb, 2.62 Nβ′, 2.85 Nβ
7.00 NNHa, 2.62 Nβ′, 2.85 Nβ
4.28 Nα
NHa
172.67 NγCO
172.67 NγCO
7.52 NNHb
7.00 NNHa
NHb
7.52, br s
γCO
172.67
C
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Table 2. continued
1
C/H
δC
δH
HMBC H−C
172.37 NCO
NOE
COSY H−1H
β
35.59 2.85, br dd J 15.9,
4.28 Nα, 8.22 NNH, 7.00 NNHa, 7.52 NNHb
4.28 Nα, 2.62 Nβ′
5.9 Hz
β′
α
2.62, dd J 15.9,
172.37 NCO
4.28 Nα, 8.22 NNH, 7.00 NNHa, 7.52 NNHb
8.22 NNH, 2.85 Nβ, 2.62 Nβ′, 8.31 GcNH
4.28 Nα, 2.85 Nβ
7.0 Hz
51.05 4.28, br q J 6.5 Hz 172.37 NCO
8.22 NNH, 2.62 Nβ′,
2.85 Nβ
CO
172.37
Leucine B (Lb)
NH
δ
7.22 overlapped
8.22 NNH, 8.57 GaNH, 4.40 Lbα, 3.76 Gaαa, 3.59 Gaα′ 4.40 Lbα
23.42 0.85, d, J 6.6 Hz
21.29 0.84, d, J 6.6 Hz
23.60 1.60, m
21.16 Lbδ′
23.42 Lbδ
1.60 Lbγ, 4.40 Lbα, 4.55 Nα
1.60 Lbγ
δ′
γ
1.60 Lbγ, 4.40 Lbα
1.60 Lbγ
0.85 Lbδ, 0.84 Lbδ′
4.40 Lb2
0.85 Lbδ, 0.84 Lbδ′
4.40 Lb2
β, β′
α
39.20 1.67, m
51.12 4.40, m
172.66 LbCO
7.22 LbNH, 8.22 NNH, 1.67 Lbβ,β′, 0.84 Lbδ′
7.22 LbNH, 1.67
Lbβ,β′
CO
172.66
a
Recorded at 300.13/75.47 MHz. Chemical shifts referenced to residual DMSO.
the CGMS Herbarium of the Universidade Federal de Mato Grosso do
Sul.
Cyclopeptide Extraction and Isolation. Bark material (2500 g)
was powdered and extracted in 95% EtOH at room temperature (RT)
and subsequently concentrated under reduced pressure. The latex (55
g) was suspended in MeOH−H2O (9:1), and the mixture extracted
successively with hexane and EtOAc. Of the resultant fractions, the
bioactive EtOAc phase was selected for further bioactivity-guided
fractionation. A 3.0 g aliquot of this phase was introduced into a
Sephadex LH-20 chromatography column (5 × 36 cm, MeOH), to
give six fractions (F1−F6).
Compound 1 (27.2 mg) was obtained as the major component of
the bioactive F2 fraction (100.8 mg) after isocratic semipreparative
HPLC using an RP-C18 column (MeCN−H2O, 37:63, v/v), at a flow
rate of 14 mL min−1.
[1−9-NαC]-Crourorb A1 (1): white, amorphous solid (H2O−
MeCN); [α]23 = −163 (c 3 × 10−2, H2O−MeCN 37%); UV
D
(MeOH) λmax (log ε) 203 (3.32), 245 (2.71) nm; IR (KBr) νmax 3300,
Figure 1. [1−9-NαC]-crourorb A1 (1), the orbitide [(cyclo)-
GaSLaGbAFGcNLb−] isolated from the latex of Croton urucurana.
1
3051, 2950, 1652, 1548; H and 13C NMR data, see Table 2; positive
HRESIMS m/z 817.4199 [M + H]+ (calcd for C37H57N10O11, m/z
817.4203); positive HRESIMS/MS data, see Table 3.
Marfey’s Analysis of the Absolute Configuration of Amino
Acid.16,40 Compound 1 (1 mg) was hydrolyzed in 1 mL of 6 N HCl
at 95 °C for 16 h. The hydrolysate was evaporated to dryness under a
N2 stream to remove traces of HCl and redissolved in 900 μL of
acetone and 1 M NaHCO3 (20 μL). A solution of N-α-(2,4-dinitro-5-
fluorophenyl)-L-alaninamide (L-FDAA, Marfey’s reagent, Sigma, 100
μL) was added, and the mixture heated at 40 °C for 1 h. The reaction
mixture was cooled to RT, quenched by adding 2 N HCl (10 μL),
dried, and dissolved in 50% aqueous CH3CN (600 μL). Five
microliters of the FDAA derivative was subjected to HPLC using a
C18 column at RT (20 °C). Aqueous CH3CN containing 4% TFA was
used as the mobile phase, with linear gradient elution (20−35%, 40
min) for L- and D-serine retention time analysis and with isocratic
elution (45% ACN) for L- and D-alanine, phenylalanine, and leucine
retention time analysis at a flow rate of 1 mL min−1. FDAA-derived
amino acids were detected by UV absorption at 340 nm.
From each separate 50 mM aqueous solution of D- or L-
configurations of A, S, F, or L, 50 μL aliquots were taken (totaling
eight different samples), and 1 M NaHCO3 (20 μL) and a 1% solution
of L-FDAA in acetone (100 μL) were added. The mixtures were
separately heated at 40 °C for 1 h, then cooled to RT, quenched by
adding 2 N HCl (10 μL), dried, and dissolved in 50% aqueous
CH3CN (600 μL). Subsequently, 5 μL of each FDAA derivative was
analyzed by HPLC. The following retention times (min) were
observed for the L-FDAA derivatives of the standard amino acids and
for the residual Marfey’s reagent, respectively: L-Ala (7.5 and 9.4), D-
Ala (8.4 and 9.4), L-Leu (13.1 and 9.4), D-Leu (18.9 and 9.4), L-Phe
The present results indicate therapeutic anticancer potential
for C. urucurana latex, corroborating its popular use in
anticancer therapy. However, further studies are necessary to
elucidate the mechanisms of action underlying the cytotoxic
effects of compound 1 against human cancer cell lines.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotation was
determined on a PerkinElmer 341 polarimeter (Na filter, λ = 589
nm). UV spectra were obtained on a USB4000 UV/vis spectropho-
tometer (Ocean Optics). IR spectra were recorded on a Nicolet iS5
FT-IR spectrometer (Thermo Scientific). NMR spectra were obtained
in DMSO-d6 (Cambridge Isotope Laboratories) on a Bruker DPX-300
spectrometer operating at 300.13 MHz (1H)/75.47 MHz (13C).
HRESIMS data were acquired in the positive ion mode on a
micrOTOF-Q II instrument (Bruker Daltonics). Sephadex LH-20 was
used for column chromatography. Reversed-phase semipreparative
HPLC separation was performed with a Shimadzu LC-6AD pump
using RP-18 silica gel (5 μm, 21.6 × 250 mm) in a Shim-Pack column
at a flow rate of 14 mL min−1, with monitoring at 270 nm.
Plant Material. Bark and red latex of Croton urucurana Baillon
(Euphorbiaceae) was collected from Campo Grande, Mato Grosso do
Sul, Brazil (20°25′13″ S/54°39′31″ W) in March 2012. The specimen
was identified by Prof. Arnildo Pott (Centro de Cien
̂
cias Biolog
́
icas e
da Saude, Universidade Federal de Mato Grosso do Sul, Campo
́
Grande, MS, Brazil), and a voucher specimen (no. 30924) deposited at
D
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Table 3. Main Fragment Ions Observed in the HRESIMS/MS Spectrum of 1 (m/z 817.4199, [M + H]+, 46.3 eV)
(12.5 and 9.4), D-Phe (16.6 and 9.4), L-Ser (8.8 and 15.1), and D-Ser
(9.2 and 15.0). The retention times (min) observed for L-FDAA
derivatives of acid hydrolysates of compound 1 and for the residual
Marfey’s reagent were as follows: L-Ala (7.4 and 9.7, respectively), L-
Leu (13.0 and 9.7), L-Phe (12.4 and 9.7), and L-Ser (8.6 and 15.3).
In Vitro Cytotoxicity Assay. The cytotoxic activities of the
extract, phases, fractions, and compound 1 were evaluated against six
human cancer cell linesnamely, 786-0 (ATCC-CRL-1932, human
kidney carcinoma), HT-29 (ATCC-HTB-38, human colon carcino-
ma), MCF-7 (ATCC-HTB-22, breast adenocarcinoma), NCI-ADR/
RES (ovary adenocarcinoma, multidrug-resistance phenotype), and
PC-03 (ATCC-CRL-1435, human prostate carcinoma), all provided
by Prof. Joao Ernesto de Carvalho (CPQBA, Universidade Estadual de
Campinas), and Hep-G2 (ATCC-HB-8065, hepatocellular carcinoma),
provided by Prof. Ilce Mara de Syllos Colus (Universidade Estadual de
́
̃
Londrina)and using the sulforhodamine B assay (purity ≥97%;
Sigma), as described by Skehan et al.41 NIH/3T3 (murine fibroblast;
ATCC CRL-1658) nontumoral cells were purchased from the Rio de
Janeiro Cell Bank.
Briefly, the cells were seeded in 96-well plates at a density of 7.5 ×
103 cells/well, and each sample was tested in triplicate at four
concentrations (0.25, 2.5, 25, and 250 μg mL−1) at 37 °C and 5% of
CO2 for 48 h. The samples were dissolved in DMSO, aliquoted, and
stored at −20 °C. Final DMSO concentration did not affect cell
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viability (0.25%). Doxorubicin hydrochloride (2 mg/mL, Libbs) was
used as the positive control. Growth inhibition by 50% (GI50) was
determined by nonlinear regression analysis using Origin 6.0 software
(OriginLab).42,43 Samples were considered inactive when GI50 values
exceeded 250 μg mL−1.
(13) Esmeraldino, L. E.; Souza, A. M.; Sampaio, S. V. Phytomedicine
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Bras. Farmacogn. 2008, 18, 587−593.
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ASSOCIATED CONTENT
* Supporting Information
■
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The Supporting Information is available free of charge on the
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prod.5b00724.
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1888.
1D and 2D NMR, HRESIMS, HRESIMS/MS, UV, and
IR spectra of compound 1 (PDF)
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Labadie, R. P. FEBS Lett. 1989, 256, 91−96.
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Bosch, J. J.; Kroes, B. H.; Beukelman, C. J.; Leeflang, B. R.; Labadie, R.
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AUTHOR INFORMATION
Corresponding Author
*Tel (W. S. Garcez): 0055-67-3345-3599. Fax: 0055-67-3345-
3552. E-mail: walmir.garcez@ufms.br.
■
(21) Auvin, C.; Baraguey, C.; Blond, A.; Lezenven, F.; Pousset, J.-L.;
Bodo, B. Tetrahedron Lett. 1997, 38, 2845−2848.
(22) Altei, W. F.; Picchi, D. G.; Abissi, B. M.; Giesel, G. M.; Flausino,
O., Jr.; Reboud-Ravaux, M.; Verli, H.; Crusca, E., Jr.; Silveira, E. R.;
Cilli, E. M.; Bolzani, V. S. Phytochemistry 2014, 107, 91−96.
(23) Altei, W. F.; Picchi, D. G.; Torres, L. B.; Bolzani, V. S.; Cilli, E.
M.; Giannini, M. J.; Giesel, G. M.; Verli, H. Annual Meeting of the
Brazilian Society on Biochemistry and Molecular Biology; 2009, 41.
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by Fundect-MS PRONEM 0088/12,
CNPq, CPq-PROPP-UFMS, and CAPES for the doctoral
grants awarded to P.M.C.-B. Thanks are also extended to Prof.
M. G. Carvalho, who kindly provided us with Marfey’s reagent
and the D- and L-amino acid standards; Prof. J. E. Carvalho and
Prof. I. M. S. Colus, for providing the cell lines; and Prof. A.
́
Pott, for his assistance in plant identification.
(25) Horsten, S. F. A. J.; van den Berg, A. J. J.; Kettenes-van den
Bosch, J. J.; Leeflang, B. R.; Labadie, R. P. Planta Med. 1996, 62, 46−
50.
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B. J. Nat. Prod. 1997, 60, 1155−1157.
(27) Mongkolvisut, W.; Sutthivaiyakit, S.; Leutbecher, H.; Mika, S.;
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