Isolation of arginase inhibitors from the bioactivity-guided fractionation of Byrsonima coccolobifolia leaves and stems

Article abstract of DOI:10.1021/np400717m

Byrsonima coccolobifolia leaf and stem extracts were studied in the search for possible leishmanicidal compounds using arginase (ARG) from Leishmania amazonensis as a molecular target. Flavonoids 1b, 1e-1g, 2a, 2b, and 2d-2f showed significant inhibitory

Full text of DOI:10.1021/np400717m

Note
pubs.acs.org/jnp
Isolation of Arginase Inhibitors from the Bioactivity-Guided
Fractionation of Byrsonima coccolobifolia Leaves and Stems
†
†
†
Lorena Ramos Freitas de Sousa, Suelem Demuner Ramalho, Marcela Carmen de Melo Burger,
†
†
†
Liliane Nebo, Joao
Monica Rosas da Costa Iemma, Caroindes Julia Correa
Maria Ines
̃
Batista Fernandes, Maria Fat
́
ima das Graca
̧
s Fernandes da Silva,
†
†
†
̂
̂
, Dulce Helena Ferreira de Souza,
‡
,†
̂
Salgueiro Lima, and Paulo Cezar Vieira*
†
Departamento de Química, Universidade Federal de Sao
Departamento de Botanica, Universidade Federal de Sao
S Supporting Information
̃
Carlos, Rod. Washington Luís, Km 235, 13565-905 Sao
̃
Carlos, SP, Brazil
̃
Carlos, SP, Brazil
‡
̂
̃
Carlos, Rod. Washington Luís, Km 235, 13565-905 Sao
*
ABSTRACT: Byrsonima coccolobifolia leaf and stem extracts
were studied in the search for possible leishmanicidal
compounds using arginase (ARG) from Leishmania amazo-
nensis as a molecular target. Flavonoids 1b, 1e−1g, 2a, 2b, and
2
d−2f showed significant inhibitory activity, with IC values
50
ranging from 0.9 to 4.8 μM. The kinetics of the most active
compounds were determined. Flavonoids 1e, 1f, 2a, 2b, and 2e
were characterized as noncompetitive inhibitors of ARG with
dissociation constants (K ) ranging from 0.24 to 3.8 μM,
i
demonstrating strong affinity. Structure−activity relationship
studies revealed some similarities in the structural features of
flavonoids related to ARG activity.
ffecting millions of people per year, leishmaniasis is one of
the first reaction in the metabolic pathway of polyamines
A
the most neglected tropical diseases and an urgent
(PAs), which are essential molecules in most living organisms,
including Leishmania parasites. Additionally, ARG is
1
3
problem in global public health according to the World Health
Organization. To overcome obstacles such as adverse reactions
and parasite resistance to available drugs against leishmaniasis, a
number of plants have been tested in the search for new front-
associated with the production of nitric oxide (NO) molecules,
14
high concentrations of which could kill the parasites. The
relationships of ARG with such molecules (PAs and NO) with
distinct properties are explained by the usage of the same
substrate, L-arginine, for the enzymatic activities in two
1
,2
line drugs.
Brazilian cerrado plants, the biodiversity of which is still
largely unknown, represent a rich source of new lead
compounds. Species such as Byrsonima coccolobifolia Kunth.
14
biosynthetic pathways.
3
Studies of mutant L. mexicana and Leishmania major parasites
(
Malpighiaceae) and Byrsonima crassa have been used in folk
lacking ARG confirmed that the ARG pathway is essential for
1
3,15,16
medicine for the treatment of stomach disorders and gastric
ulcers. Chemical study of extracts from B. crassa with an
antiulcerogenic effect has led to the isolation of flavonoids and
parasite viability and infectivity.
Importantly, the X-ray
4
,5
crystal structure of arginase from L. mexicana was solved in
recent work, which differs only in two amino acids from L.
5
13,17
biflavonoids. Furthermore, crude extracts from the leaves and
amazonensis, making them 99.4% identical.
The knowledge
of leishmanial ARG structure has revealed important features
for inhibitor interactions, thus making ARG an attractive target
stems of Byrsonima crassifolia and Byrsonima bucidaefolia were
shown to exhibit good inhibition of the growth of
promastigotes of Leishmania mexicana. Flavonoids were the
principal phytochemicals isolated from Byrsonima, which
indicates that this genus could contribute to the search for
new leishmanicidal compounds because flavonoids have
demonstrated antileishmanial activity.
these phenols were found to be inhibitors of arginase (ARG)
from Leishmania amazonensis and arginase (ARG-II) from
17
for leishmanicidal drug design.
4
In a search for new leishmanicidal compounds, extracts from
several Brazilian cerrado plants were evaluated against ARG.
The crude extracts from the leaves and stems of B. coccolobifolia
showed the most promising results, with inhibition higher than
6
,7
Recently, some of
6
0.0% against ARG at a concentration of 31.25 μg/mL. Ethyl
acetate extracts from the leaves and stems of B. coccolobifolia
showed 64% and 65% inhibition of ARG, respectively.
8
−10
mammals,
which has motivated the further study of this
class of plant secondary metabolites.
Arginase is a metalloenzyme from L. amazonensis
1
1,12
that
Received: September 10, 2013
Published: February 13, 2014
catalyzes the hydrolysis of L-arginine to L-ornithine and urea,
©
2014 American Chemical Society and
American Society of Pharmacognosy
392
dx.doi.org/10.1021/np400717m | J. Nat. Prod. 2014, 77, 392−396

Journal of Natural Products
Note
Table 1. Structures and IC50 Values of Several Flavonoids as Inhibitors of Arginase
b
compound
R1
R2
R3
R4
R5
R6
R7
IC50 (μM)
a
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
a
b
c
d
e
f
OH
OH
OH
OH
OH
OH
OH
H
OH
H
H
H
O-rha α
glc β
OH
OH
OH
OH
OH
OH
OH
OH
OAc
OCH3
OH
OH
OH
OH
OAc
OAc
H
H
H
H
H
H
H
H
H
OH
OH
OH
OH
OH
OH
OH
OH
OAc
OCH3
OH
OH
OH
OH
OAc
OAc
12.2 ± 1.8
2.0 ± 0.1
88.1 ± 9.1
223.5 ± 15.9
2.4 ± 0.2
2.1 ± 0.2
4.0 ± 0.5
55.0 ± 4.0
120.8 ± 12.1
127.9 ± 9.8
1.7 ± 0.1
1.6 ± 0.2
35.1 ± 3.2
3.7 ± 0.3
0.9 ± 0.1
4.8 ± 0.5
OH
OCH3
OCH3
OH
O-rha α
O-rha α
O-rha α
OH
OH
OH
OH
H
OH
g
h
i
OH
OH
OH
H
OH
OAc
OCH3
OH
OH
H
OAc
OCH3
OH
H
OAc
j
OCH3
H
OCH3
OH α
OH β
O-rha α
OAc α
OAc α
OAc α
OCH3
H
a
b
c
d
e
f
OH
H
H
OH
H
H
OH
OH
OAc
OH
H
H
OH
H
H
OAc
H
H
a
b
Quercitrin was used as a positive control. The values represent means of individual experiments ± SD.
Phytochemical study of these two EtOAc extracts led to the
The comparison of quercitrin (1a) (IC50 = 12.2 ± 1.8 μM)
with compound 1e (IC50 = 2.4 ± 0.2 μM) demonstrates an
improvement in inhibitory activity when the aromatic ring B of
the flavonoid has one extra hydroxy group. The observed
increase in the IC values of flavonoids with the same skeleton
as quercitrin (1a) (12.2 ± 1.8 μM) and compound 1c (88.1 ±
9.0 μM) can be related to the presence of a methoxy group
instead of a hydroxy group as in the catechol of 1a. The same
trend is seen for the glycoflavonols 1e (IC50 = 2.4 ± 0.2 μM)
and 1d (IC50 = 223.5 ± 15.8 μM), which have three
substituents in aromatic ring B, in which the replacement of
a hydroxy group with a methoxy group decreased the inhibitory
activity.
active known compounds, including flavonoids (1a, 1b, 2a,
8
,9,18−20
21
2
(
b),
(+)-syringaresinol (3), and trigonostemone
2
2
4). Compound 1a was previously isolated from a methanolic
23
extract from the leaves of B. coccolobifolia, and compounds 1b,
a, and 2b have been reported from other species in the genus
Byrsonima. Furthermore, this is the first report of compounds
50
2
4
3
and 4 in Byrsonima.
Isoquercitrin (1b), (+)-catechin (2a), and (−)-epicatechin
(2b) showed potent inhibitory activities against ARG, with IC50
values ranging from 1.6 to 2.0 μM (Table 1). Quercitrin (1a),
+)-syringaresinol (3), and trigonostemone (4) exhibited
moderate inhibitory activity, with IC50 values of 12.2 ± 1.8,
3.7 ± 1.5, and 16.6 ± 1.6 μM, respectively. The IC values of
(
1
The flavonol aglycones 1f and quercetin (1g) both showed a
50
the compounds quercitrin (1a), isoquercitrin (1b), quercetin
substantial inhibition of ARG, with IC values of approximately
50
(
1g), and kaempferol (1h) were reported previously (10, 3.8,
2.1 and 4.0 μM, respectively. However, compounds 1h, which
contains only one hydroxy group in aromatic ring B, 1i
(acetylated flavonol), and 1j (methoxylated flavonol) had less
potent effects. These compounds displayed lower activities
(IC = 55.0, 120.8, and 127.9 μM, respectively), and again this
9,24
4
.3, and ∼50 μM, respectively).
Flavonoids 1a and 1b were
characterized as noncompetitive inhibitors (K = 7.20 and 6.90
i
μM, respectively), and 1g was characterized as a mixed
inhibitor.
2
4
50
In an effort to establish a relationship of the chemical
structures of flavonoids with potent ARG inhibitory activity and
also to describe the type of inhibition for the potent inhibitors,
other flavonoids were also investigated (1c−j and 2c−f).
Catechin and quercetin were acetylated (1i, 2d−f) to analyze
the effect of substituting hydroxy groups for acetyl groups in
the flavonol and flavan-3-ol structural classes on ARG activity.
Although this class of natural products has been characterized
recently as inhibitors of recombinant ARG from L. amazonensis,
there are still only a few studies that have explored these
observation can be correlated directly with the influence of the
functional groups in the B ring. Previous in vitro studies on the
leishmania parasite showed that leishmanicidal potential was
lowered or completely lost with an increase in the number of
methoxy groups in the flavonol structure. Additionally, a
recent study showed that galangin, a flavonoid without any
7
substituents in the B ring, exhibited only low inhibition against
2
4
ARG (IC50 ≈ 100 μM).
IC values in the range 0.9 to 4.8 μM (Table 1) suggest that
50
flavan-3-ols also have important structural features that allow
these substances to bind to the enzyme. The presence of
hydroxy groups as substituents on the aromatic ring A and at
position 3 of the pyran ring of the flavonoid skeleton has been
demonstrated also to be important in relation to ARG
8
,9,24
compounds for their inhibitory effects.
The present
investigation, which searched for new ARG inhibitors, differs
from the results of previous reports in that these active
flavonoids were identified through a bioactivity-guided
approach.
24
inhibition. However, the selective acetylation of (+)-catechin
3
93
dx.doi.org/10.1021/np400717m | J. Nat. Prod. 2014, 77, 392−396

Journal of Natural Products
2a) at different positions did not show much influence on the
Note
(
decrease in Vmax values without affecting the affinity (KM).
These results reveal that flavonoids interact with ARG in a
noncompetitive manner. Kinetic analysis indicates that these
flavonoid interactions can occur in the free enzyme and also in
the enzyme−substrate complex at a site that is distinct from the
catalytic site, thus inducing changes in the shape of the active
site such that the substrate will no longer fit well.
enzyme inhibition activities (2d−f) (Table 1).
In silico studies were performed previously for some
flavonoids, demonstrating interactions with ARG enzyme
occurring between the ring B moiety and amino acids that
2
+
2+
are involved with metal bridge Mn −MnB coordination in
A
9
,24
the active site.
By the present experimental approach the
results supported such findings, showing that a catechol group
is highly important in reducing ARG activity.
Figure 1 shows Lineweaver−Burk plots for some of the most
potent compounds analyzed, providing valuable information
The dissociation constant (K ) values were obtained using
i
Dixon reciprocal plots of the initial velocities (1/v) versus a
varying concentration of inhibitor at a constant concentration
of L-arginine (Table 2).
Table 2. K Values of Some Flavonoids
i
compound
Ki (μM)
a
1
1
2
2
2
a
quercitrin
7.9
1.2
0.6
0.2
0.9
f
myricetin
a
b
e
(+)-catechin
(−)-epicatechin
3,5,7-triacetylcatechin
a
Quercitrin was used as a positive control.
Quercitrin (1a) was characterized previously as a non-
24
competitive inhibitor. The double-reciprocal plot of glyco-
flavonol 1e showed a noncompetitive inhibitory mechanism. It
is possible to deduce that inhibitor 1e preferentially binds to
the ES complex (K = 1.9 μM); in addition, 1e also interacts
is
with the free enzyme (K = 3.8 μM), as demonstrated by the
i
intersecting lines that converge to the left of the y-axis and
below the x-axis (α < 1) in the Lineweaver−Burk plot.
Overall, bioactive flavonoids with potent ARG inhibitory
activity were identified by screening the active ethyl acetate
extracts of B. coccolobifolia leaves and stems, thereby indicating
these compounds are responsible for the significant decrease in
ARG activity. The type of inhibition for the most active
flavonoids (1e, 1f, 2a, 2b, 2e) was determined by kinetic studies
showing strong affinity and potency on ARG. Compounds with
a flavan-3-ol unit represent a new subclass of noncompetitive
flavonoid ARG inhibitor.
Flavonoids 1a, 2a, 2b, 1f, and 1g were described previously
7,25,26
as inhibitors of the growth of amastigotes of L. donovani.
Additionally, quercitrin (1a) and quercetin (1g) significantly
reduced the growth of L. amazonensis in an in vivo murine
27
model of cutaneous leishmaniasis. The mechanism of action
of quercetin in L. amazonensis-infected macrophages was
28
recently related to the increase in reactive oxygen species.
Furthermore, the leishmanicidal activities of these flavonoids
may also be due to arginase inhibition, confirming these
compounds as new lead candidates in the search for
leishmanicidal drugs.
EXPERIMENTAL SECTION
■
General Experimental Procedures. The urea concentration was
detected at 600 nm on a Beckman Coulter DU 800 spectropho-
tometer. The 1D- and 2D-NMR determinations were carried out in
acetone-d and MeOH-d , using TMS as the internal reference, on a
Figure 1. Lineweaver−Burk plots indicate noncompetitive ARG
inhibition by (−)-epicatechin (2b) (A), myricetin 3-O-α-L-rhamnoside
6
4
(1e) (B), and 3,5,7-triacetylcatechin (2e) (C).
1
13
Bruker DRX-400 NMR spectrometer ( H: 400 MHz; C: 100 MHz).
MS spectra were recorded on a Bruker Daltonics micrOTOF-Q II-ESI-
TOF mass spectrometer. Isolation procedures were carried out on
silica gel 60 (Merck, 230−700 mesh) and Sephadex LH-20
about the mechanism of inhibition. In this investigation, the
kinetics of recombinant ARG were measured at pH = 9.6 and
(
Amersham Pharmacia Biotech AB). Thin-layer chromatography
2
afforded a K value of 22.6 ± 1.7 mM (R = 0.996), consistent
M
(TLC) on precoated aluminum silica 60 F254 (Merck) was used to
monitor isolation. Compounds were visualized by exposure under
UV254/366 light and by spraying with sulfuric acid−vanillin solution,
12
with the data in the literature. The double-reciprocal plots for
the evaluated compounds (1e, 1f, 2a, 2b, 2e) showed a
3
94
dx.doi.org/10.1021/np400717m | J. Nat. Prod. 2014, 77, 392−396

Journal of Natural Products
Note
followed by heating. The solvents used for extract preparation and
chromatographic fractionation were obtained from Vetec.
Plant Material. Leaves and stems of B. coccolobifolia were collected
medium was thawed at room temperature and resuspended in 30 mL
of 2 mM Tris buffer pH 7.0 with 0.3% Triton X-100 (buffer 1). The
cellular suspension was submitted to ultrasonic disruption (6 pulses of
1 min with intervals of 30 s, amplitude 20%), and the lysed cells were
centrifuged at 12 × 1000g for 20 min at 4 °C. The supernatant was
applied to a Ni-NTA Superflow column (1 mL), according to the
Qiagen protocol. The column was washed with buffer 1 plus 20 mM
imidazole (5 mL) and eluted with 250 mM imidazole in buffer 1 (5
mL). The fractions eluted from the column were pooled, dialyzed
against buffer 1, and applied to a DEAE Sepharose Fast Flow (HiTrap,
in July 2011 from the cerrado at the Federal University of Sao
̃
Carlos
(
UFSCar), Sao Carlos, SP, Brazil. Voucher specimens (8367) were
̃
deposited at the Herbarium of the Botany Laboratory (HUFSCar) at
UFSCar after identification by one of the authors (M.I.S.L.).
Extraction and Isolation. Ethanol extracts were prepared by
maceration of air-dried and powdered plant parts. The crude extracts
were obtained after drying to remove the solvents by rotatory
evaporation under reduced pressure at a temperature of 40 °C. Dried
extracts were submitted to enzymatic assays against ARG.
1
mL) anionic column previously equilibrated with column buffer 1
and eluted with a NaCl gradient (0 to 1 M) at a flow rate of 1 mL/
The ethanol extracts of the leaves (10.0 g) and stems (30.5 g) of B.
coccolobifolia exhibited ARG inhibitory activity. These crude extracts
were subjected to liquid−liquid partitioning to produce hexane,
EtOAc, and hydroalcoholic extracts. The EtOAc extracts from the
leaves (2.0 g) and stems (10.0 g) showed significant inhibition of
ARG. The leaf EtOAc extract was fractionated on a silica gel 60
column (18.0 × 5.5 cm; 1:9 acetone−hexane), affording 11 fractions
min. The protein concentration was determined by the method of
37
Bradford using the protein assay kit (BioRad) and bovine serum
albumin as standard.
Kinetic Measurements and Inhibition Assay. The activity of
recombinant arginase of L. amazonensis was defined as the amount of
1
2,38
enzyme that produced 1 μmol of urea in 1 min per mg of protein.
KM and Vmax values were determined based on the rate of the reaction
for different L-arginine concentrations (6.25, 12.5, 15.0, 25.0, 50.0,
(
BF1−BF11). Among them, BF11 (1.2 g), with 90.5% ARG inhibition,
was chromatographed over silica gel 60 (18.0 × 5.5 cm; 6:4 acetone−
hexane), leading to four subfractions (F14I−F14IV). Active
subfractions F14III (60.0 mg) and F14IV (128.0 mg), with 65.0%
and 95.0% ARG inhibition, respectively, after chromatographic
separations by Sephadex L-20 [(F14III: 56.0 × 2.0 cm; MeOH
isocratic); (F14IV: 53.0 × 4.0 and 37.0 × 1.4 cm; MeOH isocratic)],
afforded flavonoids 1a (24.9 mg) and 1b (2.0 mg). Bioactivity-guided
fractionation was performed for the stem EtOAc extract, and fraction
BC4 (4.0 g), which exhibited the highest ARG inhibition (80.0%), was
obtained by elution with 1:9 MeOH−CH Cl on silica gel 60 (50.0 ×
6
2.5, 75.0, and 100.0 mM), as determined from Michaelis−Menten
kinetic parameters and Lineweaver−Burk analysis. The samples were
evaluated against the recombinant ARG at an initial concentration of
100 μM. The IC50 values for the active samples were determined by
rate measurements for at least 10 inhibitor concentrations (1280,
1024, 512, 256, 128, 64, 16, 4.0, 1.0, and 0.25 μM). For 10 dilutions of
inhibitor, mix I was prepared using 50 μL of CHES buffer solution at
pH 9.6, 8 μL of arginase solution, and 292 μL of water. A 5 μL sample
of each inhibitor was added to 35 μL of mix I, and the reaction mixture
was incubated for 10 min at 37 °C. Then, 10 μL of L-arginine solution
was added to the reaction and incubated again for 10 min at 37 °C.
The final volume of the reaction mixture was 50 μL and contained 50
mM CHES buffer at pH 9.6 and 50 mM of the substrate L-arginine, at
pH 9.6. The second reaction was performed using an enzymatic
2
2
4.5 cm), yielding 10 subfractions. Flavonols 2a (12.0 mg) and 2b (12.0
mg), (+)-syringaresinol (3) (0.5 mg), and trigonostemone (4) (1.2
mg) were isolated by purification of subfraction 10 (35.0 mg) using a
Sephadex LH-20 column (52.0 × 1.5 cm; MeOH isocratic) and
monitoring by TLC. The isolated compounds were characterized by
1
13
NMR ( H and C, DEPT-135, HSQC, and HMBC) and mass
39
colorimetric assay with a commercially available assay kit
2
9−36
spectroscopy and compared with data published in the literature.
(Biotecnica, Brazil). To hydrolyze urea for quantification, 10 μL of
Chemicals. The following natural products were previously
the reaction mixture were added to 500 μL of reagent 1 previously
prepared (50 mM phosphate buffer, pH 6.7, 60 mM salicylate, 3.2 mM
sodium nitroprusside, and 30 000 IU urease) and incubated at 37 °C
for 10 min. Then, 500 μL of reagent 2 (140 mM NaOCl and 150 mM
NaOH) was added, and the reaction was incubated at the same
temperature for a further 10 min. Additionally, control assays were
performed without inhibitor as a negative control and in the presence
isolated in our laboratory: 3,4,6,7,3′,4′,5′-heptamethoxyflavone
2
9
30
(
1j), myricetin 3-O-α-rhamnoside (1e), catechin-3-O-α-rhamno-
31
pyranoside (2c), mearnsetin (1c), and tamarixetin 3-O-α-L-rhamno-
3
2,33
side (1d).
Myricetin (1f) was obtained by the acid hydrolysis of 1e.
Quercetin (1g) was acetylated with the acetic anhydride/pyridine/
DMAP method to produce quercetin pentaacetate (1i). 3,5,7,3′,4′-
Pentaacetylcatechin (2f), 3,5,7-triacetylcatechin (2e), and 3-acetylca-
techin (2d) were obtained by acetylation of (+)-catechin with the
3
4
35
9
of the known inhibitor quercitrin as a positive control. The enzymatic
assay was carried out in duplicate, and the urea concentration was
quantified spectrophotometrically at 600 nm. The type of inhibition
3
5,36
acetic anhydride/pyridine/DMAP method (2a).
The acetylation
and K values were determined using the same experimental approach
of (+)-catechin was monitored by TLC, and the reaction generated
partially and completely acetylated compounds, which were purified by
chromatography using a silica gel 60 column. All derivatives were fully
characterized from their spectroscopic data.
Sigma-Aldrich supplied quercitrin hydrate (1a), ≥78% (Sigma
Q3001); (+)-catechin hydrate (2a), ≥98% (Sigma C1251);
i
with three concentrations of inhibitor and a control under increasing
substrate concentrations (6.25, 12.5, 18.0, 25.0, 50.0, 60.0, and 72.0
mM). The kinetics data were analyzed by Lineweaver−Burk plot
analysis with the SigmaPlot 12.0 enzyme kinetics module.
(
−)-epicatechin (2b), ≥90% (Sigma E1753); kaempferol (1h),
ASSOCIATED CONTENT
≥
97% (Sigma 60010); quercetin hydrate (1g), ≥95% (Aldrich
37951); and quercetin 3-β-D-glucoside, ≥90% (Sigma 17793). The
enzymatic urea kit was purchased from Biotecnica (Varginha, MG,
Brazil).
■
3
*
S
Supporting Information
́
The kinetics of recombinant ARG, as represented by
Michaelis−Menten and Lineweaver−Burk plots, and Line-
weaver−Burk plots for inhibitors (1a, 2a, and 1f) are available
free of charge via the Internet at http://pubs.acs.org.
Expression and Purification of Recombinant ARG. The
recombinant enzyme was expressed and purified as described
6
previously, with some modifications. Briefly, the arginase expression
plasmid pRSET-Arg was transformed into E. coli Rosetta (DE3) pLysS
cells, and the culture was grown at 37 °C in SOB medium
supplemented with 100 μg/mL ampicillin and 34 μg/mL chlor-
amphenicol until the OD600 nm reached 0.6−0.8. Arginase expression
was induced by adding isopropyl-β-D-thiogalactopyranoside to a final
concentration of 1 mM. Incubation was extended for an additional 16
h at 30 °C. The cells were harvested by centrifugation (12 × 1000g for
AUTHOR INFORMATION
■
*
Corresponding Author
Tel/Fax: 55-16-33518350. E-mail: dpcv@ufscar.br.
Notes
1
0 min at 4 °C) and stored at −80 °C. The cell pellet from 200 mL of
The authors declare no competing financial interest.
3
95
dx.doi.org/10.1021/np400717m | J. Nat. Prod. 2014, 77, 392−396

Journal of Natural Products
Note
(
24) Manjolin, L. C.; dos Reis, M. B. G.; Maquiaveli, C. C.; Santos-
ACKNOWLEDGMENTS
■
Filho, O. A.; da Silva, E. R. Food Chem. 2013, 141, 2253−2262.
The authors wish to thank Dr. L. M. Floeter-Winter from the
Department of Physiology-IB USP for the clone provided. This
(25) Muzitano, M. F.; Cruz, E. A.; de Almeida, A. P.; Da Silva, S. A.;
Kaiser, C. R.; Guette, C.; Rossi-Bergmann, B.; Costa, S. S. Planta Med.
2006, 72, 81−83.
research project received financial support from the State of Sao
Paulo Research Foundation (FAPESP, Fundaca de Amparo a
Pesquisa do Estado de Sao Paulo) Proc. 2010-52326-9,
̃
̧
o
̃
̀
(26) Muzitano, M. F.; Tinoco, L. W.; Guette, C.; Kaiser, C. R.; Rossi-
Bergmann, B.; Costa, S. S. Phytochemistry 2006, 67, 2071−2077.
̃
(
̃
27) Muzitano, M. F.; Falcao, C. A.; Cruz, E. A.; Bergonzi, M. C.;
INBEQMEdi, and the National Council for Scientific and
Technological Development (CNPq, Conselho Nacional de
Pesquisa e Desenvolvimento), Brazil.
Bilia, A. R.; Vincieri, F. F.; Rossi-Bergmann, B.; Costa, S. S. Planta
Med. 2009, 75, 307−311.
(
28) Fonseca-Silva, F.; Inacio, J. D. F.; Canto-Cavalheiro, M. M.;
Almeida-Amaral, E. E. J. Nat. Prod. 2013, 76, 1505−1508.
(29) Ferracin, R. J.; da Silva, M. F. G. F.; Fernandes, J. B.; Vieira, P.
C. Phytochemistry 1998, 47, 393−396.
REFERENCES
■
(
(
1) da Silva-Lop
́
ez, R. E. Quim. Nova 2010, 33, 1541−1548.
(
30) Rashed, K.; Zhang, X. J.; Luo, M. T.; Zheng, Y. T.
Phytopharmacology 2012, 3, 199−207.
31) Ayres, M. C. C.; Escorcio, S. P.; da Costa, D. A.; Chaves, M. H.
Quim. Nova 2008, 6, 1481−1484.
32) Mahmoud, I. I.; Marzouk, M. S.; Moharram, F. A.; El-Gindi, M.
R.; Hassan, A. M. Phytochemistry 2001, 58, 1239−1244.
33) Son, Y. K.; Lee, M. H.; Han, Y. N. Arch. Pharm. Res. 2005, 28,
4−38.
34) Pizzolatti, M. G.; Cunha, A., Jr.; Szpoganicz, B.; de Sousa, E.;
Braz-Filho, R.; Schripsema, J. Quim. Nova 2003, 26, 466−469.
35) de Almeida, M. C. S.; Alves, L. A.; Souza, L. G. S.; Machado, L.
2) Santos, D. O.; Coutinho, C. E.; Madeira, M. F.; Bottino, C. G.;
Vieira, R. T.; Nascimento, S. B.; Bernardino, A.; Bourguignon, S. C.;
Corte-Real, S.; Pinho, R. T.; Rodrigues, C. R.; Castro, H. C. Parasitol.
Res. 2008, 103, 1−10.
3) Ratter, J. A.; Ribeiro, J. F.; Bridgewater, S. Ann. Bot. 1997, 80,
23−230.
4) Guilhon-Simplicio, F.; Pereira, M. M. Quim. Nova 2011, 34,
032−1041.
5) Sannomiya, M.; Fonseca, V. B.; da Silva, M. A.; Rocha, L. R. M.;
(
́
(
(
2
(
1
(
3
(
(
dos Santos, L. C.; Hiruma-Lima, C. A.; Souza Brito, A. R. M.; Vilegas,
W. J. Ethnopharmacol. 2005, 97, 1−6.
6) Camacho, M. D. R.; Phillipson, J. D.; Croft, S. L.; Marley, D.;
(
L.; de Matos, M. C.; de Oliveira, M. C. F.; Lemos, T. L. G.; Braz-Filho,
(
R. Quim. Nova 2010, 33, 1877−1881.
Kirby, G. C.; Warhurst, D. C. J. Nat. Prod. 2002, 65, 1457−1461.
7) Tasdemir, D.; Kaiser, M.; Brun, R.; Yardley, V.; Schmidt, T. J.;
Tosun, F.; Ruedi, P. Antimicrob. Agents Chemother. 2006, 50, 1352−
364.
8) Cruz, E. de M.; da Silva, E. R.; Maquiaveli, C. do C.; Alves, E. S.;
(36) Basak, A.; Mandal, S.; Bandyopadhyay, S. Bioorg. Med. Chem.
(
Lett. 2003, 13, 1083−1085.
̈
(37) Bradford, M. M. Anal. Biochem. 1976, 72, 248−254.
(38) Silva, E. R.; Floeter-Winter, L. M. Exp. Parasitol. 2010, 125,
152−155.
1
(
Lucon, J. F., Jr.; dos Reis, M. B.; de Toledo, C. E.; Cruz, F. G.;
(39) Fawcett, J. K.; Scott, J. E. J. Clin. Pathol. 1960, 13, 156−159.
Vannier-Santos, M. A. Phytochemistry 2013, 89, 71−77.
(
̃
9) da Silva, E. R.; Maquiaveli, C. do C.; Magalhaes, P. P. Exp.
Parasitol. 2012, 130, 183−188.
(
10) Kim, S. W.; Cuong, T. D.; Hung, T. M.; Ryoo, S.; Lee, J. H.
Pharm. Res. 2013, 36, 922−926.
(11) Colotti, G.; Ilari, A. Amino Acids 2011, 40, 269−285.
(12) da Silva, E. R.; da Silva, M. F.; Fischer, H.; Mortara, R. A.;
Mayer, M. G.; Framesqui, K.; Silber, A. M.; Floeter-Winter, L. M. Mol.
Biochem. Parasitol. 2008, 159, 104−111.
(
13) Roberts, S. C.; Tancer, M. J.; Polinsky, M. R.; Gibson, K. M.;
Heby, O.; Ullman, B. J. Biol. Chem. 2004, 279, 23668−23678.
́ ́
̃
a-Fouce, R.; Calvo-Alvarez, E.; Alvarez-Velilla, R.; Prada,
(
14) Balan
C. F.; Perez-Pertejo, Y.; Reguera, R. M. Mol. Biochem. Parasitol. 2012,
81, 85−93.
15) da Silva, M. F. L.; Zampieri, R. A.; Muxel, S. M.; Beverley, S. M.;
Floeter-Winter, L. M. PLoS One 2012, 7, e34022.
16) Iniesta, V.; Gomez-Nieto, L. C.; Corraliza, I. J. Exp. Med. 2001,
93, 777−783.
17) D’Antonio, E. L.; Ullman, B.; Roberts, S. C.; Dixit, U. G.;
Wilson, M. E.; Hai, Y.; Christianson, D. W. Arch. Biochem. Biophys.
013, 535, 163−176.
18) Kapoor, M.; Howard, R.; Hall, I.; Appleton, I. Am. J. Pathol.
004, 165, 299−307.
19) Lobo, L. T.; Castro, K. C. F.; Arruda, M. S. P.; Silva, M. N. da;
Arruda, A. C.; Muller, A. H.; Arruda, G. M. S. P.; Santos, A. S.; da
Souza, A. P. da S. F. Quim. Nova 2008, 31, 493−497.
20) Ohguchi, K.; Nakajima, C.; Oyama, M.; Iinuma, M.; Itoh, T.;
Akao, Y.; Nozawa, Y.; Ito, M. Biol. Pharm. Bull. 2010, 33, 122−124.
21) Park, J. H.; Yeon, S. W.; Cho, J. G.; Lee, D. Y.; Kim, Y. S.; Baek,
N. I. J. Korean Soc. Appl. Biol. Chem. 2010, 53, 734−739.
22) Seephonkai, P.; Sangdee, A.; Bunchalee, P.; Pyne, S. G. J. Nat.
Prod. 2009, 72, 1892−1894.
23) Lorenzi, K. C.; Rodrigues, C. M.; Sannomiya, M.; de Almeida, L.
́
1
(
(
1
(
́
2
(
2
(
̂
̈
(
(
(
(
F. R.; Souza-Brito, A. R. M.; Vilegas, W.29th Reunia
̃
́
ica. Aguas de Lindoi
́
́
a − SP; 2006; p PN-
Sociedade Brasileira de Quim
08.
1
3
96
dx.doi.org/10.1021/np400717m | J. Nat. Prod. 2014, 77, 392−396