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J. Braz. Chem. Soc., Vol. 00, No. 00, 1-9, 2014.
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Article
Leishmanicidal Activity of Brosimum glaziovii (Moraceae) and Chemical
Composition of the Bioactive Fractions by Using High-Resolution Gas
Chromatography and GC-MS
Aline Coqueiro,a Luis O. Regasini,a Gabriel M. Leme,a Luciana Polese,a
Camila T. Nogueira,b Mayara L. Del Cistia,b Marcia A. S. Graminhab and
Vanderlan da S. Bolzani*,a
Instituto de Química, Universidade Estadual Paulista (Unesp),
CP 355, 14801-970 Araraquara-SP, Brazil
a
Faculdade de Ciências Farmacêuticas, Universidade Estadual Paulista (Unesp),
14801-902 Araraquara-SP, Brazil
b
Dando continuidade às pesquisas de bioprospecção dos programas SisBiota CNPq/FAPESP e
Biota-FAPESP, o presente artigo descreve os resultados de atividade leishmanicida e determinação
da composição química das frações ativas de Brosimum glaziovii (Moraceae), por meio da
cromatografia gasosa. Os extratos e frações das folhas e galhos foram testados in vitro em cepas de
Leishmania amazonensis. As frações hexânicas das folhas e galhos apresentaram atividades potente
e moderada, respectivamente (IC50 = 3,6 e 39,1 µg mL-1). Por meio das análises de cromatografia
gasosa foi possível identificar os esteróis: campesterol, b-sitosterol e estigmasterol e os triterpenos:
α-amirina, β-amirina, acetato de β-amirina, e lupenona, além de outros constituintes apolares
principalmente ácidos graxos e seus derivados. Este é o primeiro trabalho descrito sobre a atividade
leishmanicida e composição química desta espécie.
As part of our ongoing SisBiota CNPq/FAPESP and Biota-FAPESP bioprospecting programs,
this paper deals with the leishmanicidal properties of Brosimum glaziovii (Moraceae), and
used gas chromatography analysis to determine the chemical composition of these fractions.
The extracts and fractions from the leaves and branches of B. glaziovii were screened against
Leishmania amazonensis. The hexane fractions from the leaves and branches displayed the highest
leishmanicidal activities, with IC50 = 3.6 and 39.1 µg mL-1. Using gas chromatography analysis
it was possible to identify the sterols campesterol, stigmasterol, and b-sitosterol, as well as the
triterpenes α-amyrin, β-amyrin, β-amyrin acetate, and lupenone, and other nonpolar components,
mainly fatty acids and their derivatives. This is the first report on the leishmanicidal activity and
the chemical composition of B. glaziovii.
Keywords: Brosimum glaziovii, leishmanicidal, sterol, triterpene, gas chromatography
Introduction
Neglected tropical diseases (NTDs) are a pathologically
diverse group of infections caused by a variety of
pathogens, such as viruses, bacteria, protozoa, and
helminthes. According to the World Health Organization
(WHO), 17 neglected tropical diseases exist. They are
endemic in 149 countries and affect more than 1 billion
people worldwide.1 The tropical and subtropical regions of
the globe concentrate the main countries with the lowest
*e-mail: bolzaniv@iq.unesp.br
human development index (HDI) and the highest NTDs
burden.2
Brazil ranks as the 70th country in terms of HDI; nine
of the 10 major NTDs occur in this country. Leishmaniasis,
tuberculosis, dengue, and leprosy exist in almost all the
Brazilian regions. Most cases arise in poor regions, being
the northern and northeastern Brazilian regions the most
affected.2
Leishmaniasis threatens approximately 350 million
people around the world. It is believed that as many as
12 million people are currently infected with this pathogen.
Roughly 1-2 million new cases appear every year.3 The
A
2
Leishmanicidal Activity of Brosimum glaziovii (Moraceae) and Chemical Composition
human immunodeficiency virus (HIV) pandemic has
contributed to raising the number of leishmaniasis cases
in endemic areas.4 In Brazil, more than 26,000 cases of
cutaneous leishmaniasis (CL) emerged between 1988 and
2009;5 more than 70,000 cases of visceral leishmaniasis
(VL) arose between 1980 and 2008, with 3,800 deaths.6
Currently, leishmaniasis treatment relies on
chemotherapy, which poses limitations such as toxicity,
difficult administration route, and lack of efficacy in
endemic areas. Despite the efforts devoted to finding
new drugs that can fight against Leishmania spp.,
leishmaniasis treatment still depends on the use of
pentavalent antimonials (sodium stibogluconate and
meglumine antimoniate), first developed over 60 years
ago. Unfortunately, the emergence of resistant strains has
limited the usefulness of these antimonials. Amphotericin
B (and its liposomal formulation, Ambisome), pentamidine
isethionate, miltefosine, and paromomycin constitute
alternative medications, but their toxicity and high prices
have limited their use.7
Brazilian biodiversity is among the richest in the
world. Indeed, Brazil is home to many plant species with
unknown potential. These plants could find appplication
in the search for natural products to treat health problems,
like NTDs.
Plants belonging to the family Moraceae Cecropia pachystachya Mart., 8 Ficus mucusa, 9 and
Pourouma guianensis,10 among others, possess antiprotozoal
activities. In particular, Brosimum glaziovii (Moraceae) is
endemic in Brazil, but habitat loss threatens this species.
The absence of reports on the chemical composition
and biological potential of this plant has motivated the
phytochemical and biological investigation that is the
subject of this article.
To our knowledge, only three species of the same genus
as B. glaziovii have been phytochemically examined:
B. gaudichaudii, whose roots contain coumarins, chalcones,
cinnamic and dihydrocinnamic acid derivatives, triterpenes,
and sterols;11-13 B. acutifolium, which possesses flavonoids
and lignoids; 14-18 and B. rubescens Taubert, whose
heartwood accumulates coumarins.19
As part of the SisBiota CNPq/FAPESP and
Biota‑FAPESP biodiscovery programs, which aim to
search for new hits and lead compounds from the Brazilian
flora, this study evaluated the leishmanicidal activity of
Brozimum glaziovii (Moraceae) and used high-resolution
gas chromatography (HRGC) and gas chromatographymass spectrometry (GC-MS) to analyze the chemical
composition of the bioactive extracts. HRGC constitutes
a promising tool to identify the chemical profile of
complex nonpolar extracts and fractions. It is an alternative
J. Braz. Chem. Soc.
methodology to aid the construction of the inventory and
the characterization of the biodiversity of São Paulo state,
which should be useful when exploring mechanisms for its
conservation and sustainable use.
Experimental
Plant material
The leaves and branches of Brosimum glaziovii
were collected from the Botanic Garden of São Paulo
(São Paulo, Brazil) in October 2006 and identified by
Dr Inês Cordeiro (IBt-SMA). A voucher specimen (col.
S. Romaniuc Neto 1298) was deposited at the State
Herbarium “Maria Eneida P. Kaufman” of the Institute
of Botany (São Paulo, Brazil).
Preparation of the extracts
Air-dried and powdered leaves (0.85 kg) and branches
(2.5 kg) were exhaustively extracted by maceration with
ethanol at room temperature. After filtration, the solvent was
evaporated at low temperature (< 40 °C) and under reduced
pressure, to yield the crude extract from the leaves (35.3 g)
and branches (57.3 g). Part of the crude extract from the
leaves (10 g) and branches (17 g) was dispersed in water/
methanol 4:1 (600 mL) and successively partitioned with
hexane (200 mL, 3×), ethyl acetate (200 mL, 3×), and
n-butanol (200 mL, 3×), to afford four fractions from the
leaves, namely the hexane (1.5 g), ethyl acetate (1.2 g),
n-butanol (0.5 g), and the lyophilized aqueous methanol
(0.7 g) fractions, as well as four fractions from the branches
- the hexane (2.5 g), ethyl acetate (6.1 g), n-butanol (2.1 g),
and the lyophilized aqueous methanol (2.2 g) fractions.
Samples of the ethanol extracts and of the hexane, ethyl
acetate, n-butanol, and the lyophilized aqueous methanol
fractions were further assayed for their leishmanicidal action.
The hexane fractions were analyzed by gas chromatography
coupled to a flame ionization detector (GC-FID) and gas
chromatography-mass spectrometry (GC-MS).
Sample cleanup for HRGC analysis (GC-FID)
Samples of the hexane fraction from the leaves
and branches (10.0 mg) were dissolved in chloroform
(3.0 mL) and percolated through a chromatographic
column consisting of celite/norit (1:1, 100.0 mg) + silica
gel (200.0 mg); the fractions were eluted with chloroform
(10.0 mL). After evaporation to dryness at room temperature,
the eluate was dissolved in hexane/ethyl acetate (7:3) and
analyzed by HRGC, in triplicate.
Vol. 00, No. 00, 2014
Coqueiro et al.
3
GC-FID conditions
Parasite cultures
The hexane fractions were analyzed by HRGC on
a Varian model CP-3800 gas chromatograph equipped
with split injector [initial splitless; 0.75 min (1:50);
2.00 min (1:20)] at 260 ºC. The flame ionization detector
was set at 310 and 290 ºC for the SPB-5 and SPB‑50
columns, respectively. The injected volume was 2.0 µL.
SPB-50 (cross-linked 50% phenylmethylsiloxane,
30 m × 0.25 mm × 0.25 µm) and SPB-5 (cross-linked 5%
phenylmethylsiloxan 30 m × 0.25 mm × 0.25 µm) capillary
columns were employed. In the case of SPB-50, the column
temperature was 280 ºC (isotherm). For SPB‑5, a column
temperature program was used - the initial temperature was
250 ºC, maintained for 12 min, followed by a temperature
rise to 280 ºC at 6 ºC min-1. Then, the temperature was kept
at 280 ºC for additional 30 min. Nitrogen was employed as
the carrier gas at an average linear velocity of 1 mL min-1.
The triterpenes and sterols were identified by comparison
of the relative retention (RR) of the samples with the RR of
authentic standards. Cholesterol was used as internal standard.
Promastigotes of the Leishmania amazonensis MPRO/
BR/1972/M1841-LV-79 strain were maintained at 28 ºC in
liver-infusion tryptose medium (LIT) supplemented with
10% heat-inactivated fetal calf serum (FCS). LIT medium
was prepared by mixing 10 mg mL-1 hemin (bovine, type I)
(1 mL) with a solution containing NaCl (4.0 g), KCl (0.4 g),
Na2PO4 (8.0 g), glucose (2.0 g), liver infusion broth (5.0 g),
and tryptose (5.0 g) at pH 7.0 (900 mL).
Authentic triterpene and sterol standards
The triterpenes and sterols used as standards were
previously isolated from Lauraceae and Rubiaceae
species, including Alibertia macrophylla, A. sessilis,
Licaria rodriguesii, L. subbulata, and Aniba parviflora. The
molecular structures of these compounds were identified
by the 1H and 13C nuclear magnetic resonance (NMR) and
the MS techniques.20,21
GC-MS conditions
Coupled gas chromatography-mass spectrometry
(GC‑MS) analysis was performed on an Agilent
Technologies 7890A gas chromatograph coupled to a 5975C
mass selective detector (Agilent Technologies) under the
following instrumental conditions: DB-5 capillary column
(cross-linked 5% diphenyl/95% dimethyl polysiloxane,
30 m × 0.25 mm × 0.25 µm, Agilent Technologies Inc.,
USA). Helium was used as the carrier gas at a flow rate of
1.00 mL min-1. The injection volume was 1 μL; the split
ratio was 20:1. The oven temperature was increased to
50 °C and held at this temperature for 2 min. Then, the
temperature was raised to 250 °C at a rate of 8 °C min-1,
to 300 ºC at a rate of 3 °C min-1, and to 310 °C at a rate
of 3 °C min-1. The total run time was 47 min. The mass
spectra were obtained by electron ionization (EI) at 70 eV.
All registered peaks were identified by comparison of the
mass spectra with those available in the NIST library.
Evaluation of the leishmanicidal activity
An antileishmanial assay using promastigote forms
of the Leishmania amazonensis MPRO/BR/1972/
M1841-LV-79 strain was performed by employing the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrasodium
bromide (MTT) colorimetric method described by Santos
et al.22 At the end of the exponential growth phase (4-dayold culture forms), the cultured promastigotes were
seeded in 96-well microplates at 8 × 106 parasites mL-1.
The B. glaziovii extracts and fractions were dissolved in
dimethyl sulfoxide (DMSO; the highest concentration
was 1.4%, which was not hazardous to the parasites, as
assessed previously), added to the parasite suspension
to final concentrations between 1.6 and 100 µg mL-1,
and incubated at 28 ºC for 72 h. The assays were carried
out in triplicate. The reference drug was pentamidine
isethionate purchased from Sigma-Aldrich (St Louis,
MO, USA). To this end, a 16.7 mg mL-1 stock solution
of the reference drug was prepared in sterile deionized
water and added to the parasite suspension, to obtain final
pentamidine isethionate concentrations between 1.6 and
100 µg mL-1. Leishmanicidal effects were assessed by
the MTT method with modifications.22 The 50% mean
inhibitory concentration (IC50) of the tested compounds
and the positive control were determined by calculating
the percentage of cytotoxicity.
Evaluation of the cytotoxicity on J774 macrophages
To evaluate the cytotoxicity of the B. glaziovii extracts
and fractions on J774 macrophage lineages, the latter
cells were grown in 25 cm2 culture flasks containing 5 mL
of Roswell Park Memorial Institute (RPMI) medium
supplemented with 10% bovine fetal serum, kept in an
incubator at 37 °C and 5% CO2 atmosphere, and transferred
to a new medium on a weekly basis. After removal of the
medium, the cell monolayer was removed from the flask with
a scraper and transferred to a 15 mL conical tube containing
1 mL of RPMI medium (RPMI 1640 medium consisting
4
Leishmanicidal Activity of Brosimum glaziovii (Moraceae) and Chemical Composition
of Gibco® powder, distilled water, sodium bicarbonate,
Hepes buffer, bovine fetal serum, and antibiotics); the
concentration of the cells was set to 1 × 105 cells mL-1.
Cell viability was evaluated by addition of the vital dye
trypan blue (0.02%). The cell suspension (200 µL) was
distributed in TPP 96-well plates and incubated at 37 °C
and 5% CO2 for 24 h. The supernatant was removed from
the plate; then, 97 µL of RPMI and 3 µL of the extracts and
fractions that had activity < 100 µg mL-1 were diluted with
sterile water and added in decreasing concentrations (250
to 1.5 μg mL-1), in triplicate. Next, the cells were incubated
at 37 °C and 5% CO2 for 24 h. Pentamidine was used as the
reference drug. After incubation, 10 µL of MTT/phenazine
methosulfate (PMS) solution was added, and the cells were
incubated at 37 °C and 5% CO2 for 75 min. Then, 100 μL
of sodium dodecyl sulfate (SDS) was added, which was
followed by incubation at room temperature for 30 min.
The absorbance at 540 nm was measured in a microplate
reader (Elisa reader, Readwell Touch/Robonik). The results
were expressed in LC50 (lethal concentration that killed
50% of the cells).
The selectivity index (SI), an important characteristic to
define hit compounds, was calculated by dividing the values
obtained for the cytotoxic activity against macrophages
J774 by the results achieved during leishmanicidal actions
(SI = LC50 / IC50) for the most active extracts and fractions.
Results and Discussion
To expand the current knowledge about the plants
of the Moraceae family, in this work we evaluated the
antileishmanial potential of extracts and fractions from
the leaves and branches of B. glaziovii (Moraceae) and
investigated the chemical profile of the active fractions
J. Braz. Chem. Soc.
by high-resolution gas chromatography (HRGC) and
gas chromatography-mass spectrometry (GC-MS).
Antileishmanial assays of the extracts and fractions
from the leaves and branches of B. glaziovii against the
promastigote form of L. amazonensis helped us to achieve
our goals. Table 1 summarizes the main results.
Among all the tested fractions and extracts, the nonpolar
hexane fraction from the leaves of B. glaziovii displayed
the highest activity against L. amazonensis, with IC50 of
3.6 ± 0.34 µg mL-1. This result was close to that obtained with
the positive control pentamidine (IC50 = 4.0 ± 0.26 µg mL‑1),
the drug of choice to treat cutaneous leishmaniasis in South
America.23 The activity of the hexane fraction from the leaves
of B. glaziovii was higher than that of the corresponding
ethanol extract (IC50 = 82.9 ± 0.48 µg mL‑1). This evidenced
that fractionation potentiated the activity by concentrating
the most active compound(s) in the hexane fraction.
The ethyl acetate fraction from the leaves of B. glaziovii
had lower biological activity than the hexane fraction
(51.8 ± 2.26 µg mL-1). Nevertheless, these data indicated
that B. glaziovii contains antileishmanial compounds with
different polarities.
As for the leishmanicidal action of the branches of
B. glaziovii, fractionation also potentiated the activity,
as verified by comparison of the activities of the hexane
and ethyl acetate fractions (IC 50 = 39.1 ± 1.15 and
48.7 ± 1.87 µg mL-1, respectively) with that of the crude
ethanol extract (IC50 > 100 µg mL-1), considered inactive.
Hence, the branches of B. glaziovii also presented
leishmanicidal activity, albeit almost 10-fold lower than
the leaves.
Regarding cytotoxicity evaluation, we calculated the
selectivity index (SI), a relevant characteristic to define
hit compounds, for the ethanol extracts and the hexane
Table 1. Leishmanicidal and cytotoxic activities of the extracts and fractions from B. glaziovii in µg mL-1
Extracts and fractions
Leaves
Ethanol
Hexane
Ethyl acetate
n-Butanol
Aqueous methanol
Branches
Ethanol
Hexane
Ethyl acetate
n-Butanol
Aqueous methanol
Pentamidinea
a
Positive control.
Antiprotozoal activity (IC50)
Leishmania amazonensis
Cytotoxicity (LC50)
Macrophage JJ74
Selectivity index (SI)
82.9 ± 0.48
3.6 ± 0.34
51.8 ± 2.26
> 100
> 100
266.50 ± 4.95
129.25 ± 1.06
200.75 ± 7.42
–
–
3.21
35.90
3.87
–
–
> 100
39.1 ± 1.15
48.7 ± 1.87
> 100
> 100
4 ± 0.26
–
127.25 ± 1.06
200.50 ± 2.12
–
–
7.5 ± 0.35
–
3.25
4.12
–
–
1.87
Vol. 00, No. 00, 2014
Coqueiro et al.
and ethyl acetate fractions from the leaves and branches of
B. glaziovii. To this end, we divided the cytotoxic activity
of the extract or fraction against J774 macrophages
(LC50) by its leishmanicidal activity (SI = LC50 / IC50).
We employed macrophages to determine SI, because
they are the main host cells for Leishmania (Table 1).
Using the LC50 value of 129.3 µg mL-1 found for J774
macrophages treated with the hexane fraction from the
leaves of B. glaziovii (which was the most active fraction),
we calculated an SI of 35.9 for L. amazonensis, which
is almost twenty times higher than that of the control
drug pentamidine (SI = 1.9, Table 1). Therefore, this
fraction possesses good selectivity for L. amazonensis
promastigote forms rather than macrophages. The SI
values determined for the other active extracts and
fractions (ethanol extract and ethyl acetate fraction from
the leaves and the hexane and ethyl acetate fractions from
the branches) indicated that they were less selective than
the hexane fraction from the leaves, but they still were
twice more selective for the parasite than the control drug
pentamidine (SI = 1.9). Hence, these extracts and fractions
are also attractive for further chemical characterization.
The biological activities of nonpolar extracts have often
been attributed to complex mixtures of triterpenoid and/or
steroid compounds.24,25
5
Gas chromatography coupled with mass spectrometry
(GC-MS) is the most powerful technique to characterize
nonpolar compounds in a mixture, so it constitutes an
alternative to the preliminary screening of complex plant
extracts. However, this hyphenated analytical technique
is still inaccessible to many research groups. On the other
hand, GC-FID analysis is rapid, simple, relatively cheap,
and more readily available, so it is potentially applicable
as a preliminary procedure.21
Aiming to identify the steroids and triterpenes present
in the most active fractions from the leaves and branches of
B. glaziovii, we used high-resolution gas chromatography
(HRGC), a fast method that does not require prederivatization. The GC-FID method we employed in this
study involved two different columns: SPB-5 and SPB-50.
The low polarity, inertness toward organic compounds, and
high temperature limit of these columns make them ideal
for analysis of underivatized semi-volatile plant extracts.
By using the SPB-5 and SPB-50 columns to analyze
the hexane fraction from the leaves of B. glaziovii, it was
possible to obtain chromatograms with well-resolved peaks,
which enabled proper identification of some components
by RR values and comparison of these values with those
of authentic samples (Figures 1A, 1B, 1C, and 1D). The
RR values were calculated by dividing the retention time
Figure 1. Gas chromatogram (HRGC) of B. glaziovii. (A) Leaves; SPB-5 column [IS: internal standard (cholesterol) (tR = 18.60 min); (1) campesterol
(tR = 20.95 min); (2) β-sitosterol (tR = 23.35 min); (3) β-amyrin (tR = 24.32 min), (4) α-amyrin (tR = 25.51 min), and (5) β-amyrin acetate (tR = 27.53 min)].
(B) Leaves; SPB-50 column [IS: internal standard (cholesterol) (tR = 13.06 min); (1) campesterol (tR = 16.19 min); (2) β-sitosterol (tR = 19.09 min);
(3) β-amyrin (tR = 23.26 min), (5) β-amyrin acetate (tR = 25.22 min), and (4) α-amyrin (tR = 26.22 min)]. (C) Branches; SPB-5 column [IS: internal
standard (cholesterol) (tR = 18.53 min); (1) campesterol (tR = 20.90 min); (6) stigmasterol (tR = 21.75 min); (2) β-sitosterol (tR = 23.35 min); (3) β-amyrin
(tR = 24.33 min); (7) lupenone (tR = 24.92 min); (4) α-amyrin (tR = 25.56 mim), and (5) β-amyrin acetate (tR = 27.50 min)]. (D) Branches; SPB-50 column
[IS: internal standard (cholesterol) (tR = 13.05 min); (1) campesterol (tR = 16.15 min); (6) stigmasterol (tR = 17.13 min); (2) β-sitosterol (tR = 19.06 min);
(3) β-amyrin (tR = 23.20 min); (5) β-amyrin acetate (tR = 25.23 min), and (4) α-amyrin (tR = 26.26 mim), and (7) lupenone (tR = 26.26 min)].
6
Leishmanicidal Activity of Brosimum glaziovii (Moraceae) and Chemical Composition
(tR) of the compound by the retention time of the standard
for both columns. Co-injection with authentic standards
additionally confirmed the presence of each triterpene or
sterol and avoided misclassifications due to overlapping of
the retention zones.
The HRGC results (Table 2) attested to the major
occurrence of the sterols campesterol (1), and β-sitosterol
(2) and of the triterpenes β-amyrin (3), α-amyrin (4) and
β-amyrin acetate (5) in the hexane fractions from the
leaves and branches of B. glaziovii; besides these the sterol
stigmasterol (6), and the pentacyclic triterpene lupenone (7)
were also present in the hexane fraction from the branches
of B. glaziovii.
Table 2. Chemical composition of the hexane fractions from the leaves
and branches of B. glaziovii as assessed by HRGC analysis (SPB-5 and
SPB-50 columns)
Standard compounds
Leaves
Branches
RRa
RRb
α-Amyrin
+
+
1.381
2.015
β-Amyrin
+
+
1.318
1.799
Lupeol
–
–
1.389
1.859
Taraxerol
–
–
1.285
1.750
α-Amyrin acetate
–
–
1.572
2.246
β-Amyrin acetate
+
+
1.486
1.927
Lupeol acetate
–
–
1.582
2.265
Bauerenyl acetate
–
–
–
2.536
Taraxerol acetate
–
–
1.464
1.928
Friedelanoyl acetate
–
–
1.823
2.949
Friedelin
–
–
1.580
2.776
Lupenone
–
+
1.341
2.011
α-Amyrenone
–
–
1.341
1.940
β-Amyrenone
–
–
1.279
1.727
Germanicone
–
–
1.264
1.658
Campesterol
+
+
1.131
1.231
Stigmasterol
–
+
1.175
1.302
β-Sitosterol
+
+
1.263
1.482
SPB-5 column; SPB-50 column. RR: relative retention related to the
internal standard (cholesterol); (+): presence of the compound; (–):
absence of the compound.
a
b
The mixture of α/β-amyrin triterpenes exists in
many medicinal plants and accounts for many bioactive
properties, including analgesic, antimicrobial, and antiinflammatory actions.26 These compounds can underlie the
activity exhibited by the hexane fractions from B. glaziovii.
Interestingly, although these substances occur in the hexane
fractions from both the leaves and branches, the fraction
from the leaves showed stronger activity, suggesting that
other compounds also contribute to the antileishmanial
properties.
J. Braz. Chem. Soc.
To prove the identity of the substances identified by
GC‑FID (by comparison with the authentic compounds)
and to obtain the full profile of the hexane fractions from the
leaves and branches of B. glaziovii, we also performed the
GC-MS analysis for the hexane fractions (Supplementary
Information). We identified the substances on the basis of
the NIST library and by comparison with literature data.
Only the compounds with 90% of similarities with the
database or higher than that were taken into account for
our analysis. The GC-MS analysis of the hexane fractions
of leaves and branches are given in the Table 3. Besides
the confirmation of the triterpenes and sterols previously
identified by HRGC, the GC-MS allowed us to detect a
further series of compounds constituted mainly of long
chain fatty acids and its derivatives.
The hexane fraction of leaves mainly contains
palmitic acid (n-hexadecanoic acid) and its derivative
(hexadecanoic acid, ethyl esther); stearic acid derivatives
[(12-octadecadienoic acid (Z,Z)-; 9,12,15-octadecatrienoic
acid, (Z,Z,Z)-] and octadecanoic acid, ethyl ester); linoleic
acid, ethyl ester and the diterpene alcohol‑phytol. For the
hexane fraction of branches the most abundant fatty acids
were the palmitic acid derivative (hexadecanoic acid, ethyl
esther); the stearic acid derivatives [(octadecanoic acid, ethyl
ester and 9,12,15-octadecatrienoic acid, (Z,Z,Z)-] and linoleic
acid, ethyl ester. Some fatty acids, like palmitic acid,27 among
others28 have been shown to have antiprotozoal activity.
These results demonstrated that, despite the similarities
between the hexane fractions from the leaves and branches
of B. glaziovii, these fractions exhibited different chemical
profiles and antileishmanial activity. Taken together, these
findings corroborated that plant tissues produce compounds
in different ways, which can be relevant when considering
the chemical interface between plants and the surrounding
environment.29
The preliminary phytochemical screening by HRGC
proved to be a useful tool to identify secondary metabolites,
in this case, the triterpenes and sterols presented in
complex extracts and fractions of B. glaziovii, in special
the bioactive hexane fraction, which the chemical profile
was successfully mapped (Figure 2).
Further bio-guided studies regarding the isolation and
antileishmanial evaluation of the pure compounds are
necessary to identify the bioactive compounds or a possible
synergism between the molecules present in the active
fractions of B. glaziovii.
Conclusions
Neglected tropical diseases are a recognized public
health concern affecting mostly the impoverished regions of
Vol. 00, No. 00, 2014
7
Coqueiro et al.
Table 3. Compounds identified in the hexane fractions from the leaves and branches of B. glaziovii using gas chromatography coupled with electron
ionization mass spectrometry (GC-MS)
Compounds
Molecular formula
Molecular weight
Leaves
Branches
Phenol, 4,4’-(1-methylethylidene)bis-; 95a; 95b
C15H16O2
228
X
X
n-Hexadecanoic acid; 99 ; 99
C16H32O2
256
X
X
Tetradecanoic acid, ethyl ester; 90b
C16H32O2
256
–
X
a
b
Cyclohexene, 4-(4-ethylcyclohexyl)-1-pentyl-; 95
C19H34
262
–
X
2-Pentadecanone, 6,10,14-trimethyl-; 91a
C18H36O
268
X
–
Pentadecanoic acid, ethyl ester; 99
C17H34O2
270
–
X
Hexadecanoic acid, methyl ester; 98b
C17H34O2
270
–
X
C16H22O4
278
–
X
9,12,15-Octadecatrienoic acid, (Z,Z,Z)-; 99ª; 96
C18H30O2
278
X
X
1,2-Benzenedicarboxylic acid, mono(2-ethylhexyl) ester; 91a; 90b
C16H22O4
278
X
X
9,12-Octadecadienoic acid (Z,Z)-; 99ª; 99
C18H32O2
280
X
X
12-Methyl-E,E-2,13-octadecadien-1-ol; 93a
C19H36O
280
X
–
Cyclopropaneoctanal, 2-octyl-; 97
C19H36O
280
–
X
2-Methyl-Z,Z-3,13-octadecadienol; 91a
C19H36O
280
X
–
Ethyl 9-hexadecenoate; 95ª; 99
C18H34O2
282
X
X
C18H36O2
284
X
X
C20H40O
296
X
X
Heptadecanoic acid, ethyl ester; 98 ; 99
C19H38O2
298
X
X
9,12,15-Octadecatrienoic acid, ethyl ester, (Z,Z,Z)-; 99ª; 99b
C20H34O2
306
X
X
Linoleic acid, ethyl ester; 99ª; 99
C20H36O2
308
X
X
Ethyl oleate; 98b
C20H38O2
310
–
X
C20H40O2
312
X
X
4,8,12,16-Tetramethylheptadecan-4-olide; 93
C21H40O2
324
X
–
Nonadecanoic acid, ethyl ester; 93ª; 96b
C21H42O2
326
X
X
Nonadecanoic acid, ethyl ester; 95b
C21H42O2
326
–
X
C24H48
336
X
X
C30H48O
424
X
–
Fern-7-en-3β-ol; 90a
C30H50O
426
X
–
Vitamin E; 99a
C29H50O2
430
X
–
C26H52O2
396
–
X
C30H50
410
–
X
C30H50O
426
–
X
b
b
Dibutyl phthalate; 90
b
b
b
b
b
Hexadecanoic acid, ethyl ester; 99 ; 98
a
b
Phytol; 91a; 95b
a
b
b
Octadecanoic acid, ethyl ester; 99 ; 99
a
b
a
Cyclodocosane, ethyl-; 96a
4,4,6a,6b,8a,11,12,14b-Octamethyl-1,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,14,
14a,14b-octadecahydro-2H-picen-3-one; 99a
Ethyl tetracosanoate; 95
b
Squalene; 99
b
Lanosterol; 93b
Percentage of similarity of the compounds of leaves with NIST library; percentage of similarity of the compounds of branches with NIST library.
a
the world. In the American continent, Brazil is the country
that is most impacted by leishmaniasis. This country also
relies on wide biodiversity, which constitutes a superb
source of chemical diversity. By using rational screening,
it is possible to identify hits and leads that will be essential
for further research into the development of potential drugs.
The chemical and pharmacological potential of many
plants remains unexplored. HRGC proved to be a rapid,
simple, and relatively cheap preliminary screening tool to
b
characterize plant mixtures in a reliable way. This technique
enabled the identification of some triterpenes and sterols
present in the bioactive extracts from Brosimum glaziovii,
traced as campesterol (1), β-sitosterol (2), β-amyrin (3),
α-amyrin (4), β-amyrin acetate (5), stigmasterol (6) and
lupenone (7). Although the identified metabolites have
been known for some time, this is the first time that their
presence has been detected in the species B. glaziovii. The
GC-MS analysis allowed the confirmation of the substances
8
Leishmanicidal Activity of Brosimum glaziovii (Moraceae) and Chemical Composition
J. Braz. Chem. Soc.
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