Anti-trypanosoma activity and synergistic effects of natural and semi-synthetic triterpenes and predominant cell death through autophagy in amastigote forms

Article abstract of DOI:10.21577/0103-5053.20170103

Chagas’ disease is a parasitic disease with unsatisfactory treatment, mainly in chronic stage. This study aimed to evaluate the trypanocidal activity and action mechanisms of α/β-amyrin and its semi-synthetic derivatives, together with four isolated natural triterpenes, tested against trypomastigote and amastigote forms. The structure-activity relationship was suggested and cytotoxicity was measured. In general, greater polar compounds may have improved the selectivity to the protozoan. Action mechanisms were only performed for the amastigotes of Trypanosoma cruzi by evaluating the ultrastructural alterations, membrane permeability, mitochondrial membrane potential and cell volume, since the majority of compounds displayed promising antiamastigote activities. Triterpenes promoted changes on mitochondrial membrane potential and ultrastructural features that suggest autophagy processes. Both combinations between α/β-amyrin and 3-O-acetyl-11-oxo-α/β-amyrin and 3-O-acetyl-α/β-amyrin with benznidazole displayed synergistic effects against amastigotes and antagonistic effects on LLCMK₂ cells. The antiamastigote activities, chemical derivatization, drug combinations and action mechanisms revealed to be crucial approaches toward this chronic disease.

Full text of DOI:10.21577/0103-5053.20170103

http://dx.doi.org/10.21577/0103-5053.20170103
J. Braz. Chem. Soc., Vol. 28, No. 12, 2473-2489, 2017.
Printed in Brazil - ©2017 Sociedade Brasileira de Química
0103 - 5053 $6.00+0.00
Article
Anti-Trypanosoma Activity and Synergistic Effects of Natural and Semi-Synthetic
Triterpenes and Predominant Cell Death through Autophagy in Amastigote Forms
Gleison D. P. Bossolani,a Tânia Ueda-Nakamura,a Sueli O. Silva,a Benedito P. Dias Filho,a
Tulio O. G. Costa,b Raúl H. R. Quintanilla,c Sabrina T. Martinez,c Valdir F. Veiga-Junior,b,d
Angelo C. Pinto†,c,d and Celso V. Nakamura*,a
aDepartamento de Ciências Básicas da Saúde, Universidade Estadual de Maringá (UEM),
Av. Colombo, 5790, 87020-900 Maringá-PR, Brazil
bDepartamento de Química, Instituto de Ciências Exatas, Universidade Federal do Amazonas (UFAM),
Av. Rodrigo Octávio, 6200, 69077-000 Manaus-AM, Brazil
cDepartamento de Química Orgânica, Instituto de Química, Universidade Federal do Rio de Janeiro (UFRJ),
Av. Athos da Silveira Ramos, 149, 21941-909 Rio de Janeiro-RJ, Brazil
dInstituto Militar de Engenharia (IME), 22290-270 Rio de Janeiro-RJ, Brazil
Chagas’disease is a parasitic disease with unsatisfactory treatment, mainly in chronic stage. This
study aimed to evaluate the trypanocidal activity and action mechanisms of α/β-amyrin and its semi-
synthetic derivatives, together with four isolated natural triterpenes, tested against trypomastigote
and amastigote forms. The structure-activity relationship was suggested and cytotoxicity was
measured. In general, greater polar compounds may have improved the selectivity to the protozoan.
Action mechanisms were only performed for the amastigotes of Trypanosoma cruzi by evaluating
the ultrastructural alterations, membrane permeability, mitochondrial membrane potential and cell
volume, since the majority of compounds displayed promising antiamastigote activities. Triterpenes
promoted changes on mitochondrial membrane potential and ultrastructural features that suggest
autophagy processes. Both combinations between α/β-amyrin and 3-O-acetyl-11-oxo-α/β-amyrin
and 3-O-acetyl-α/β-amyrin with benznidazole displayed synergistic effects against amastigotes and
antagonistic effects on LLCMK2 cells. The antiamastigote activities, chemical derivatization, drug
combinations and action mechanisms revealed to be crucial approaches toward this chronic disease.
Keywords: triterpenes, Trypanosoma cruzi, synergistic effects, amastigote, autophagy
Introduction
far from the ideal due to their several side effects, the limited
efficacy against the genetically heterogeneous parasite, the
long-term therapy, and the well-known poor activity in the
late chronic phase.2,3 In addition, low investments toward
drug discovery research for Chagas’ disease contribute to
the slow advancements in the development of new drugs.4,5
Several studies have been conducted to find novel
active compounds against T. cruzi. The search of natural
products is promising source for the discovery of novel
drugs, mainly for the neglected diseases.Another approach
is that plant-derivative synthetic compounds may have
an increased antiparasitic activity and a reduced toxicity
through the modifications in their original chemical
structure.6,7 Triterpenes are widely distributed in nature
and isolated from a variety of plant sources, and more
than 20,000 triterpenes have been isolated and identified
Chagas’ disease, caused by the flagellate protozoan
Trypanosoma cruzi, is a chronic systemic parasitosis that
affects about 8 million people worldwide, mostly in Latin
America where it is considered endemic.1 Symptomatic
Chagas’ disease is mainly associated with cardiac and
enteric consequences that may lead to increased rates
of morbidity and mortality.2 Unfortunately, there is no
satisfactory treatment for the chronic stage of the disease
and new anti-T. cruzi drug candidates are needed. The
current available drug therapy for Chagas’disease relies on
two nitro derivatives: benznidazole and nifurtimox. Both are
*e-mail: cvnakamura@uem.br
†In memoriam

2474
Anti-Trypanosoma Activity and Synergistic Effects of Natural and Semi-Synthetic Triterpenes
J. Braz. Chem. Soc.
from nature.8 These compounds have shown a wide
spectrum of pharmacologic activities, such as antibacterial,
antiviral, antifungal, anti-oxidative, anti-inflammatory,
anti-ulcer, anti-hyperlipidemic, hepatoprotective, as well
as anti-cancer.9,10
performance liquid chromatography (HPLC). These two
analyses, performed and published previously, were used
to define, together with proton nuclear magnetic resonance
(1H NMR), the amount of these α- and β-amyrins in this
mixture was 66 and 34%, respectively.
Studies with terpenes revealed promising antileishmanial
and trypanocidal activities that leads to different types of
cellular death mechanisms induced by these compounds.
Furthermore, different classes of terpenes have been
demonstrated desirable synergistic effects when combined,
since drug combination may improve the parasitic activity
with lower dosage of these substances, in addition to
reducing their undesirable toxic effects.11-17 The search
for synergic compounds can improve the efficiency of
the treatment, reduce their toxicities on mammalian cells
and diminish the selection of resistant mutations.12,18-20
Alternative strategies have been designed to identify
candidates among the already available drugs in the market
that could be used in combination to provide synergic
effects and improve the chemotherapy efficacy.Antifungal
agents, tricyclic antidepressants and several antipsychotics
have been studied in combination with benznidazole in
order to search new combination therapy approaches.19-21
After the discovery of several biological activities of
ursolic acid and their derivatives, the search for sustainable
sources of triterpenes with promising biological activities
has increased in order to improve their original biological
activities. Amyrins, hydroxylated monounsaturated
alcohols obtained from several triterpenic skeletons, mainly
from ursane and oleanane, are commonly observed in
apolar extracts of plants and abundantly available in oil
resins that is produced by Burseraceae species, well-known
incense, frankincense or breu.22-25
3-O-Fumaryl-α/β-amyrin (2)
A solution of α/β-amyrin (0.30 g, 0.703 mmol) in
diclorometane (CH2Cl2), maleic anhydride (0.55 g,
5.6 mmol) and 4-dimethylaminopyridine (DMAP, 0.02 g,
0.164 mmol) was refluxed for 24 h. Then, the mixture was
concentrated and subjected to liquid-liquid extraction.
Column chromatography (silica gel 60, Merck, hexane:ethyl
acetate, 95:5) of the crude residue was performed to give
expected pure compound (0.33 g, 0.629 mmol), with yield
of 89%, and appearance light yellow; IR (KBr) v / cm-1
3126 (O–H), 1738 (C=O, ester), 1686 (C=O, acid),
1308 (C(=O)–O, ester); 13C NMR (75 MHz, CDCl3) was
compared with literature data,27 dC (ppm), α isomer, d 15.6
(C-25), 15.6 (C-24), 16.8 (C-26), 18.2 (C-6), 23.5 (C-11),
23.7 (C-30), 26 (C-27), 26.1 (C-15), 26.9 (C-2), 26.9
(C-16), 28.1 (C-23), 28.4 (C-28), 31.1 (C-20), 32.5 (C-7),
32.5 (C-17), 33.3 (C-29), 34.7 (C-21), 36.8 (C-10), 37.1
(C-22), 38.1 (C-1), 39.8 (C-8), 39.8 (C-4), 41.7 (C-14), 46.7
(C-19), 47.2 (C-18), 47.5 (C-9), 55.2 (C-5), 85.1 (C-3),
121.4 (C-12), 129.1 (C’2), 137.8 (C’3), 145.3 (C-13), 164.1
(C’4), 168 (C’1); β isomer, 15.7 (C-24), 15.7 (C-25), 16.8
(C-26), 17.5 (C-29), 18.2 (C-6), 21.4 (C-30), 23.2 (C-27),
23.3 (C-11), 26.6 (C-16), 26.6 (C-15), 28.1 (C-2), 28.1
(C-28), 28.7 (C-23), 31.2 (C-21), 32.8 (C-7), 33.8 (C-17),
36.8 (C-10), 38.3 (C-1), 38.3 (C-4), 39.6 (C-19), 39.6
(C-20), 40 (C-8), 41.5 (C-22), 42.1 (C-14), 47.6 (C-9), 55.2
(C-5), 59 (C-18), 85.1 (C-3), 124.1 (C-12), 139.7 (C-13);
HRMS (ESI) m/z [M – H]– observed 523.3791; C34H51O4
requires 523.3787.
This study evaluated the antiprotozoal activities of
twelve triterpenoid and combinatorial effects, as well as the
cytotoxicity against mammalian cells. The structure-activity
relationship (SAR) between the chemical structure and
biological properties of the compounds was also suggested.
Ultrastructural changes, integrity of the mitochondria and
cellular membrane and cell volume were also studied to
investigate some action mechanisms of the triterpenes.
3-O-Succinyl-α/β-amyrin (3)
To obtain a solution of 3-O-succinyl-α/β-amyrin (3),
succinic anhydride (0.56 g, 5.6 mmol) and DMAP
(0.02 g, 0.164 mmol) were added in α/β-amyrin (0.30 g,
0.703 mmol) in CH2Cl2, and refluxed for 24 h. Then,
the mixture was concentrated and subjected to liquid-
liquid extraction. It was obtained a white solid (0.35 g,
0.664 mmol) with yield of 94%; IR (KBr) v / cm-1 3443
(O–H), 1732 (C=O, ester), 1713 (C=O, acid), 1308
(C(=O)–O, ester); 13C NMR (75 MHz, CDCl3) was
compared with literature data,27 dC (ppm), α isomer, d 15.7
(C-24), 15.7 (C-25), 16.8 (C-26), 17.5 (C-29), 18.2 (C-6),
21.4 (C-30), 23.2 (C-27), 23.5 (C-11), 26.6 (C-15), 26.6
(C-16), 28 (C-23), 28 (C-28), 28.7 (C-2), 29.3 (C’2), 29.3
(C’3), 31.2 (C-21), 32.8 (C-7), 33.7 (C-17), 36.7 (C-10),
Experimental
Syntheses
α/β-Amyrin (1)
The binary mixture of triterpenoids α/β-amyrin was
isolated from Protium resin.26 The high purity of this
mixture was considered based on the only two peaks
in the analyses of gas chromatography (GC) and high

Vol. 28, No. 12, 2017
Bossolani et al.
2475
38.4 (C-1), 39.6 (C-4), 39.6 (C-19), 39.6 (C-20), 40 (C-8),
41.5 (C-22), 42 (C-14), 47.6 (C-9), 55.2 (C-5), 59 (C-18),
81.6 (C-3), 124.3 (C-12), 139.6 (C-13), 171.9 (C’1), 177.2
(C’4); β isomer, 15.5 (C-24), 15.5 (C-25), 16.8 (C-26), 18.2
(C-6), 23.5 (C-11), 23.7 (C-30), 26 (C-16), 26 (C-27), 26.1
(C-15), 26.9 (C-2), 27.9 (C-23), 28.4 (C-28), 29.4 (C’2),
29.4 (C’3), 31.1 (C-20), 32.5 (C-7), 32.5 (C-17), 33.3
(C-29), 34.7 (C-21), 36.8 (C-10), 37.1 (C-22), 38.2 (C-1),
39.8 (C-4), 39.8 (C-8), 41.7 (C-14), 46.8 (C-19), 47.2
(C-18), 47.5 (C-9), 55.2 (C-5), 81.5 (C-3), 121.6 (C-12),
145.2 (C-13), 171.9 (C’1), 177.1 (C’4); HRMS (ESI) m/z
[M – H]– observed 525.3969; C34H53O4 requires 525.3949.
(C-25), 16.6 (C-26), 16.8 (C-24), 17.5 (C-29), 18.2 (C-6),
21.4 (C-30), 23.2 (C-11), 23.4 (C-27), 26.6 (C-16), 28.1
(C-2), 28.1 (C-28), 28.2 (C-23), 28.7 (C-15), 31.3 (C-21),
32.8 (C-7), 33.7 (C-17), 36.7 (C-10), 38.4 (C-1), 39.6 (C-4),
39.6 (C-19), 39.6 (C-20), 40 (C-8), 41.5 (C-22), 42 (C-14),
47.6 (C-9), 55.3 (C-5), 59 (C-18), 83.1 (C-3), 124.3 (C-12),
129 (C’3), 129.9 (C’6), 131.1 (C’2), 131.4 (C’7), 132.7
(C’4), 133.4 (C’5), 139.6 (C-13), 168 (C’1), 171.4 (C’8);
β isomer, 15.7 (C-25), 16.8 (C-24), 16.8 (C-26), 18.2 (C-6),
23.6 (C-30), 23.7 (C-11), 25.9 (C-27), 26.2 (C-16), 28.1
(C-23), 28.4 (C-15), 28.4 (C-28), 28.6 (C-2), 31 (C-20),
31.1 (C-21), 32.7 (C-17), 32.8 (C-7), 33.4 (C-29), 36.8
(C-10), 37.1 (C-22), 38.5 (C-1), 39.6 (C-4), 39.6 (C-8),
41.7 (C-14), 46.8 (C-19), 47.5 (C-9), 47.6 (C-18), 55.4
(C-5), 83.1 (C-3), 121.6 (C-12), 129 (C’3), 129.9 (C’6),
131.1 (C’2), 131.4 (C’7), 132.7 (C’4), 133.4 (C’5), 145.2
(C-13), 168 (C’1), 171.4 (C’8); HRMS (ESI) m/z [M – H]–
observed 573.3930; C38H53O4 requires 573.3943.
3-O-Acetyl-α/β-amyrin (4)
A solution of α/β-amyrin (0.5 g, 1.17 mmol), acetic
anhydride (3.0 mL) and DMAP (0.01 g, 0.082 mmol) was
refluxed for 30 min. Then, the mixture was concentrated and
subjected to liquid-liquid extraction with CH2Cl2 in water
(CH2Cl2/H2O). This derivate binary mixture was obtained
as a white solid (0.54 g, 1.15 mmol) with yield of 98%;
IR (KBr) v / cm-1 1736 (C=O, ester), 1243 (C(=O)–O, ester);
13C NMR (75 MHz, CDCl3) was compared with literature
data,28,29 dC (ppm), α isomer, d 15 (C-25), 16.6 (C-26),
16.8 (C-24), 17.4 (C-29), 18.2 (C-6), 21.3 (C-30), 23.1
(C-11), 23.4 (C-27), 25.9 (C-2), 26.6 (C-16), 28.1 (C-28),
28.2 (C-23), 28.7 (C-15), 31.3 (C-21), 32.8 (C-7), 33.7
(C-17), 36.7 (C-10), 37.7 (C-4), 38.5 (C-1), 39.6 (C-19),
39.6 (C-20), 40 (C-8), 41.5 (C-22), 42 (C-14), 47.7 (C-9),
55.2 (C-5), 59 (C-18), 80.9 (C-3), 124.3 (C-12), 139.6
(C-13); β isomer, 15.7 (C-25), 16.8 (C-24), 16.8 (C-26),
18.2 (C-6), 21.3 (C’2), 23.4 (C-11), 23.6 (C-2), 26.1 (C-27),
26.2 (C-16), 26.6 (C-30), 26.9 (C-28), 28.1 (C-23), 28.4
(C-15), 31 (C-20), 32.5 (C-17), 32.6 (C-7), 33.4 (C-29),
34.8 (C-21), 36.8 (C-10), 37.1 (C-22), 37.7 (C-4), 38.2
(C-1), 39.6 (C-8), 41.5 (C-14), 46.8 (C-19), 47.2 (C-18),
47.5 (C-9), 55.3 (C-5), 80.7 (C-3), 121.6 (C-12), 145.2
(C-13), 170.9 (C’1); GC-MS (EI), m/z 468 [M]+ (4), 218
(100), 203 (22-50).
3-O-Acetyl-11-oxo-α/β-amyrin (6)
To obtain a solution of 3-O-acetyl-11-oxo-
α/β-amyrin (6), acetic anhydride (3 mL), acetic acid and
tert-butyl chromate (3 mL) were added in the solution of
3-O-acetyl-α/β-amyrin (0.20 g, 0.427 mmol), and refluxed
for 6 h. Then, the mixture was concentrated and subjected
to liquid-liquid extraction with CH2Cl2/H2O and oxalic acid
(5%). The oxidation yield was 97%; 13C NMR (75 MHz,
CDCl3) was compared with literature data,29,30 dC (ppm),
α isomer, d 16.5 (C-25), 16.7 (C-24), 17.4 (C-26), 17.4
(C-29), 18.5 (C-6), 21.1 (C-30), 21.2 (C’2), 23.5 (C-2),
23.6 (C-27), 27.2 (C-16), 27.4 (C-15), 28 (C-23), 28.8
(C-28), 30.9 (C-21), 32.8 (C-7), 33.9 (C-17), 36.8 (C-10),
38 (C-4), 38.8 (C-1), 39.2 (C-19), 39.3 (C-20), 40.9 (C-8),
43.6 (C-22), 45.1 (C-14), 55 (C-9), 59 (C-5), 61.4 (C-18),
80.6 (C-3), 130.4 (C-12), 165 (C-13), 171 (C’1), 199.6
(C-11); β isomer, 16.4 (C-25), 16.6 (C-24), 17.4 (C-26),
18.6 (C-6), 23.4 (C-2), 23.5 (C-27), 23.5 (C-30), 26.4
(C-15), 26.4 (C-16), 28 (C-23), 28.7 (C-28), 31 (C-20), 32.3
(C-17), 32.6 (C-7), 33 (C-29), 34.4 (C-21), 36.5 (C-22),
36.9 (C-10), 38 (C-4), 38.7 (C-1), 43.3 (C-8), 45 (C-19),
45.4 (C-14), 47.5 (C-18), 55 (C-5), 61.5 (C-9), 80.6 (C-3),
128 (C-12), 170.6 (C-13), 200.1 (C-11); GC-MS (EI), m/z
482 [M]+ (4), 232 (60-100), 273 (80-100).
3-O-Phthaloyl-α/β-amyrin (5)
A solution of α/β-amyrin (0.30 g, 0.703 mmol) in
CH2Cl2, phthalic anhydride (0.83 g, 5.6 mmol) and DMAP
(0.02 g, 0.164 mmol) was refluxed for 24 h. Then, the
mixture was concentrated and subjected to liquid-liquid
extraction on CH2Cl2/H2O. Column chromatography
(silica gel 60, Merck, hexane:ethyl acetate, 95:5) of the
crude organic residue was performed to give a white solid
(0.37 g, 0.644 mmol), with yield of 92%; IR (KBr) v / cm-1
3460 (O–H), 1713 (C=O, ester), 1641 (C=O, acid), 1300
(C(=O)–O, ester); 13C NMR (75 MHz, CDCl3) was
compared with literature data,27 dC (ppm), α isomer, d 15.7
3-O-Succinyl-11-oxo-α/β-amyrin (7)
A solution of 3-O-succinyl-α/β-amyrin (0.20 g,
0.380 mmol), acetic anhydride (3 mL) and acetic acid
tert-butyl chromate (3 mL) was refluxed for 6 h. The mixture
was concentrated and subjected to liquid-liquid extraction
with CH2Cl2/H2O and oxalic acid (5%). The oxidation yield
was 97%; 13C NMR (75 MHz, CDCl3) was compared with

2476
Anti-Trypanosoma Activity and Synergistic Effects of Natural and Semi-Synthetic Triterpenes
J. Braz. Chem. Soc.
literature data,27,30 dC (ppm), α isomer, d 16.5 (C-25), 16.7
(C-24), 17.4 (C-26), 17.4 (C-29), 18.5 (C-6), 21.1 (C-30),
23.4 (C-27), 27.2 (C-16), 27.4 (C-15), 28 (C-23), 28 (C-28),
28.8 (C-2), 29 (C’3), 29.3 (C’2), 31 (C-21), 32.4 (C-7), 33.9
(C-17), 36.8 (C-10), 38.1 (C-4), 38.8 (C-1), 39.2 (C-19), 39.3
(C-20), 40.9 (C-8), 43.6 (C-22), 45.1 (C-14), 55 (C-9), 59
(C-5), 61.4 (C-18), 81.2 (C-3), 130.3 (C-12), 165.1 (C-13),
171.8 (C’4), 177.5 (C’1), 199.7 (C-11); β isomer, 16.4
(C-25), 16.6 (C-24), 17.4 (C-26), 18.6 (C-6), 23.4 (C-2),
23.5 (C-27), 23.5 (C-30), 26.4 (C-15), 26.4 (C-16), 28
(C-23), 28.7 (C-28), 29 (C’3), 29.3 (C’2), 30.9 (C-20), 32.3
(C-17), 32.8 (C-7), 33.2 (C-29), 34.4 (C-21), 36.5 (C-22),
36.9 (C-10), 38 (C-4), 38.7 (C-1), 43.3 (C-8), 45.1 (C-19),
45.4 (C-14), 47.6 (C-18), 55 (C-5), 61.5 (C-9), 81.2 (C-3),
128 (C-12), 170.8 (C-13), 171.8 (C’1), 177.5 (C’4), 200.2
(C-11); HRMS (ESI) m/z [M – H]– observed 539.3714;
C34H51O5 requires 539.3742
Lup-20(29)-en-3-one (10)
For a solution of Protium resin26 (0.100 g) in acetone
(10 mL), Jones reagent was slowly added until green-blue
color. Then, isopropanol (2 mL) was added and, after
10 min, the product was washed with aqueous solution
of oxalic acid (10%, 3 × 10 mL) and water (3 × 10 mL),
dried with sodium sulfate (Na2SO4) and the solvent
evaporated, under vacuum. The product (85 mg) was
subjected to separation by semi-preparative HPLC, thus
obtaining a white solid (10 mg), which was characterized
as lup-20(29)-en-3-one; 13C NMR (100 MHz, CDCl3) was
compared with literature data.32,33 d 14.6 (C-27), 15.9
(C-26), 16.1 (C-25), 18.2 (C-28), 19.4 (C-30), 19.8 (C-6),
21.2 (C-24), 21.58 (C-11), 25.23 (C-12), 26.8 (C-23), 27.5
(C-15), 29.9 (C-21), 33.7 (C-7), 34.3 (C-2), 35.6 (C-16), 37
(C-10), 38.3 (C-13), 39.7 (C-1), 40.1 (C-22), 40.9 (C-8), 43
(C-17), 43.1 (C-14), 47.5 (C-4), 48.1 (C-19), 48.3 (C-18),
49.9 (C-9), 55 (C-5), 109.5 (C-29), 151 (C-20), 218.5
(C-3); GC-MS (EI) C30H48O m/z (%) 424 (32) [M]+•, 409
(20), 313 (27), 245 (25), 218 (34), 205 (100), 189 (40), 109
(94), 95 (80), 81 (60).
3-O-Phthaloyl-11-oxo-α/β-amyrin (8)
To obtain the solution of 3-O-phthaloyl-11-oxo-
α/β-amyrin (8), acetic anhydride (3 mL) and acetic
acid tert-butyl chromate (3 mL) were added in the
3-O-phthaloyl-α/β-amyrin (0.20 g, 0.348 mmol), and
refluxed for 6 h. Then, the mixture was concentrated and
subjected to liquid-liquid extraction with CH2Cl2/H2O and
oxalic acid (5%). The oxidation yield was 97%; 13C NMR
(75 MHz, CDCl3) was compared with literature data,27,30
dC (ppm), α isomer, d 16.5 (C-25), 16.8 (C-24), 17.5
(C-26), 17.5 (C-29), 18.5 (C-6), 21.1 (C-30), 23 (C-27),
27.2 (C-16), 27.4 (C-15), 28.1 (C-23), 28.1 (C-28), 28.8
(C-2), 32.8 (C-7), 33.9 (C-17), 33.9 (C-21), 36.9 (C-10),
38.4 (C-1), 39.2 (C-20), 39.3 (C-19), 40.9 (C-4), 40.9 (C-8),
43.8 (C-22), 45.2 (C-14), 55.2 (C-9), 59 (C-5), 61.4 (C-18),
82.8 (C-3), 128.9 (C’3), 129.9 (C’6), 130.3 (C-12), 130.8
(C’2), 131.7 (C’7), 133.3 (C’4), 133.3 (C’5), 165.4 (C-13),
171.3 (C’1), 171.3 (C’8), 199.9 (C-11); β isomer, 16.4
(C-25), 16.8 (C-24), 17.5 (C-26), 18.6 (C-6), 23.4 (C-27),
23.4 (C-30), 26.1 (C-16), 26.4 (C-15), 28.1 (C-23), 28.1
(C-28), 28.8 (C-2), 31 (C-20), 32.6 (C-7), 33.8 (C-29), 33.9
(C-17), 33.9 (C-21), 36.9 (C-10), 38.4 (C-1), 40.9 (C-4),
40.9 (C-22), 43.6 (C-8), 45.2 (C-19), 45.4 (C-14), 55.2
(C-5), 59 (C-18), 61.6 (C-9), 82.8 (C-3), 128.9 (C-12),
128.9 (C’3), 129.9 (C’6), 130.8 (C’2), 131.7 (C’7), 133.3
(C’4), 133.3 (C’5), 171.3 (C-13), 171.3 (C’1), 171.3
(C’8), 200.4 (C-11); HRMS (ESI) m/z [M – H]– observed
587.3708; C38H51O5 requires 587.3742.
Betulinic acid (11)
This natural compound was previously isolated and
characterized by our collaborator from Gustavia augusta L.
(Lecythidaceae family) and provided a sample to this
study.34
Lanosta-8,24-dien-3-one (12)
Lanosta-8,24-dien-3-ol (50-60%) was purchased from
Sigma and purified as in Kavtaradze et al.35 Subsequently,
the lanosta-8,24-dien-3-ol was oxidized with the Jones
reagent in order to obtain the lup-20(29)-en-3-one.
13C NMR (100 MHz, CDCl3) was compared with literature
data.36,37 d 15.8 (C-18), 17.8 (C-26), 19 (C-21), 19.9 (C-6),
20.4 (C-19), 21.2 (C-29), 21.5 (C-11), 24.4 (C-30), 24.9
(C-23), 25.9 (C-27), 26.8 (C-28), 27.6 (C-7), 28.3 (C-16),
29.8 (C-15), 30.9 (C-12), 34.7 (C-2), 35.5 (C-1), 35.7
(C-20), 36 (C-22), 37.2 (C-10), 44.2 (C-13), 47.4 (C-4),
49.8 (C-14), 50.2 (C-17), 51.6 (C-5), 125.3 (C-24), 131.1
(C-25), 132.7 (C-9), 134.8 (C-8), 218.6 (C-3); GC-MS
(EI), m/z (%) 424 (25) [M]+•, 409 (100), 257 (55), 245
(25), 109 (39).
Equipments
Mass spectra were performed in a gas chromatograph,
Agilent 6890, equipped with a mass selective detector
(MS) (with electron impact ionization, EI, 70 eV). Samples
were injected using split ratio 1/10. The silica capillary
column DB-5 (Agilent J&W, 30 m × 0.25 mm i.d.) coated
Lup-20(29)-ene-3β,6β,16β-triol (9)
This natural compound was previously isolated
and characterized by our collaborator31 from
Combretum leprosum and provided a sample to this study.

Vol. 28, No. 12, 2017
Bossolani et al.
2477
with 5% phenylpoly (methylsiloxane) (0.25 μm film
thickness) was used. The operating temperatures were as
follows: injector, 280 °C; ionization chamber, 230 °C; and
transfer line, 280 °C. The carrier gas was helium with flow
1 mL min-1. NMR spectra (1H and 13C) were recorded on
a Bruker (300 and 400 MHz for 1H; 75 and 100 MHz for
13C) spectrometer, and CDCl3 was used as solvent and an
internal reference.Analysis by HPLC was performed on an
Agilent liquid chromatograph, model HP 1100, equipped
with UV detector. Columns used were: Zarbax 300SB-C18
analytical (4.6 × 150 mm, 5 μm) and Zorbax 300SB-C18
semi-preparative (9.4 × 250 mm, 5 μm). As mobile phase
was used acetonitrile and acid water (pH 4, with acetic
acid) in the proportions: 88:12, 90:10 and 95:5 (% v/v),
with flow 0.7 mL min-1 (analytical) and 3.0 mL min-1
(semi-preparative). Analysis was monitored at 200 nm.
HRESI-MS analysis was performed on a mass spectrometer
Waters MIcromass Q-TOF Micro (Milford, MA, USA),
equipped with an ESI. The infrared (IR) spectra were
recorded on a Fourier transform infrared spectrophotometer
(FTIR) model Magna IR 760.
the medium that contained the parasites was removed, the
cells were washed in phosphate-buffered saline (PBS),
and DMEM with or without increasing concentrations of
the compounds was added. The cells were maintained for
96 h at 37 °C in a 5% CO2 atmosphere.After the incubation
period, the glass coverslips were fixed with methanol and
Giemsa staining and permanently mounted with Entellan
(Merck, Darmstadt, Germany). Under a light microscope,
a total of 200 mammalian cells were counted and the
percentage of the infected cells and number of amastigotes
per cell were determined. The results were expressed as the
survival index. The control infected without treatment was
considered as 100%, and the results for the treated-groups
were comparatively evaluated, and the concentrations that
inhibits 50% (IC50) and 90% (IC90) were determined.
Cytotoxicity assay
Cytotoxicity was assessed to determinate the cell
viability by using a MTT (3-[4,5-dimethylthiazol-2-yl]-
2,5-diphenyltetrazolium bromide) assay.39 This colorimetric
assay is based on the ability of the viable mitochondria
to convert MTT, a water-soluble tetrazolium salt, into an
insoluble purple-colored formazan precipitate. LLCMK2
cell line was plated and incubated for 24 h at 37 °C with
5% CO2. Afterwards, the medium was removed and the
cells were treated with different concentrations of the
compounds and incubated for 96 h at 37 °C with 5% CO2
atmosphere. The cell wells with fresh medium and 10%
SFB without treatment were included as controls.After 96 h
of incubation, the cells were washed in PBS, and 50 μL of
MTT (2 mg mL-1) were added to each well. The formazan
crystals were solubilized in DMSO (dimethyl sulfoxide),
and the absorbance was read at 570 nm in a microplate
reader (BioTek, Power Wave XS).All the experiments were
carried out in duplicate. The means were determined from
at least three experiments. The selectivity index (SI) was
calculated by CC50 (cytotoxic concentration for 50% of the
cells) of the LLCMK2 divided by IC50 of the amastigote
forms of T. cruzi.
Activity on trypomastigotes
In order to obtain tissue culture-derived trypomastigote
forms, LLCMK2 cells (Macaca mulatta epithelial kidney
cells) were cultivated in DMEM (Dulbecco’s Modified
Eagle Medium, pH 7.4, Gibco Invitrogen Co., Grand
Island, NY), diluted in 10% SFB (bovine fetal serum) in
a 5% CO2 atmosphere at 37 °C. Trypomastigotes were
harvested from the supernatant of infected LLCMK2 and
added to 96-well plates at 1 × 107 parasites mL-1 in the
presence and absence of increasing concentrations of the
triterpenoids up to the maximum concentration of 100 μM.
This assay was incubated at 37 °C in a 5% CO2 atmosphere.
The trypomastigote motility was determined by using the
Pizzi-Brener method.38 Briefly, 5 μL aliquots from each
well were removed and deposited on slides, coverslipped,
and analyzed immediately. Motility reflected parasite
viability. The concentration that killed 50% of the parasites
(EC50) was calculated.
Haemolytic assay
Activity on amastigotes
The cytotoxicity of the twelve compounds was
also evaluated against human red blood cells. Blood
(A+ type) from a healthy human donor was collected,
defibrinated, and washed in glycosylated saline in order
to remove free hemoglobin from the defibrinization
process. Red blood cells were added to Eppendorfs with
6% glycosylated saline with different concentrations of
triterpenoids (50-2000 μM), and then incubated for 3 h at
37 °C. The supernatant was transferred to 96-well plates
In order to evaluate the activity of triterpenoids
against the intracellular forms of the parasite, LLCMK2
cells were deposited on glass coverslips in 24-well
microplates at a concentration of 2.5 × 105 cells mL-1.
After the monolayer formation, the trypomastigote forms
(10 trypomastigotes/1 LLCMK2 ratio) were added to each
well, and the plates were incubated for 24 h. After that,

2478
Anti-Trypanosoma Activity and Synergistic Effects of Natural and Semi-Synthetic Triterpenes
J. Braz. Chem. Soc.
and read at 550 nm in a microplate reader. To calculate the
percentage of hemolysis, 1% Triton X-100 was used as a
positive control.
cacodylate buffer, and the cell monolayer was detached
and collected and, then, pos-fixed in 1% osmium tetroxide
and 0.8% potassium ferrocyanide, prepared in a cacodylate
buffer. The parasites were washed, and then dehydrated in
growing concentrations of acetone (50, 70, 80, 90, 95 and
100%), gradually embedded in Epon resin and polymerized
at 60 °C. Ultra-thin sections of the samples were obtained
with an ultramicrotome cut and stained with uranyl acetate
and lead citrate. A JEOL JM1400 transmission electron
microscope was used to examine the ultrastructure of the
parasites.
Drug combination assay
To verify the combinatory effects between triterpenes
and the combination of one of them with benznidazole
against the amastigote forms and LLCMK2 cells, three
compounds were randomly chosen (α/β-amyrin, 3-O-acetyl-
α/β-amyrin and 3-O-acetyl-11-oxo-α/β-amyrin). Two drug
interaction assays between triterpenes were performed
by using amastigote forms and combinations between
α/β-amyrin with 3-O-acetyl-11-oxo-α/β-amyrin, in
different concentrations. The compound 3-O-acetyl-
α/β-amyrin was only combined with benznidazole
(2.5-40.0 μM) to evaluate the synergy with the standard
drug. As described above, the same experimental assay
of cytotoxicity against nucleated cells and antiamastigote
activity of T. cruzi was carried out. The experimental design
consists in combinations of at least four concentrations of
each drug arranged in a checkerboard at a 1:2 concentration
ratio. To evaluate the combinational effect, the combination
index (CI) method proposed by Chou and Talalay40 was
adopted. The data were mathematically calculated with
the combination index
Evaluation of the mitochondrial membrane potential and
cell membrane integrity
To associate the ultrastructural alterations with the
action mechanisms of the triterpenes, both mitochondrial
membrane potential (ΔΨm) and cell membrane integrity
were evaluated in amastigotes treated with triterpenoids
by using flow cytometry. The same five compounds
and the higher synergistic combination found between
α/β-amyrin and 3-O-acetyl-11-oxo-α/β-amyrin, assessed
by electron microscopy were also assessed through
the flow cytometry. Therefore, T. cruzi parasitic forms
(1 × 107 amastigotes mL-1), either treated or not with
the IC50 and IC90 for 24 h with five different compounds
(α/β-amyrin, 3-O-phthaloyl-α/β-amyrin, 3-O-acetyl-
11-oxo-α/β-amyrin, 3-O-succinyl-11-oxo-α/β-amyrin and
lup-20(29)-en-3-one) and with the combination between
α/β-amyrin and 3-O-acetyl-11-oxo-α/β-amyrin, were
assessed with electron microscopy. The cell membrane
integrity was evaluated by using PI (propidium iodide),
a probe that binds to DNA (deoxyribonucleic acid) in
ruptured membrane cells. After treatment, the parasites
were washed and incubated with 0.2 mg mL-1 PI for 10 min
to verify the cell membrane integrity. Data acquisition
and analysis were performed by using a FACSCaliburTM
flow cytometer (Becton-Dickinson, Rutherford, NJ,
USA) equipped with the CellQuest software (Joseph
Trotter, The Scripps Research Institute, La Jolla, CA,
USA). A total of 10,000 events were acquired in the
previously established region that corresponds to the
parasites.Alterations in the PI fluorescence were quantified
as the increase percentage in the fluorescence compared
with the control (untreated parasites). To evaluate the
mitochondrial membrane potential (ΔΨm), the rhodamine
123 (Rh123) was used as a fluorescent probe, which
accumulates within mitochondria. The amastigotes
previously treated for 24 h were washed and incubated
with 5 mg mL-1 Rh123 for 15 min to verify alterations
in ΔΨm, and data acquisition was obtained with a flow
CI = [IC50drug A combined/IC50drug A alone] +
[IC50drug B combined/IC50drug B alone
]
(1)
(based on the sum of division data of the IC50 of the drugA
combined with B divided by IC50 of the drug A when used
separately, plus the division data of the IC50 of the drug B
combined with A by IC50 when used alone) in which CI
values less, equal, and more than 1 indicate combinatorial
effects of synergism, additive, and antagonism, respectively.
The data were also graphically expressed in an isobologram.
Transmission electron microscopy (TEM)
To evaluate the effect of the compounds on the
morphology of intracellular amastigote forms by TEM,
the compounds 3-O-phthaloyl-α/β-amyrin, 3-O-succinyl-
11-oxo-α/β-amyrin and lup-20(29)-en-3-one, as well as the
compounds α/β-amyrin and 3-O-acetyl-11-oxo-α/β-amyrin
separately, and in combination, were chosen. Thus, a flask
of culture LLCMK2 cells with a well-formed monolayer
and previously infected with 1 × 107 trypomastigotes mL-1
was either treated or not with concentrations of the IC50
and IC90 of triterpenes for 24 h. The cells were collected
and fixed with 2.5% glutaraldehyde prepared in 0.1 M

Vol. 28, No. 12, 2017
Bossolani et al.
2479
cytometer. Changes in Rh123 fluorescence were quantified
by using an index variation (IV) obtained by the equation
(MT − MC) / MC, where MT is the median fluorescence
for treated parasites and MC is the median fluorescence
of control parasites. Negative IV values (‑IV) correspond
to the depolarization of the mitochondrial membrane and
positive IV values (+IV), hypolarization process. The
carbonyl cyanide 3-chlorophenylhydrazone (CCCP) with
a dosage of 100 μM was used as a positive control for the
Rh123 experiments, and digitonin (40 μM) was used as a
positive control for the PI experiments.
Maringá (acceptance 293/2006 COPEP-UEM). In order to
obtain tissue culture-derived trypomastigote forms, blood
was weekly collected through cardiac puncture, the Swiss
mice were obtained in the Central Animal Facility of the
State University of Maringá (UEM).All the procedures were
carried out in accordance with the guidelines established
by the Ethics Committee on Animal Experiments of UEM,
stating the detailed approved protocol for this experiment
(acceptance 074/2011-CONCEA).
Results
Cell volume determination
Amyrins were obtained from Burseraceae family
that produces Brazilian oil resins, such as the breu resin
exudate that is obtained from species Protium hebetatum
and P. paniculatum. These triterpenes are promising raw
materials for the semi-synthesis of triterpene derivatives,
since these compounds are easily purified and available
in high amounts throughout Amazon region.25 However,
the isomers are not easy to separate,26 and have usually
been studied as a binary mixture, especially α-amyrin and
β-amyrin, from oleanane and ursane skeletons, respectively.
In the present study, seven derivatives were obtained
from this binary mixture [(1) α/β-amyrin]: (2) 3-O-fumaryl-
α/β-amyrin, (3) 3-O-succinyl-α/β-amyrin, (4) 3-O-acetyl-
α/β-amyrin, (5) 3-O-ftaloyl-α/β-amyrin, (6) 3-O-acetyl-
11-oxo-α/β-amyrenone, (7) 3-O-acetyl-11-oxo-α/β-amyrin
and (8) 3-O-ftaloyl-11-oxo-α/β-amyrin. To improve
the discussion on SAR of the triterpenes, four natural
triterpenes previously isolated were also studied: three
pentacyclic lupane compounds [(9) lup-20(29)-en-
3-one, (10) 3β,6β,16β-trihydroxylup-20(29)-ene and
(11) betulinic acid], together with a tetracyclic dammarane
[(12) lanosta-8,24-dien-3-one]. All compounds were
used to determine their trypanocidal activities and action
mechanisms against trypomastigote and amastigote forms
(Figure 1). The semi-synthesis reactions of triterpenic
derivatives obtained by α/β-amyrin are shown in Figure 2.
The amastigotes (1 × 107 parasites mL-1) were treated
for 24 h with IC50 and IC90 of α/β-amyrin, 3-O-phthaloyl-
α/β-amyrin, 3-O-acetyl-11-oxo-α/β-amyrin, 3-O-succinyl-
11-oxo-α/β-amyrin and lup-20(29)-en-3-one, and the
combination between α/β-amyrin and 3-O-acetyl-
11-oxo-α/β-amyrin. The parasites were collected with
centrifugation, washed twice in PBS, resuspended in
PBS, and directly analyzed through fluorescence-activated
cell sorting by using a BD FACSCalibur flow cytometer
(Becton-Dickinson, Rutherford, NJ, USA). A total of
10,000 events were acquired in a region previously
established for the parasites. The parasites treated with
actinomycin D (20.0 mM) were used as a positive control.
Histograms were generated and the analyses were
performed by using the CellQuest software, in which the
FSC (forward scatter) parameters represent the cell volume.
Statistical analysis
The experiments were carried out in different occasions,
and the statistical analysis was performed with the
GraphPad Prism 5.0 software with one-way analysis of
variance (ANOVA) test.Values of p < 0.05 were considered
significant.
Ethics
Activity against trypomastigote forms
For the hemolytic assay, voluntary donations of human
blood were obtained according to the Declaration of Helsinki
(Ethical principles for medical research involving human
beings). The donors were informed about the purpose of the
study and provided with the written consent before blood
collection. The blood was collected through brachial vein
puncture by a trained professional with appropriate material
and medical support. All procedures were conducted as
described in the specific protocol approved by the Ethics
Committee on Human Beings of the State University of
In general, the treatment with triterpenoids was
not effective in the nonproliferative forms of T. cruzi.
For all the compounds, it was not observed 50%
activity against trypomastigotes even up to the assessed
maximum concentration (100 μM), except for compounds
(2) 3-O-fumaryl-α/β-amyrin, (3) 3-O-succinyl-
α/β-amyrin and (5) 3-O-phthaloyl-α/β-amyrin.
Compounds (3) 3-O-succinyl-α/β-amyrin and
(5) 3-O-phthaloyl-α/β-amyrin displayed a moderate activity
with an IC50 values of 74.6 and 86.3 μM, respectively. Only

2480
Anti-Trypanosoma Activity and Synergistic Effects of Natural and Semi-Synthetic Triterpenes
J. Braz. Chem. Soc.
Figure 1. Chemical structures of the triterpenes tested for trypanocidal activity: (1) α/β-amyrin, (2) 3-O-fumaryl-α/β-amyrin, (3) 3-O-succinyl-α/β-amyrin,
(4) 3-O-acetyl-α/β-amyrin, (5) 3-O-phthaloyl-α/β-amyrin, (6) 3-O-acetyl-11-oxo-α/β-amyrin, (7) 3-O-succinyl-11-oxo-α/β-amyrin, (8) 3-O-phthaloyl-
11-oxo-α/β-amyrin, (9) lup-20(29)-en-3-one, (10) lup-20(29)-ene-3β,6β,15β-triol, (11) betulinic acid and (12) lanosta-8,24-dien-3-one; α isomer: R3 = CH3,
R4 = H; β isomer: R3 = H, R4 = CH3.
the compound (2) 3-O-fumaryl-α/β-amyrin exhibited a
good activity, with an IC50 of 25.2 μM (Table 1).
compound (6) 3-O-acetyl-11-oxo-α/β-amyrin showed a
moderate activity with an IC50 of 55.3 μM, and among
the other eleven triterpenes, the IC50 values varied from
3.2 to 21.8 μM. Based on the CC50 and IC50 values, the
SI was determined to compare toxicity for the parasite
when compared to the host cells. The higher selectivity
indexes indicate more toxicity to the protozoan than
mammalian cells. Compounds (5) 3-O-phthaloyl-
Activity against amastigote forms
Unlike the antitrypomastigote activity, all triterpenoid
compounds exhibited an excellent activity, inhibiting
the parasite intracellular multiplication. After 96 h, only

Vol. 28, No. 12, 2017
Bossolani et al.
2481
Figure 2. Semi-synthesis of triterpene derivatives, reagents and conditions: (a) maleic anhydride, DMAP, CH2Cl2, reflux, 24 h; (b) succinic anhydride,
DMAP, CH2Cl2, reflux, 24 h; (c) acetic anhydride, DMAP, reflux, 30 min; (d) phthalic anhydride, DMAP, CH2Cl2, reflux, 24 h; and (e) acetic anhydride,
acetic acid, tert-butyl chromate, reflux, 6 h.
Table 1. Trypanocidal activity and cytotoxicity on mammalian cells of twelve triterpenoid compounds (with standard deviation): (1) α/β-amyrin,
(2) 3-O-fumaryl-α/β-amyrin, (3) 3-O-succinyl-α/β-amyrin, (4) 3-O-acetyl-α/β-amyrin, (5) 3-O-phthaloyl-α/β-amyrin, (6) 3-O-acetyl-11-oxo-α/β-amyrin,
(7) 3-O-succinyl-11-oxo-α/β-amyrin, (8) 3-O-phthaloyl-11-oxo-α/β-amyrin, (9) lup-20(29)-en-3-one, (10) lup-20(29)-ene-3β,6β,15β-triol, (11) betulinic
acid and (12) lanosta-8,24-dien-3-one
Trypomastigote
EC50 (24 h) / μM
> 100
Amastigote
IC50 (96 h) / μM
20.2 2.0
3.2 0.6
LLCMK2
CC50 (96 h) / μM
136.3 7.7
16.2 1.1
Erytrocyte
H (3 h) / %
8.6 1.1
SI
CC50/IC50
6.7
Compound
1
2
25.2 0.6
86.3 1.3
> 100
51.1 0.3
3.4 1.3
5.1
3
10.1 1.8
21.2 4.9
6.1 2.9
150.4 4.3
135.8 6.2
104.6 2.5
103.6 6.7
195.5 5.9
150.3 5.5
556.5 11.3
29.6 0.7
14.9
6.4
4
12.6 1.2
27.5 3.4
5.5 3.2
5
74.6 3.8
> 100
17.1
1.9
6
55.3 4.0
9.7 1.4
7
> 100
23.0 1.4
82.4 1.9
5.1 1.6
20.2
15.5
25.5
5.0
8
> 100
9.7 3.0
9
> 100
21.8 4.2
5.9 0.2
10
11
12
Bz
> 100
24.0 0.4
25.7 1.4
10.5 2.2
49.5 3.4
> 100
9.9 0.1
102.2 3.6
137.1 7.2
1,235.3 15.1
10.4
12.5
132.8
> 100
11.0 5.5
9.3 0.9
5.1 2.1
Values of concentration are represented in μM. IC50: inhibitory concentration of 50%; EC50: effective concentration of 50%; CC50: cytotoxic concentration
of 50%; H: percentage of hemolysis at concentration of 2000 μM; SI: selectivity index between LLCMK2 cells and amastigote forms; significant differences
between activity of the compounds and control cell growth, ANOVA (p < 0.0001).

2482
Anti-Trypanosoma Activity and Synergistic Effects of Natural and Semi-Synthetic Triterpenes
J. Braz. Chem. Soc.
α/β-amyrin, (7) 3-O-succinyl-11-oxo-α/β-amyrin and
(9) lup-20(29)-en-3-one were 17.1 to 25.5-fold more active
against amastigotes than the host cells. The compounds
(2) 3-O-fumaryl-α/β-amyrin, (6) 3-O-acetyl-11-oxo-
α/β-amyrin and (10) lup-20(29)-ene-3β,6β,15β-triol were
the least selective, with SIs of 5.1, 1.9, and 5.0 (Table 1).
Comparing the triterpenoids’ antiamastigote activity with
benznidazole, the standard drug displayed an IC50 of 9.3 μM
to the intracellular parasites of the vertebrate host cells.
In addition, the combination of (4) 3-O-acetyl-α/β-amyrin
and benznidazole also showed synergistic interaction with
a CI of 0.81. The above described combinations were
also performed in LLCMK2 cells to verify variation on
cells. Satisfactory results were also obtained because both
combinations exhibited antagonistic effects in LLCMK2
cells, with a CI of 1.35 and 1.4, respectively (Figure 3).
Ultrastructural and morphological alterations
Cytotoxicity in mammalian cells
TEM images of the intracellular replicative amastigotes
treated with triterpenoids revealed morphological alterations
due to the lack of uniformity of the plasma membrane (white
arrow head, Figures 4 and 5). Furthermore, alterations
were observed in the ultrastructure mitochondrion-
kinetoplast complex (Figures 4C, E and F, and Figure 5E),
including rupture of the mitochondrion (white star,
Figure 4E), multiple cytoplasmic vacuolization structures
(Figure 5B) that accumulate lipids in these inclusions
(white star, Figure 5C) and a formation of the membrane
within the kinetoplast (black arrow head, Figure 5E). The
most evident feature observed in treated-amastigotes was
an induction of the formation of autophagosomes (black
arrow, Figures 4 and 5) and autophagosome-like structures
such as: well-developed endoplasmic reticulum (white
arrow, Figures 4 and 5), which surrounds cytoplasmic
structures and organelles, Golgi complex extension
(white arrow head, Figure 5C) and concentric myelin-like
membrane structures (black arrow head, Figure 4G).
Most the triterpenes displayed a low cytotoxicity
against the mammalian host cells (LLCMK2) incubated for
96 h. The most toxic compounds were (2) 3-O-fumaryl-
α/β-amyrin and (10) lup-20(29)-ene-3β,6β,15β-triol with
CC50 values of 16.2 and 29.6 μM, respectively, and the lowest
toxic compound was (9) lup-20(29)-en-3-one with a CC50 of
556.5 μM.Among the nine remaining triterpenes, the lowest
toxic compounds were observed with a CC50, which ranged
from 102.2 to 195.5 μM. The benznidazole demonstrated the
lowest toxicity in relation to the triterpenes. The toxicity in
erythrocytes was lower than nucleated cells. After 3 h, the
hemolysis rate for the lowest hemolytic compounds varied
from 3.4 to 27.5% with the highest tested concentration
(2.000 μM). However, the compounds (2) 3-O-fumaryl-
α/β-amyrin and (8) 3-O-phthaloyl-11-oxo-α/β-amyrin
showed the highest hemolytic activity with 51.1 and 82.4%
of hemolysis rate (Table 1).
Drug combination assay
Evaluation of the mitochondrial membrane potential and
cell membrane integrity
Because of the higher antimastigote activity of the
triterpenes, the aim of this study was to evaluate the
drug combination assay only in intracellular forms. The
evaluation of the combinatorial effects with combination of
the compounds (1) α/β-amyrin and (6) 3-O-acetyl-11-oxo-
α/β-amyrin revealed a synergistic activity with a CI of 0.78.
For the cell membrane integrity assay, alterations
were not verified in the membrane permeability in
T. cruzi intracellular forms through the incubation with
triterpenoids. The low number of high fluorescence events
(upper left and upper right quadrants) shows cells with
Figure 3. Combinatorial effects between (1) α/β-amyrin with (6) 3-O-acetyl-11-oxo-α/β-amyrin and benznidazole with (4) 3-O-acetyl-α/β-amyrin
on amastigotes of T. cruzi and LLCMK2 cells. Isobolograms (a) and (b) show the combinatorial effects of (1) α/β-amyrin with (6) 3-O-acetyl-11-oxo-
α/β-amyrin and (4) 3-O-acetyl-α/β-amyrin with benznidazole against amastigotes, respectively; (c) and (d) illustrate combination between (1) α/β-amyrin
with (6) 3-O-acetyl-11-oxo-α/β-amyrin and (4) 3-O-acetyl-α/β-amyrin with benznidazole on LLCMK2 cells, respectively. Points below the additivity
effect line indicate a synergistic effect and above indicate an antagonistic effect (combination index (CI)).

Vol. 28, No. 12, 2017
Bossolani et al.
2483
Figure 5. Transmission electron images of amastigotes forms of
T. cruzi treated with triterpenes for 24 h. (A)-(F) correspond to treated-
amastigote with an IC90 of the compounds (1) α/β-amyrin (97.3 μM),
(5) 3-O-phthaloyl-α/β-amyrin (86.3 μM), (6) 3-O-acetyl-11-oxo-
α/β-amyrin (112.5 μM), (7) 3-O-succinyl-11-oxo-α/β-amyrin (90.6 μM),
(9) lup-20(29)-en-3-one (95.9 μM), and drug combination between
(1) α/β-amyrin (38.3 μM) with (6) 3-O-acetyl-11-oxo-α/β-amyrin
(47.2 μM), respectively. Key: n, nucleus, k, kinetoplast, and m,
mitochondrion. Bar = 1 μm.
Figure 4. Transmission electron images of amastigotes forms of T. cruzi
treated with triterpenes for 24 h. (A) Control; (B)-(G) correspond
to treated-amastigotes with IC50 of the compounds (1) α/β-amyrin
(20.2 μM), (5) 3-O-phthaloyl-α/β-amyrin (6.1 μM), (6) 3-O-acetyl-
11-oxo-α/β-amyrin (55.3 μM), (7) 3-O-succinyl-11-oxo-α/β-amyrin
(9.7 μM), (9) lup-20(29)-en-3-one (21.8 μM), and drug combination
between (1) α/β-amyrin (7.1 μM) with (6) 3-O-acetyl-11-oxo-α/β-amyrin
(14.5 μM), respectively; n: nucleus, k, kinetoplast, m, mitochondrion, and
f, flagellum. Bar = 1 μm.
fluorescence, which indicates a mitochondrial membrane
hyperpolarization (Figure 7). CCCP caused a high Rh123
fluorescence intensity reduction in comparison to untreated
cells (data not shown). In general, the treated-amastigotes
with IC90 dosage of the triterpenoids displayed higher
indexes of IV values than IC50, indicating dose-dependent
effects. According to the higher IV values, treatment with
(1) α/β-amyrin and (6) 3-O-acetyl-11-oxo-α/β-amyrin
combined with IC90 (Table 2) was the most pronounced
mitochondrial depolarization.
normal membrane permeability due to blockage of the
entrance of PI inside the cells. In the three experiments
with amastigote forms, the effects of the treatment with
triterpenes with IC50 and IC90 were very similar to those
untreated parasites (Figure 6). The digitonin (positive
control) promoted a loss of the cell membrane integrity
with 93.9% PI-stained positive cells in the upper left and
upper right quadrants.
Differently from the assay above described, some
alterations were verified in ΔΨm. The histograms showed
a decrease in total Rh123 fluorescence intensity for all the
compounds mainly treated with IC90, thus resulting in a
mitochondrial depolarization. However, (1) α/β-amyrin
combined (6) 3-O-acetyl-11-oxo-α/β-amyrin with
IC50 that showed an enhancement of the total Rh123
Cell volume determination
The amastigote forms treated with triterpenes, as
previously described, were analyzed for FSC parameters
through the flow cytometry. The positive control showed
a substantial cell volume reduction by a decrease of
the fluorescence intensity in the FSC parameters (data

2484
Anti-Trypanosoma Activity and Synergistic Effects of Natural and Semi-Synthetic Triterpenes
J. Braz. Chem. Soc.
Figure 6. Cell membrane integrity assay by flow cytometry in amastigote forms of T. cruzi treated with triterpenes for 24 h and stained with PI. (a) Untreated
amastigotes; (b) positive control; histograms (c), (e), (g), (i) (k) and (m) correspond to treated-amastigotes with IC50, and (d), (f), (g), (h), (j) (l) and
(n) treated-amastigotes with IC90 of the compounds (1) α/β-amyrin, (5) 3-O-phthaloyl-α/β-amyrin, (6) 3-O-acetyl-11-oxo-α/β-amyrin, (7) 3-O-succinyl-
11-oxo-α/β-amyrin, (9) lup-20(29)-en-3-one and drug combination between (1) α/β-amyrin with (6) 3-O-acetyl-11-oxo-α/β-amyrin, respectively. Percentage
of PI-stained positive cells is shown in the upper right and left quadrants. Typical histograms of at least three independent experiments are shown.
not shown). As illustrated in Figure 8, the cell volume
maintenance was observed by two concentrations (IC50
and IC90) tested for mammalian stage T. cruzi parasites
with five selected compounds and for the (1) α/β-amyrin
with (6) 3-O-acetyl-11-oxo-α/β-amyrin in combination.
All histograms showed a similar effect compared with the
untreated control group for all the compounds and two
selected concentrations by maintaining the fluorescence
intensity in the FSC parameters.
the treatment with triterpenoids and benznidazole
displayed an excellent anti-T. cruzi activity against
the relevant clinically intracellular amastigote forms,
which are responsible for the chronic phase infection
maintenance through parasite persistence and parasite
load in target organs.21,41 These features revealed to
be crucial even at asymptomatic phase, since the host
can develop autoimmunity due to the presence of the
parasite.42 Thus, the aim of treatment in this stage should
eliminate the amastigotes or reduce the parasite load.
The weaker antitrypomastigote activity observed in this
study with terpenes is well-reported in several previous
researches.11-17,42,43
Discussion
Considering modern drug research, natural products
and plant-derivative terpenoids may offer good sources
designs and discoveries for a new drug. In this study,
Several investigations relate that terpenes may arrest
the cell division cycle and, in turn avoiding the parasite

Vol. 28, No. 12, 2017
Bossolani et al.
2485
Figure 7. Mitochondrial membrane potential assay in amastigote forms of T. cruzi treated with IC50 and IC90 of the triterpenes for 24 h and stained with
Rh123. (a)-(f)Amastigotes treated with triterpenoids (1) α/β-amyrin, (5) 3-O-phthaloyl-α/β-amyrin, (6) 3-O-acetyl-11-oxo-α/β-amyrin, (7) 3-O-succinyl-
11-oxo-α/β-amyrin, (9) lup-20(29)-en-3-one and drug combination between (1) α/β-amyrin with (6) 3-O-acetyl-11-oxo-α/β-amyrin, respectively. Arrows
correspond to the concentration tested and control group (untreated parasites) that are also shown. Typical histograms of at least three independent
experiments are presented.
Table 2. Mitochondrial potential assay in parasitic forms of Trypanosoma cruzi treated with following triterpenoids for 24 h and stained with Rh123:
(1) α/β-amyrin, (5) 3-O-phthaloyl-α/β-amyrin, (6) 3-O-acetyl-11-oxo-α/β-amyrin, (7) 3-O-succinyl-11-oxo-α/β-amyrin, (9) lup-20(29)-en-3-one and drug
combination between (1) α/β-amyrin with (6) 3-O-acetyl-11-oxo-α/β-amyrin
a
Median of the fluorescence (Rdh 123)
IV
Compound
IC50 / μM
165.5
155.4
160.2
116.2
140.8
355.5
250.3
IC90 / μM
134.5
135.8
143.8
136.2
120.8
77.0
IC50 / μM
–0.3
IC90 / μM
–0.5
1
5
–0.4
–0.5
6
–0.4
–0.4
7
–0.5
–0.5
9
–0.4
–0.5
1 + 6
Control
+0.4
0.0
–0.7
250.3
0.0
aIV = (MT – MC)/MC, where MT corresponds to the median of the fluorescence for treated parasites and MC for control parasites; Rh123: rhodamine 123;
IC50: inhibitory concentration of 50%; IIV: index variation; –IV: negative IV values; +IV: positive IV values; C90: inhibitory concentration of 90%.
proliferation. Lozano et al.44,45 reported a more triterpenoid
resistence against trypomastigote forms may be related
with their non-multiplying capacity. Despite the low
antitrypomastigote activity, these compounds are promising
candidates for therapeutic targets in the chronic stage.
It is well-known that benznidazole displays a limited
effectiveness for the chronic stage; even being the best
current available drug in the market.
Aiming to obtain compounds with synergistic properties
against chronic illness, great interactions were obtained
with synergic activities by combining (1) α/β-amyrin
with (6) 3-O-acetyl-11-oxo-α/β-amyrin, as well as
(4) 3-O-acetyl-α/β-amyrin combined with the standard
drug benznidazole against intracellular forms.Additionally,
these two combinations were performed in LLCMK2,
and antagonism effects were obtained that indicate a
satisfactory reduction of toxicity in the mammalian cells.
In a recent study, Izumi et al.12 evaluated the combinational
effects against trypomastigotes between copalic acid
and β-caryophyllene in which a strong synergism was
found with FICI (fractional inhibitory concentration
index) values of 0.12. In addition, combination therapies
with benznidazole can allow the decrease of the dosage of
the standard drug for treating Chagas’ disease and, thus,
its side-effects might be alleviated and may extend the
period of the treatment with benznidazole.21 The lowest

2486
Anti-Trypanosoma Activity and Synergistic Effects of Natural and Semi-Synthetic Triterpenes
J. Braz. Chem. Soc.
Figure 8. Cell volume determination of amastigotes treated with triterpenoids for 24 h and analyzed by flow cytometer. The histograms show the relationship
between the number of cells (counts) and FSC considered as a function of cell size. Gray-filled areas represent untreated cells. In each plot, the black line
with unfilled areas represents treated-parasites. Histograms (a), (c), (e) (g), (i) and (k) correspond to treated-amastigotes with IC50 and (b), (d), (f), (h), (j) and
(l) treated-amastigotes with IC90 of the compounds (1) α/β-amyrin, (5) 3-O-phthaloyl-α/β-amyrin, (6) 3-O-acetyl-11-oxo-α/β-amyrin, (7) 3-O-succinyl-
11-oxo-α/β-amyrin, (9) lup-20(29)-en-3-one and drug combination between (1) α/β-amyrin with (6) 3-O-acetyl-11-oxo-α/β-amyrin, respectively.
cytotoxicity of benznidazole in vitro demonstrated in this
study does not occur in vivo.
triterpenic core and the position insertion of these
radicals is determinant for the activity. For instance,
comparing the four ursane and oleanane-type triterpenoid
derivatived from triterpenoid mixture of (1) α/β-amyrin,
it may be observed that (1) α/β-amyrin was the less
effective and (2) 3-O-fumaryl-α/β-amyrin was the
highest active from the twelve triterpenoids assessed
against two T. cruzi developmental forms in the host
and LLCMK2 cells. These data can be explained by an
insertion of a radical 3-O-fumaryl at C-3 position for
(2) 3-O-fumaryl-α/β-amyrin in which its radical shows
four oxygen atoms, possibly due to the formation of the
intramolecular hydrogen that consistently increases its
polarity and activity in comparison with (1) α/β-amyrin.
Such feature may also suggest the high cytotoxicity of
(2) 3-O-fumaryl-α/β-amyrin. The low presence of oxygen
atoms in the substituent groups at C-3 may also explain the
lower activity of (1) α/β-amyrin, (6) 3-O-acetyl-11-oxo-
α/β-amyrin and (9) lup-20(29)-en-3-one.
Previousresearchesreportedthatthepresenceofhydroxyl
group at C-3 and carboxyl group at C-17 is important for
the parasitic activity.47 For this reason, (11) betulinic
acid was more effective than (9) lup-20(29)-en-3-one.
The lowest cytotoxicity of (9) lup-20(29)-en-3-one and
the lowest antiamastigote activity of (7) 3-O-succinyl-
11-oxo-α/β-amyrin may be explained by oxidizing the C-3
position and R2 position. Furthermore, the increase of the
Toxicity data indicated that (9) lup-20(29)-en-3-one
is one of the less toxic compounds, and (2) 3-O-fumaryl-
α/β-amyrin and (10) lup-20(29)-ene-3β,6β,15β-triol
showed the strongest toxicity to LLCMK2 cells. The
other compounds exhibited low and similar toxicities.
Additionally, all the compounds tested showed non-toxicity
properties in blood erythrocytes at very high concentrations
of 2.000 μM, even though the compounds (2) 3-O-fumaryl-
α/β-amyrin and (8) 3-O-phthaloyl-11-oxo-α/β-amyrin
displayed high hemolysis rates. It has already been reported
that ursane, lupane- and oleanane-type triterpenes are
considered as relatively non-toxic drugs.46 Furthermore, it
has been reported that some natural pentacyclic triterpenoid
derivatives such as oleanolic, ursolic and betulinic acids are
a thousand-fold more active than the natural compounds.45
Based on this study, four compounds were the most
selective to the protozoan [(5) 3-O-phthaloyl-α/β-amyrin,
(6) 3-O-succinyl-11-oxo-α/β-amyrin, (8) 3-O-phthaloyl-
11-oxo-α/β-amyrin and (9) lup-20(29)-en-3-one], only
(9) lup-20(29)-en-3-one is a natural isolated compound,
and other compounds are triterpenic derivative, thereby
promising compounds may be obtained by chemical
derivatization.
Aiming to establish the SAR, it may be suggested that
the more insertion of polar groups in the fundamental

Vol. 28, No. 12, 2017
Bossolani et al.
2487
polarity of the compounds, nature and orientation of the
substituents at C-3 and C-17 position may affect the activity
of the triterpenes, mainly at C-3 position in the majority
compounds of this study. Indeed, the positions C-3 and
C-17 are considered a target of chemical derivatization
for the insertion of the different substituents.47 In addition,
the presence of hydroxyl groups in different positions at
C-3, C-6 and C-15 of (10) lup-20(29)-ene-3β,6β,15β-triol
as well as spatial configuration and several other factors
may have influenced to the greater antiamastigote and
cytotoxic activity.
The ultrastructural alterations, visualized by TEM of
the T. cruzi intracellular parasites, revealed changes in
the parasite shape, as well as ultrastructural alterations
in the mitochondrion-kinetoplast complex and multiple
cytoplasmic vacuolization. No changes were observed in
the cell membrane permeability with the flow cytometry.
These results can be associated with the absence of
the rupture plasma membrane not seen by TEM. The
plasma membrane rupture is modulated in response to
the programmed cell death by necrosis, which is also
characterized by the subsequent loss of the intracellular
content and an increase in cell volume,19,48 thereby the
present data exclude cell death by necrosis. Furthermore,
amastigotes did not undergo any modification of the cell
volume in the present study.
Except for combination of (1) α/β-amyrin with
(6) 3-O-acetyl-11-oxo-α/β-amyrin at IC50 concentration,
the flow cytometry analyses of triterpenoid-treated
parasites showed a loss of the ΔΨm with a decrease in
Rh123 fluorescence intensity, resulting in mitochondrial
depolarizing effects, mainly the treatment with IC90. The
most pronounced depolarization found was obtained with
the IC90 dosage with the combination between compounds
(1) α/β-amyrin with (6) 3-O-acetyl-11-oxo-α/β-amyrin.
In contrast, IC50 dosage of the combination between
(1) α/β-amyrin with (6) 3-O-acetyl-11-oxo-α/β-amyrin
displayed a mitochondrial membrane hyperpolarization
that enhanced the total Rh123 fluorescence intensity. Part
of these results may be correlated with ultrastructural
mitochondrial alterations confirmed by TEM analyses,
resulting in mitochondrial dysfunctions.The depolarization
indicated an enhancement in the proton permeability
through the inner mitochondrial membrane, the inhibition
of the electron transport, or decrease in mitochondrial
substrate transport/oxidation, which would impair proton
pumping efflux and adenosine triphosphate synthesis,
resulting in the parasite cell death. Specific mechanisms
may be involved in the hyperpolarizing phenomena of the
ΔΨm, such as complex II respiratory chain inhibition in
the protozoan.49
The remarkable ultrastructural alterations seen
by TEM were the formation of autophagosomes
and autophagosome-like structures, such as dilated
endoplasmic reticulum profiles and Golgi complex
alterations, which surround cytoplasmic structures, and
the formation of concentric myelin-like figures. These
features are morphologically characterized as signs of
the hallmark cell death of autophagy, in which their
cellular components, such as the cytoplasm, organelles
and protein aggregates, are catabolized for recycling
as a pro-survival protection mechanism.50 Not all the
triterpenes induced the formation of autophagosome-like
structures, which may suggest an autophagic process
[(9) lup-20(29)-en-3-one]. For this reason, cell death by
apoptosis can also be involved. Izumi et al.12 reported that
some terpenes found in copaiba oils promoted parasite
death by different mechanisms, including oxidative stress
and autophagy. Thus, other studies should be carried out
to further investigate the possible mechanisms involved
in the cell death.
Conclusions
This study showed that triterpenes are promising
compounds due to their excellent antiamastigote activities.
Most compounds exhibited a low toxicity to the mammalian
cells. The SAR of the triterpenes suggested that the
insertion of more numbers of polar substituent groups
may lead to greater selective compounds to the protozoan,
thereby chemical derivatization can optimize the search of
biological promising compounds with low toxicity to the
host. Our results reinforce the importance of evaluating
compounds with synergistic effects against the parasite
and antagonistic effects against LLCMK2 cells, being
an important alternative to treat the chronic stage of
disease. Finally, the main cell death mechanism induced
by triterpenoids against amastigotes suggests autophagy.
Acknowledgments
The authors thank to FAPERJ, FAPEAM, CNPq and
CAPES.
References
1. Rassi Jr., A.; Rassi, A.; Marin-Neto, J. A.; Lancet 2010, 375,
1388.
2. Buckner, F. S.; Urbina, J. A.; Int. J. Parasitol. 2012, 2, 236.
3. Urbina, J. A.; Campo, R.; Trends Parasitol. 2003, 19, 495.
4. Chatelain, E.; Konar, N.; Drug Des. Dev. Ther. 2015, 9, 407.
5. Chatelain, E.; J. Biomol. Screening 2015, 20, 22.

2488
Anti-Trypanosoma Activity and Synergistic Effects of Natural and Semi-Synthetic Triterpenes
J. Braz. Chem. Soc.
6. Ulla, A.; Baratto, L. C.; Paula, R. C.; Silva, L. H. V.; Soares,
26. Dias, M. O.; Hamerski, L.; Pinto, A. C.; Quim. Nova 2011, 34,
704.
M. J.; Oliveira, B. H.; J. Braz. Chem. Soc. 2016, 27, 1245.
7. Silva Júnior, E. N.; Jardim, G. A. M.; Menna-Barreto, R. F. S.;
Castro, S. L.; J. Braz. Chem. Soc. 2014, 25, 1780.
8. Thimmappa, R.; Geisler, K.; Louveau, T.; O’Maille, P.; Osbourn,
A.; Annu. Rev. Plant Biol. 2016, 65, 225.
27. Barros, F. W.; Bandeira, P. N.; Lima, D. J.; Meira, A. S.;
de Farias, S. S.;Albuquerque, M. R.; dos Santos, H. S.; Lemos,
T. L.; de Morais, M. O.; Costa-Lotufo, L. V. Pessoa, C. O.;
Bioorg. Med. Chem. 2011, 19, 1268.
9. Mazumder, K.; Tanaka, K.; Fukase, K.; Molecules 2013, 18,
8929.
28. Okoye, N. N.; Ajaghaku, D. L.; Okeke, H. N.; Ilodigue, E. E.;
Nworu, C. S.; Okoye, F. B.; Pharm. Biol. 2014, 52, 1478.
29. Agrawal, P. K.; Jain, D. C.; Prog. NMR Spectr. 1992, 24, 1.
30. Barnes, R. A.; Pereira, A. L.; Scofield, T. C. V.; Brás Filho, R.;
Pinto, A. C.; Chem. Pharm. Bull. 1984, 32, 3674.
31. Facundo, V. A.; Andrade, C. H. S.; Silveira, E. R.; Braz-Filho,
R.; Hufford, C. D.; Phytochemistry 1993, 32, 411.
32. Cota, B. B.; Siqueira, E. P.; Oliveira, D. M.; Alves, T. M. A.;
Sobral, M. E. G.; Rabello, A.; Zani, C. L.; Braz. J. Pharmacog.
2012, 22, 1253.
10. Laszczyk, M. N.; Planta Med. 2009, 75, 1549.
11. Leite, A. C.; Ambrozin, A. R. P.; Fernandes, J. B.; Vieira, P. C.;
Silva, M. F. G. F.;Albuquerque, S.; Planta Med. 2008, 74, 1795.
12. Izumi, E.; Ueda-Nakamura, T.;Veiga-Junior,V. F.; Pinto,A. C.;
Nakamura, C. V.; J. Med. Chem. 2012, 12, 2994.
13. Ramírez-Macías, I.;Marín, C.;Chahboun, R.;Messouri, I.;Olmo,
F.; Rosales, M. J.; Gutierrez-Sánchez, R.;Alvarez-Manzaneda,
E.; Sánchez-Moreno, M.; Am. Soc. Trop. Med. Hyg. 2012, 87,
481.
33. Carpenter, R. S.; Sotheeswaran, S.; Sultanbawa, M. U. S.;
Ternai, B.; Org. Magn. Reson. 2015, 14, 462.
14. Santos, A. O.; Ueda-Nakamura, T.; Dias Filho, B. P.;
Veiga-Junior,V. F.; Nakamura, C.V.; Evid. Based Complement.
Alternat. Med. 2012, DOI: 10.1155/2012/898419.
15. Santos,A. O.; Izumi, E.; Ueda-Nakamura, T.; Dias-Filho, B. P.;
Veiga-Junior, V. F.; Nakamura, C. V.; Mem. Inst. Oswaldo Cruz
2013, 108, 59.
34. Leão De Souza,A. D.; Souza,A. D. L.; Rocha,A. F. I.; Pinheiro,
M. L. B.; Andrade, C. H. S.; Queiroz, A. L. A. G.; Santos,
M. P. S. S.; Quim. Nova 2001, 24, 439.
35. Kavtaradze, L. K.; Manley-Harris, M.; Nicholson, B. K.;
Steroids 2004, 69, 697.
16. Santos, V. A. F. F. M.; Leite, K. M.; Siqueira, C. M.; Regasini,
L. O.; Martinez, I.; Nogueira, C. T.; Galuppo, M. K.; Stolf, B. S.;
Pereira, A. M. S.; Cicarelli, R. M. B.; Furlan, M.; Graminha,
M. A. S.; Molecules 2013, 18, 1053.
36. Nana, F.; Sandjo, L. P.; Keumedjio, F.; Ambassa, P.; Malik,
R.; Kuete, V.; Rincheval, V.; Choudhary, M. I.; Ngadjui, B. T.;
J. Braz. Chem. Soc. 2012, 3, 482.
37. Mendes, C. C.; Sandes, L. Q.; Cruz, F. G.; Roque, N. F.; Chem.
Biodiversity 2009, 6, 1463.
17. Izumi, E.; Ueda-Nakamura, T.; Veiga-Junior, V. F.; Nakamura,
C. V.; Planta Med. 2013, 79, 952.
38. Brener, Z.; Rev. Inst. Med. Trop. São Paulo 1962, 4, 386.
39. Mosmann, T.; J. Immunol. Methods 1983, 16, 55.
40. Chou, T. C.; Talalay, P.; Adv. Enzyme Regul. 1984, 22, 27.
41. Urbina, J. A.; J. Euk. Microbiol. 2015, 62, 149.
42. Leon, J. S.; Engman, D. M.; Int. J. Parasitol. 2001, 31,
555.
18. Castro, S. L.; Batista, D. G. J.; Batista, M. M.; Batista, W. D. A.;
Souza, E. M.; Menna-Barreto, R. F. S.; Oliveira, G. M.;
Salomão, K.; Silva, C. F.; Silva, P. B.; Nazar, M.; Soeiro, C.;
Mol. Biol. Int. 2011, DOI: 10.4061/2011/306928.
19. Rodrigues, J. H. S.; Ueda-Nakamura, T.; Corrêa, A. G.; Sangi,
D. P.; Nakamura, C. V.; PLoS One 2014, 9, 85706.
20. Martins, T. A. F.; Diniz, L. F.; Nascimento, A. F. S.; Caldas, S.;
Caldas, I. V.; Andrade, I. M.; Ribeiro, I.; Bahia, M. T.; PLoS
One 2015, 9, 4807.
43. Ferreira, D. S.; Esperandim, V. R.; Alonso, M. P.; Kuehn,
T. C. C.; Júnior, J. C. P.; Cunha, W. R.; Silva, M. L. A.;
Albuquerque, S.; Exp. Parasitol. 2013, 134, 455.
44. Lozano, E.; Barrera, P.; Romina, S.; Nieto, I. V.; Tonn, M. C.;
Kemmerling, U.; Mortara, R. A.; Sosa, M. A.; Parasitol. Int.
2012, 61, 628.
21. Castro, J.A.; De Mecca, M. M.; Bartel, L. C.; Hum. Exp. Toxicol.
2006, 25, 471.
22. Sonar, V. P.; Corona, A.; Distinto, S.; Maccioni, E.; Meleddu,
R.; Fois, B.; Floris, C.; Malpure, N.V.;Alcaro, S.; Tramontano,
E.; Cottiglia, F.; Eur. J. Med. Chem. 2017, 130, 248.
23. Silva Júnior, W.; Pinheiro, J.; Moreira, C.; Rüdiger,A.; Barbosa,
E.; Lima, E.; Veiga Júnior, V. F.; Silva Júnior, A.; Aragão, C.;
Lima, A.; J. Therm. Anal. Calorim. 2017, 127, 1757.
24. deAlmeida, P. D. O.; Boleti,A. P.A.; Rüdiger,A. L.; Lourenço,
G. A.; Veiga Junior, V. F.; Lima, E. S.; Yasukawa, K.; Evid.
Based Complement. Alternat. Med. 2015, 2015, 10.
25. Rüdiger, A. L.; Veiga-Junior, V. F.; Chem. Biodiversity 2013,
10, 1142.
45. Lozano, E.; Barrera, P.; Tonn, C.; Nieto, M.; Sartor, T.; Sosa,
M. A.; Parasitol. Int. 2012, 61, 275.
46. Xia, Q.; Zhang, H.; Sun, X.; Zhao, H.; Zhu, L. W. D.;Yang, G.;
Shao, Y.; Zhang, X.; Mao, X.; Zhang, L.; She, G.; Molecules
2014, 19, 17478.
47. Isah, M. B.; Ibrahim, M. A.; Mohammed, A.; Aliyu, A. B.;
Masola, B.; Coetzer, T. H. T.; Parasitol. 2015, 143, 1219; Leite,
J. P.; Oliveira,A. B.; Lombardi,A. J.; Filho, J. D. S.; Chiari, E.;
Biol. Pharm. Bull. 2006, 29, 2307; Ferreira, D. S.; Esperandim,
V. R.; Toldo, M. P.; Saraiva, J.; Cunha, W. R.; Albuquerque, S.;
Parasitol. Res. 2010, 106, 985.

Vol. 28, No. 12, 2017
Bossolani et al.
2489
48. Menna-Barreto, R. F.; Salomão, K.; Dantas, A. P.; Santa-Rita,
R. M.; Soares, M. J.; Barbosa, H. S.; Castro, S. L.; Micron 2009,
40, 157.
Diks, S. H.; Spek, C. A.; Carmen, V. F.; Peppelenbosch, M. P.;
PLoS One 2012, 7, 4536; Schontal,A.A.; Scientifica 2012, DOI:
10.6064/2012/857516; Adade, M. A.; Oliveira, I. R. S.; Pais,
J.A. R.; Souto-Padrón, T.; Toxicon 2013, 69, 227; Meira, C. S.;
Guimarães, E. T.; Bastos, T. M.; Moreira, D. R.; Tomassini,
T. C.; Ribeiro, I. M.; Santos, R. R.; Soares, M. B.; Parasitology
2013, 140, 1811; Veiga-Santos, P.; Reignault, L. C.; Huber, K.;
Bracher, F.; De Souza, W.; De Carvalho, T. M.; Parasitology
2014, 141, 814.
49. Tiuman, T. S.; Ueda-Nakamura, T.; Alonso, A.; Nakamura,
C. V.; BMC Microbiol. 2014, 14, 152; Sen, N.; Das, B. B.;
Ganguly, A.; Mukherjee, T.; Tripathi, G.; Bandyopadhyay, S.;
Rakshit, S.; Sem, T.; Majumder, H. K.; Cell Death Differ. 2004,
11, 924; Mehta,A.; Shaha, C.; J. Biol. Chem. 2004, 279, 11798.
50. Chaabane, W.; User, S. D.; El-Gazzah, M.; Jaksik, R.; Sajjadi,
E.; Rzeszowska-Wolny, J.; Los, M. J.; Arch. Immunol. Ther. Exp.
2013, 61, 43; Queiroz, K. C. S.; Milani, R.; Ruela-de-Sousa,
R. R.; Fuhler, G. M.; Justo, G. Z.; Zambuzzi, W. F.; Duran, N.;
Submitted: March 20, 2017
Published online: June 6, 2017