Differential lethal action of C17:2 and C17:0 anacardic acid derivatives in Trypanosoma cruzi – A mechanistic study

Article abstract of DOI:10.1016/j.bioorg.2020.104068

The n-hexane extract from leaves of Schinus terebinthifolius (Anacardiaceae) induced 100% of death of trypomastigote forms of T. cruzi at 300 μg/mL and was subjected to a bioactivity-guided fractionation to afford a C17:2 derivative of anacardic acid [6-(8′Z,11′Z)-heptadecadienyl-salicylic acid, 1]. Additionally, compound 1 was subjected to hydrogenation procedures to afford a C17:0 derivative (6-heptadecanyl-salicylic acid, 1a). Compounds 1 and 1a were effective in killing trypomastigote forms of T. cruzi with IC₅₀ values of 8.3 and 9.0 μM, respectively, while a related compound, salicylic acid, was inactive. Furthermore, no cytotoxicity was observed for the highest tested concentration (CC₅₀ > 200 μM) for all evaluated compounds. Due to the promising results, the mechanism of parasite death was investigated for compounds 1 and 1a using flow cytometry and spectrofluorimetry. The cell membrane permeability assay with SYTOX Green indicated that compound 1 significantly altered this parameter after 40 min of incubation, while compound 1a caused no alteration. Considering that the hydrogenation rendered a differential cellular target in parasites, additional assays were performed with 1a. Despite no permeabilization of the plasma membrane, compound 1a induced depolarization of the electric potential after two hours of incubation. The mitochondria of the parasite were also affected by compound 1a, with depolarization of the mitochondrial membrane potential, and reduction of reactive oxygen species (ROS) levels. The Ca²⁺ levels were not affected during the time of incubation. Considering that the mitochondrion is a single organelle in Trypanosoma cruzi for ATP generation, compounds affecting the bioenergetic system are of interest for drug discovery against Trypanosomatids.

Full text of DOI:10.1016/j.bioorg.2020.104068

Journal Pre-proofs
Differential lethal action of C17:2 and C17:0 anacardic acid derivatives in
Trypanosoma cruzi - a mechanistic study
Eric Umehara, Thais A. Costa Silva, Viviane M. Mendes, Rafael C.
Guadagnin, Patricia Sartorelli, Andre G. Tempone, João Henrique G. Lago
PII:
S0045-2068(20)31365-1
DOI:
https://doi.org/10.1016/j.bioorg.2020.104068
YBIOO 104068
Reference:
To appear in:
Bioorganic Chemistry
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Revised Date:
Accepted Date:
13 April 2020
27 June 2020
29 June 2020
Please cite this article as: E. Umehara, T.A. Costa Silva, V.M. Mendes, R.C. Guadagnin, P. Sartorelli, A.G.
Tempone, J. Henrique G. Lago, Differential lethal action of C17:2 and C17:0 anacardic acid derivatives in
Trypanosoma cruzi - a mechanistic study, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.
2020.104068
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1
Differential lethal action of C17:2 and C17:0 anacardic acid
derivatives in Trypanosoma cruzi - a mechanistic study
Eric Umeharaa, Thais A. Costa Silvaa, Viviane M. Mendesb,
Rafael C. Guadagninc, Patricia Sartorellic, Andre G. Temponeb,
João Henrique G. Lagoa,*
aCenter of Natural Sciences and Humanities, Federal University of ABC, Santo
Andre, SP, 09210-180, Brazil
bCentre for Parasitology and Mycology, Instituto Adolfo Lutz, São Paulo, SP,
01246-000, Brazil
cInstitute of Environmental, Chemical and Pharmaceutical Sciences, Federal
University of São Paulo, Diadema, SP, 09972-270, Brazil
*corresponding author
E-mail address: joao.lago@ufabc.edu.br (J.H.G. Lago)

2
ABSTRACT
The n-hexane extract from leaves of Schinus terebinthifolius (Anacardiaceae)
induced 100% of death of trypomastigote forms of T. cruzi at 300 g/mL and was
subjected to a bioactivity-guided fractionation to afford a C17:2 derivative of
anacardic acid [6-(8’Z,11’Z)-heptadecadienyl-salicylic acid, 1]. Additionally,
compound 1 was subjected to hydrogenation procedures to afford a C17:0
derivative (6-heptadecanyl-salicylic acid, 1a). Compounds 1 and 1a were effective
in killing trypomastigote forms of T. cruzi with IC50 values of 8.3 and 9.0 μM,
respectively, while a related compound, salicylic acid, was inactive. Furthermore,
no cytotoxicity was observed for the highest tested concentration (CC50 > 200 µM)
for all evaluated compounds. Due to the promising results, the mechanism of
parasite death was investigated for compounds 1 and 1a using flow cytometry and
spectrofluorimetry. The cell membrane permeability assay with SYTOX Green
indicated that compound 1 significantly altered this parameter after 40 min of
incubation, while compound 1a caused no alteration. Considering that the
hydrogenation rendered a differential cellular target in parasites, additional assays
were performed with 1a. Despite no permeabilization of the plasma membrane,
compound 1a induced depolarization of the electric potential after two hours of
incubation. The mitochondria of the parasite were also affected by compound 1a,
with depolarization of the mitochondrial membrane potential, and reduction of
reactive oxygen species (ROS) levels. The Ca2+ levels were not affected during
the time of incubation. Considering that the mitochondrion is a single organelle in
Trypanosoma cruzi for ATP generation, compounds affecting the bioenergetic
system are of interest for drug discovery against Trypanosomatids.

3
Keywords: Schinus terebinthifolius; anacardic acid derivatives; Trypanosoma
cruzi; mitochondrial membrane potential; plasma membrane

4
1. INTRODUCTION
Chagas Disease (CD), also known as American Trypanosomiasis, is
caused by the flagellated protozoan Trypanosoma cruzi. Endemic areas of CD
include 21 countries in Latin America, including Brazil, Mexico, Argentina, and
Bolivia [1]. Due to migratory processes, this problem was also evidenced in Europe
and North America [2]. WHO estimates that about six million people are infected
with T. cruzi and more than 70 million people are at risk of transmission [3]. The
available therapy, limited to nifurtimox and benznidazole, shows severe adverse
side effects [4].
Schinus terebinthifolius (Anacardiaceae) is a plant species used in folk
medicine for treatment of ulcers, breathing problems, diarrhea, arthritis, as well as
an antiseptic and anti-inflammatory remedy [5, 6]. Previous studies reported the
isolation of different bioactive metabolites including antitumoral flavonoids and
antiprotozoal tirucallane triterpenoids [7, 8] from this plant. As part of a continuous
study for discovery of anti-trypanosomal metabolites from S. terebinthifolius, the
present study reports the bioactivity-guided fractionation of bioactive n-hexane
extract from its leaves which afforded a C17:2 derivative of anacardic acid [6-
(8’Z,11’Z)-heptadecadienyl-salicylic acid, 1]. Anacardic acid derivatives inhibited
the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of T.
cruzi, considered a potential target for antitrypanosomal agents [9]. Therefore,
mechanistic studies are important tools in the lead optimization process, proving
information about new biochemical targets for structure-activity relationships and
modification of derivatives [10].

5
Besides the antitrypanosomal activity and mammalian cytotoxicity assays,
we studied the lethal mechanisms of compound 1 and its hydrogenated derivative
(6-heptadecanyl-salicylic acid, 1a) in the parasite, with focus in the plasma
membrane (permeability and electric potential - ΔΨp), mitochondrion (ROS levels,
mitochondrial membrane potential – ΔΨm) and in the calcium levels.
2. MATERIAL AND METHODS
2.1. General experimental procedures
NMR spectra were recorded on a Varian INOVA 500 spectrometer (Palo
Alto, CA, USA), operating at 500 and 125 MHz for 1H and 13C nuclei, respectively.
CDCl3 (Aldrich) was used as solvent and TMS (Aldrich) as the internal standard.
Chemical shifts are reported in  units (ppm) and coupling constants (J) in Hz. High
resolution electrospray ionization mass spectra (HRESIMS) were recorded on a
Bruker Daltonics MicrOTOF-Q IITM ESI-Qq-TOF (negative ionization mode). Low
resolution electron ionization mass spectra (LREIMS) were measured at 70 eV on
a Finnigan-Mat INCOS50 quadrupole spectrometer. Silica gel 60 (Merck, 63–210
mesh) and Sephadex LH-20 (Amersham Biosciences) were used for the column
chromatographic separation, while silica gel 60 PF254 (Merck) was used for
analytical and thin-layer chromatography (TLC) (0.25 mm). Semi-preparative
HPLC chromatography separations were performed on an Ultimate 3000 system
(Dionex, Sunnyvale, CA, USA) equipped with a quaternary pump system, a PDA
detector, and a Phenomenex reversed-phase RP-C18 column (250 × 10.0 mm, 5
μm). Salicylic acid was purchased from Aldrich and purified by recrystallization

6
using H2O. All solvents used for column chromatography were of analytical grade
(CAAL) while those used for HPLC separations were of HPLC grade (Tedia).
2.2. Plant material
S. terebithifolius leaves were collected on October 15th, 2017 at the Instituto
de Botanica, São Paulo, Brazil (registration code at SISGEN A4123E4). The plant
material was identified by Prof. Dr. Maria Claudia Marx Young and a voucher
specimen (SP272591) has been deposited in the Herbarium of Institute of
Biosciences, University of São Paulo, SP, Brazil.
2.3. Extraction and isolation
Fresh leaves of S. terebinthifolius (125 g) were dried at 35C during 120 h
and powdered to afford 38 g of plant material, which was exhaustively extracted
with n-hexane. After removal of the solvent under reduced pressure, 1 g of crude
extract was obtained. Part of this material (500 mg) was submitted to column
chromatography using a silica gel column eluted with increasing amounts of EtOAc
in n-hexane to afford 140 fractions (10 mL each), which were pooled together in
seven groups (A – G) after TLC analysis. After evaluation of activity against
trypomastigote forms of T. cruzi, groups A – D, F and G were inactives, while group
E (20 mg) showed to be composed by bioactive compounds. Thus, part of this
group (18 mg) was purified by semi-preparative RP-HPLC (eluent MeOH:H2O 9:1
+ 0.1% of HOAc, flow rate 2.0 mL/min, detection at  = 235 nm) to afford pure
compound 1 (12.6 mg).

7
6-(8’Z,11’Z)-heptadecadienyl-salicylic acid (1). White amorphous solid. RRt
(HPLC): 13.2 min. HRESIMS [M-H]- m/z 371.2588 (calc. to C24H35O3: 371.2586,
error: 0.54 ppm). 1H NMR (500 MHz, CDCl3), /ppm: 7.30 (dd, J = 8.3 and 7.6 Hz,
H-4), 6.83 (d, J = 8.3 Hz, H-3). 6.74 (d, J = 7.6 Hz, H-5), 5.39 – 5.29 (m, H-8’, H-
9’, H-11’, and H-12’), 2.96 (t, J = 8.0 Hz, H-1’), 2.76 (t, J = 6.5 Hz, H-10’), 2.06 –
2.00 (m, H-7’ and H-3’),1.56 (m, H-2’), 1.25 (br s, H-3’ – H6’ and H-14’ – H-16’),
13
0.88 (t, J = 6.9 Hz, H-17’). C NMR (125 MHz, CDCl3), /ppm: 175.2 (COOH),
163.2 (C-2), 147.4 (C-6), 134.8 (C-4), 130.2 (C-8’), 130.1 (C-9’), 127.9 (C-11’ and
C-12’), 122.5 (C-5), 115.6 (C-3), 111.0 (C-1), 36.3 (C-1’), 31.9 (C-2’), 31.5 (C-3’),
29.7 – 29.4 (C-4’ – C-6’ and C-13’ – C-15’), 27.2 (C-7’), 25.6 (C-10’), 22.5 (C-16’),
14.0 (C-17’).
2.4. Hydrogenation of compound 1
In a high-pressure reactor (stainless steel) compound 1 (5.0 mg) and Ni-
Raney catalyst (10.0 mg) was added. After addition of H2 (18 atm), the reaction
mixture was stirred for 3 h at 120 °C. Thus, the crude product was dissolved in
EtOAc and the catalyst removed by filtration on Celite. After evaporation of the
solvent under reduced pressure, 3.5 mg of compound 1a were obtained.
6-Heptadecanyl-salicylic acid (1a). White amorphous solid. HRESIMS [M-H]- m/z
1
375.2919 (calc. to C24H39O3: 375.2899, error: 5.3 ppm). H NMR (500 MHz,
CDCl3), /ppm: 7.30 (dd, J = 8.3 and 7.6 Hz, H-4), 6.83 (d, J = 8.3 Hz, H-3). 6.74

8
(d, J = 7.6 Hz, H-5), 2.96 (t, J = 8.0 Hz, H-1’), 1.56 (m, H-2’), 1.25 (br s, H-3’ – H-
16’), 0.88 (t, J = 6.9 Hz, H-17’). 13C NMR (125 MHz, CDCl3), /ppm: 175.2 (COOH),
163.2 (C-2), 147.4 (C-6), 134.8 (C-4), 122.5 (C-5), 115.6 (C-3), 111.0 (C-1), 36.3
(C-1’), 31.9 (C-2’), 31.5 (C-3’), 29.7 – 29.4 (C-4’ – C-15’), 22.5 (C-16’), 14.0 (C-
17’).
2.5. Experimental animal models
Female and male BALB/c mice were obtained by the animal breeding facility
at the Instituto Adolfo Lutz, São Paulo State, Brazil. The animals were maintained
in sterilized boxes with absorbent material under a controlled environment and
received water and food ad libitum. Female BALB/c mice were used to obtain
peritoneal macrophages, and male for maintenance of the T. cruzi strain. Animal
procedures were performed with the approval of the Research Ethics Commission
(CTC 2017 / 72J), in agreement with the Guide for the Care and Use of Laboratory
Animals from the National Academy of Sciences.
2.6. Parasites and mammalian cell maintenance
T. cruzi trypomastigotes (Y strain) were maintained routinely in Rhesus
monkey kidney cells (LLC-MK2 – ATCC CCL 7), using RPMI-1640 medium
supplemented with 2% FBS at 37 ºC in a 5% CO2-humidified incubator.
Alternatively, male BALB/c mice were infected with trypomastigotes to obtain blood
derived parasites. The murine conjunctive cells (NCTC clone 929, ATCC) were
maintained in RPMI-1640 medium supplemented with 10% FBS at 37 ºC in a 5%

9
CO2 – humidified incubator. Peritoneal macrophages were obtained from BALB/c
mice by washing them with RPMI-1640 medium supplemented with 10% FBS and
were maintained at 37 ºC in a 5% CO2-humidified incubator.
2.7. Determination of the antitrypanosomal activity – bioactivity-guided
fractionation
Trypomastigotes were counted in a Neubauer hemocytometer and seeded
at 1 × 106 cells per well in 96-well microplates. The crude n-hexane extract from
the leaves of S. terebinthifolius and groups A – G (see experimental part) were
added at 300 μg/mL for 24 h at 37°C in a 5% CO2-humidified incubator. The
trypomastigote viability (in percentage) was determined by visual reading under
light inverted microscope (Nikon). The results were expressed as follow: a) 100%
of parasite death – excellent activity, b) 50% of parasite death – moderate activity,
and c) 0% of parasite death – inactive material.
2.8. Determination of 50% inhibitory concentration (IC50) against T. cruzi
The activity of compounds 1, 1a and salicylic acid was evaluated in vitro
against trypomastigote forms of T. cruzi, obtained from previously infected LLC-
MK2 cells. The parasites were counted in a hemocytometer, seeded at 1 x 106
cells/well (96-well plates) and incubated with the compounds (200 – 1.56 µM)
during 24 h at 37 ºC in a 5% CO2-humidified incubator, using benznidazole as the
standard drug. A negative control was included in the experiment (compound plus
medium). Trypomastigote viability was determined by the resazurin (Sigma) assay

10
and the IC50 values were calculated as described in the literature [11]. The optical
density was determined using a FilterMax F5 (Molecular Devices – San Jose –
California - USA) at 570 nm.
2.9. Determination of cytotoxic concentration (CC50) against mammalian
cells
The cytotoxicity assay of the compounds 1, 1a and salicylic acid was
performed against NCTC cells-clone 929. The cells (6 x 104 cells/well) were
seeded and incubated with compounds (200 – 1.56 µM) for 48 h at 37 ºC in a 5%
CO2 incubator. A blanked sample was included in the experiment (compound plus
medium). The 50% cytotoxic concentration (CC50) was determined by the MTT
assay [12]. The optical density was determined using a FilterMax F5 (Molecular
Devices – San Jose – California - USA) at 570 nm. Selectivity index (SI) values
were calculated for trypomastigotes using the equation: CC50 against NCTC
cells/IC50 against parasites forms.
2.10. Evaluation of T. cruzi cell membrane alterations caused by compounds
1 and 1a
2.10.1. Evaluation of cell membrane permeability
The action of compounds 1 and 1a in the cell membrane permeability were
evaluated for T. cruzi trypomastigotes (2 x 106 cells/well), obtained as described
above. The parasites were washed and incubated in the dark with 1 μM SYTOX
Green probe (Molecular Probes) in HANKS' balanced salts solution (HBSS;

11
Sigma-Aldrich) supplemented with 10 mM D-glucose (HBSS+Glu) [13]. Tested
compounds were added (t = 0 min) at IC50 concentration and the fluorescence
levels were measured every 20 min for up to 120 min. The maximum
permeabilization was obtained with the addition of 0.5% Triton X-100.
Fluorescence intensity was determined using a fluorometric microplate reader
(FilterMax F5 Multi-Mode Microplate Reader-Molecular Devices) with excitation
and emission wavelengths of 485 and 520 nm, respectively. Three independent
experiments were performed. Samples were tested in triplicate.
2.10.2. Evaluation of plasma membrane electric potential (ΔΨp)
Determination of the ΔΨp alteration was performed by measuring the
increase in absorbance of bis-(1,3-diethylthiobarbituric acid) trimethine oxonol
[DiSBAC2(3)] (Invitrogen) as described in literature [11]. Parasites (2 x 106
cells/well) were washed with HBSS+Glc as previously described, treated with
compound 1a (IC50 value) and gramicidin D (0.5 mg/mL) (Sigma-Aldrich) as a
positive control of the assay and incubated at 37°C for 120 min. Untreated
trypomastigotes were used as negative control. In sequence, 0.2 µM of
DiSBAC2(3) was added and incubated at 37°C for 10 min. The samples were
analyzed in an Attune NxT Flow Cytometer (Thermo Fisher Scientific). Three
independent experiments were performed, each one with duplicate samples.
2.11. Evaluation of T. cruzi mitochondrial alterations
2.11.1. Evaluation of the mitochondrial membrane potential (ΔΨm)

12
Mitochondrial membrane potentials (ΔΨm) were determined by flow
cytometry (Attune NxT flow cytometer – ThermoFisher) using the cationic lipophilic
dye 5,5´,6,6´-tetrachloro-1,1´,3,3´-tetraethylbenzimidazole carbocyanide iodide
(JC-1, ThermoFisher) and the ratio between red/green fluorescence intensities
(BL-2/BL-1; 590 nm/530 nm) was calculated [14, 15]. Briefly, T. cruzi
trypomastigotes (2 x 106 cells/well) were treated with compound 1a (at IC50 value)
and 100 μM of the mitochondrial uncoupled carbonyl cyanide 3-
chlorophenylhydrazone (CCCP, Sigma) (positive control) for 2 and 4 h of
incubation at 37ºC. Next, the parasites were washed in HBSS+Glc, resuspended
with JC-1 dye (10 μM) and incubated in the dark for 10 min at 37ºC. After that, the
samples were washed in HBSS+Glc and analyzed using an Attune NxT Flow
Cytometer (Thermo Fisher Scientific). Three independent experiments were
performed, each one with duplicate samples. Non-treated parasites were used as
negative control of the assay.
2.11.2. Evaluation of ROS levels
ROS levels were evaluated in T. cruzi trypomastigotes treated with
compound 1a. The parasites (2 x 106 cells/well) were washed with HBSS + Glu as
described above and treated with compound 1a (at IC50 value) for 120 min at 37
ºC in a 5% CO2-humidified incubator. After that, the cell-permeant 2',7'-
dichlorodihydrofluorescein diacetate H2DCFDA (20 μM) was added and the
trypomastigotes incubated for 15 min. Fluorescence intensity was evaluated using
a fluorometric microplate reader (FilterMax F5 Multi-Mode Microplate Reader-
Molecular Devices) with excitation and emission wavelengths of 485 and 520 nm,

13
respectively [16]. Azide was used as a positive control and untreated
trypomastigotes as the negative control. Three independent experiments were
performed, each one with duplicate samples.
2.12. Evaluation of Intracellular calcium (Ca2+) levels
Cytosolic Ca2+ levels were analyzed in T. cruzi trypomastigotes. Previously,
the parasites (2 x 106 cells/well) were pretreated with 5 μM of Fura-2 AM (Molecular
Probes) in PBS, for 40 min at 37°C in the dark. Next, T. cruzi trypomastigotes were
washed and treated with compound 1a (at IC50 value). The fluorescence levels
were measured at 20, 60 and 120 min using a fluorometric microplate reader
(FilterMax F5 Multi-Mode, Molecular Devices) with excitation and emission
wavelengths of 360 and 500 nm, respectively. Maximum levels of calcium were
obtained using 0.5% Triton X-100 and untreated parasites were used as negative
control [17].
2.13. Statistical analysis
IC50 and CC50
represent the mean of three independent
values
representative assays tested in duplicate and were calculated using a sigmoid
dose-response curves in Graph–Pad Prism 5.0 software (GraphPad Prism
software 6.0, San Diego, CA, USA). Mechanism of action results were analyzed
using ANOVA test turkey for significance (p < 0.05).
3. RESULTS AND DISCUSSION

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The n-hexane extract from leaves of S. terebinthifolius (Anacardiaceae)
exhibited activity against T. cruzi trypomastigotes (100% of death at 300 g/mL).
This extract was subjected to a bioactivity-guided procedure using silica gel to
afford seven groups (A – G). After evaluation of antitrypanosomal activity, group G
resulted in higher potential against T. cruzi trypomastigotes (100% of death at 300
g/mL) and was purified by semi-preparative RP-HPLC to afford pure compound
1. HRESIMS analysis indicated deprotonated [M-H]- ion peak at m/z 371.2588,
corresponding to molecular formula C24H36O3. 1H NMR spectrum showed signals
attributed to an aromatic ring at  7.30 (dd, J = 8.3 and 7.6 Hz, H-4), 6.83 (d, J =
8.3 Hz, H-3), and 6.74 (d, J = 7.6 Hz, H-5). Additionally, the presence of an
unsaturated side chain was proposed due to the signals at range  5.39 – 5.29 (m,
H-8’, H-9’, H-11’ and H-12’), 2.96 (t, J = 8.0 Hz, H-1’), 1.56 (m, H-2’), 2.06 – 2.00
(m, H-7’ and H-3’), 1.25 (br s, H-3’ – H-6’ and H-14’ – H16’), and at  0.88 (t, J =
6.9 Hz, H-17’). 13C and DEPT NMR spectra of both compounds showed the peaks
assigned to a carbonyl carbon at  175.2, to an aromatic ring at  163.2 – 111.0
(C-1 to C-6), to a long side chain at  29.7 – 29.4 (C-4’ – C-6’ and C-13’ – C-15’)
and a methyl group at  14.0 (C-17’). Additionally, signals of sp2 carbons were
observed at  130.2, 130.1 and 127.9, attributed to the carbons of two non-
conjugated cis double bond. The positioning of double bonds at C-8’ and C-11’
was determined by analysis of LREIMS spectrum of obtained product after reaction
using Me2S2 (m/z 295, 355, 369, 429, and 503 fragments). Finally, comparison of
NMR data with those reported in literature [18], enabled the identification of 6-
(8’Z,11’Z)-heptadecadienyl-salicylic acid, a C17:2 derivative of anacardic acid, as
showed in Figure 1. Previous studies have described the occurrence of this

15
compound in Pentaspadon officianalis [19], Spondias mombin [20] and Ginkgo
biloba [21].
Compound 1 was effective in killing the trypomastigotes of T. cruzi with an
IC50 value of 8.3 μM and no cytotoxicity at the highest tested concentration (> 200
µM), as shown in Table 1. Considering the selectivity index (SI), the ratio between
activity against T. cruzi and mammalian cytotoxicity in NCTC cells, it was possible
to obtain a value of >24. As previously described [22], a chemically related C15:2
anacardic acid containing the double bonds at the same positions (C-8’ and C-11’)
and configuration (Z) of C17:2 derivative isolated in the present work was
completely inactive against trypomastigote forms of T. cruzi. This result suggests
that the extension of side chain plays an important role in the antitrypomastigote
potential.
Due to this promising result with the extracellular form of the parasite, an
important parasite stage in the acute phase of the disease [23], we decided to
evaluate the lethal action of the compound using the probe SYTOX Green. This
probe binds in the nuclear DNA, and is impermeant to live cells, making it a useful
indicator of cells integrity within a population [13] Compound 1 induced an
.
increased fluorescence level in treated parasites, as a result of the alteration in the
cell membrane permeability after 40 min of incubation (Figure 2). Some pore-
forming compounds can alter the plasma membrane permeability, causing osmotic
imbalance and consequently, altering the homeostasis of the cell [11]. Compound
1 altered the permeability of T. cruzi membrane in a short period of incubation and
consequently, the intracellular organelles were presumable harmed, making
additional studies of mechanism of action with compound 1 needless.

16
Additionally, compound 1 was subjected to catalytic hydrogenation
1
procedure to afford compound 1a. As evidenced by H NMR, the spectrum of
compound 1a presented similarities with that recorded to compound 1, except for
the absence of signals at  5.39 – 5.29, assigned to hydrogens H-8’, H-9’, H-11’
and H-12’, linked to sp2 carbons. Similarly, the 13C NMR and DEPT spectra showed
aromatic carbons at 111.0 – 163.2 (C-1 to C-6), a carbonyl group at  175.2,
methylene carbons of a saturated side chain at  31.9 – 22.5 (C-2’ – C-16’), and a
methyl group at  14.0 (C-17’). Finally, HRESIMS analysis of compound 1a
indicated the deprotonated [M–H]- ion peak at m/z 375.2919, corresponding to
molecular formula C24H40O3, confirming the structure of 6-(8’Z,11’Z)-heptadecanyl-
salicylic acid (Figure 1).
As showed in Table 1, compound 1a was also effective in killing the
trypomastigotes with an IC50 value of 9.0 μM. This derivative also showed no
cytotoxicity to mammalian cells at the highest tested concentration (CC50 > 200
μM). As observed, the IC50 values of both compounds 1 and 1a were very similar
and both showed SI values higher than 20. Comparing the structures of compound
1/1a and their activities against trypomastigote forms of T. cruzi, it is possible to
infer that the presence of alkyl chain is crucial for the anti-trypanosomal activity,
which was proven by the lack of activity of salicylic acid. These results could be
associated, at least in part, by the similar lipophilicity of related compounds 1 (log
P = 6.59) and 1a (log P = 7.11) in comparison to that of salicylic acid (log P = 1.24)
[24].
Based on these results, the mechanism of parasite death caused by
compound 1a was also investigated. As observed in Figure 3, the process of
hydrogenation interfered with the action on the plasma membrane of the parasite.

17
Differently from the results of compound 1, the hydrogenated derivative 1a showed
no alteration of the membrane permeability, with similar fluorescence levels to
those of untreated cells. Although compound 1a induced no permeabilization, this
derivative caused depolarization of the plasma membrane electric potential after
two hours of incubation (Figure 4), as detected with the DiSBAC2(3) probe. This
probe allows detection of subtle membrane changes related to ionic variations.
DiSBAC2(3) enters depolarized cells and bind to intracellular proteins, generating
enhancement of the fluorescence levels [25]. These changes in the plasma
membrane can alter the transportation of molecules to the cell, metabolites,
nutrients, ions and the pH controls [26, 27]. Previous studies [28] showed that
anacardic acid mediated alterations in membrane potential and pH gradient of the
liposomal membranes, corroborating our results. The alteration in the plasma
membrane potential caused by compound 1a was followed by the depolarization
of the mitochondrion after four hours of incubation as shown in Figure 5. Shorter
incubation time (two hours) was not enough to depolarize the mitochondrion (data
not shown).
In Trypanosomatids, mitochondrion is a single organelle responsible for the
ATP and ROS generation and consequently, energy production for the parasite,
thus being an important therapeutic target [29 – 32]. Although no statistical
significance (p < 0.05) was observed for the ROS levels between 1a-treated and
untreated parasites, compound 1a induced minor alterations of ROS (Figure 6) as
a consequence of the depolarization of the mitochondria (Figure 5) an important
site of physiological generation of ROS [29]. Similarly, the Ca2+ levels were not
altered after incubation time with compound 1a, as showed in Figure 7.

18
4. CONCLUSION
In the present study, 6-(8’Z,11’Z)-heptadecadienyl-salicylic acid (1) was
isolated from S. terebinthifolius and exhibited in vitro activity against
trypomastigote forms of T. cruzi, which could be caused by the alteration of the
plasma membrane permeability. Aiming to evaluate the effect caused by the
presence of double bonds at the side chain, compound 1 was subjected to a
catalytic hydrogenation procedure to obtain the derivative 1a (6-heptadecanyl-
salicylic acid), which showed an anti-trypanosomal potential similar to the natural
product 1. However, the investigation of the mechanism of action of the
hydrogenated compound 1a shown a different lethal effect, affecting the
mitochondrion of the parasite instead of the plasma membrane, targeted by
compound 1. Therefore, the obtained results suggest that these differences might
be associated to the presence of the lipophilic side chain (unsaturated or saturated
derivatives), since salicylic acid showed no in vitro activity against trypomastigotes
of T. cruzi.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgments
Authors are thankful for financial support and fellowships provided by
CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior), CNPq
(Conselho Nacional de Desenvolvimento Científico e Tecnológico) and FAPESP

19
(Fundação de Amparo à Pesquisa do Estado de São Paulo – projects 2018/07885-
1 and 2018/10279-6). This publication is part of the activities of the Research
Network Natural Products against Neglected Diseases (ResNetNPND):
http://www.uni-muenster.de/ResNetNPND/.
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22
Table 1. Anti-T. cruzi activity (trypomastigote forms) and mammalian cytotoxicity
(NCTC cells) of 6-(8’Z,11’Z)-heptadecadienyl-salicylic acid (1), isolated from S.
terebithifolius, 6-heptadecanyl-salicylic acid (1a) derivative, salicylic acid and
positive control benznidazole.
IC50 (M) ± SD
8.3 ± 0.7
CC50 (M)
> 200
SI
1
> 24
> 22
1a
9.0 ± 1.2
> 200
salicylic acid
benznidazole
NA
> 200
> 200
-
> 36
5.5 ± 2.2
± SD - standard deviation; IC50 - 50% effective concentration; CC50- 50% cytotoxic
concentration; SI- selectivity index.

23
11'
8'
12'
9'
1 R =
17'
1'
10'
OH
O
R
2
1
3
4
OH
1a R =
6
5
salicilic acid R = H
Figure 1. Chemical structures of compound 1, isolated from S. terebinthifolius, its hydrogenated derivative 1a and salicylic acid.

24
150
100
50
1
C +
C -
***
0
0
0
0
4
0
0
0
0
0
2
6
8
0
2
3
1
1
1
Time (min)
-50
Figure 2. Evaluation of plasma membrane permeability with the probe SYTOX
green in T. cruzi trypomastigotes treated with compound 1 (IC50 value) at 120
min. Positive control (C +)of the assay were trypomastigotes treated with TX-
100 (0.5%) (maximum permeabilization – positive control) and untreated
parasites, negative control (C-), were used as negative control. To show the
viability of the cells were add to each sample TX-100 at the time 130 min. A
representative experiment is shown. *** p < 0.0001

25
150
100
50
1a
C +
C -
0
0
0
0
0
0
0
0
0
2
4
6
8
0
2
3
1
1
1
Time (min)
-50
Figure 3. Evaluation of plasma membrane permeability with the probe SYTOX
green in T. cruzi trypomastigotes treated with compound 1a (IC50 value) at 120
min. Positive control (C+) of the assay were trypomastigotes treated with TX-
100 (0.5%) (maximum permeabilization) and untreated parasites were the
negative control (C-). To show the viability of the cells were add to each sample
TX-100 at the time 130 min. A representative experiment is shown.

26
6000
4000
2000
0
**
-
a
1
+
C
C
Figure 4. Plasma membrane electric potential (ΔΨp) analysed with DISBAC2 (3)
probe in T. cruzi trypomastigotes treated for two hours with compound 1a.
Controls of the assay were trypomastigotes treated with gramicidin (0.5 µg/mL),
positive control (C+) and untreated parasites, negative control (C-).
Fluorescence was quantified by calculating the mean percentages of untreated
(0%) and gramicidin-treated (100%) parasites. ** p < 0.001. A representative
experiment is shown.

27
2.0
1.5
1.0
0.5
0.0
***
-
a
1
+
C
C
Figure 5. Mitochondrial membrane potential evaluated in T. cruzi
trypomastigotes treated with compound 1a for four hours labeled with JC-1
probe (0.2 μM). The fluorescence was measured in a flow cytometer (ATTUNE)
after 4 hours of incubation with the compound. Negative control (C -) - minimum
(untreated) and positive control (C +) - maximum (CCCP – 100 µg/mL treated)
alteration in the mitochondrial membrane potential (depolarization) were
obtained. Fluorescence was quantified by calculating the ratio between the
channels BL2/BL1. *** p < 0.05. A representative experiment is shown.

28
150
100
50
0
a
+
-
1
C
C
Figure 6. ROS levels in T. cruzi trypomastigotes treated with compound 1a for
2 hours and labeled with H2DCf- DA. The fluorescence intensity was detected
using a fluorometric microplate reader (FilterMax F5 Multi- Mode Microplate
Reader) with excitation and emission wavelengths of 485 and 520 nm,
respectively. Azide (10 µM) was used as positive control (C+) and untreated
parasites were used as negative control (C-).

29
200
150
100
50
0
20
60
120
0
-
a
+
1
C
C
Figure 7. Ca2+ levels evaluated in T. cruzi trypomastigotes treated with
compound 1a (IC50 value). Fura-2 AM probe fluorescence was measured
spectrofluorimetrically (excitation 360nm and emission 500nm) after 20, 60, 120
min of incubation. Untreated trypomastigotes, negative control (C-) and treated
with TX-100 (0.5%), positive control (C+) were used to obtain minimal and
maximal fluorescence, respectively. Fluorescence is reported as normalized
data for untreated trypomastigotes (100%).