Caatinga plants: Natural and semi-synthetic compounds potentially active against Trichomonas vaginalis

Article abstract of DOI:10.1016/j.bmcl.2016.03.061

Trichomonas vaginalis causes trichomoniasis; the most common but overlooked non-viral sexually transmitted disease worldwide. The treatment is based at 5′-nitroimidazoles, however, failure are related to resistance of T. vaginalis to chemotherapy. Caatinga is a uniquely Brazilian region representing a biome with type desert vegetation and plants present diverse biological activity, however, with few studies. The aim of this study was to investigate the activity against T. vaginalis of different plants from Caatinga and identify the compounds responsible by the activity. A bioguided fractionation of Manilkara rufula was performed and four major compounds were identified: caproate of α-amyrin (1b), acetate of β-amyrin (2a), caproate of β-amyrin (2b), and acetate of lupeol (3a). In addition, six derivatives of α-amyrin (1), β-amyrin (2) and lupeol (3) were synthesized and tested against the parasite. Ursolic acid (5) reduced about 98% of parasite viability after 2 h of incubation and drastic ultrastructural alterations were observed by scanning electron microscopy. Moreover, 5 presented high cytotoxicity to HMVII and HeLa cell line and low cytotoxicity against Vero line at 50 μM (MIC against the parasite). Metronidazole effect against T. vaginalis resistant isolate was improved when in association with 5.

Full text of DOI:10.1016/j.bmcl.2016.03.061

Accepted Manuscript
Caatinga plants: Natural and semi-synthetic compounds potentially active
against Trichomonas vaginalis
Patrícia de Brum Vieira, Nícolas Luiz Feijó Silva, Gloria Narjara Santos da
Silva, Denise Brentan Silva, Norberto Peporine Lopes, Simone Cristina Baggio
Gnoatto, Márcia Vanusa da Silva, Alexandre José Macedo, Jaume Bastida,
Tiana Tasca
PII:
S0960-894X(16)30281-5
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.03.061
BMCL 23704
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
12 November 2015
14 March 2016
15 March 2016
Please cite this article as: Vieira, P.d.B., Silva, N.L.F., Silva, G.N.S., Silva, D.B., Lopes, N.P., Gnoatto, S.C.B.,
Silva, M.V.d., Macedo, A.J., Bastida, J., Tasca, T., Caatinga plants: Natural and semi-synthetic compounds
potentially active against Trichomonas vaginalis, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://
dx.doi.org/10.1016/j.bmcl.2016.03.061
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1
Caatinga plants: natural and semi-synthetic compounds potentially active
against Trichomonas vaginalis
Patrícia de Brum Vieiraa, Nícolas Luiz Feijó Silvaa, Gloria Narjara Santos da Silvab,
Denise Brentan Silvac,d, Norberto Peporine Lopesd, Simone Cristina Baggio Gnoattob,
Márcia Vanusa da Silvae, Alexandre José Macedof,g, Jaume Bastidah, Tiana Tascaa,g*
aLaboratório de Pesquisa em Parasitologia, Faculdade de Farmácia, Universidade
Federal do Rio Grande do Sul, Porto Alegre, RS, 90610-000, Brasil; Emails:
patriciasbrum@yahoo.com.br; nicolaslfsilva@gmail.com; tiana.tasca@ufrgs.br.
bLaboratório de Fitoquímica e Síntese Orgânica, Faculdade de Farmácia, Universidade
Federal do Rio Grande do Sul, Porto Alegre, RS, 90610-000, Brasil; Emails:
narjarags@gmail.com; simone.gnoatto@ufrgs.br.
cLaboratório de Produtos Naturais e Espectrometria de Massas (LAPNEM),
Universidade Federal de Mato Grosso do Sul (UFMS), Campo Grande, MS, 79090-
900, Brasil; Email: denise.brentan@ufms.com.
dNúcleo de Pesquisas em Produtos Naturais e Sintéticos, Faculdade de Ciências
Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP,
14040-903, Brasil; Emails: npelopes@fcfrp.usp.br .
eCentro de Ciências Biológicas e Departamento de Bioquímica, Universidade Federal
de Pernambuco, Recife, PE, 50670-901, Brasil; Email: Márcia.vanusa@ufpe.br.
fFaculdade de Farmácia e Centro de Biotecnologia, Universidade Federal do Rio
Grande do Sul, Porto Alegre, RS, 90610-000, Brasil; Email:
Alexandre.jose.macedo@gmai.com.
gInstituto Nacional do Semi-Árido (INSA), Núcleo de Biprospecção da Caatinga
(NBioCaat), Campina Grande, Pernambuco, Brasil.

2
hDepartment of Natural Products, Plant Biology and Soil Science, Faculty of Pharmacy,
Barcelona, Catalonia, 08028, Spain; Email: jaumebastida@ub.edu.
*Author to whom correspondence should be addressed, email tiana.tasca@ufrgs.br;
Phone: +55 51 3308 5325; Fax: +55 51 3308 5437.
Abstract
Trichomonas vaginalis causes trichomoniasis; the most common but overlooked non-
viral sexually transmitted disease worldwide. The treatment is based at 5’-
nitroimidazoles, however, failure are related to resistance of T. vaginalis to
chemotherapy. Caatinga is a uniquely Brazilian region representing a biome with type
desert vegetation and plants present diverse biological activity, however, with few
studies. The aim of this study was to investigate the activity against T. vaginalis of
different plants from Caatinga and identify the compounds responsible by the activity. A
bioguided fractionation of M. rufula was performed and four major compounds were
identified: caproate of α-amyrin (1b), acetate of β-amyrin (2a), caproate of β-amyrin
(2b), and acetate of lupeol (3a). In addition, six derivatives of α-amyrin (1), β-amyrin (2)
and lupeol (3) were synthesized and tested against the parasite. Ursolic acid (5)
reduced about 98% of parasite viability after 2h of incubation and drastic ultrastructural
alterations were observed by scanning electron microscopy. Moreover, 5 presented
high cytotoxicity to HMVII and HeLa cell line and low cytotoxicity against Vero line at 50
µM (MIC against the parasite). Metronidazole effect against T. vaginalis resistant
isolate was improved when in association with 5.
Keywords: Trichomonas vaginalis; Caatinga plants; triterpenes; ursolic acid;
cytotoxicity.

3
Abbreviations: 1H or 13C NMR, proton or carbon nuclear resonance magnetic; CDCl3,
deuterated chloroform; TMS, tetramethylsilane; EI, electron ionization; MS, mass
spectroscopy; GC, gas chromatography; RI, retention index; IR, infrared spctra; IPA,
Instituto Agronômico de Pernambuco; VLC, vaccum liquid chromatography; EtOAc,
ethyl acetate; MeOH, methanol; TLC, thin liquid chromatography; DMAP, 4-
dimethylaminopyridine; (1), α-amyrin; (2), β-amyrin; (3), lupeol; (1a), acetate of α-
amyrin; (1b), caproate of α-amyrin; (2a), acetate of β-amyrin; (2b), caproate of β-
amyrin; (3a), acetate of lupeol; (3b), caproate of lupeol; DMSO, dimethyl sulfoxide;
TYM, trypticase-yeast maltose medium; SEM, scanning electron microscopy; MTT, 3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
Trichomonas vaginalis is a flagellated protozoan that causes trichomoniasis, the
number one non-viral sexually transmitted disease worldwide.1 The parasite colonizes
the human tract and causes vaginitis in women and urethritis in men. Serious
consequences are related to trichomoniasis, including cervical and prostate cancers
increased risks,2 adverse pregnancy outcomes and an increased susceptibility to
human immunodeficiency virus acquisition.3 Metronidazole, an antibiotic of the
nitroimidazole class, is the first-line treatment for trichomoniasis. Nevertheless, T.
vaginalis resistance to metronidazole is rising4 and requires new strategies to treat this
neglected infection. Natural products play an important role in the search for new active
drugs.
Caatinga is a semi-arid region of Brazil Northeastern with a great diversity of
plants and animals, however it is yet poorly studied and its potential is underestimated.
The plants Croton nummularius Baill., Senna lechriosperma H.S.Irwin & Barneby, and
Manilkara rufula (Miq.) H.J. Lam, Blumea are native and endemic of Northeast of
Brazil. Several biological activities have been attributed to plants of Croton, Senna and
Manilkara genera, such as anti-inflammatory and analgesic,5 anti-microbial, anti-

4
parasitic, anti-HIV and antitumoral.6 Moreover, anti-T. vaginalis activity of M. dissecta
crude extract was reported.7 The necessity of studies in this area is requiring, because
there is a scarcity of studies about Caatinga plants.
The miscellaneous of activities related to Manilkara sp. may be attributed to the
presence of a range of secondary metabolic in these species, such as saponins,
flavonoids, tannins, and triterpenes.8 These compounds present a variety of biological
activities, such as anti-inflammatory, antiviral, antibacterial, antifungal, and antitumor.9
Moreover, triterpenes showed potential activity against protozoa, such as Leishmania,
Trypanosoma, Plasmodium and Trichomonas vaginalis.10,11 Triterpenes are important
candidates for designing lead compounds for new active agents development, as
showed by Guimarães et al.12 Moreover, structural modifications approaches help to
improve biological activities against etiological agents and, nowadays, have showed an
increasing interest.
In order to investigate the potential against T. vaginalis, leaves and branches of
C. nummularius, M. rufula and S. lechriosperma were collected at Parque Nacional do
Catimbau (PARNA do Catimbau), Pernambuco, Brazil (837’S 3708’W) in February
2012 with authorization of SISBIO 16.806 Instituto Chico Mendes de Conservação da
Biodiversidade (ICMBio). Voucher specimens have been deposited in the Herbarium of
the Instituto Agronômico de Pernambuco (IPA) as C. nummularius (IPA 86868), M.
rufula (IPA 84889) and S. lechriosperma (IPA 84959). The dried leaves and branches of
the three plants were powered and extracted with ethanol:water. Polar components
were removed by liquid-liquid extraction with acetate and n-butanol. Non-polar fraction
was subjected to a sequential vacuum liquid chromatography (VLC).13
In addition, six derivatives of α-amyrin (1), β-amyrin (2), and lupeol (3) were
synthesized14 (Figure 1A) and the compounds identification was performed by IR, 1H
and 13C NMR, and HR-ESI-MS15-20. The data were compared with previous study.21

5
R2
R3
R4
(A)
R1
HO
HO
3
1: R1 = Me, R2 = H, R3 = Me, R4 = Me
2: R1 = H, R2 = Me, R3 = Me, R4 = Me
4: R1 = H, R2 = Me, R3 = Me, R4 = COOH
5: R1 = Me, R2 = H, R3 = Me, R4 = COOH
(B)
i
O
R =
(1a)
(1b)
HO
HO
HO
RO
R =
1
O
O
i
(2a)
R =
(2b)
R =
RO
O
2
i
O
(3a)
(3b)
R =
RO
R =
3
O
Reagents and conditions: (i) dichloromethane, commercial anhydride (acetic or caproic
anhydride), DMAP, reaction time of 3 h
Figure 1.

6
The parasites used in this study were Trichomonas vaginalis 30236
metronidazole-sensitive isolate from ATCC and TV-LACM2R metronidazole-resistant
isolate (fresh clinical isolates from female patients from Laboratório de Análises
Clínicas e Toxicológicas, Faculdade de Farmácia, UFRGS, Brazil; project with ethical
approval by UFRGS Ethical Committee, number 18923). Trophozoites were cultured as
previously described by Diamond.22 Anti-T. vaginalis assays were performed.23
Moreover, the minimum inhibitory concentration (MIC) of ursolic acid (5) was
determined by eightfold dilution, ranging of 100 – 0.78 µM. After 24 h of incubation, the
viability was evaluated by counting in hemocytometer with exclusion dye trypan blue
0.2% and comparing with parasites untreated. After MIC determination, parasites were
inoculated to fresh TYM medium and MIC was confirmed. The effect of 5 on parasite
growth and viability was performed.24 In addition, the viability of the parasites was
assessed using fluorescein diacetate (FDA) (Sigma, USA) and propidium iodide (PI)
(Sigma, USA). Parasites were treated or not with 5 at 50 μM for 0, 2, 4, 6, and 12 h and
stained with 10 μg/mL of FDA and 10 μg/mL of PI for 20 min for fluorescence analysis.
In order to verify parasite ultrastructure changes induced by 5, scanning
electron microscopy (SEM) was performed as described in previous study with T.
vaginalis.25 Also, the cytotoxicity of UA to HMVII (vaginal epithelial melanoma cell),
HeLa (cervical cancer cell), and VERO (kidney epithelial cells) lines was performed.26
The association of 5 and metronidazole was performed against TV-LACM2R isolate at
6.25 or 12.5 μM of 5 in association or not with 15 and 73 μM metronidazole. All the
experiments were performed in triplicate and with at least three independent cultures (n
= 3). Student’s t test was chosen for comparisons between two groups. The results are
expressed as the mean ± SEM of at least three individual experiments. P < 0.05 was
considered a statistically significant difference. Analyses were performed using
Statistical Package for the Social Sciences (SPSS) software v.14.

7
Ethanolic extracts of leaves and branches of C. nummularius and S.
lechriosperma showed a slight reduction (about 40%) of T. vaginalis viability at 1.0
mg/mL (Figure 2A). Studies demonstrated that different species of Croton and Senna
present a range of biological activities,5 however, we did not find reports about anti-T.
vaginalis activity of Senna sp. nor Croton sp.
(A)
100
80
60
40
20
0
CTL DMSO MTZ
CL
CB
ML
MB
SL
SB
(B)
100
80
60
40
20
0
CTL
DMSO
MTZ
ML-5.16.13 ML-5.16.40
Figure 2.

8
In the case of M. rufula extracts (leaves and branches), at 1.0 mg/mL, the
parasite viability was reduced up to 100% and 80% (Figure 2A), respectively, showing
a promising activity of M. rufula extracts against T. vaginalis. These results agreed with
the literature, which was demonstrated anti-T. vaginalis activity of M. dissecata.7 Taking
into account the potential of M. rufula extract against the parasite, the crude extract of
this plant was bioguided fractionated. After, repeated column chromatography on silica
gel was performed, two major triterpene-enriched fractions were obtained (ML-5.16.13
and ML-5.16.40). These fractions promoted a low reduction of parasite viability, 26.3%
and 40.1%, respectively (Figure 2B). Regarding the scarcity of chemical studies from
M. rufula, GC-MS and NMR analysis were performed to identify the mainly compounds
in the triterpene-enriched fractions. Taking into account that these compounds are
largely found in nature and their structures are already known, the M. rufula
compounds identification was based in literature comparison: 1b, 2a, 2b, and 3a
(Figure 1B).27 Despite these compounds are known, it is the first time that these
compounds were isolated and identified in M. rufula, highlighting the importance of
such study of plants from Caatinga region. Many plants from Caatinga show a great
potential, however, the phytochemical and biological properties from the most of these
plants have never been studied before.
In attempting to improve M. rufula fractions biological activity and to
characterize the activity of isolated compounds, six derivatives based on 1, 2, and 3
(Figure 2A) structures were synthesized: 1a, 1b, 2a, 2b, 3a, and 3b (Figure 2B). This
strategy is considered a hot research topic in medicinal chemistry over ten decades
and demonstrated the potential of natural compounds derivatives as leading
compounds.11,28 The anti-T. vaginalis activity of the six synthesized derivatives, the
standards 1, 2, and 3 and derivatives acids oleanolic (4) and ursolic acid (5) was
performed. As demonstrated at Table 1, semi-synthetic compounds demonstrated a low
reduction of the parasite viability. The compound 3b presented the lowest reduction of

9
T. vaginalis viability, 28.6% at 100 µM, in comparison with negative control. The other
compounds reduced about 40% of trophozoite viability. Although data from literature
demonstrate that derivatives from different skeleton have different biological activity,29
herein, we demonstrated that derivatives obtained from three different skeleton showed
similar anti-T. vaginalis activities. The compounds 1, 2, 3 and 4 demonstrated a weak
reduction of T. vaginalis viability, representing a reduction of less than 25% the parasite
viability at 100 µM (Table 1).
Table 1. Anti-trichomonal activity of standards and semi-synthesized
compounds. Results are mean ± SD of three different experiments performed in
triplicate.
Compounds
T. vaginalis viability at 100 µM
(mean ± SD)a
Control
MTZ
100.00 ± 0.00
0.00 ± 0.00
α-amyrin (1)
76.10 ± 1.59
84.30 ± 6.23
117.80 ± 16.80
78.30 ± 8.94
0.00 ± 0.00
β-amyrin (2)
Lupeol (3)
Oleanolic acid (4)
Ursolic acid (5)
Acetate of α-amyrin (1a)
Caproate of α-amyrin (1b)
Acetate of β-amyrin (2a)
Caproate of β-amyrin (2b)
Acetate of lupeol (3a)
Caproate of lupeol (3b)
56. 40 ± 2.46
56.20 ± 0.05
54.50 ± 1.06
62.80 ± 3.61
59.80 ± 4.47
71.40 ± 8.36
aT. vaginalis viability was determined by comparison with negative control. MTZ is a positive control.
In contrast, 5 reduced 100% the T. vaginalis viability at the same concentration
(100 µM) of other evaluated compounds and the MIC was 50 µM (Figure 3A). As
demonstrated by Cos et al.30 pure compounds are considered relevant and selective
when present IC50-values below 25 µM. Nevertheless, T. vaginalis is an extracellular

10
organism and an endpoint more robust is required for activity assays; therefore, the
anti-T. vaginalis activity is usually expressed as MIC-values,31 because it is necessary
a compound that totally abolish the parasite growth, avoiding further growth. In this
sense, 5 presented a relevant and selective activity against T. vaginalis, demonstrating
a potential for new alternative to treat trichomoniasis. To overcome subjective counting
mistakes of trophozoite counting under microscopy observation, parasites at MIC and
one concentration above were inoculated in fresh TYM medium and the viability
assessed each 24 h during 144 h. After 24 h of incubation in new medium, the
parasites at 50 and 100 µM of compound 5 did not grow, confirming that 50 µM is a
real MIC and the effect was antiproliferative and nonreversible, since parasite growth
was inhibited after removal of the compound from medium (Figure 3A inset).
1.0
Control
100 M
50 M
(A)
150
0.5
100
0.0
24
48
72
96
120
144
Time (hours)
50
0
CTL DMSO 0.78 1.56 3.12 6.25 12.5
25
50
100
Concentration (M)

11
(B)
1.5
1.0
0.5
0.0
CTL
UA - 50 M
1.0
0.5
0.0
0
2
4
6
8
10
12
Time (hours)
0
24
48
72
96
Time (hours)
(C)
UA_0 h –
C - P1
UA_2 h –
P1
P1
UA_4 h
– P1
UA_6 h
– P1
UA_12 h
– P1
F
D
A
P
I

12
(D)
100
75
50
25
0
CTL
0
2
4
6
12
Time (hours)
Figure 3.
The effect of 5 on parasite growth and viability was investigated by kinetic
growth. After 24 h of incubation, untreated organisms (control) exhibited the classical
growth peak. The treated organisms (50 µM of 5), in turn, did not present the classical
growth peak and, after 12 h of incubation, this compound totally abolished parasite
growth and only trichomonads debris could be observed (Figure 3B). To increase the
accuracy of kinetic growth curve obtained by counting parasites in hemocytometer, flow
cytometry assay was performed. The parasites treated or not with ursolic acid were
evaluated at different times of incubation (2, 4, 6, and 12 h) and stained simultaneously
with FDA and PI. Trophozoites with intact membranes retain FDA dye and were
considered as viable organisms. In turn, parasites with compromised membranes
incorporate PI dye and were assumed as nonviable. As can be seen in Figure 3C,
untreated parasites (control condition) presented only FDA labeling. Trichomonas
vaginalis treated with 5 exhibited FDA labeling, however, in comparison with control
condition (untreated parasites), treated organisms presented much less labeling
(Figure 3D). The next experiments performed with 5, the incubation time chosen was 2

13
h, because the organisms were burst and only debris was found in times longer than 2
h.
Furthermore, to evaluate morphological and ultrastructural effects of 5 on T.
vaginalis, trophozoites were treated (or not, as control) with this compound and
analyzed by SEM, as shown in Figure 4. Untreated trophozoites presented a typical
shape, the teardrop morphology (Figure 4A). After 2 h of treatment with 5, drastic
effects on parasite membrane were observed (Figure 4B-D). Firstly, the typical shape
was disrupted and trophozoites became rounded (Figure 4B). Next, it was observed
membrane projections and holes. In addition, undulating membrane and flagella was
displayed (Figure 4C-D). As explained before, the treatment with 5 destroyed the
trophozoites and incubation times longer than 2 h was not possible. SEM assay of T.
vaginalis demonstrated that 5 treatment caused critical alterations, such as the
appearance of rounded and wrinkled trophozoites and significant membrane damage.
These features indicate that the parasite is in process of death, and have been
previously reported for trichomonads treated with other compounds, such lycorine and
candimine,32,33 and hydroxiquinuclidine derivative.34

14
Figure 4.
Taking into account that treatment constitutes a major therapeutic challenge,
because approximately 10% of T. vaginalis clinical isolates present some level of
resistance to metronidazole,4 new alternatives to treat resistant isolates were
necessary. The TV-LACM2R isolate is resistant to metronidazole (MIC is 73 µM)35 and
low concentration of metronidazole (15 µM) reduced about 80% of trophozoite viability.
Compound 5 was able to totally reduce TV-LACM2R viability at 12.5 µM (Figure 5A),
demonstrating a great potential of this natural derivative against resistant T. vaginalis
isolates. Furthermore, 5 was evaluated with 15 µM of metronidazole and at this
concentration, the compound significantly improved metronidazole effect against TV-
LACM2R isolate, indicating a successful synergic effect (Figure 5B).

15
(A)
100
75
50
25
0
CTL DMSO 0.78 1.56 3.12 6.25 12.5
25
50
100
Concentration (M)
(B)
100
75
50
25
*
0
-
-
-
-
+
-
-
+
-
-
-
-
-
+
-
+
-
-
+
+
-
-
+
-
5 12.5 M
5 6.25 M
MTZ 73 M
MTZ 15 M
-
+
-
-
+
-
-
-
-
+
+
+
Figure 5
Taking into account the potential of 5 against T. vaginalis sensitive and resistant
to metronidazole isolates, the in vitro cytotoxicity of this compound was evaluated by
MTT assay against HMVII and HeLa cancer cell lines and Vero normal cell line. As

16
showed in Figure 6, after 24 h of incubation, 5 at 50 µM reduced cancer cell lines
viability about 60 and 50%, respectively. After 48 h of incubation, the compound
showed higher toxicity to these cells, reducing about 90% of cell viability. These
findings agreed with earlier studies that demonstrated a great potential of 5 and
derivatives against cancer.28 Furthermore, the viability of Vero cells, normal cell line,
was performed and about 30% of cell viability was reduced. In order to compare the
activity anti-parasite versus cytotoxicity, the selectivity index (SI) of 5 was calculated as
the ratio of cytotoxicity to anti-parasite activity (SI = CC50 cell/IC50 T. vaginalis). The
effectiveness of a compound is indicated by selectivity indices ≥10.36 Thus, low SI
indicates high in vitro cytotoxicity of the compound and the lack of selectivity for the
parasites.
HMVII - 24 h
HMVII - 48 h
HeLa - 24 h
HeLa - 48 h
Vero - 24 h
Vero - 48 h
100
75
50
25
0
Control
Triton-X 100
DMSO
5
Figure 6
Despite 5 demonstrated a promising anti-T. vaginalis activity, this compound
presented a low SI against cancer and normal cell lines (Table 2), thereby, showing not

17
to be selective. Nevertheless, a compound that displayed a high cytotoxicity against
mammalian cells should not be abandoned. It is important to emphasize however, that
selectivity indices are important for general orientation, but must not be the unique
criterion to decide whether a compound should be left aside or forwarded to animal
model to continue the search for a new bioactive molecule. As demonstrated in
different studies, some compounds showed very favorable SI in vitro assays, however,
come out to be inactive or very toxic to animals.37-39
Table 2. Comparison of anti-T. vaginalis activity and mammalian cells effects.
Results are representative of three independent experiment.
Condition
MIC (µM) IC50 (µM)
CC50 (µM)
SIa
HMVII
Vero
HMVII
Vero
Ursolic acid
50
35.3
24 h
48 h
24 h
48 h
35.9
24 h
48 h
1.97
24 h
48 h
1.02
145.9
69.5
31.9
4.13
0.90
aSI – selectivity index.
In conclusion, herein we demonstrated the potential of M. rufula from Caatinga
region against T. vaginalis. Collectively, this study provides in vitro evidence that 5 is a
potential alternative therapeutic choice for treating this pathogen. This compound is a
natural compound that can be obtained from a byproduct from juice industry in large
and cheap scale and can be used as prototype to development of new derivatives even
more potent.
Acknowledgement
This work was supported by the NANOBIOTEC-Brazil program from
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Brazil

18
(grant 23038.019022/2009-68) to AJM, Conselho Nacional de Desenvolvimento
Científico e Tecnológico (CNPq), Brazil (grant 474930/2012-2) to T.T, and FAPESP
(Process: 2009/54098-6 and 2012/18031-7) to NPL and DBS. Patrícia B. Vieira
receives scholarship from CAPES; Alexandre J. Macedo (grant 305951/2012-2) and
Tiana Tasca (grant 307447/2014-6) are supported by researcher fellowships from
CNPq. The authors thank Centro de Microscopia Eletrônica (CME/UFRGS) for
technical assistance in electron microscopy.

19
References and notes
1. WHO, Global incidence and prevalence of selected curable sexually transmitted
infections: 2008: World Health Organization, Department of Reproductive Health and
Research, 2012 ISBN 978 92 4 150383 9.
2. Sutcliffe, S.; Neace, C.; Magnuson, N. S.; Reeves, R., Alderete, J. F., Trichomonosis,
a common curable STI, and prostate carcinogenesis - a proposed molecular
mechanism, PLoS Pathog., 2012, 8, e1002801.
3. Poole, D. N., McClelland, R. S., Global epidemiology of Trichomonas vaginalis, Sex.
Transm. Infect., 2013, 89, 418.
4. Schwebke, J. R., Barrientes, F. J., Prevalence of Trichomonas vaginalis isolates with
resistance to metronidazole and tinidazole, Antimicrob. Agents Chemother., 2006, 50,
4209.
5. Salatino, A.; Salatino, M. L. F., Negri, G., Traditional uses, chemistry and
pharmacology of Croton species (Euphorbiaceae), J. Brazil Chem. Soc., 2007, 18, 11.
6. Fernandes, C. P.; Correa, A. L.; Lobo, J. F.; Caramel, O. P.; de Almeida, F. B.;
Castro, E. S.; Souza, K. F.; Burth, P.; Amorim, L. M.; Santos, M. G.; Ferreira, J. L.;
Falcao, D. Q.; Carvalho, J. C., Rocha, L., Triterpene esters and biological activities
from edible fruits of Manilkara subsericea (Mart.) Dubard, Sapotaceae, Biomed Res.
Int., 2013, 2013, 280810.
7. Desrivot, J.; Waikedre, J.; Cabalion, P.; Herrenknecht, C.; Bories, C.; Hocquemiller,
R., Fournet, A., Antiparasitic activity of some New Caledonian medicinal plants, J.
Ethnopharmacol., 2007, 112, 7.
8. Rhourri-Frih, B.; Renimel, I.; Chaimbault, P.; André, P.; Herbette, G., Lafosse, M.,
Pentacyclic triterpenes from Manilkara bidentata resin. Isolation, identification and
biological properties, Fitoterapia, 2013, 88, 101.

20
9. Shai, L. J.; McGaw, L. J.; Aderogba, M. A.; Mdee, L. K., Eloff, J. N., Four pentacyclic
triterpenoids with antifungal and antibacterial activity from Curtisia dentata (Burm.f)
C.A. Sm. leaves, J. Ethnopharmacol., 2008, 119, 238.
10. Innocente, A. M.; Vieira, P. B.; Frasson, A. P.; Casanova, B. B.; Gosmann, G.;
Gnoatto, S. C., Tasca, T., Anti-Trichomonas vaginalis activity from triterpenoid
derivatives, Parasitol. Res., 2014, 113, 2933.
11. Silva, G. N.; Maria, N. R.; Schuck, D. C.; Cruz, L. N.; de Moraes, M. S.; Nakabashi,
M.; Graebin, C.; Gosmann, G.; Garcia, C. R., Gnoatto, S. C., Two series of new
semisynthetic triterpene derivatives: differences in anti-malarial activity, cytotoxicity and
mechanism of action, Malar. J., 2013, 12, 89.
12. Guimaraes, A. G.; Serafini, M. R., Quintans-Junior, L. J., Terpenes and derivatives
as a new perspective for pain treatment: a patent review, Expert Opin. Ther. Pat., 2014,
24, 243.
13. The general VLC procedure consisted in the use of a silica gel 60 A (6−35 μm)
column with a height of 4.0 cm and a variable diameter according to the amount of
sample (2.5 cm for 1000 mg; 1.5 cm for 100 mg). Compounds were eluted using n-
hexane gradually enriched with ethyl acetate (EtOAc) and then EtOAc gradually
enriched with methanol (MeOH) reaching a maximum concentration of 15%. Fractions
of 15 mL were collected, monitored by TLC (silica gel 60 F254 plates and
anisaldehyde-sulphuric acid's reagent were used) and combined according to their
profiles.
14. α-amyrin (1), β-amyrin (2), and lupeol (3) derivatives were synthesized by the
addition of the appropriate anhydride (0.032 mmol, 9 Eq) and DMAP (0.021 mmol, 2
Eq) to 1, 2 or 3 (0.011 mmol) in dichloromethane (1.0 mL) and reacted for three hours
without refluxing. Column chromatography of formed product was performed to give the
expected pure compounds. TLC was performed on silica gel 60 F254 plates (Merck)
and anisaldehyde-sulphuric acid's reagent was used.

21
15. Data for 3-O-Acetyl-α-amirine (compound 1a): white solid. Yield 71%. IR (ATR cm-
1): 2961 (C-H); 1733 (C=O); 1447 (C-O); 1241 (C-O-C). 1H-NMR (CDCl3, ): 0.73 (s,
6H, H26 and H28); 0.81 (s, 9H, H23, H24 and H25); 0.85 (s, 3H, H30); 0.91 (s, 3H H29); 0.94
(s, 3H, H27); 1.00 (t, 2H, H1); 1.19 (t, 2H, H16); 1.22 (t, 2H, H15); 1.25 (t, 2H, H22); 1.30 (t,
1H, H20); 1.45 (t, 2H, H7); 1.98 (s, 3H, H32); 4.44 (dd, 1H, H3); 5.06 (t, 1H, H12). 13C-
NMR (CDCl3,): 14.73 (C-25); 15.73 (C-26); 15.85 (C-29); 16.50 (C-6); 17.23 (C-30);
22.22 (C-32); 22.32 (C-24); 22.36 (C-11); 22.39 (C-23); 22.59 (C-2); 25.59 (C-27);
27.06 (C-28); 27.74 (C-15); 30.23 (C-21); 31.85 (C-16); 32.70 (C-7); 35.67 (C-17);
36.68 (C-20); 37.44 (C-10); 37.45 (C-4); 38.59 (C-1); 38.63 (C-19); 39.01 (C-22); 40.52
(C-8); 41.06 (C-14); 46.52 (C-9); 54.14 (C-5); 58.08 (C-18); 79.88 (C-3); 123.54 (C-12);
146.62 (C-13); 171.12 (C-31). HR-ESI-MS m/z: 491.3849 [M+Na]+ (calcd. for
C32H52O2Na+ 491.3865, error 3.2 ppm).
16. Data for 3-O-Caproyl-α-amirine (compound 1b): white solid. Yield 77%. IR (ATR cm-
1): 2929 (C-H); 1729 (C=O); 1450 (C-O); 1245 (C-O-C). 1H-NMR (CDCl3, ): 0.73 (t, 6H,
H28 and H36); 0.76 (m, 1H, H19); 0.80 (s, 9H, H23, H24 and H25); 0.83 (s, 3H, H26); 0.84 (d,
3H, H30); 0.91 (d, 3H, H29); 0.94 (s, 3H, H27); 1.18 (t, 1H, H5); 1.25 (m, 8H, H1, H16, H21
and H34); 1.30 (t, 1H, H20); 1.48 (t, 2H, H15); 1.56 (t, 2H, H6); 1.57 (t, 2H, H7); 1.75 (d,
1H, H18); 1.84 (t, 2H, H33); 1.97 (t, 2H, H2); 1.84 (t, 2H, H11); 2.29 (t, 1H, H9); 2.42 (t, 2H,
H32); 4.43 (dd, 1H, H3); 5.06 (t, 1H, H12). 13C-NMR (CDCl3, ): 15.68 (C-36); 17.49 (C-
26); 18.57 (C-29); 18.62 (C-6); 19.24 (C-30); 19.96 (C-34); 23.16 (C-23 and C-24);
24.06 (C-11); 24.96 (C-2); 25.13 (C-27); 25.39 (C-33); 26.58 (C-28); 28.36 (C-15);
29.80 (C-21); 30.50 (C-34); 33.00 (C-16); 33.10 (C-7); 34.61 (C-32); 35.46 (C-17);
36.61 (C-20); 38.61 (C-10); 39.47 (C-4); 40.13 (C-1); 41.36 (C-19); 41.74 (C-22); 43.26
(C-8); 43.82 (C-14); 49.32 (C-9); 57.01 (C-5); 60.67 (C-18); 82.25 (C-3); 126.07 (C-12);
149.48 (C-13); 159.69 (C-31). HR-ESI-MS m/z: 547.4438 [M+Na]+ (calcd. for
C36H60O2Na+ 547.4491 error 9.7 ppm).

22
17. Data for 3-O-Acetyl-β-amirine (compound 2a): white solid. Yield 88%. IR (ATR cm-
1): 2947 (C-H); 1729 (C=O); 1462 (C-O); 1247 (C-O-C). 1H-NMR (CDCl3, ): 0.83 (s,
3H, H28); 0.87 (s, 12H, H23, H24, H29 and H30); 0.96 (s, 6H, H25 and H27); 1.13 (t, 3H, H5
and H16); 1.25 (d, 2H, H19); 1.33 (t, 2H, H1); 1.41 (t, 2H, H7); 1.52 (t, 2H, H15); 1.58 (t,
4H, H6 and H22); 1.62 (t, 4H, H11 and H21); 1.86 (t, 2H, H2); 1.96 (t, 2H, H9 and H18); 2.05
(s, 3H, H32); 4.50 (dd, 1H, H3); 5.18 (t, 1H, H12). 13C-NMR (CDCl3, ): 15.70 (C-25);
16.84 (C-26); 16.94 (C-6); 18.40 (C-32); 21.67 (C-23 and C-24); 23.67 (C-11); 23.82
(C-2); 26.10 (C-27); 26.27 (C-28); 27.05 (C-15); 28.18 (C-29); 28.54 (C-30); 31.23 (C-
16); 32.63 (C-20); 32.72 (C-21); 33.48 (C-17); 34.87 (C-7); 36.98 (C-10); 37.31 (C-4);
37.86 (C-1); 38.39 (C-8); 39.94 (C-22); 41.84 (C-14); 46.92 (C-19); 47.36 (C-18); 47.70
(C-9); 55.39 (C-5); 81.29 (C-3); 121.86 (C-12); 140.65 (C-13); 176.82 (C-31). HR-ESI-
+
MS m/z: 469.4004 [M+H]+ (calcd. for C32H53O2 469.4040, error 7.7 ppm).
18. Data for 3-O-Caproyl-β-amirine (compound 2b): white solid. Yield 75%. IR (ATR cm-
1): 2917 (C-H); 1723 (C=O); 1454 (C-O); 1248 (C-O-C). 1H-NMR (CDCl3, ): 0.91 (m,
12H, H25, H26, H28 and H36); 0.97 (s, 6H,H29 and H30); 1.13 (s, 6H, H23 and H24); 1.33 (s,
14H, H1, H5, H7 H16, H27, H34 and H35); 1.64 (t, 10H, H2, H19, H21, H22 and H33); 1.86 (t,
2H, H11); 1.95 (t, 1H, H18); 2.36 (t, 3H, H32); 4.49 (dd, 1H, H3); 5.17 (t, 1H, H12). 13C-
NMR (CDCl3, ): 14.00 (C-36); 15.56 (C-25); 18.31 (C-26); 19.69 (C-6); 22.43 (C-35);
23.61 (C-11); 24.69 (C-23, C-24, C-27 and C-33); 25.97 (C-2); 26.14 (C-28); 26.93 (C-
30); 28.05 (C-15); 28.41 (C-29); 31.37 (C-16, C-17, C-22 and 34); 33.34 (C-7); 34.74
(C-21 and C-32); 37.71 (C-1); 39.93 (C-4, C-8 and C-10); 41.62 (C-14); 46.71 (C-19);
47.21 (C-18); 47.51 (C-9); 55.16 (C-5); 80.46 (C-3); 121.60 (C-12); 141.75 (C-13);
179.65 (C-31). HR-ESI-MS m/z: 547.4509 [M+Na]+ (calcd. for C36H60O2Na+ 547.4491
error 3.3 ppm).
19. Data for 3-O-Acetyl-lupeol (compound 3a): white solid. Yield 82%. IR (ATR cm-1):
2940 (C-H); 1732 (C=O); 1635 (C=C); 1472 (C-O); 1243 (C-O-C). 1H-NMR (CDCl3, ):

23
0.72 (t, 1H, H5); 0.78 (s, 9H, H25, H26 and H28); 0.87 (s, 3H, H27); 0.91 (s, 3H, H23); 0.96
(s 3H, H24); 1.11 (m, 2H, H12); 1.14 (t, 1H, H18); 1.19 (t, 4H, H1 and H15); 1.32 (m, 6H,
H7, H16 and H21); 1.41 (m, 2H, H11); 1.54 (m, 2H, H6); 1.58 (t, 2H, H22); 1.61 (s, 3H, H30);
1.84 (m, 2H, H2); 1.97 (s, 3H, H32); 2.30 (m, 2H, H9 and H19); 4.40 (s, 1H, H3); 4.50 (brs,
1H, H29b); 4.62 (brs, 1H, H29a). 13C-NMR (CDCl3, ): 14.41 (C-25); 15.93 (C-26); 16.15
(C-27); 16.46 (C-6); 18.00 (C-32); 18.16 (C-30); 19.21 (C-11); 20.90 (C-28); 21.31 (C-
23); 23.67 (C-24); 25.05 (C-2); 27.39 (C-12); 27.95 (C-15); 29.79 (C-21); 34.16 (C-7);
35.52 (C-16); 37.04 (C-10); 37.76 (C-13); 38.30 (C-4); 39.96 (C-1); 40.80 (C-22); 42.75
(C-8); 42.96 (C-14); 47.97 (C-19); 48.24(C-18); 50.29 (C-9); 55.10 (C-5); 80.90 (C-3);
109.19 (C-29); 150.75 (C-20); 171.03 (C-31). HR-ESI-MS m/z: 491.3847 [M+Na]+
(calcd. for C32H52O2Na+ 491.3865 error 3.7 ppm).
20. Data for 3-O-Caproyl-lupeol (compound 3b): white solid. Yield 100%. IR (ATR cm-
1): 2926 (C-H); 1727 (C=O); 1639 (C=C); 1451 (C-O); 1256 (C-O-C). 1H-NMR (CDCl3,
): 0.72 (t, 1H, H5); 0.77 (t, 6H, H28 and H36); 0.83 (s, 17H, H12, H23, H24, H25, H26 and
H27); 0.87 (m, 2H, H13 and H18); 0.96 (t, 2H, H16); 1.14 (m, 2H, H11); 1.18 (m, 2H, H34);
1.26 (m, 12H, H1, H6, H7, H15, H21 and H35); 1.57 (m, 6H, H2, H22 and H33); 1.61 (s, 3H,
H30); 2.20 (t, 1H, H19); 2.22 (t, 1H, H9); 2.28 (t, 2H, H32); 4.40 (s, 1H, H3); 4.50 (brs, 1H,
H29b); 4.62 (brs, 1H, H29a). 13C-NMR (CDCl3, ): 13.97 (C-36); 14.52 (C-25); 15.97 (C-
26); 16.17 (C-27); 16.57 (C-6); 22.42 (C-30); 23.74 (C-11); 24.84 (C-35); 25.01 (C-23
and C-24); 25.09 (C-28); 27.43 (C-2); 27.96 (C-33); 28.96 (C-15); 29.82 (C-12); 31.48
(C-21); 34.20 (C-34); 34.83 (C-32); 35.57 (C-7); 37.08 (C-16); 37.83 (C-10); 38.03 (C-
13); 38.36 (C-4); 39.66 (C-1); 40.00 (C-22); 40.84 (C-8); 42.82 (C-14); 42.94 (C-17);
48.01 (C-19); 48.23 (C-18); 50.27 (C-9); 55.37 (C-5); 80.44 (C-3); 108.98 (C-29);
150.82 (C-20); 179.27 (C-31). HR-ESI-MS m/z: 1049.9237 [2M+H]+ (calcd. for
+
C72H121O4 1049.9259 error 2.1 ppm).

24
21. Mahato, S. B., Kundu, A. P., 13C NMR Spectra of pentacyclic triterpenoids—a
compilation and some salient features, Phytochemistry, 1994, 37, 1517.
22. Diamond, L. S., The establishment of various trichomonads of animals and man in
axenic cultures, J. Parasitol., 1957, 43, 488.
23. Parasites with a cellular density 1.0 x 105 trophozoites/mL were treated with crude
extract and fractions (1.0 mg/mL) or semi-synthesized compounds (100 µM). Three
controls were carried out: negative control (parasites only), DMSO 0.62% (vehicle for
solubilization), and positive control (100 µM metronidazole, Sigma-Aldrich, St. Louis,
MO, USA). After 24 h of incubation at 37 C and 5.0 % CO2, the number of viable
parasites was accessed by counting using a hemocytometer. The results were
expressed as the percentage of trophozoite viability compared with untreated parasites
after 24 h of incubation, considering motility, normal morphology and exclusion by
trypan blue dye (0.2%).
24. After MIC confirmation, parasites in a cellular density of 1.0 x 105 trophozoites/mL
were treated or not with compound 5 at MIC value (50 μM) and incubated for 2, 4, 6,
12, 24, 48, 72, and 96 h. The parasite number was determined by counting in
hemocytometer. The results were expressed as trophozoite number per milliliter
comparing with negative control (untreated organisms).
25. In a cellular density of 1.0 x 105 trophozoites/mL parasites were treated or not with
compound 5 at 50 μM for 4 h. Organisms were harvested and fixed in 2.5% (v/v)
glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2); post-fixed in 1.0% OsO4 in
0.1 M sodium cacodylate buffer (pH 7.2), and dehydrated in acetone. Critical-point-
dried with CO2 and coated with gold-palladium. The samples were examined with a
JEOL 6060 scanning electron microscope.
26. The cell lineages HMVII (vaginal epithelial melanoma cell), HeLa (cervical cancer
cell), and VERO (kidney epithelial cells) were grown and maintained in RPMI and

25
DMEM medium, supplemented with 10% fetal bovine serum (FBS) at 37 C and 5%
CO2. For this assay, 3.0 x 104 or 1.0 x 104 cells per well were seeded in 96-well
microtiter plates overnight. After this period, the medium was replaced with fresh
medium containing or not (control condition) compound 5 at 50 μM. Positive control
(0.2% Triton X-100) was added. The cytotoxicity was evaluated after 24 or 48 h of
incubation. After incubation period, media were suctioned; 0.5 mg/mL MTT solution
was added and incubated for 1 h. The solution was removed and the insoluble purple
formazan was dissolved in DMSO and read at 570 nm.
27. Shiojima, K.; Arai, Y.; Masuda, K.; Takase, Y.; Ageta, T., Ageta, H., Mass Spectra of
Pentacyclic Triterpenoids, Chem. Pharm. Bull., 1992, 40, 1683.
28. Hua, S. X.; Huang, R. Z.; Ye, M. Y.; Pan, Y. M.; Yao, G. Y.; Zhang, Y., Wang, H. S.,
Design, synthesis and in vitro evaluation of novel ursolic acid derivatives as potential
anticancer agents, Eur. J. Med. Chem., 2015, 95, 435.
29. Gauthier, C.; Legault, J.; Girard-Lalancette, K.; Mshvildadze, V., Pichette, A.,
Haemolytic activity, cytotoxicity and membrane cell permeabilization of semi-synthetic
and natural lupane- and oleanane-type saponins, Bioorgan. Med. Chem., 2009, 17,
2002.
30. Cos, P.; Vlietinck, A. J.; Berghe, D. V., Maes, L., Anti-infective potential of natural
products: how to develop a stronger in vitro 'proof-of-concept', J. Ethnopharmacol.,
2006, 106, 290.
31. Vieira, P. B.; Giordani, R. B.; Macedo, A. J., Tasca, T., Natural and synthetic
compound anti-Trichomonas vaginalis: an update review, Parasitol. Res., 2015, 114,
1249.
32. Giordani, R. B.; Vieira, P. B.; Weizenmann, M.; Rosemberg, D. B.; Souza, A. P.;
Bonorino, C.; De Carli, G. A.; Bogo, M. R.; Zuanazzi, J. A., Tasca, T., Lycorine induces

26
cell death in the amitochondriate parasite, Trichomonas vaginalis, via an alternative
non-apoptotic death pathway, Phytochemistry, 2011, 72, 645.
33. Giordani, R. B.; Vieira, P. B.; Weizenmann, M.; Rosemberg, D. B.; Souza, A. P.;
Bonorino, C.; De Carli, G. A.; Bogo, M. R.; Zuanazzi, J. A., Tasca, T., Candimine-
induced cell death of the amitochondriate parasite Trichomonas vaginalis, J. Nat. Prod.,
2010, 73, 2019.
34. Rocha, D. A.; de Andrade Rosa, I.; Urbina, J. A.; de Souza, W., Benchimol, M., The
effect of 3-(biphenyl-4-yl)-3-hydoxyquinuclidine (BPQ-OH) and metronidazole on
Trichomonas vaginalis: a comparative study, Parasitol. Res., 2014, 113, 2185.
35. Butler, S. E.; Augostini, P., Secor, W. E., Mycoplasma hominis infection of
Trichomonas vaginalis is not associated with metronidazole-resistant trichomoniasis in
clinical isolates from the United States, Parasitol. Res., 2010, 107, 1023.
36. Mahlo, S. M.; McGaw, L. J., Eloff, J. N., Antifungal activity and cytotoxicity of
isolated compounds from leaves of Breonadia salicina, J. Ethnopharmacol., 2013, 148,
909.
37. Schmidt, T. J.; Brun, R.; Willuhn, G.; Khalid, S. A., Anti-trypanosomal activity of
helenalin and some structurally related sesquiterpene lactones, Planta Med., 2002, 68,
750.
38. Mohammed, R. S.; Abou Zeid, A. H.; El Hawary, S. S.; Sleem, A. A.; Ashour, W. E.,
Flavonoid constituents, cytotoxic and antioxidant activities of Gleditsia triacanthos L.
leaves, Saudi J. Biol. Sci., 2014, 21, 547.
39. Fernandes, C. R.; Turatti, A.; Gouvea, D. R.; Gobbo-Neto, L.; Diniz, A.; Ribeiro-
Silva, A.; Lopes, N. P.; Garcia, S. B., The protective role of Lychnophora ericoides Mart.
(Brazilian arnica) in 1,2-dimethylhydrazine-induced experimental colon carcinogenesis,
Nutr. Cancer, 2011, 63, 593.

27
Figure legends
Figure 1. (A) Structure of α-amyrin (1), β-amyrin (2), lupeol (3), oleanolic (4) and ursolic
(5) acids. (B) Synthesis of α-amyrin (1a-b), β-amyrin (2a-b) and lupeol (3a-b)
derivatives.
Figure 2. Anti-T. vaginalis activity of (A) crude extracts at 1.0 mg/mL of Croton
nummularius, Manilkara rufula and Senna lechriosperma leaves and branches. CL – C.
nummularius leaves; CB – C. nummularius branches; ML – M. rufula leaves; MB – M.
rufula branches; SL – S. lechriosperma leaves; SB – S. lechriosperma branches; (B)
fractions of M. rufula leaves at 1.0 mg/mL. CTL – control condition (trophozoites
untreated); MTZ – metronidazole (positive control); DMSO – vehicle control. Bars
represent the mean ± SD of three different experiments (parasite suspensions)
performed in triplicate.
Figure 3. (A) MIC determination of UA against T. vaginalis 30236 isolate. Inset: MIC
confirmation by counting trophozoite number after incubation on TYM fresh medium.
(B) Kinetic growth curve of UA (50 µM), treated trophozoite in comparison to control
(untreated parasites). (C) Dot plots of parasite viability treated or not with UA label with
FDA-PI. (D) Viability quantification from flow cytometry assy. Results are mean ± SD of
three different experiments (parasite suspensions) performed in triplicate.
Figure 4. SEM of T. vaginalis under UA treatment 50 μM. (A) a typical untreated
trophozoite displaying a tear-drop shape, four anterior flagella (af), undulating

28
membrane (um) and axostyle (ax); (B-D) after UA treatment for 2 h trophozoites
displayed a profound membrane alterations.
Figure 5. MIC determination of metronidazole against T. vaginalis TV-LACM2R isolate
(A). Combinatory effect of UA and metronidazole against TV-LACM2R isolate (B).
*MTZ and UA association statistically different from 15 µM MTZ treatment. Results are
mean ± SD of three different experiments (parasite suspensions) performed in
triplicate.
Figure 6. Effect of 5 on the viability of HMVII, HeLa and Vero lines at 50 μM.
Compound 5 was highly cytotoxic to HMVII and HeLa lines, while to Vero line it
presented low cytotoxicity. Controls represent cells only in medium, without 5. Results
are mean ± SD of three different experiments performed in triplicate.

29
Graphical abstract