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Phytomedicine
journal homepage:www.elsevier.de/phymed
Infections
Article history: Leishmaniasis and Chagas disease are infectious diseases caused by parasite Leishmania sp. and Trypanosoma cruzi,
Received 3 September 2013 respectively, and are included among the most neglected diseases in several underde-veloped and developing countries, with
Received in revised form 25 October 2013 an urgent demand for new drugs. Considering the antiparasitic potential of MeOH extract from leaves of Casearia sylvestris
Accepted 14 January 2014 Sw. (Salicaceae), a bioguided fractionation was conducted and afforded four active clerodane diterpenes (casearins A, B, G,
and J). The obtained results indicated a superior efficacy of tested casearins against trypomastigotes of T. cruzi, with IC 50
Keywords:
values ran-ging from 0.53 to 2.77 g/ml. Leishmania infantum promastigotes were also susceptible to casearins, with IC 50
Casearia sylvestris
Salicaceae values in a range between 4.45 and 9.48 g/ml. These substances were also evaluated for mam-malian cytotoxicity against
Clerodane diterpenes NCTC cells resulting in 50% cytotoxic concentrations (CC 50 ) ranging from 1.46 to 13.76 g/ml. Additionally, the action of
Leishmania casearins on parasite membranes was investigated using the fluorescent probe SYTOX Green. The obtained results
Trypanosoma cruzi demonstrated a strong interaction of casearins A and B to the plasma membrane of T. cruzi parasites, corroborating their
Casearins higher efficacy against these parasites. In contrast, the tested casearins induced no alteration in the permeability of plasma
membrane of Leishmania parasites, suggesting that biochemical differences between Leishmania and T. cruzi plasma
membrane might have contributed to the target effect of casearins on trypomastigotes. Thus, considering the importance of
studying novel and selective drug candidates against protozoans, casearins A, B, G, and J could be used as tools to future drug
design studies.
Introduction countries (WHO 2010). The chemotherapy for these diseases is unsatisfactory
in terms of lack of effectiveness and also the unde-sirable side effects
Casearia sylvestris a plant popularly known as guac atonga, is associated with long term treatment with discovered drugs (Schmidt et al.
geographically distributed throughout Latin America (Lorenzi and Matos 2012). Medicinal plants have been used for the treatment of cutaneous
2002) and has been used in traditional medicine as anti-inflammatory, anti- leishmaniasis by rural peo-ple (Fournet et al. 1992; Franca et al. 1996) and
ulcer, anti-ophidian and anti-tumor (Ferreira et al. 2011). The chemical have attracted more interest from the scientific community. Several
composition of C. sylvestris has been char-acterized by clerodane type compounds isolated from Brazilian plants have been described with potent
diterpenes, known as casearins A-X which showed pronounced antitumor anti-trypanossomal and antileishmanial activities (Schmidt et al. 2012).
activity (Itokawa et al. 1990; Morita et al. 1991; Carvalho et al. 1998; Wang However, only eleven derivatives displayed comparable activity to standard
et al. 2009a; Santos et al. 2010). Clerodane diterpenes with different drugs used in therapy to treat these tropical diseases, as could be seen in Table
stereochemistry from casearins, named casearvestrins A-C and other 1.
clerodane diterpenes have also been described from leaves of C. sylvestris
(Oberlies et al. 2002; Santos et al. 2007; Wang et al. 2009b). Mesquita et al. (2005) evaluated extracts of thirteen medici-nal plants from
the Brazilian Cerrado biome for antileishmanial (Leishmania donovani) and
Protozoan diseases as Leishmaniasis and Chagas disease are main health antitrypanosomal activities and discov-ered that the leaves extract of C.
and socioeconomic problems in many developing sylvestris exhibited activity at 100 g/mL. However, no active compounds have
been isolated so far.
Corresponding author. Tel.: +55 11 3319 3300; fax: +55 11 4043 6428. E-mail address: Thus, the present work describes for the first time the
psartorelli@unifesp.br (P. Sartorelli).
antileish-manial and antitrypanosomal activities of casearins
0944-7113/$ see front matter 2014 Elsevier GmbH. All rights reserved. A, B, G, and J
http://dx.doi.org/10.1016/j.phymed.2014.01.004
D.D. Bou et al. / Phytomedicine 21 (2014) 676681 677
Table 1
Brazilian plants and isolated compounds which displayed antiparasitic activity (anti-trypanossomal and antileishmanial) and comparison of IC 50 values with those reported to positive controls.
isolated from the MeOH extract from the leaves of C. sylvestris using caserins G (77 mg) and J (51 mg). Fraction A12 (300 mg) was also submitted
bioguided fractionation procedures. Additionally, the effect on the to CC Sephadex LH-20 to yield casearin B (12 mg). Finally, fractionation of
permeability of plasma membrane of L. infantum and T. cruzi was evaluated. A13 (526 mg) over Sephadex LH-20, followed by prep TLC (hexanes 7:
EtOAC 3) afforded casearin A (43 mg) (Fig. 1).
Plant material
Mammalian cells
Leaves of C. sylvestris were collected from a single tree at the Atlantic
The mammalian cells (NCTC clone 929) and LLC-MK2 were
Forest area in So Paulo city, SP, Brazil (coordinates 23 5308.86S, 46
maintained in M-199 medium without phenol red and supple-mented with
4010.45O), in October, 2012. The identification was made by Dr. Oriana
10% fetal bovine serum at 37 C in a 5% CO2 humidified incubator.
Fvero (Universidade Presbiteriana Mackenzie-SP).
Leaves of C. sylvestris (290 g), were dried and grounded, and then In order to determine the 50% inhibitory concentration (IC 50 ), the isolated
extracted using MeOH for 3 days, to afford 11.1 g of crude extract. The active casearins were dissolved in 100% MeOH, serially diluted in culture medium
MeOH extract was suspended in MeOH:H2 O 2:1. After partition using and incubated with parasites at con-centrations in the range between 0.1 and
150 g/ml. The maximal concentration of MeOH in plate wells was 0.5% and
hexanes, CH2 Cl2 and EtOAc, were obtained the three phases which were
internal controls were used to detect possible solvent toxicity to parasites.
evaluated to anti-trypanosomal and anti-Lesihmania activities.
Leishmania. The isolated casearins were incubated with pro-mastigotes of
6
L. (L.) infantum seeded at 1 10 /well in 96 well plates. After 48 h, the
Separation, purification and identification of chemical viability of promastigotes was measured by the mitochondrial activity using
constituents the MTT assay at 570 nm (Tada et al. 1986). Pentamidine was used as a
positive control.
The active hexane phase (6.4 g) was subjected to SiO 2 col-umn Trypanosoma cruzi. Trypomastigotes of T. cruzi (Y strain) obtained from
chromatography gel eluted with increasing amounts of EtOAc in hexane (9:1 LLC-MK2 cells were counted in a Neubauer hemo-cytometer and seeded at 1
to 1:9) to give 23 fractions (A1A23). The active fractions A11, A12 and A13 106 /well in 96-well microplates. Ater 24 h, the viability of trypomastigotes
were submitted to new chromato-graphic steps guided by the antiparasitic was measured by the mito-chondrial activity using the MTT assay at 570 nm
activity. Thus, fraction A11 (380 mg) was fractionated over Sephadex LH-20, (Lane et al. 1996). Benznidazole was used as the standard drug.
eluted with MeOH and followed by SiO2 prep TLC (hexanes 8: EtOAC 2) to
afford
678 D.D. Bou et al. / Phytomedicine 21 (2014) 676681
12 16
13
11
20 14
MeO
1 9
17 15
2 10 8
3 5 7
4 6
O
19 R2
18
O
R1 O O
casearin R1 R2
A CH3CO2 OH ()
G CH3CO2 H
J n-C3H7CO2 OH ()
B CH3CO2 CH3CO2 ()
Fig. 1. Structures of casearins isolated from C. sylvestris.
Determination of the cytotoxicity 13
C NMR spectra of these fractions
against mammalian cells
showed in general from 29 to 31
The in vitro selectivity index signals, similar to those reported for
casearins in the literature.
(CC50 /IC50 ) was determined using
13
NCTC cells, which were seeded at Characteristic signals in C NMR
4 spectra were observed at 57
6 10 /well in 96 well plates and
M-199 medium as described above. (CH3 ) indicating the presence of a
The mammalian cells were methoxy group in the structure, and
incubated with casearins and the
72 (CH) for the carbinolic carbon
standard drugs pentamidine and
(C2) in all isolated com-pounds.
benznidazole for 48 h at 37 C in a The signals at 75 (CH) were
5% CO2 humidified incubator. The assigned to carbons attached to
viability of cells was determined by hydroxyl groups (C6), except to
the MTT assay as described above casearin G, which displayed the
and the 50% cytotoxic methylenic carbon (C6) at 34. The
concentration (CC50 ) was double bond at C-3 and C-4 also
calculated. characteristic of casearins was
observed at 123 and 142
Spectrofluorimetric detection of respectively. The chemical shifts at
disruption of the parasites 53 (C-5) and 35 (C-10) were
plasma membrane similar to those described for
casearins and analogous
Late growth-phase (non- compounds. The signals of
stationary) promastigotes were methynic carbons at 95 (C-18) and
washed in PBS, seeded at 2 97 (C-19) indi-cate the existence of
6 the diacetalic ring system C, while
10 /well and incubated with 1 M
those at 170174 suggest the
SYTOX Green for 15 min at 24 presence of acetate and butanoate
C (Mangoni et al. 2005). Casearins groups as substituents.
were added, and the fluorescence
was measured every 20 min for 60
min total. The maximum
permeabilization was obtained with
0.1% Triton X-100. Fluorescence Determination of
intensity was determined using a the 50% inhibitory
fluorimetric microplate reader concentration and
(FilterMax F5 Multi-Mode mammalian
Microplate Reader-Molecular cytotoxicity
Devices) with excitation and emis-
sion wavelengths of 485 and 520 Casearins were incubated for 24
nm, respectively. The following h with T. cruzi trypomastigo-tes and
internal controls were used in the 48 h with promastigotes of L. (L.)
evaluation: (i) the background infantum. The viability was
fluorescence of casearins at the determined by the colorimetric
respective wavelengths, (ii) the assay of MTT. All tested sub-
possible interference of DMSO, stances killed 100% of parasites at
(iii) untreated promastigotes, and the highest tested concentration
(iv) medium without any cells. (150 g/ml); casearins A, B, G and J
Samples were tested in triplicate. showed IC50 values in a range
between 0.53 and 2.7 g/ml against
T. cruzi trypomastigotes (Table 2).
The most promising activity was
Results observed for casearin J, which
showed an IC50 value of 0.53 g/mL.
Isolation and identification of Leishmania promastig-otes were
casearins also susceptible to casearins
resulting in IC50 values ranging
from 4 to 9 g/ml (Table 2).
The chromatographic
Similarly to T. cruzi assay, the most
procedures carried out to active active substance against Leishmania
fractions A11, A12 and A13 was casearin J, which showed an
allowed the isolation of four pure IC50 value of 4.45 g/ml (Table 2).
substances. The Casearins were also tested
for cytotoxicity after
incubation with NCTC cells
for 48 h. The viability was
also determined using the
MTT assay. Casearins
D.D. Bou et al. / Phytomedicine 21 (2014) 676681 679
Table 2
Antileishmanial, antitrypanosomal and cytotoxicity effects of casearins isolated from C. sylvestris.
Casearins IC50 ( g/ml) promastigotes IC50 ( g/ml) trypomastigotes CC50 ( g/ml)
L. (L.) infantum T. cruzi Cell NCTC
95% CI 95% CI 95% CI
A 6.34* (4.0110.01) 1.37* (1.071.75) 2.62 (1.923.59)
B 9.48* (7.4312.09) 2.77* (2.632.93) 13.76 (9.1920.58)
J 4.45* (3.935.04) 0.53* (0.350.81) 1.46 (1.361.58)
G 5.93* (5.596.29) 1.57* (1.232.00) 7.54 (5.849.74)
95% CI = 95% confidence interval; Leishmania (promastigotes) pentamidine IC 50 0.22 g/ml; T. cruzi (trypomastigotes) benznidazole IC50 114.68 g/ml. *p < 0.05 (compared to standard drug).
showed CC50 values in a range between 1 and 13 g/ml (Table 2). It was also among the tested substances was significant (p < 0.001). Methanol at 0.5%
possible to note that casearin J was the most cytotoxic substance. was used as internal control and showed no alteration in permeability of
membranes. Casearins showed minimum alter-ation in the permeability of
Leishmania membrane when compare to fully (100%) permeabilized parasites
Plasma membrane permeabilization after 60 min, as follows: (i) casearin J = 6%; (ii) casearin G = 2.8%; (iii)
casearin B = 4.6% and (iv) casearin A = 4%. According to ANOVA test, the
The activity of casearins on parasites membrane of trypomasti-gotes and difference among the tested substances was significant (p < 0.001).
promastigotes was evaluated using the fluorescent probe SYTOX green (Fig.
2). The isolated casearins resulted in an intense and time-dependent increase
in fluorescence levels when incu-bated with T. cruzi parasites. When
Discussion
compared to fully permeabilized (100%) parasites, casearins showed the
following percentage of permeability alteration on T. cruzi membranes after
Clerodane diterpenes have been isolated from different plant species such
60 min incuba-tion: (i) casearin J = 29%; (ii) casearin G = 58%; (iii) casearin
as Baccharis tricuneata (Wagner et al. 1978), Zuelania guidonia (Khan et al.
B = 80% and (iv) casearin A = 87%. According to ANOVA test, the difference
1990), Laetia procera (Jullian et al. 2005) and Laetia corymbulosa (Beutler et
al. 2000). These derivatives are char-acteristic of the genus Casearia and have
been previously described from several species such as Casearia rupestris
(Vieira-Jnior et al. 2011), Casearia obliqua (Vieira-Jnior et al. 2009) and
Casearia nigrencens (Williams et al. 2007). However, characteristic diter-
penes from C. sylvestris are known as caseaerins/casearvestrins and display
promising biological activities, such as chemopreventive effect and anti-tumor
action (Prieto et al. 2013; Ferreira et al. 2010 ). The structural identification of
casearins A, B, G and J, isolated in this work was performed by comparing the
1 13
data obtained from the spectral H and C NMR with the literature data
(Itokawa et al. 1990; Morita et al. 1991; Carvalho et al. 1998).