Parasitol Res
DOI 10.1007/s00436-013-3659-x
ORIGINAL PAPER
Treatment with triterpenic fraction purified from Baccharis
uncinella leaves inhibits Leishmania (Leishmania )
amazonensis spreading and improves Th1 immune response
in infected mice
Eduardo Seiji Yamamoto & Bruno Luiz S. Campos & Márcia Dalastra Laurenti &
João H.G. Lago & Simone dos Santos Grecco & Carlos E. P. Corbett & Luiz Felipe D. Passero
Received: 23 September 2013 / Accepted: 16 October 2013
# Springer-Verlag Berlin Heidelberg 2013
Abstract The current medications used to treat leishmaniasis
have many side effects for patients; in addition, some cases of
the disease are refractory to treatment. Therefore, the search
for new leishmanicidal compounds is indispensable. Recently,
it was demonstrated that oleanolic- and ursolic-containing
fraction from Baccharis uncinella leaves eliminated the
promastigote and amastigote forms of Leishmania
(Leishmania ) amazonensis and L . (Viannia ) braziliensis
without causing toxic effects for J774 macrophages. Thus,
the aim of the present work was to characterize the therapeutic
effect of the triterpenic fraction in L . (L .) amazonensis infected BALB/c mice. Oleanolic- and ursolic acidcontaining fraction was extracted from B. uncinella leaves
using organic solvents and chromatographic procedures. L.
(L .) amazonensis -infected BALB/c mice were treated
intraperitoneally with triterpenic fraction during five
consecutive days with 1.0 and 5.0 mg/kg of triterpenic
fraction, or with 10.0 mg/kg of amphotericin B drug. Groups
of mice treated with the triterpenic fraction, presented with
decreased lesion size and low parasitism of the skin—both of
E. S. Yamamoto : B. L. S. Campos : M. D. Laurenti :
C. E. P. Corbett : L. F. D. Passero (*)
Laboratório de Patologia de Moléstias Infeciosas, Departamento de
Patologia da Faculdade de Medicina, da Universidade de São Paulo,
Av. Dr. Arnaldo, 455Cerqueira César 01246-000 São Paulo, Brazil
e-mail: felipepassero@yahoo.com.br
J. H. Lago
Departamento de Ciências Exatas e da Terra, Universidade Federal
de São Paulo, 09972-270 Diadema, São Paulo, Brazil
S. d. S. Grecco
Centro de Ciências Naturais e Humanas, Universidade Federal do
ABC, 09210-180 Santo André, São Paulo, Brazil
which were associated with high amounts of interleukin-12
and interferon gamma. The curative effect of this fraction was
similar to amphotericin B-treated mice; however, the final
dose, required to eliminate amastigotes, was lesser than
amphotericin B. Moreover, triterpenic fraction did not cause
microscopic alterations in liver, spleen, heart, lung, and kidney
of experimental groups. This work suggests that this fraction
possesses compounds that are characterized by leishmanicidal
and immunomodulatory activities. From this perspective, the
triterpenic fraction can be explored as a new therapeutic agent
for use against American Tegumentar Leishmaniasis.
Introduction
Leishmaniasis is an infectious disease caused by a protozoal
parasite belonging to the Leishmania genus (Silveira et al.
2009). In the New World, there are two subgenera, and
different species are capable of infecting humans and animals,
causing visceral and cutaneous leishmaniasis (Rittig and
Bogdan 2000; Campos et al. 2008). This diversity among
species has led to the development of a variety of cutaneous
lesions, ranging from a single ulcer that is able to heal
spontaneously, to numerous lesions that affect the cutis and
mucosa, and which do not spontaneously heal, consequently
requiring specific treatments. Although different species cause
the disease and induce distinct lesion phenotypes, the specific
treatment used to target the disease is based on pentavalent
antimonials and, as the second-line form of the drug,
amphotericin B (Santos et al. 2008; Sundar and Chakravarty
2013).
Pentavalent antimony is likely reduced in vivo to trivalent
antimonials, which is more toxic for promastigote and
amastigote forms of the parasite (Frézard et al. 2009).
Parasitol Res
Although this treatment has long been used, the mechanism of
action is still regarded as controversial, and some studies have
indicated that different biochemical pathways of the parasite
can be affected. It is possible that the inhibition of ATP
detected in Leishmania sp. at the intracellular level can be a
direct result of antimony interference in glycolysis. Moreover,
some alterations in the transporters of nucleosides may also
result from the binding of antimony to the ribose moiety,
forming stable complexes with adenine and thus interfering
with the nucleoside transporters (Aït-Oudhia et al. 2009).
Antimonials also seem to interfere with enzymes associated
with DNA replication, such as DNA topoisomerase (Walker
and Saravia 2004). Amphotericin B is a polyene macrolide
antibiotic, which is recommended when antimonial treatment
fails. This antibiotic has a great affinity for ergosterol, the
major cell membrane sterols present in Leishmania parasites
(Saha et al. 1986). Thus, amphotericin B forms a complex
with membrane sterols and alters their permeability through
the uncontrolled loss of ions by forming barrel-shaped
transmembrane pores, leading to cell lysis (Mishra et al.
2007).
In spite of the undeniable leishmanicidal activity observed
among both drugs, patients frequently present with many side
effects during treatment. These side effects include local pain
and systemic effects such as nausea, vomiting, weakness,
myalgia, abdominal colic, diarrhea, skin rashes,
hepatotoxicity, nephrotoxicity, and cardiotoxicity (Rath et al.
2003); each of these undesired effects leads patients to
abandon treatment. Therefore, it is crucial that compounds
with lesser toxicity profiles and stronger leishmanicidal effects
be found.
Plants possess diverse secondary metabolites that display a
great array of pharmacological activity, with the potential to be
used against leishmaniasis (Brenzan et al. 2007; Passero et al.
2013). The Baccharis genus (Asteraceae) is characterized by
a broad array of microbicidal activity (Muelas-Serrano et al.
2000), which can be associated with the diversity of its
compounds, such as triacontane derivatives, diterpenoids,
triterpenoids, flavonoids, and chromenes (Bohlmann et al.
1979; Labbe et al. 1986; Zdero et al. 1988; Jarvis et al.
1991; Verdi et al. 2005). Studies have shown that the
flavonoids, lactone and triterpenoids, from Baccharis
dracunculifolia and Baccahris retusa (Silva et al. 2009;
Grecco et al. 2012) were active against different forms of
promastigote and amastigote Leishmania sp. pointed out for
the chemotherapeutical potential of compounds present in this
genus.
Recently, Passero et al. (2011) demonstrated that the
fraction containing the triterpenes (oleanolic and ursolic acids)
purified from B . uncinella was not toxic for the J774
macrophages and presented activity against the amastigote
forms of Leishmania (Leishmania ) amazonensis and
Leishmania (Viannia ) braziliensis . Furthermore, the
incubation of infected macrophages treated with high
triterpene fraction concentrations increased the production of
nitric oxide, suggesting that in addition to having a direct
effect on the amastigote forms, these triterpenes triggered a
possible immunostimulatory mechanism within the infected
cells. Thus, the aim of the present work was to analyze the
therapeutic effect of this fraction on the treatment of
experimental American Tegumentar Leishmaniasis caused
by L. (L.) amazonensis.
Materials and methods
General
Silica gel (230–400 mesh; Merck Corporation, Whitehouse
Station, NJ, USA) was used for column chromatographic
separation, while silica gel 60 PF254 (Merck Corporation)
was used for analytical (0.25 mm) thin layer chromatography
(TLC). All chemicals employed were of analytical reagent
grade. Nuclear magnetic resonance (NMR) spectra
(500 MHz) were recorded on a Bruker model DRX-500
NMR spectrometer (Bruker Corporation, Rheinstetten,
Germany) with chloroform-d (CDCl3) as a solvent and
tetramethylsilane (TMS) as the internal standard. Chemical
shifts (δ) were recorded in parts per million (ppm) relative to
TMS.
Plant material
Aerial parts of Baccharis uncinella DC. were collected in the
“Campos de Altitude” region of the Campos do Jordão, São
Paulo, SP, Brazil, in June 2005. The plant was authenticated
by Dr Oriana A. Fávero, and a voucher specimen was
deposited at the herbaria of the Prefeitura Municipal de São
Paulo (PMSP) with the reference number PMSP8983.
Extraction and fractionation
The dried and powdered aerial parts of B. uncinella (430 g)
were exhaustively extracted with MeOH (5×1 L) at room
temperature, and the resulting solutions were filtered and
concentrated under vacuum to obtain the MeOH extracts
(25.1 g). This crude extract was dissolved in MeOH/H2O
(1:2 v/v; 500 mL) and partitioned with n-hexane. After the
parts were dried over Na2SO4 and the solvent was evaporated
under reduced pressure, 18 g of the n-hexane phase were
obtained. Part of this material (8.3 g) was subjected to SiO2
column chromatography and was eluted with increasing
amounts of EtOAc in n-hexane to give 77 fractions, which
were then pooled together in six groups (I–VI) after TLC
analysis. Group IV was composed of a triterpenic fraction
Parasitol Res
(715 mg), which were characterized as oleanolic and ursolic
acids.
Animals and parasites
Eight-week-old male BALB/c mice obtained from the Animal
Facility of the School of Medicine of São Paulo University
(FMUSP), Brazil, were maintained in the laboratory during
the experiments, in accordance with the institutional
guidelines regarding the welfare of experimental animals
and with the approval of the Animal Ethics Committee of
São Paulo University (322/12).
L . (L.) amazonensis (MHOM/BR/1973/M2269) parasites
were isolated from a patient in Pará state, north of Brazil, who
presented with anergic diffuse leishmaniasis. The parasite was
identified using monoclonal antibodies and isoenzyme
electrophoretic profiles at the Leishmaniasis Laboratory of the
Evandro Chagas Institute (Belém, Pará state, Brazil). This
parasite was grown in Roswell Park Memorial Institute-1640
medium—RPMI 1640 (Gibco®, Life Technologies, Carlsbad,
CA, USA), supplemented with 10 % heat-inactivated fetal
bovine serum, 10 μg/mL of gentamicin, and 1,000 U/mL of
penicillin (R10) at 25 °C. Promastigote forms in the stationary
phase of growth were used.
Infection and treatment
BALB/c mice were infected subcutaneously into the hind
footpad with 106 promastigote forms of L. (L.) amazonensis.
Fifteen days after infection, the groups of mice were treated
intraperitoneally during five consecutive days with 1.0 mg/kg
and 5.0 mg/kg of triterpenic fraction purified from B. uncinella.
In addition, one group was treated with 10 mg/kg of
amphotericin B using the same protocol of triterpene treatment.
Control mice (infected) were injected intraperitoneally only with
vehicle solution (phosphate-buffered saline; PBS). Healthy mice
were injected subcutaneously and intraperitoneally with PBS. At
5 weeks postinfection (PI), animals were killed, and the skin
parasitism, cellular immune response, and histological alterations
of the internal organs (heart, lung, liver, spleen, and kidneys)
were analyzed.
Parasite load
The parasite load in the skin lesion was determined using the
quantitative limiting-dilution assay, as previously described
(Passero et al. 2010). Briefly, the infected footpad fragments
from the different groups were aseptically excised and were
homogenized in Schneider’s medium. The cellular
suspensions were subjected to 12 serial dilutions with four
replicate wells. The number of viable parasites was
determined based on the highest dilution that promastigotes
could be grown after 10 days of incubation at 25 °C.
Analysis of cellular immune response
The popliteal lymph nodes of all groups were collected and
macerated, and the cells were counted under Trypan blue
exclusion. The cell suspensions (2×105/well) were cultured
in R10 under stimulation with 5.0 μg of whole antigen of L.
(L.) amazonensis or 1.0 μg of concanavalin A, which was
used as a positive control. After 72 h, the supernatants of the
different groups were collected, and the amounts of
interleukin (IL)-4, IL-12, and interferon gamma (IFN-γ)
(BD, Franklin Lakes, NJ, USA) were quantified by sandwich
enzyme-linked immunosorbent assay in accordance with the
manufacturer’s recommendations.
Statistical analysis
The results were expressed as the mean ± standard deviation
of three independent experiments and the nonparametric
Mann-Whitney U test was used to compare lesion size,
parasite load and cytokine production between the groups.
Differences were considered statistically significant at a 5%
significance level (p < 0.05). GraphPad Prism 5 (GraphPad
Software, Inc., La Jolla, CA,USA) was used to analyze the
results. Differences were considered statistically significant
when p< 0,05.
Results
Nuclear Magnetic Resonance
The n-hexane phase of the MeOH extract from the aerial
parts of B . uncinella was subjected to several
chromatographic steps to yield a fraction that was analyzed
by TLC, revealing one homogeneous spot. Its 1H-NMR
spectrum (CDCl3) displayed several overlapping signals at
δ2.3–0.8, which were characteristic of aliphatic hydrogens, as
well as two signals at δ 3.3 and 2.6, which were characteristic
of allylic H-18 hydrogen of oleanane/ursane triterpenoids
(Kontogianni et al. 2009). Analyzing the integration values
of these signals, it is possible to establish that the proportion
of oleanane/ursane is 6:1. The 13C and DEPT 135° NMR
spectra of the mixture revealed differences in the chemical
shifts of the olefinic carbons C-12 (δ123.2/125.8) and C-13
(δ144.8/138.0), as well as differences in the signals attributed
to the methyl groups C-18 (δ42.6/52.7), C-19 (δ47.3/38.7),
and C-20 (δ 31.4/38.4). Therefore, comparisons of these
NMR data with those reported in the literature (Seebacher
et al. 2003) allowed for the identification of oleanolic and
ursolic acids in the triterpenic fraction from the aerial parts of
B. uncinella.
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Lesion size, parasite load, and histological alterations
The infected group presented with an increased lesion size
between 2 and 5 weeks PI, although mice treated with 5.0 mg/
kg of the triterpenic fraction showed smaller sized lesions
from 2 to 5 weeks PI (P <0.05), while mice treated with
1.0 mg/kg of the triterpenic fraction presented with a moderate
lesion size from 2 to 5 weeks PI (P <0.05). Treatment with
amphotericin B also inhibited the growth of cutaneous lesions
in mice (Fig. 1).
The parasitism in mice treated with 1.0 and 5.0 mg/kg of
the triterpenic fraction and 10 mg/kg of amphotericin B
presented with lesser skin parasitism when compared to the
infected and untreated group. In addition, the group treated
with 5.0 mg/kg of the triterpenic fraction presented with a
significantly reduced parasite load in the skin compared to
groups treated with 1.0 mg/kg of the triterpenic fraction and
amphotericin B (Fig. 2).
In addition, treatment with 1.0 and 5.0 mg/kg of the
triterpenic fraction did not alter the architecture of the liver,
heart, lungs, and kidneys when compared to infected mice as
showed in Fig. 3. In addition, it was verified that the treatment
with 1.0 and 5.0 mg/kg of the triterpenic fraction increased the
regions of white pulp when compared to infected infected
mouse spleen, suggesting an immunomodulatory action of
triterpenic acids.
Analysis of immune response in BALB/c mice
BALB/c mice treated with 1.0 and 5.0 mg/kg of the triterpenic
fraction presented with significant increases in IL-12 (Fig. 4b)
and IFN-γ (Fig. 4a) levels as compared to mice treated with
amphotericin B and the control groups (P <0.05). The levels
of IL-4 (Fig. 4c) observed in the triterpenic fraction-treated
groups were similar to those of infected mice; however,
Fig. 1 Lesion developments in mice subjected to treatment with 1.0 and
5.0 mg/kg of the triterpenic fraction purified from B. uncinella leaves.
*P <0.05 compared to the lesion size of mice treated with the triterpenic
fraction and amphotericin B. **P <0.05 compared to the lesion size of
mice treated with amphotericin B and 5.0 mg/kg of the triterpenic fraction
Fig. 2 Analysis of the parasite load in the skin of the different
experimental groups. *P <0.05 compared to skin parasitism of the
infected group; •P <0.05 compared to skin parasitism of the groups
treated with 1.0 mg/kg of triterpene and amphotericin B
treatment with amphotericin B caused a decrease in the levels
of IL-4 compared to infected mice (P <0.05).
Discussion
A previous study conducted by Passero et al. (2011)
demonstrated that a fraction containing oleanolic and ursolic
acids was not cytotoxic for J774 macrophages, and it
eliminated the intracellular amastigote of L. (L.) amazonensis
and L . (V.) braziliensis with the participation of nitric oxide.
In the present manuscript, the therapeutic efficacy of this
fraction was evaluated in vivo using a murine model of
American Tegumentar Leishmaniasis.
The group of infected mice that received treatment with 1.0
or 5.0 mg/kg of the triterpenic fraction demonstrated an
inhibition of lesion size, which was accompanied by the
elimination of skin parasitism. This effect can be associated
with the direct effect of oleanolic and ursolic acids on the
promastigote and amastigote forms of L. (L.) donovani, L.
(L .) amazonensis , and L . (V.) braziliensis , as previously
indicated (Peixoto et al. 2011; Passero et al. 2011; Musayeib
et al. 2013). Although these studies examined the
antileishmanial activity of triterpenes in vitro, none of the work
to date has characterized the therapeutic potential of these
triterpenes in leishmaniasis in vivo. Therefore, oleanolic and
ursolic acids triterpenes possess therapeutic effects, which were
similar to those found after administering amphotericin B.
Although similar protective effects had been detected in the
triterpene- and amphotericin B-treated groups, the total dose
administrated to decrease parasitism was different, as
exemplified by the fact that in order to inhibit skin parasitism
to 86 %, a total dose of 1.25 mg of amphotericin B needed to
be injected in mice, while to inhibit approximately 93 % of the
parasitism, a total dose of 0.625 mg of the triterpene fraction
was required (in mice treated with 5.0 mg/kg of triterpenic
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Fig. 3 Histological analysis of spleen, liver, heart, lung, and kidney of mice submitted to the treatment with triterpenic fraction. Magnification: ×20
fraction). These findings indicated that the most effective or
selective compounds used to treat cutaneous leishmaniasis
will demand lower concentrations and/or fewer cycles of drug
administration. In addition, mice treated with the triterpenic
fraction did not present with any alterations to their heart,
lungs, spleen, liver, or kidneys. This result was expected, as
previous works have demonstrated that oleanolic and ursolic
acids are nontoxic among experimental animals and humans
(Liu 1995; Liu 2005). In addition, these triterpenes have
been used as a component in a number of agents, serving
Fig. 4 IFN-γ (a ), IL-12 (b ), and IL-4 (c ) levels produced by
mononuclear cells from infected mice, as well as from mice receiving
1.0 mg/kg of the triterpenes, 5.0 mg/kg of the triterpenes, and
amphotericin B. *P <0.05 compared to the cytokine production of the
infected group; •P <0.05 compared to the cytokine production of the
groups treated with 1.0 and 5.0 mg/kg of the triterpenes
Parasitol Res
different purposes in Asiatic countries (reviewed in Liu 1995),
highlighting the possibility that this class of compounds might
be of interest in a number of drug developments (Ferreira et al.
2010). Studies in the area of oncology also demonstrated that
oleanolic and ursolic acids are highly selective for cancer cells
when compared to normal cells (Lúcio et al. 2011; Sannigrahi
et al. 2012), reinforcing the absence of toxicity of these
triterpenes to mammals.
In leishmaniasis, an elevation in the Th1 cytokines is
essential for eliminating intracellular amastigotes and for
developing resistance, as IL-12 can trigger the differentiation
and activation of the subset of CD4+ T-lymphocytes that
secrete IFN-γ, which, in turn, activate the infected
macrophages, consequently eliminating intracellular parasites
(Rocha et al. 2007; Ota et al. 2008; Kaur et al. 2008). After
treatment with the triterpene fraction, high amounts of both of
these cytokines could be detected, suggesting that in addition
to the direct effect they exert on Leishmania parasites, these
compounds could modulate immune cells to produce high
concentrations of these beneficial cytokines (Masihi 2003).
It is possible that these triterpenes, once administered in
infected mice, can alter some immunological pathways and
increase Th1 cytokines.
Conversely, treatment with amphotericin B did not
stimulate the production of IL-12 and IFN-γ; however,
diminished concentrations of IL-4 were verified after
treatment. The IL-4 cytokine is associated with the Th2
immune response and, consequently, with disease
progression (Nakaya et al. 2011; Passero et al. 2012;
Carvalho et al. 2012). Therefore, the low levels of IL-4
observed among amphotericin B-treated mice may be
important in reducing the pathology, which was mediated
by the amastigote forms. In triterpene-treated and infected
groups, similar levels of IL-4 have been found; however, the
presence of this cytokine did not interfere with the production
of IL-12 and IFN-γ. In addition to their leishmanicidal effect,
these data indicate that oleanolic and ursolic acids can
beneficially modulate the immunological response of
BALB/c mice.
In this study, we demonstrated that the fraction containing
oleanolic and ursolic acids inhibited the development of skin
lesions, decreased skin parasitism, and increased levels of IL12 and IFN-γ in infected BALB/c mice. In addition, any
toxic effects were verified, suggesting that oleanolic and
ursolic acids can be studied as a prototype drug in
the development of new pharmaceuticals used against
leishmaniasis and other neglected diseases since, in this
experimental study, the fraction was more effective than
amphotericin B.
Acknowledgments This work was supported by grant number 2012/
03903-9 from the São Paulo Research Foundation and HCFMUSPLIM50.
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