Advances in Bioscience and Biotechnology, 2013, 4, 304-315 ABB
http://dx.doi.org/10.4236/abb.2013.42A041 Published Online February 2013 (http://www.scirp.org/journal/abb/)
Seco-limonoid 11α,19β-dihydroxy-7-acetoxy-7-
deoxoichangin promotes the resolution of Leishmania
panamensis infection
Diana Granados-Falla1,2, Carlos Coy-Barrera3, Luis Cuca3, Gabriela Delgado1*
1Immunotoxicology Research Group, Department of Pharmacy, Sciences Faculty, Universidad Nacional de Colombia Bogotá, Bo-
gotá, Colombia
2Biotechnology Doctorate Program, School of Sciences, Universidad Nacional de Colombia, Medellin, Colombia
3Natural Products Group, School of Sciences, Universidad Nacional de Colombia, Bogotá, Colombia
Received 3 January 2013; revised 3 February 2013; accepted 10 February 2013
ABSTRACT
The high morbidity generated by the infection caused
by parasites of the genus Leishmania, make of this
infection into one of the vector-borne infectious dis-
eases most relevant worldwide, which added to the
fact that the drugs used for its treatment are far from
be optimal and considering that prophylactic appro-
aches (such as the development of a vaccine) still
seems far from being achieved, make of the search for
new therapeutic alternatives for safe and effective
treatment of this disease one of the most accurate ap-
proaches to the control of this disease. In this study
we evaluated the antileishmanial and immunomodu-
latory activity of the compound 11α,19β-dihydroxy-
7-acetoxy-7-deoxoichangin (a seco-limonid molecule)
through: 1) evaluation of its cytotoxicity over pro-
mastigotes and axenic amastigo tes of L. (V) panamen-
sis, 2) determination of its ability to induce the con-
trol of in vitro infection, using infected murine cells
(J774.2) and human dendritic cells (hDCs), 3) quan-
tifying the levels of pro-inflammatory cytokines, (iv)
evaluating the expression of cell markers associated
with hDCs maturation, and (v) determinating the
production of nitric oxide free radicals (NO). In this
regard, this seco-limonoid exhibited an antileishma-
nial activity represented in the reduction of in vitro
infection in J774.2 cells and hDCs, with a EC50 of 7.9
µM (4.48 µg/mL) and 25.5 µM (14.39 µg/mL), respec-
tively, and additionally, we observed an increase on
the production of IL-12p70, TNF-α and NO, as also,
in the number of hDCs HLA-DR-positive in treated
infected hDCs. These findings suggest that anti-leish-
manial activity of this compound could be associated
with the potential “reactivation” of phagocytic cell
that is “paralyzed” by the infection, generating an
immune phenotype associated with protection.
Keywords: Leishmaniasis; Treatment;
Immunomodulation; Seco-Limonoid
1. INTRODUCTION
Leishmaniasis is a parasitic disease of global public
relevance, which is transmitted by the bite of sandfly
species belonging to the genus Lutzomyia or Phleboto-
mus, infected with promastigotes of the genus Leishma-
nia. According to reports getting for the World Health
Organization (WHO), the transmission of the pathogens
responsible for this disease occurs endemically in at least
98 countries, primarily affecting developing countries
located in tropical and subtropical regions around the
world [1,2]. Annually, at least 1.5 million of new cases of
the cutaneous form of the disease (CL) and approxima-
tely 500,000 new cases of the visceral leishmaniasis (VL)
are reported around the world, being the VL form the
clinical presentation responsible for the deaths associated
with the pathogen and the CL form the responsible of its
high levels of morbidity [3].
Chemotherapy against leishmaniasis is based on the
administration of pentavalent antimonial salts (as first-
choice drugs) or the use of formulations of pentamidine®,
pa ro momyc i n ®, miltefosine® or amphotericin B®, as
second-choice drugs. However, the mechanism of action
of these drugs has not been well clarified yet, and this
added to the fact that none of them was originally de-
signed for the treatment of this disease and limitations
associated with their administration routes [4,5], high
costs and duration of the treatment, the emergence of
parasites resistant to these drugs [3,6] and the high toxic-
ity of some formulations, make necessary and urgent the
develop of safer and more effective therapeutic alterna-
*Corresponding author.

D. Granados-Falla et al. / Advances in Bioscience and Biotechnology 4 (2013) 304-315 305
tive to the better control this disease [1,7]. Among the
elucidated to date in relation to the mechanism of action
postulated for the drugs mentioned above, it have been
found that the formulations of pentavalent antimony salts
act as pro-drugs, needing for that, their reduction to tri-
valent form to perform their action, which is proposed to
be directed against the trypanothione reductase and zinc-
finger protein (proteins that are related with the protec-
tion of the parasites from oxidative damage and toxic
heavy metals, and with the process of replication and
repair of DNA, respectively) [8,9]. On the other hand,
the mechanism of action of pentamidine (second drug of
choice for the treatment of this disease, used when pa-
tients cannot tolerate the antimonial drugs, or in cases of
resistance to these drugs) is associated with the accumu-
lation and altering of the function of the kinetoplast, as
well as the collapse of the membrane potential of the
microorganism [10,11], while the mechanism of action of
amphotericin B (a macrolide used for treatment of mu-
cocutaneous and visceral leishmaniasis) is associated
with its capability to bind to the ergosterol molecule (the
major sterol of the Leishmania parasite membrane), lead-
ing to the formation channels that would alter the per-
meability of the cell membrane [12].
Recently, the study of compounds derived from plant
species has become in a major tool for the developed of
novel therapeutic agents against several infectious dis-
eases, such as leishmaniasis [13-15], and in this regard,
several research groups have screened a large number of
natural compounds, with the aim of find potential che-
mical entities (prototypes) that can be used for the de-
velopment of more effective and safer alternative thera-
pies against these pathogens [16-18].
In previous studies, our research group demonstrated
that the compound 11α,19β-dihydroxy-7-acetoxy-7-de-
oxoichangin (a seco-limonoid) exhibits an antileishma-
nial specific activity against the intracellular form of the
parasite (corresponding to the most relevant stage for
studying and designing possible therapeutic targets
against this pathogen) [19,20]. This compound was iso-
lated from the vegetal species Raputia heptaphylla, a spe-
cies belonging to the family Rutaceae, and whose geo-
graphical distribution is concentrated in tropical areas of
Central and South America (mainly reports its location in
Costa Rica, Panama, Colombia, and forested areas of
Brazil and Peru). Species belonging to the genus Rapu-
tia have been characterized by the present of abundant
secondary metabolites (primarily alkaloids and limonoid),
being an example of this, the studies carried out in Rapu-
tia prateermisa (other species of the genus Raputia),
whose leaves have been used in decoction for treatment
of cutaneous leishmaniasis and chagas disease in selvatic
areas of Brazil, and whose phytochemical study showed
that limonoids compounds are an important part of its
composition to the secondary metabolites level [21], be-
ing them possibly responsible for some of the biological
activities attributed to these plants.
In this study, we sought to confirm the antileishmanial
activity of this seco-limonoid using for in vitro assays,
murine macrophages and human dendritic cells (hDCs)
infected with this protozoan, as also, we wanted to char-
acterize its possible immunomodulatory effect on anti-
gen-presenting cells (APCs), which are a critical popula-
tion for the resolution of natural infections by parasites of
the genus Leishmania, observing that the compound pre-
sents an antileishmanial activity associated with an im-
munomodulatory effect, that it is evident by the differen-
tial induction on the increase in the production of soluble
pro-inflammatory mediators, such as IL-12p70 and TNF-
α, and a significant increase in the expression of MHC
class II molecules as also in the levels of nitric oxide
(NO) on infected cells exposed to the seco-limonoid
compared with infected cells without treatment.
2. MATERIALS AND METHODS
2.1. Cells
RPMI 1640 (Gibco BRL-Life Technologies Inc., Grand
Island, NY, USA) medium was used for culturing the
phagocytic cell line J774.2 (a murine macrophage cell
line derived from the murine J774A.1 cells, which are
monocyte/macrophage cells, obtained from reticulum
cell sarcoma of ascites in BALC/c mouse, which are sus-
ceptible to the infection with Leishmania parasites), hu-
man dendritic cells (hDCs) derived from peripheral
blood monocytes (obtained from healthy volunteers) and
the promastigotes of Leishmania (Viannia) panamensis
(L. (V) panamensis), which were either untransfected
(MHOM/88CO/UA140) or transfected (MHOM/88/CO/
UA140irGFP) with a plasmid (ir) encoding green fluo-
rescent protein (GFP) (kindly donated by Dr. Sara
Robledo from the Program for the Study and Control of
Tropical Diseases; PECET-Programa de Estudio y Con-
trol de Enfermedades Tropicales, of the Universidad de
Antioquia, Colombia [22]). The RPMI medium was sup-
plemented with 2 mM L-glutamine (Gibco BRL-Life
Technologies Inc.), 1% non-essential amino acids, 1000
U/ml penicillin, 0.1 mg/ml streptomycin, 0.25 ug/ml
amphotericin B (Sigma Chemical Co., St. Louis, MO,
USA), 24 mM sodium bicarbonate (Sigma Chemical
Co.), and 25 mM HEPES (Gibco BRL-Life Technologies
Inc.). The culture media were enriched with 10% fetal
bovine serum (FBS) (Microgen, Bogotá, Colombia) for
the J774.2 cells and parasites or with AB negative human
plasma that was filtered and inactivated (Lonza Walkers-
ville, MD, USA) for the culture of hDCs.
The mammalian cells were incubated at 36˚C - 37˚C in

D. Granados-Falla et al. / Advances in Bioscience and Biotechnology 4 (2013) 304-315 306
an atmosphere of 5% CO2 and 90% humidity, whereas
the promastigotes of L. (V) panamensis were grown at
25˚C - 26˚C.
The axenic amastigotes were produced from the cul-
ture of promastigotes of L. (V) panamensis in RPMI me-
dium enriched with 20% FBS at pH 5.4, and differentia-
tion was achieved by the gradual exposure of the cells to
increases in temperature (29˚C, 32˚C, and 34˚C).
It should be note that depending on the assay, the
monocytes used to differentiate hDCs were derived from
2 different sources of peripheral blood taken, either of
them, from healthy donors. For the in vitro infection as-
says, the monocytes were obtained by leukopheresis and
density gradient centrifugation of a single donor sample
(that was screened for HIV-1, hepatitis B, hepatitis C,
HTLV-1 and HTLV-2) (Lonza Walkersville, MD, USA),
whereas to evaluate the compound-induced immuno-
modulation, the monocytes were obtained from he-
parinized blood samples from healthy volunteers without
and with a medical history of active cutaneous presenta-
tion of the disease prior the treatment of the disease (six
(6) and seven (7) individuals, respectively), and whose
samples were taken after the explanation and subsequent
signing of the informed consent (approval by the Ethics
Committee, act 04 of June 2009 at the Faculty of Sci-
ences of the Universidad Nacional de Colombia).
Briefly, for the monocytes derived from blood of vol-
unteers without and with a medical history of leishmani-
asis, the samples were centrifuged at 2500 rpm for 15
minutes, and the leuko-platelet layer (buffy coat) was
collected and diluted in RPMI-1640 (Gibco BRL-Life
Technologies Inc.). Subsequently, the cell suspension
was used to prepare the density gradient by adding 1
volume of the ficoll-hypaque reagent (Invitrogen, USA)
to 2 volumes of diluted blood sample, and the gradients
were centrifuged for 30 minutes at 3000 RPM at room
temperature, producing a layer of peripheral blood
mononuclear cells (PBMCs), which was collected and
washed by centrifugation at 2000 RPM for 10 minutes
and at 2000 RPM for 7 minutes. Then, the PBMCs were
cultured for 2 h at 37˚C in sterile Petri dishes (TPP,
Techno Plastic Products, Switzerland) using the previ-
ously described culture medium. Ended the incubation
time, the non-adherent cells were removed, and the ad-
herent cells were incubated in RPMI-1640 medium sup-
plemented with recombinant human GM-CSF at 1ng/mL
(BD Biosciences, San Diego, USA) and human recom-
binant IL-4 at 1 ng/mL (BD Biosciences) for 5 - 7 days.
The differentiation process of the hDCs, was monitored
by evaluation of the cell morphology using an inverted
microscope and the purity of hDCs was evaluated using
flow cytometry. Finally, the yield and the cell viability
were determined using trypan blue dye staining, and the
cells were counted in a Neubawer chamber [23].
2.2. Compound
The plant material from the bark of Raputia heptaphylla
used in this study was collected in Albán (Cundinamarca)
in Colombia (a specimen of this species rests in the Na-
tional Herbarium of Colombia, Institute of Natural Sci-
ences, Universidad Nacional de Colombia, under the
code COL. 511102). Briefly, to obtain the crude ethano-
lic extract, the dried plant material was subjected to ex-
traction by percolation in 96% ethanol at room tempera-
ture, and the organic solvent was removed by distillation
under reduced pressure in a rotavapor. Next, the metabo-
lites were extracted from the ethanolic extract, and the
purification of the seco-limonoid was performed using
conventional chromatographic methods [18]. To eluci-
date the structure of this seco-limonoid compound (Fig-
ure 1(a)), spectroscopic methods were used (including
ultraviolet, infrared (IR), nuclear magnetic resonance
(NMR) of hydrogen and carbon, circular dichroism, and
nuclear Overhauser enhancement spectroscopy (NOESY)
experiments), as also data reported in the literature of
related seco-limonoid.
Furthermore, the extracts, fractions, and the com-
pounds isolated from the plant material as well, the cul-
ture medium used were subjected to evaluation for the
presence of endotoxins using the LAL test and the re-
agent Pyrogent™ Plus Gel Clot LAL assay (Lonza
Walkersville, MD, USA). This procedure was performed
to ensure that any functional regulation of the hDCs in-
fected with L. (V) panamensis and treated with the com-
pound was due to the treatment and not to the presence
of contaminating lipopolysaccharide (LPS) in the mate-
rial. For this purpose, the experimental procedures were
made according to the instructions recommended by
Lonza.
2.3. Assays to Evaluate the Effects of the
Cytotoxic Activity on Mammalian
Phagocytic-Cell Targets of Infection with
L. (V) panamensis
Using the resazurin test for cell metabolism detection,
the toxicity of the seco-limonoid was evaluated accord-
ing to its ability to damage murine macrophage cells
(J774.2) and hDCs [24]. Briefly, 2 × 104 J774.2 cells or 3
× 104 hDCs were exposed to different concentrations of
the compound [sequential 1:4 dilutions were used, using
as maximum concentration 354.5 μM (200 μg/mL) and a
minimum of 22 μM (12.5 ug/mL)] for 72 h at 36˚C -
37˚C in an atmosphere containing 5% CO2 and 90% hu-
midity. The controls included cells exposed to the re-
agents that were used to solubilize the seco-limonoid
[dimethylsulfoxide (DMSO), ethanol, and chloroform]
and cells cultured in the absence of the compound. After
the 72 h of incubation period, the resazurin solution was

D. Granados-Falla et al. / Advances in Bioscience and Biotechnology 4 (2013) 304-315 307
(a) (b)
Figure 1. (a) correspond to the seco-limonoid compound struc-
ture (11α,19β-dihydroxy-7-acetoxy-7-deoxoichangin-C28H36
O12, 565.2285) and (b) shows the cytotoxic activity of the
seco-limonoid on monocyte/macrophage cells derived from
mice-J774.2 cells (open circles) and hDCs (filled circles). The
graph depicts the average percentage viability ± SEM of the
cells exposed to different concentrations of the compounds.
added at a final concentration per well of 44 μM (Sigma-
Aldrich), and after 4 h of incubation at 36˚C - 37˚C, the
plates were read using a spectrofluorometer at λexc 535
nm and λemi 590 nm (Tecan, Genios, Salzburg, Austria).
The lethal concentration 50% (LC50) of the seco-limo-
noid which corresponds to the concentration at which
50% of the cells die, was determined using the statistical
software package GraphPad Prism version 5.00 Demo
(GraphPad Software, USA). Each assay was performed
in duplicate in at least three independent experiments.
2.4. Antileishmanial Activity E valuation
2.4.1. Antileis hmanial Activi ty a gainst P romastigotes
of L. (V) panamensis
The in vitro antileishmanial effect of seco-limonoid on
the extracellular flagellar form of the parasite was evalu-
ated by culturing promastigotes of L. (V) panamensis
(expressing GFP) in the presence of different concentra-
tions of the compound, using for this experiment, seco-
limonoid concentrations lower than the LC50 (that had
previously been determined in cytotoxicity assays over
cells of mammals). Briefly, 2 × 105 promastigotes were
incubated for 72 h at 25˚C - 26˚C in the presence of
106.3 μM (60 μg/mL), 26.6 μM (15 μg/mL), 6.6 μM
(3.75 μg/mL), 1.1 μM (0.63 μg/mL), 0.55 μM (0.31
μg/mL) or 0.28 μM (0.16 ug/mL) of seco-limonoid. As a
positive control for the direct cytotoxicity of the com-
pound against the extracellular form of the parasite, the
promastigotes were exposed to different concentrations
of pentamidine isethionate (Pentacarinat®, Sanofi-Av-
entis, France) at 1:4 dilutions starting at a maximum
concentration of 5 μg/mL to a minimum concentration of
1 × 10−5 μg/mL, using the same experimental conditions
as those used in the evaluation tests of the activity of the
compound. Next, the viability of the parasites was de-
termined by flow cytometry (FacsCantoII) (BD Biosci-
ences) and resazurin metabolic assay (Sigma Chemical
Co.), where the reduction in the fluorescence emission
from the promastigotes and the decrease in their meta-
bolic activity, was associated with non-viable parasites.
2.4.2. Antileishmanial Activity agains t the
Intracellular Form of the Parasite
The antileishmanial effect on intracellular amastigotes
was determined by in vitro APC infection assays using
J774.2 cells and hDCs. 2 × 105 cells were infected with
promastigotes of L. (V) panamensis (constitutively ex-
pressing GFP) at ratios between 1:40 and 1:50 (cell per
parasites) for 6 h, time after which the non-internalized
parasites were removed by washing with RPMI-1640
medium, and once the infection was established, the cells
were exposed to various concentrations of seco-limonoid
[consecutive 1:4 dilutions were used starting from a
maximum concentration of 53.2 μM (30 μg/mL) down to
a minimum concentration of 0.81 μM (0.47 μg/mL)] for
48 h at 36˚C in an atmosphere of 5% CO
2. Similarly,
infected cells treated with 2000 μg/mL, 500 μg/mL, 250
μg/mL, 125 μg/mL, 40 μg/mL, and 20 μg/mL of sodium
stiboglucon ate® (Ryan Laboratories, Colombia) were
used as positive controls for the resolution of the in vitro
infection. Completed the incubation times, the super-
natants were collected and cryopreserved at −20˚C until
they were required for quantification of the cytokines
associated with inflammatory processes. The J774.2 cells
were transferred to flow cytometry tubes to quantify the
percentage of infected cells by flow cytometry (Fac-
sCanto II, BD, San José, CA, USA), and thereby, the
fluorescence emitted by the fluorescent parasites was
captured in the green channel using a 530/30 nm filter,
and was used to distinguish the infected cells from the
uninfected population. Because of the complexity of
hDCs, clear differences among the infected cells popula-
tion and uninfected cells were not easily observed using
flow cytometry, due mainly at the characteristic auto-
fluorescence of this kind of cells, factor for which is lim-
ited the analysis using this technique; therefore, light
microscopy with slides containing Giemsa-stained cell
preparations were used to evaluate infection on hDCs.
For this assay, the protocol described above was used to
evaluate the antileishmanial activity against the intracel-
lular form of the pathogen; however, the hDCS were
placed onto the chamber slides, containing 16 wells
(Nunc, Rochester, NY, USA) instead of 96-well flat-
bottom plates. Free parasites, uninfected cells, infected
cells in the absence of the compound, and infected cells
exposed to the conventional treatment (pentavalent an-
timonials) were used as controls for each of the assays.
In these assays, reductions in the percentage of infected
cells and/or in the emitted fluorescence by the internal-
ized parasites were the parameters that were indicative of
the parasiticidal activity of the compounds. Antileishma-
nial activity against the promastigotes and intracellular

D. Granados-Falla et al. / Advances in Bioscience and Biotechnology 4 (2013) 304-315 308
amastigotes was evaluated in at least three independent
experiments.
Using these assays, the effective concentration 50
(EC50), which corresponds to the concentration that re-
duces the parasite burden by 50% in experimentally in-
fected phagocytes, was determined, and the division of
the values obtained from the cytotoxic antileishmanial
activity on the intracellular pathogens [Lethal Concentra-
tion 50 (LC50)/Effective Concentration 50 (EC50)] cor-
responds to the selectivity index (SI), a parameter related
to the selectivity and safety of the tested molecule. For
the compounds with potential leishmanicidal activity,
arbitrary SI values have been reported, considering a
value ≥ 2 to be significant.
2.4.3. Antileishmanial Activity against Axenic
Amastigotes
The evaluation of antileishmanial activity against the
axenic forms of the parasite was performed by seeding 2 -
5 × 105 axenic amastigotes per well in 96-well flat-bot-
tom plates (TPP, Switzerland) which were exposed to
different concentrations of the compound for 72 h at
36˚C, 5% CO2 and 90% humidity (using the concentra-
tions mentioned previously for the evaluation of activity
against intracellular amastigotes). Pentamidine isethi-
onate® and sodium stibogluconate® were used as treat-
ment controls, and cells that were not exposed to treat-
ment or compounds were included in the assays, as nega-
tive controls. After the incubation period, the viability of
the cells was determined using the indirect resazurin
metabolic assay (Sigma-Aldrich, St. Louis, USA), fol-
lowing the protocol described in a previous section.
2.5. Immunomodulatory Activity Assessment
2.5.1. Determination of Nitric Oxide (NO)
J774.2 cells or hDCs (4 × 105 cells) were infected at a
ratio of between 1:40 and 1:50 with promastigotes of L.
(V) panam ensis (not transfected parasites) for 6 h at
36˚C - 37˚C in an atmosphere with 5% CO2 and 90%
humidity. Once the experimental infection was estab-
lished, the non-internalized parasites were removed by
gentle washes using a sterile saline solution, followed by
treatment of the cells with the concentrations of the seco-
limonoid mentioned previously (see protocols for the
evaluation of antileishmanial activity). Sodium stiboglu-
conate® was used as control drug (as described previ-
ously). Similarly, a sample of cells was treated with 10
μg/mL phorbol myristateacetate for 24 h as a positive
control for the induction of NO, as also, a sample of cells
not exposed to any type of treatment (with and without
infection) was used to determine the basal levels of NO
production for each population. The NO production was
monitored after 48 h of treatment with the seco-limonoid
or the control drug, and latter, the cells was transferred to
flow cytometry tubes and staining with 4 μM DAF-FM
diacetate solution (Invitrogen, USA) for 1 h at 36˚C -
37˚C. Subsequently, the cells were washed, with the aim
to remove the unincorporated probe and the readings
were performed in a BD FacsCanto II flow cytometer
using the blue laser (λexc 488 nm) as the excitation source
and the 530/30 nm filter for detection.
2.5.2. Quantification of Cy tokines in
Supernatants Cultures
Briefly, 15 µL of a mixture comprising capture beads for
each of the cytokines to be evaluated (IL-6, IL-10,
MCP-1, IFN-γ, TNFα, and IL-12p70 for the J774.2 cells
or IL-8, IL-1β, IL-6, IL-10, TNF and IL-12p70 for the
hDC) was mixed with 15 µL of the PE detection reagent
and 50 µL of the culture supernatants. The mixtures were
incubated at ambient temperature for 2 - 3 h protected
from the light, and after this incubation period, the sam-
ples were washed to remove the unbound PE detection
antibody. On the other hand, using the same conditions
mentioned before, standards for each cytokine were run
simultaneously. The readings were performed using the
FacsCanto II flow cytometer (BD Biosciences, San
Diego, CA, USA) and the BD FACSDiva (BD Biosci-
ences) acquisition software. The data were analyzed us-
ing the FCAP ArrayTM (Soft Flow) software.
2.5.3. Evaluation of Cell Surface Marker Expression
For the phenotypic analysis of the hDCs, the surface
marker expression was evaluated using the following
antibodies: clone L243, (APC-conjugated anti-HLA-DR),
clone DCN46 (FITC-conjugated anti-CD209), clone
MφP9 (PerCP-conjugated anti-CD14), clone HB15e (PE-
conjugated anti-CD83), and clone L307.4 (PE-conju-
gated anti-CD80). The acquisition and analysis were
performed using a FacsCanto II (BD, Biosciences) flow
cytometer and the acquisition software FACSDiva (BD
Biosciences).
2.6. Statistical Analyses
The results from each experimental protocol were ana-
lyzed according to the parameters for each technique and
were validated by comparison with the established con-
trols for each assay. Therefore, the data obtained allowed
the determination of the lethal concentration 50 (LC50)
and the effective concentration 50 (EC50) as expected
according to the technique used. Using nonparametric
tests, it was possible to evaluate differences between the
experimental groups, according to the parameters of the
technique employed. For these analyses, the statistical
software package GraphPad Prism version 5.00 Demo
(GraphPad Software, USA) was used.

D. Granados-Falla et al. / Advances in Bioscience and Biotechnology 4 (2013) 304-315 309
3. RESULTS
3.1. Cytotoxic Activity on Mammalian Cells
Targeted by L. (V) panamensis Infection
The cytotoxic activity of the seco-limonoid on cells of
the murine monocyte/macrophage lineage (the murine
macrophage cell line J774.2) and hDCs was determined
using the resazurin metabolic assay, which indirectly
demonstrates the viability of a cell population through
evaluating of the metabolism of the non-fluorescent in-
dicator (resazurin) to a fluorescent molecule (resorufin)
[25]. In these assays, we observed a decreased suscepti-
bility of the murine cells to the toxicity induced by the
seco-limonoid, determining an LC50 of 368.5 µM (207.9
µg/mL) for the J744.2 cells versus 116.8 µM (65.89
µg/mL) for the hDCs. The cytotoxic effect induced by
the greatest concentration of the compound observed in
both cell lines was statistically significant (P value <
0.0001).
The cell viability results in these cell populations ex-
posed to varying concentrations of the seco-limonoid are
shown in Figure 1(b), where it’s shows that to achieve
similar death levels in the hDCs (filled circles) as in the
J774.2 cells (empty circles) a lower concentration of the
compound is required. Moreover, pentavalent antimony-
als (sodium stibogluconate) did not induce toxicity, as
demonstrated by the resazurin metabolic assay, which
could not determine an LC50 for the two cell populations
evaluated (LC50 > 2000 µg/mL).
3.2. Antileishmanial Activity
With the aim of evidencing whether previously demon-
strated activity of seco-limonoid was conserved in the
different stages of the pathogen, tests were developed on
the extracellular form (promastigotes), as well as the
intracellular and axenic amastigotes forms. The parasites
used in the present study belong to the genus Leishmania,
subgenus Viannia, specie L. panamensis, which is one of
the most important epidemiological species in South
America, being the primarily responsible for the cases of
cutaneous leishmaniasis in Colombia. The data obtained
show that the activity of the seco-limonoid compound
observed here appears to be directed specifically against
the intracellular form of the pathogen (intracellular
amastigotes) determining an EC50 of 7.9 µM (4.48
µg/mL) and 25.5 µM (14.39 µg/mL) using the in vitro
infection model of J774.2 cells and hDCs, respectively.
Figure 2 shows the representative results of the in vitro
infection assays using infected murine cells (panel 2a) or
hDCs (panels 2b and 2c). In the panel a of the Figure 2,
the broken line of the histogram, and panel b, are the
infected cells without treatment, while, the solid lines in
panel 2a and the panel c correspond to cells exposed to
30 µg/mL of the seco-limonoid.
Here, we show that the in vitro infection is controlled
when cells were treated with compound, as demonstrated,
by the decrease in the percentage of fluorescent cells
(62.5% in untreated infected cells versus 10.32% in in-
fected cells treated with seco-limonoid at 30 µg/mL) as
also, was observed in the slides stained with Giemsa so-
lution a decrease in uninfected cells in relation to the
total cell population (Figures 2(d) and (e)). Similarly,
Figure 3(a) shows the antileishmanial effect on axenic
amastigotes of L. (V) panamensis, with an EC50 of 86.3
µM (48.7 µg/mL), demonstrating a concentration depen-
dent response, which was not observed on the flagellar
form of the pathogen (statistically significant differ-
ences were found when comparing the response obtained
in both stages with 60 µg/ml of the compound, being
most susceptible to the effect of the compound the axenic
amastigotes form that promastigotes with a P value of
0.0024) (Figure 3(b)). Figure 3(c) shows the results
obtained with pentamidine drug on the flagellar ex-
tracellular form of the pathogen (promastigotes) deter-
mining an EC50 of 0.6 µg/ml Table 1 shows a summary
of the cytotoxicity results and the efficacy observed in
the tests described.
In this respect, an antileishmanial effect was demon-
strated for the amastigote forms of the parasite (being
mostly susceptible the intracellular form that the axenic);
finding that the concentration of compound required to
control 50% of the population for the intracellular stage
of the parasite was lower (4.48 and 14.39 µg/mL for the
infected J774.2 and hDCs that were treated with the
compound, respectively) than for the axenic stage (48.7
µg/mL), which allows us to suggest that the compound,
in addition to exhibiting antiparasitic activity capable of
controlling the pathogen, may be acting on some meta-
bolic pathways of APCs, leading to the complete resolu-
tion of the infection, possibly by the recovery of the an-
timicrobial activity that was silenced by the pathogen.
The data indicate that this compound needs to be me-
tabolized to carry out its activity or its antileishmanial
activity is associated with an event caused by the immu-
nomodulation of APCs by this molecule.
3.3. Immunomodulatory Activity
3.3.1. Nitric Oxi d e (NO) Production
Given the importance of the free nitrogen radicals in the
control of intracellular infectious agents (such as
Leishmania spp.) in this study, we determined the levels
of NO in infected murine J774.2 macrophages and hDCs
treated with the seco-limonoid by flow cytometry using
the indicator DAF-FM diacetate (4-amino-5-methyl-
amino- 2’,7’-difluorofluorescein diacetate) as a probe. In
both in vitro infection models (J774.2 and hDCs) the
seco-limonoid induced an increase in NO production in
nfected and treated cells (with significant differences for i

D. Granados-Falla et al. / Advances in Bioscience and Biotechnology 4 (2013) 304-315 310
(a)
(b) (c)
(d) (e)
Figure 2. Cells infected with L. (V) panamensis and treated with seco-limonoid. (a) corre-
spond to a histogram (MFI detected by 530/30 filter) of uninfected murine macrophages
(continuous gray line), macrophages infected with L. (V) panamensis transfected with GFP
without exposure to treatment after 48 h of infection in vitro (broken line) and macrophages
infected and treated with 30 µg/ml of the seco-limonid (continuous black line); (b) and (c)
correspond to hDCs infected with promastigotes of L. (V) panamensis without exposure to
any treatment and exposed to 30 µg/ml of the seco-limonid after 72 h of infection in vitro,
respectively, showing the internalized parasites observed by light microscopy on Giemsa-
stained slides; (d) and (e) correspond to the graphs of concentration versus response in both
in vitro models of infection treated with compound.
Table 1. Cytotoxic and antileishmanial activity on murine macrophages J774.2 cells and human dendritic cells.
Murine Macrophages
Compound L. (V) panamensis
promastigotes
L. (V) panamensis
axenic amastigotes(cell line J774.2)
Human Dendritic
Cells (hDCs)
EC50 (µg/mL) EC50 (µg/mL) LC50 (µg/mL)EC50 (µg/mL)SI LC50 (µg/mL) EC50 (µg/mL)SI
Sodium stibogluconate >1000 3018 >2000 40.17 >50 >2000 210 >10
seco-limonoid >60 48.7 207.9 4.483 46 65.89 14.39 4.6
the case of hDCs treated) in a concentration-dependent
manner when compared to the levels of NO in the un-
treated infected cells (Figure 4). Therefore, despite the
variability in the response, in the murine cells and the

D. Granados-Falla et al. / Advances in Bioscience and Biotechnology 4 (2013) 304-315 311
(a)
(b)
(c)
Figure 3. (a) shows the cytotoxic activity of seco-li-
monoid on axenic amastigotes (the graph shows the
average percentage viability ± SD); while (b) and (c)
correspond to cytotoxic activity of the seco-limonoid
and Pentacarinat® against the extracellular flagellar
form of the parasite (promastigotes). The graphs show
the average percentage mortality ± SD.
hDCs, there is a marked tendency toward NO production
at the highest concentration of seco-limonoid, which
reinforces the concept of its potential immunomodula-
tory activity. It should be noted that the compound used
in this study, as well the culture media used here for the
maintenance of the cell populations described before, did
not yield positive results in the determination of endo-
(a)
(b)
Figure 4. The diagrams correspond to nitric oxide (NO)
production by murine macrophages (J774.2) (a) and human
dendritic cells (hDCs) (b) The graph depicts the mean of the
mean fluorescence intensity (MFI) and its SEM.
toxin assay (commercial kit which present a sensibility
range of 0.06 EU/mL)
3.3.2. Quantification of Cytokines by Flow Cytometry
To evaluate the possible immunomodulatory effect of the
seco-limonoid, we quantified cytokines associated with
inflammatory processes using the cytometric bead array
(CBA) method for flow cytometry. Thereby, IL-6, IL-10,
MCP-1, IFN-γ, TNF, and IL-12p70 were assayed in the
supernatants of the J774.2 cells, and IL-8, IL-1β, IL-6,
IL-10, TNFα, and IL-12p70 were assayed in the super-
natants of the hDCs from patients with active disease
(cutaneous presentation) and control volunteers with no
history of the disease. In this regard, we determined that
the seco-limonoid modulates cytokine secretion selec-
tively in cells infected with L. (V) pan amensis in the two
in vitro infection models used (see Figures 5 and 6)

D. Granados-Falla et al. / Advances in Bioscience and Biotechnology 4 (2013) 304-315 312
Figure 5. Correspond to levels of cytokines present in the supernatans of murine cells J774.2.
Figure 6. Correspond to cytokines levels in the supernatans of hDCs.
preferentially to the pro-inflammatory phenotype. Nota-
bly, despite the trend observed, significant differences
between the two in vitro models when compared to the
untreated infected cells were not observed.
Furthermore, the evaluation on the production of solu-
ble mediators involved in inflammatory processes in
cells derived from active leishmaniasis patients and the
control volunteers demonstrated that the hDCs derived
from patients with active disease produced more of these
cytokines compared with the control group of unexposed
patients; however, because of the variability of the re-
sults, significant differences were not observed.
3.3.3. E valuation o f Cell Surfa ce Markers by Flow
Cytometry
To explore the possible immunomodulatory effect of the
seco-limonoid on hDCs (critical cell population for the
development of adequate immune response for the reso-
lution of this disease) the expression of cell surface
markers associated with the maturation of the hDCs was

D. Granados-Falla et al. / Advances in Bioscience and Biotechnology 4 (2013) 304-315 313
assessed. The cells were labeled with commercial mono-
clonal antibodies in the presence of HLA-DR, DC-SIGN,
and the co-stimulatory molecule human CD83, allowing,
in this way the evaluation of the basic phenotypic char-
acteristics of the hDCs population of infected that was
treated with the compound. The results demonstrate that
in infected cells treated with 15 µg/mL of the seco-li-
monoid, the expression of HLA-DR was enhanced (sig-
nificant differences between the untreated infected cells
and the infected cells treated with the compound in the
two experimental groups, P value < 0.05) (Figure 7).
Similar results were obtained when evaluating the ex-
pression of the DC-SIGN molecule and CD83 (data not
shown); however, no significant differences were ob-
served for these molecules, when comparing the un-
treated infected cells with the treated cells. It is notewor-
thy that the number of positive events in the hDCs de-
rived from patients with active disease was less than the
number of positive events in the hDCs derived from the
control volunteers for the three molecules tested; how-
ever, despite this marked tendency, significant differ-
ences between the two experimental groups of individu-
als were not observed.
4. DISCUSSION
The current first-choice treatment for leishmaniasis is
based on the administration of pentavalent antimonial
salts, which is still used despite their associated [7,11]
adverse effects, as well as, the appearance of drug-re-
sistant parasite [6,26] and the high cost associated with
patient care. Also, despite the existence of second-line
drugs (as amphotericin B, hexadecilphosphocholine, pen-
tamidine isethionate, and paromomycin sulfate), these
also have presented some drawbacks (such as adverse
effects induced in the treated individuals and the high
costs involved in the access to certain formulations).
Considering this, it acquires even more relevance the ur-
gent need for the development of new therapeutic agents
for the treatment of this disease [5]. For many years, the
study of compounds derived from natural products has
been an important source of information for the design
and/or discovery new therapeutic alternatives for the
control of diverse diseases, such as leishmaniasis disease
[13,17]. Therefore, various research studies have focused
on the search for new, safer, and more effective bioactive
molecules against leishmaniasis.
In this study, we confirmed the antileishmanial activity
(a) (b)
hDCS Infected hDCS Treated infected hDCs
Basal with 30 μg/mL of seco-limonoid compound
(c)
Figure 7. (a) and (b) shows the expression of the HLA-DR molecule in hDCs derived from individuals with and without clinical
history of Leishmania infection, respectively. **correspond to significant differences finding when comparing the response found it
with the basal response with a P value < 0.05, *significant differences with a P value < 0.01. (c) corresponds to histograms repre-
senting the HLA-DR expression in hDCs.

D. Granados-Falla et al. / Advances in Bioscience and Biotechnology 4 (2013) 304-315 314
of a seco-limonoid (11α,19β-dihydroxy-7-acetoxy-7-de-
oxoichangin), which was isolated from the bark of Rapu-
tia heptaphylla (Rutaceae family), which was previously
described by Coy and colleagues [18]. We suggest here a
possible mechanism for the antiparasitic activity of this
compound that is related to the immunomodulatory ef-
fect on APCs.
In this regard and taking into account the information
currently available regarding the parasite responsible for
leishmaniasis and the information regarding its verte-
brate host [20,27-29] suggest that immunomodulation is
an attractive and promising tool [7,30,31] for developing
new therapies for the adequate control of intracellular
pathogens, such as Leishmania spp. [32]. Here, we ob-
served that the seco-limonoid exhibits a specific activity
against intracellular amastigotes of Leishmania and that
this activity is possibly mediated or promoted by the
immunomodulatory effect that this compound induces
only in infected cells (depending on their interaction with
pathogen). Thereby, this seco-limonoid has been shown
to be a bioactive molecule exhibiting an interesting spe-
cific projection (because it would only modulate the mi-
crobicidal activity of infected cells) that can be used for
the design of a leishmanicidal therapy.
The immunomodulatory potential of the seco-limonoid
11α,19β-dihydroxy-7-acetoxy-7-deoxoichangin is sup-
ported by the production of nitric oxide and the expres-
sion of HLA-DR, which is down-regulated following
infection and exhibits a tendency to recover after expo-
sure to this seco-limonoid. It is also important to high-
light the importance of developing studies to confirm this
activity in vivo (results using the hamster model Meso-
cricetus auratus have demonstrated the resolution of
experimental cutaneous lesions of leishmaniasis).
Notably, for many years, numerous molecules exhibi-
ting microbicidal activity has been reported; however,
these molecules have not been developed into drug pro-
totypes. One of the reasons for this delay is the difficulty
in establishing the mechanism of action, which rein-
forces the importance of this study with respect to the
immunomodulatory potential of the seco-limonoid in in-
fected cells.
Furthermore, this study enhances the importance of
considering the highly complex life cycle of the patho-
gen responsible for leishmaniasis [33]. When developing
in vitro screens to study bioactive molecules for the con-
trol of this microorganism, there is a necessity for de-
veloping assays to evaluate possible antiparasitic activi-
ties against parasites of the genus Leishmania at the dif-
ferent life cycle stages of this pathogen. It is particularly
important to develop antiparasitic therapies against the
form of the pathogen responsible for the disease in the
vertebrate host during the clinical manifestations of the
disease [34,35], and is in this way, that in the present
study we confirmed the efficacy of a seco-limonoid
(triterpene) [18] which exhibits activity against only the
intracellular form of the parasite, leading to a decrease
and subsequent resolution of the infection in treated in-
fected cells. We demonstrated that this activity is related
to an immunomodulatory mechanism of action in which
the compound induces an apparent “reactivation” of the
“paralyzed” APC caused by the survival mechanisms
developed by the pathogen.
5. ACKNOWLEDGEMENTS
We express our gratitude to the Immunotoxicology Research Group,
the Natural Plant Products Group of the Universidad Nacional de Co-
lombia, and the PECET of the Universidad de Antioquia. This project
was financed by Bogotá Research Division (DIB) at the Universidad
Nacional de Colombia (projects Hermes No. 15099 and No. 16105) and
the Colombian Institute for the Development of Science and Technol-
ogy “Francisco Jose de Caldas” (project No.110151928476).
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