Parasitology International 67 (2018) 627–636

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Parasitology International
journal homepage: www.elsevier.com/locate/parint

Chemoprevention of Leishmaniasis: In-vitro antiparasitic activity of T

dibenzalacetone, a synthetic curcumin analog leads to apoptotic cell death
in Leishmania donovani☆
Indira Singh Chauhana, G. Subba Raob, Jai Shankarc, Lalit Kumar Singh Chauhanc,
⁎
Govind J. Kapadiad,1, Neeloo Singha,
a
Biochemistry Division, CSIR Central Drug Research Institute, Jankipuram Extension, Sitapur Road, Lucknow 226031, India
b
Global Biotechnology Resource Center, 145 Rosewood Drive, Streamwood, IL 60107, USA
c
Transmission Electron Microscopy, CSIR Indian Institute of Toxicology Research, Mahatma Gandhi Marg, Lucknow 226001, India
d
Department of Pharmaceutical Sciences, College of Pharmacy, Howard University, Washington, DC 20059, USA

A R T I C LE I N FO A B S T R A C T

Keywords: Curcumin is the major phenolic compound found in turmeric, a dry powder of rhizomes and roots of the plant,
Leishmania donovani Curcuma longa L., which is widely used as spice and food colorant around the world, and in herbal medicinal
A curcumin analog dibenzalacetone (DBA) practice in Asian countries. The present study reports the leishmanicidal activity of trans-dibenzalacetone (DBA),
Apoptosis a synthetic monoketone analog of curcumin, against Leishmania donovani parasites. We for the first time report
the antiproliferative effect of a curcumin analog (DBA) on the intracellular amastigotes of L. donovani, the
clinically more relevant stage of the parasite than its promastigotes stage. The leishmanicidal effect of DBA was
further confirmed by scanning and transmission electron microscopies. Cell growth was arrested in G0/G1 phase
with increased concentration of cytosolic calcium and dissipation of mitochondrial membrane potential. Further,
the unique trypanothione/trypanothione reductase (TR) system of Leishmania cells was significantly inhibited
by DBA. This economically synthesizable simple monoketone analog of curcumin has the potential for field use
against visceral leishmaniasis which is currently widespread in tropical and subtropical developing countries of
the world. In conclusion, we have identified an analog of curcumin for potential applications against leishma-
niasis, based on its strong antiparasitic activity and low toxicity. This curcumin analog compares favorably, at
least in vitro, with the existing medication miltefosine.

1. Introduction leishmaniasis is caused by a protozoan parasite which they named

Trypanosomatids are a family of kinetoplastid protozoa comprised
of flagellated protists characterized by the presence of a single fla-
gellum [1]. Leishmaniasis is one of the three major human parasitic
diseases caused by trypanosomatids, the other two being sleeping
sickness caused by Trypanosoma brucei and Chagas disease caused by
T. cruzi. Leishmania donovani is transmitted to humans by sandflies and
responsible for visceral leishmaniasis (VL), the fatal form if untreated,
in 88 tropical and subtropical countries in every continent except
Australia and Antarctica [2, 3]. There is evidence for its prevalence
among early Egyptians dating back to 3500 BC by recent detection of L.
donovani DNA in the bone tissue samples from ancient mummies of the
era [4]. In 1903, Leishman and Donovan were the first to report that
Leishmania donovani [5]. Being an opportunistic infection with up to
70% of adult leishmaniasis related to HIV infection, it is the second-
largest parasitic disease in the world (after malaria) with 500,000 new
cases of VL reported annually [6]. VL has been classified by WHO as one
of the most neglected tropical diseases which impacts mostly the
poorest populations and for which no adequate therapy currently exists
[7]. The orally effective drugs, such as miltefosine, a phosphocholine
derivative and sitamaquine, an aminoquinoline have found limited
success in India, Brazil and Kenya, all exhibit serious toxic side-effects
and develop resistance [8–11]. Recent trend has been to employ com-
bination therapy, such as antimonials or miltefosine with antileismanial
antibiotics to overcome drug resistance [12]. However, most of these
therapies are not practical for field use in treating VL in economically-

☆
This article is dedicated to Prof. Govind J. Kapadia
⁎
Corresponding author.
E-mail address: neeloo888@yahoo.com (N. Singh).
1
Deceased.

https://doi.org/10.1016/j.parint.2018.06.004
Received 2 February 2018; Received in revised form 27 April 2018; Accepted 8 June 2018
Available online 18 June 2018
1383-5769/ © 2018 Elsevier B.V. All rights reserved.
I.S. Chauhan et al.
challenged developing countries. Thus, there is urgent need for new
orally effective, affordable drugs without toxicity and drug resistance in
managing VL infection and improving quality of life of large popula-
tions afflicted with this debilitating parasitic disease in both developing
and developed countries of the world.
Curcumin [(1E,6E)-1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-hepta-
diene-3,5-dione] is the principal phenolic compound responsible for the
yellow color of turmeric, a dry powder of rhizomes and roots of the
perennial plant, Curcuma longa L. belonging to the ginger family,
Zingiberaceae [13]. Over the centuries, turmeric has been used ex-
tensively as a spice; food colorant and fabric dye, and in herbal medical
practice in India, China and other Asian countries [14]. The European
Union and the US Food and Drug Administration consider curcumin,
designated as yellow food color E100, safe for human consumption
(http://www.feingold.org/Research/PDFstudies/colors.pdf). It is a
strong antioxidant exhibiting remarkable pharmacological properties,
such as anti-inflammatory, antimicrobial and cancer chemoprevention
through a variety of mechanisms involving multiple targets [15–17]. In
clinical trials, curcumin exhibited no adverse effects in humans at high
doses (8-12 g/day) [16, 18]. However, instability, poor cellular uptake
and bioavailability of curcumin and its naturally occurring analogs,
curcumin II (demethoxycurcumin) and curcumin III (bisdemethox-
ycurcumin) have hindered their development into clinically useful
drugs to treat systemic leishmaniasis [19]. Since antioxidant capacity of
curcumin and its analogs with β-diketone [20–23] or monoketone [24,
25] moiety in their structure is considered essential for their biological
activities, numerous curcumin analogs with improved stability, anti-
oxidant capacity, cell penetration and bioavailability profile have been
synthesized for structure-antioxidant/biological activity studies [19,
25–27]. Previous investigations have demonstrated antiparasitic activ-
ities of curcumin [21, 28–30] and related synthetic curcuminoids [31,
32].
The average daily intake of turmeric in India is estimated to be
38–190 mg (equivalent to 0.38–9.5 mg/day of curcumin) where it is
widely used in popular curry dishes for its unique flavor and signature
bright yellow color [33, 34]. In some rural areas of this country, its
consumption is as high as 3.8 g/day. Effect of curcumin on systemic
leishmaniasis appears to be dose dependent: in high doses it has anti-
parasitic effect and in low doses it increases parasite load in critical
organs and exacerbates VL [34, 35]. Formation of reactive oxygen
species (ROS) and imbalance of calcium homeostasis account for high
doses of curcumin inducing cell death of L. donovani [21]. In contrast,
curcumin in low doses suppresses interferon γ and nitric oxide pro-
duction by activation of the anti-inflammatory protein called PPARγ
which results in suppression of the body's initial acute inflammatory
immune response against invading parasite and helps them evade the
host immune attack [35]. Thus, currently there is an inconclusive re-
search on the effect of dietary intake of curcumin in systemically
Leishmania infected population in India, especially those in rural areas.
However, topical ointments and skin injections of turmeric and cur-
cumin have proven to be helpful in the treatment and prevention of
cutaneous leishmaniasis [36].
The present study describes the leishmanicidal activity of (1E, 4E)-
1,5-diphenyl-1,4-pentadien-3-one (trans-dibenzalacetone, DBA), a
simple, synthetic monoketone analog of curcumin, against promasti-
gotes and intracellular amastigotes of L. donovani. The potential me-
chanism of leishmanicidal activity of DBA was studied by cell cycle
progression and ultrastructure of treated parasites. These leishmani-
cidal results are of particular interest since DBA is a simple monoketone
curcumin analog easily synthesized in high yield (> 85%) from in-
expensive ingredients by a one-step process at room temperature and
has potential for economically treating VL currently wide-spread among
poor populations of the world.
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2. Materials and methods
2.1. Synthesis of trans-Dibenzalacetone (DBA)
DBA was synthesized as described earlier [37, 38] by the one-step
Aldol condensation reaction of benzaldehyde in ethanol with acetone
under basic conditions utilizing aqueous sodium hydroxide solution at
room temperature with a yield of > 85%. Recrystallization from aqu-
eous alcohol twice yielded DBA as bright yellow crystals, melting point
103–104 °C and its purity established by thin-layer chromatography, as
described earlier [39]. The chemical structure of DBA was confirmed by
infrared (strong signal for the presence of carbonyl group at
1649 cm−1with none attributable to hydroxyl group), ultraviolet (λmax
326 nm, log εmax4.53 for conjugated carbonyl compound), 1H-nuclear
magnetic resonance (vinyl protons doublet of doublets, 16 Hz at 7.74
and 7.09 ppm, each for 2H for the benzylic protons and for the protons
α to the carbonyl carbon, respectively) and mass spectra (molecular
ion, M+ at m/z 234, M+–H ion at m/z 233 and ion at m/z 131 from
cleavage at carbonyl carbon leading to ion at m/z 103 from loss of CO)
[39, 40].
2.2. Parasite culture and measurement of cell viability
Promastigotes and intracellular amastigotes of L. donovani strain
(MHOM/IN/80/DD8) used in this study were routinely cultured as
described previously [41]. Stock solution of DBA (1 mg/ml) was made
in 0.1% (v/v) of DMSO and stored in aliquots at 4 °C. The drug was
diluted in culture media immediately prior to each experiment to pro-
vide series of drug concentrations (0, 10, 20, 40, 80 and 160 μg/ml), as
needed.
Resazurindye/AlamarBlue® (7-hydroxy-3H-phenoxazin-3-one-10-
oxide) (Invitrogen, Cat. No. DAL1025, Carisbad, CA) was employed for
measuring parasite (promastigotes) viability. Logarithmic phase pro-
mastigotes of L. donovani (50,000 cells/200 μl/well) were seeded in 96-
well microtiter plates (Greiner, Bio-one, Germany) in the presence of
increasing concentrations (0–40 μg/ml) of DBA, incubated at 25 °C for
24 h. Miltefosine was used as the standard drug. AlamarBlue® (20 μl)
was added to each well and the plate was further incubated at 25 °C for
4 h. Absorbance was measured in a microplatereader (Biotek, Epoch)
using λ = 570 nm as test wavelength (resorufin) and λ = 600 nm as
reference wavelength (resazurin) serving as blank (normalized to the
600 nm value). The percent of cell-viability of DBA treated parasites
was analyzed by following equation: [untreated control λ
(570–600 nm) – treated set λ (570–600 nm)]/untreated control λ
(570–600 nm) × 100.
2.3. In vitro evaluation of antileishmanial activity of DBA in intracellular
amastigotes
The BALB/c mouse macrophage cell line J774A.1 was infected with
stationary phase promastigotes of L. donovani as previously described
[41]. Infected macrophages were treated with increasing concentra-
tions (0–20 μg/ml) of DBA, incubated at 37 °C in 5% CO2 for 24 h. After
indicated incubation time, infected macrophages treated with or
without DBA were fixed with ice-cold methanol on slides which were
then submerged in 10% (v/v) Giemsa staining solution (Thomas Baker)
for 45 min, followed by water rinse and let dry. The slides were viewed
on an inverted bright field microscope (IX73 Inverted Microscope,
Olympus). At least 100 macrophages per well were counted from du-
plicate cultures and the percentage of infected macrophages were cal-
culated using the formula [42]: % reduction = number of amastigotes
per 100 macrophages (treated samples)/number of amastigotes per 100
macrophages (infected control) × 100.
In vitro antileishmanial activity was expressed as the concentration
inhibiting parasite growth by 50% (IC50) and was analyzed by plotting
percentage of cell viability versus log growth concentration of DBA.
I.S. Chauhan et al. Parasitology International 67 (2018) 627–636

(caption on next page)

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Fig. 1. [A] Cell viability assessment by AlamarBlue® (7-Hydroxy-3H-phenoxazin-3-one-10-oxide) shows reduction in viability of promastigotes treated with different
concentrations (0, 10, 20 and 40 μg/ml) of DBA for 24 h. (a) Graph shows percentage of cell viability against log value of DBA concentrations (μg/ml). (b) Changes of
medium color associated with AlamarBlue®reduction from blue (oxidized) to pink (reduced) in triplicate wells. [1] Control (cells without DBA) shows pink color and
[2 and 3] promastigotes treated with different concentrations of DBA show blue color where a blue color in the well was interpreted as no cell growth, and pink color
was scored as growth. The data are presented as mean ± standard deviation of three independent experiments. [B] In vitro analysis of antileishmanial activity of
DBA in intracellular amastigotes: Macrophages (4000 cells/well, final volume 200 μl) were infected with promastigotes of L. donovani in a ratio of 8:1 (parasites/
macrophages) and infected macrophages were treated with increasing concentrations (0, 5, 10, 15 and 20 μg/ml) of DBA for 24 h. After 24 h, treated or untreated
cells were stained with Giemsa stain and the slides were viewed on an inverted bright field microscope (IX73 Inverted Microscope, Olympus). The 50% inhibitory
concentration (IC50) was obtained by plotting the graph of percentage of cell viability against log value of DBA concentrations (μg/ml). The results were taken as the
mean of duplicate experiments and p < .05 has been considered as the level of significance. [C] Chemical structure of (a) curcumin [(1E,6E)-1,7-bis(4-hydroxy-3-
methoxyphenyl)-1,6-heptadiene-3,5-dione] (b) a curcumin analog, trans-dibenzalacetone (DBA) [(1E,4E)-1,5-diphenyl-1,4-pentadien-3-one]. [D](a) Cytotoxicity in
macrophages: Macrophages (50,000 cells/well, final volume 200 μl) were treated with increasing concentrations (0, 10, 20, 40, 80 and 160 μg/ml) of DBA for 24 h.
After 24 h, the viability of the macrophages was estimated by AlamarBlue® cell viability reagent. The 50% cytotoxic concentration (CC50) was determined by
logarithmic regression analysis using GraphPrism 5 software. (b) Growth in the medium is indicated by change in color from blue to pink. Results are presented as
mean ± SD; n = 3 and p < .05 has been considered as the level of significance. [E] PI uptake analysis: histograms show that treatment of Leishmania parasites with
DBA leads to (PI-positive cells M2) cell death in a concentration-dependent manner [a, b, c and d represent 0, 10, 20 and 40 μg/ml respectively]. All data depicted in
this figure are representative of at least three replicates. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of
this article.)

2.4. In vitro cytotoxicity against BALB/c mouse macrophages 2.7. Ultrastructural analysis by transmission electron microscopy (TEM)

The BALB/c mouse macrophage cell line J774A.1 was seeded in 96-
well culture plates (104 cells/200 μl/well). The plates were incubated
overnight in a CO2 incubator in an atmosphere of 95% air and 5%
CO2at 37 °C. DBA in different concentrations (0–160 μg/ml) was dis-
pensed in triplicate and the remaining three wells were used as con-
trols. After 24 h, 20 μl of AlamarBlue® was added to each well and the
plate was further incubated at 37 °C for 4 h. Absorbance was measured
in a microplate reader as described before [43]. The 50% cytotoxic
concentration (CC50) for the above mentioned assays was determined
by logarithmic regression analysis using GraphPrism 5 software.
2.5. Propidium iodide (PI) uptake assay
Logarithmic phase promastigotes of L. donovani (106 cells/2 ml/
well) were seeded in 6-well microtiter plates in the presence of different
concentrations (0–40 μg/ml) of DBA and incubated at 25 °C for 24 h.
The cells were then centrifuged (3500 x g for 10 min), washed once
with PBS (pH 7.2), re-suspended in 50 μg/ml final concentration of PI
(Sigma-Aldrich), and incubated for 30 min in the dark at room tem-
perature. Unbound PI was removed by washing and samples were
processed on a FACSCalibur flow cytometer (BD Bioscience, San Jose,
CA, USA) and analyzed using CellQuest Pro software. Twenty thousand
events from each sample were acquired to ensure adequate data and at
least three independent experiments were carried out [44, 45].
2.6. Analysis of topological alterations by scanning electron microscopy
(SEM)
Logarithmic-phase L. donovani promastigotes (2 × 107 cells/ml)
were incubated with IC50 dose (17.80 μg/ml) of DBA for 24 h at 25 °C in
M199 medium. The parasites were then washed with PBS (pH 7.2) prior
to fixing in 2.5% glutraldehyde in sodium cacodylate buffer (pH 7.2) for
2 h at 4 °C. Fixed parasites were centrifuged at 3500 x g for 10 min and
washed 3 times with 0.1 M sodium cacodylate buffer and post-fixed in
1% osmium tetraoxide for 2 h. Post-fixed parasites were further washed
with sodium cacodylate and dehydrated in ascending acetone series
(15, 30, 60 and 100%), embedded in araldite-DDSA mixture and baked
at 60 °C for 48 h. The baked blocks were cut (60–80 nm thick) by an
ultramicrotome (Leica EM UC7, Vienna, Austria), mounted on copper
grids, and double stained with uranyl acetate and lead citrate. The
stained sections were examined by TEM (TECNAI G2 SPIRIT, FEI,
Netherland) equipped with GatanOrius camera at 80 KV [41].
2.8. Cell cycle analysis
In order to determine the changes induced by DBA on the cell cycle,
2 × 106 cells/ml of log phase L. donovani promastigotes were treated
with different concentrations (0–40 μg/ml) of DBA for 24 h and were
fixed by incubation in 70% ethanol and 30% PBS for 1 h at 4 °C. Fixed
cells were washed in 1 ml PBS and re-suspended in 1 ml PBS with
100 μg/ml of RNAse A (Cat. No.EN0531, Fermentas) and PI (10 μg/ml).
The cells were then incubated at 35 °C for 45 min and analyzed using
FACSCalibur flow cytometer. Cell cycle distribution was modeled using
ModFit LT for Mac V3.0. Ten thousand events from each sample were
acquired to ensure adequate data and at least three independent ex-
periments were run [46].

2.9. Analysis of externalized phosphatidylserine (PS)

Logarithmic phase promastigotes of L. donovani were incubated in
the presence of DBA washed with PBS (pH 7.2) prior to fixing in 2.5%
Double staining with Annexin-V and PI was used to measure the
glutraldehyde in sodium cacodylate (Ladd Research Industries, USA)
apoptotic effects of DBA on the plasma membrane of L. promastigotes
buffer (pH 7.2) for 4 h at 4 °C. Fixed samples were washed with sodium
(Annexin V-FITC Apoptosis Detection kit, Sigma). The log phase L.
cacodylate buffer (0.1 M) three times and the suspensions were placed
donovani promastigotes (2 × 106 cells/ml) were treated with different
on poly-L-lysine-coated glass slides and allowed to adhere for 15 min at
concentrations (0–40 μg/ml) of DBA for 24 h and treated cells were
room temperature. The samples were post-fixed in 1% osmium tetra-
analyzed on a FACSCalibur flow cytometer and 10,000 events from
oxide (OsO4) in dark at 4 °C for 4 h, washed and dehydrated in ethanol
each sample were acquired to ensure adequate data and at least three
series from 15 to 100%. Dehydrated samples were critical point dried
independent experiments were conducted [41].
by K850 Critical Point Dryer (Quorum Technologies Ltd., UK) and taken
on stubs for coating by gold sputter coater (SC7620, mini sputter coater,
Quorum 1040 Technologies Ltd., UK). DBA treated parasites were ex- 2.10. Terminal Deoxynucleotidyltranferase(TdT)-mediated dUTP -nick end
amined under scanning electron microscope (Quorum FEG 450, FEI, labeling (TUNEL) assay
Netherland).
DNA fragmentation within L. donovani promastigotes was analyzed
by Terminal Deoxynucleotidyltranferase(TdT)-mediated dUTP Nick

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I.S. Chauhan et al.
Fig. 2. Scanning electron microscopy (SEM) shows morphological alterations in (b) promastigotesof L. donovani treated with the IC50value (17.80 μg/ml) of DBA for
24 h (a) as compared to untreated cells. Scale bars 5 μm and 10 μm.
End Labeling (TUNEL) using an APO-BRDU flow cytometry assay kit
(Sigma)as described previously [41]. Promastigotes were incubated
with different concentrations (0–40 μg/ml) of DBA at 25 °C for 24 h,
and treated cells were analyzed for fluorescence on a FACSCalibur flow
cytometer equipped with dual pass FITC/PI filter set. CellQuest Pro
software was used for flow cytometer data acquisition and analysis
which represented three independent experiments with 10,000 events
from each sample to ensure adequate data.
2.11. Monitoring of changes in intracellular Ca2+ levels
Changes in intracellular calcium levels were measured by the
fluorometric dye fluo-4 acetoxymethyl ester (Fluo 4-AM) (Cat. No·F-
14201, Molecular Probes) [47, 48]. Logarithmic phase promastigotes of
L. donovani (1 × 106 cells, final volume 2 ml/well) were seeded in 6-
well microtiter plates in the presence of different concentrations
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Parasitology International 67 (2018) 627–636
(0–40 μg/ml) of DBA and incubated at 25 °C for 24 h. Then, the cells
were centrifuged (3500 x g for 5 min), washed once with PBS (pH 7.2),
re-suspended in 5 μl of Fluo-4 AM (1 mM) and incubated for 30 min in
the dark at room temperature. After washing, samples were processed
on a FACSCalibur flow cytometer and analyzed using CellQuest Pro
software. Ten thousand events from each sample were acquired to en-
sure adequate data and at least three independent experiments were
run.
2.12. Measurement of mitochondrial membrane potential (Δψm)
Mitochondrial damage upon treatment with DBA in L. donovani
promastigotes was assessed by flow cytometry using a cell-permeable
dye, MitoTracker deep red (Molecular Probes, USA) [49]. L. donovani
promastigotes were treated with different concentrations (0–40 μg/ml)
of DBA for 24 h, washed in PBS and loaded in dark for 30 min with
I.S. Chauhan et al.
MitoTracker (10 μM) as per manufacturer's instructions. Analysis for
mean fluorescence intensity (MFI) was done using a FACSCalibur flow
cytometer and CellQuestPro software. Ten thousand events from each
sample were acquired to ensure adequate data and at least three in-
dependent experiments were run.
2.13. Measurement of intracellular non-protein thiols
The probe, 5-chloromethylfluorescein-diacetate (CMFDA,
cellTracker™ Green CMFDA, Cat.No·C7025, Molecular Probes) was
employed to measure intracellular non-protein thiols [50]. The log
phase L. donovani promastigotes (2 × 106 cells/ml) treated with DBA
(0–40 μg/ml) for 24 h were collected. The cell pellets were then washed
with PBS, incubated with CMFDA in the dark for 15 min at 37 °C and
analyzed for fluorescence in the FL1 channel, equipped with a 530/
30 nm band pass filter by using a FACSCalibur flow cytometer and
analyzed by CellQuestPro software. Ten thousand events from each
sample were acquired to ensure adequate data and at least three in-
dependent experiments were carried out.
2.14. Statistical analysis
Each experiment was performed at least three times and results
expressed as mean ± SD. The data were analyzed by one-way analysis
of variance using GraphPad Prism program. Multiple comparisons of
the means were made through two-way analysis of variance (ANOVA)
with a Newman–Keuls post-hoc test. Statistically significant difference
between sample and control were designated as: *to denote P < .05
and **to denote P < .01 levels of confidence while NS denotes dif-
ference that is statistically not significant.
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Parasitology International 67 (2018) 627–636
Fig. 3. Evaluation of ultrastructural damage by
transmission electron microscopy (TEM):
Promastigotes of L. donovani were incubated with the
IC50 value (17.80 μg/ml) of DBA for 24 h: [A] control
(untreated); [B] DBA treated cells, n: nucleus; k: ki-
netoplast; m: mitrochondria; pf: pocket flagellar; pm:
plasma membrane; G: golgi body; g: glycosome; a:
acidocalcisomes; ER: endoplasmic reticulum; sm:
sub-membrane microtubules; v: contractile vacuole.
Scale bars 1.0 μm and 0.5 μm.
3. Results
3.1. In vitro activity of DBA against L. donovani and mammalian
cytotoxicity
The antileishmanial activity of DBA was tested initially against
promastigotes of L. donovani. Promastigotes which resulted in the loss
of parasite viability in a concentration-dependent manner (Fig. 1A).
The DMSO solvent employed had no effect on the parasite viability. The
IC50 value of DBA for intracellular amastigotes (7.43 ± 1.88 μg/ml,
Fig.1B) was significantly lower than the IC50 value determined for
promastigotes (17.80 ± 1.42 μg/ml, Fig. 1A) and was closer to that
reported for standard drug miltefosine (0.01–10.9 μg/ml). Chemical
structures of curcumin (a) and DBA (b) are shown in Fig. 1C. DBA was
also tested against the BALB/c mouse cell line J774A.1 to determine
whether the doses used for IC50 determination for intracellular amas-
tigotes were toxic to the cell. This revealed that the 50% cytotoxic dose
(CC50) was higher than the IC50 dose for intracellular amastigotes
(Fig. 1D) with a SI (selectivity index) value of 15.34. To determine the
mechanism of cell death triggered by DBA, we evaluated plasma
membrane integrity in treated promastigotes stained with PI which
diffuses across permeable membranes and binds to nucleic acids. This
method has been applied earlier by other investigators [44, 45]. As
shown in Fig. 1E, increased cell death along the x-axis (M2) by the
gradual shifting of the dead cell population (increased MFI) was ob-
served with increased concentration of DBA. Treated promastigotes
were stained with PI in a concentration-dependent manner. A sharp
increase in PI positive (M2) cells from counts 19.12 ± 0 at 10 μg/ml to
counts 48.70 ± 0 at 20 μg/ml and thereafter gradual decrease from
counts 44.70 ± 2.84 at 40 μg/ml after 24 h-treatment were observed.
I.S. Chauhan et al. Parasitology International 67 (2018) 627–636

Fig. 4. [A] DBA causes cell cycle arrest in the G0/G1 phase of L. donovani promastigotes. Cells were treated with different concentrations [a, b, c and d represent 0,
10, 20 and 40 μg/ml respectively] of DBA for 24 h and stained with propidium iodide (PI). The DNA content was analyzed using flow cytometry with Cell Quest
software. Untreated cells were used as control. (e) The bar graph shows mean values and S.D. (*P < .05; **P < .01 compared with the control group, n = 3), NS:
not significant. [B] Externalization of phosphatidylserine in DBA treated promastigotes: L. donovani promastigotes were incubated with different concentrations [a, b,
c and d represent 0, 10, 20 and 40 μg/ml respectively] of DBA for 24 h and analyzed by flow cytometry. After indicated incubation time, a significant number of
membrane compromised cells were stained positively by Annexin V- FITC, PI (upper-right quadrant LA), and only PI positive (upper-left quardrant N). Undamaged
cells were unstained with Annexin V-FITC/PI (bottom left quadrant L). (e) The bar graph shows mean values and S.D. (**P < .01 compared with the control group,
n = 3). [C] DBA-induced DNA fragmentation in promastigotes of L. donovani was analyzed by TUNEL assay using flow cytometry. Increasing concentrations of DBA
showed the increase number of cells (R2) stained by anti-BrdU which have moved from non apoptotic population (G0/G1 and G2 phase) to the apoptotic population
(S phase). Where (a), (b), (c) and (d) show promastigotes treated with different concentrations (0, 10, 20 and 40 μg/ml) of DBA respectively. All data depicted in this
figure are representative of at least three replicates. [D] Measurement of DBA induced changes in intracellular Ca2+ level: Cells were treated with different
concentrations [a, b, c and d represent 0, 10, 20 and 40 μg/ml respectively] of DBA for 24 h and stained with fluo-4 AM. The cytosolic Ca2+ level was analyzed using
flow cytometry with Cell Quest software. Untreated cells were used as control. All data depicted in this figure are representative of at least three replicates.

Untreated cells served as control and showed no cell death.
3.2. DBA induced morphological and ultrastructural changes
For investigation of cell death induced by DBA in promastigotes,
analysis by SEM and TEM was conducted. SEM revealed that DBA
caused morphological alterations in the promastigote forms of L.
donovani compared with untreated parasites, which showed typical
characteristics with an elongated shape, long flagella and smooth cell
surfaces (Fig. 2a). The Fig. 2b shows alterations in shape and size and
cellular disintegration in the DBA-treated parasites. These cells ex-
hibited shrinking and rounding-up responses and in some cases, mem-
brane protrusions resembling surface blebs and ruffling of the plasma
membrane. This type of morphological effect has previously been

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I.S. Chauhan et al. Parasitology International 67 (2018) 627–636

Fig. 5. [A] Changes in mitochondrial membrane potential following treatment with DBA. L. donovani promastigotes were incubated with different concentrations [a,
b, c and d represent 0, 10, 20 and 40 μg/ml respectively] of DBA for 24 h, loaded in dark for 30 min with Mitotracker (10 μM) and analyzed by flow cutometry. All
data depicted in this figure are representative of at least three replicates. [B] Intracellular level of the glutathione (GSH) in L. donovani promastigotes treated with
different concentrations [a, b, c and d represent 0, 10, 20 and 40 μg/ml respectively] of DBA for 24 h and its fluorescence was measured using a flow cytometer. All
data depicted in this figure are representative of at least three replicates.

shown by other investigators [51]. promastigotes.

In normal promastigotes (Fig. 3 A), all features observed by TEM
were characteristic of Leishmania such as single reticulated mitochon-
3.4. Analysis of externalized phosphatidylserine
dria on the periphery of the cell, an oval nucleus, glycosomes, the rod
shaped kinetoplast disc, and the paraflagellar rod within the flagellar
To investigate the mechanism of DBA-induced apoptotic effects in
pocket [52]. Longitudinal section of the promastigote revealed a deep
Leishmania cells, double staining with Annexin V-FITC and PI was em-
flagellar pocket. The basal body of the flagellum and the closely asso-
ployed. This double staining allows differentiation between early
ciated kinetoplast disc were positioned at the bottom of the flagellar
apoptotic (Annexin V-FITC positive, A), late apoptotic (Annexin V-FITC
pocket as commonly seen in promastigotes. Ultrastructural analysis of
and PI positive, LA), necrotic (PI positive, N) and live cells (unstained)
the DBA-treated promastigotes (Fig. 3B) revealed severe damage to the
[53]. Late exponential-phase promastigotes of L. donovani were treated
parasite mitochondrion. Promastigotes treated with the IC50 con-
with different concentrations (10, 20 and 40 μg/ml) of DBA for 24 h, co-
centration (17.80 ± 3 μg/ml) of DBA for 24 h showed different degrees
stained with PI and Annexin V-FITC, and analyzed by flow cytometry.
of morphological changes which induced loss of parasite viability and
The percentage of DBA treated promastigotes that were positive for
severe loss of cristae and matrix were also seen. Moreover, the matrix
Annexin-V (A + LA) which indicated phosphatidylserine exposure,
displayed a washed-out appearance, indicating a decrease in the elec-
gradually increased, to 7.24 ± 0.24%, 12.66 ± 0.42% and
tron density, Golgi disruption and extensive cytoplasmic vacuolization.
17.36 ± 0.41% at 10, 20and 40 μg/ml, respectively (Fig. 4B). The PI is
an impermeable fluorescent probe that enters cells only when the
permeability of plasma membrane is lost. The number of cells that were
3.3. Cell cycle analysis
PI-positive (upper-left quadrant, N) gradually increased from
8.35 ± 1.68%, 22.92 ± 0.70% and 36.03 ± 0.94% at 10, 20 and
To assess the role of different concentrations (10, 20 and 40 μg/ml)
40 μg/ml, respectively (Fig. 4B). These observations suggested that
of DBA in mediating G0/G1 arrest, cell cycle analysis was performed
these cells were considered to be necrotic. Untreated cells were pri-
based on PI staining using flow cytometry (42). As shown in Fig. 4A,
marily FITC Annexin-V and PI negative, indicating that they were vi-
DBA (at 10, 20 and 40 μg/ml concentrations) induced a marked in-
able and not undergoing apoptosis or necrosis.
crease (~2 fold) in the number of cells in the G0/G1 phase (G1:
43.60 ± 0.98%, 53.65 ± 0.56% and 78.41 ± 0.65%), and simulta-
neous decrease (~3 fold) in both S phase (S: 39.80 ± 1.27%, 3.5. Terminal Deoxynucleotidyltranferase(TdT)-mediated dUTP Nick end
31.84 ± 1.56% and 16.05 ± 0.21%) and G2/M phase labeling (TUNEL) assay
(16.60 ± 2.26%, 14.51 ± 2.13% and 5.54 ± 0.86%) compared with
respective controls (G0/G1: 37.20 ± 1.55%, S: 48.18 ± 2.39% and The TUNEL assay was performed to verify whether DBA was able to
G2/M: 14.62 ± 3.94%). At higher concentration of DBA (40 μg/ml) we induce DNA fragmentation in promastigotes of L. donovani.
found 2.66 ± 0.38% sub-G1 peak which is characteristic of apoptosis Promastigotes when incubated for 24 h with DBA at concentrations of
(Fig. 4Ad). The concentration-dependent effect on G0/G1 arrest in 10, 20 and 40 μg/ml showed small changes in the MFI (1.29 ± 0.03%,
promastigotes was largely at the expense of S phase cells, with lesser 1.91 ± 0.09% and 1.60 ± 0.06% TUNEL-positive cells) when com-
change in the G2/M-phase cell population compared to the untreated pared with untreated promastigotes (0.34 ± 0.04%) (Fig. 4C).

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Increasing the concentrations of DBA showed increased number of cells
(as stained by anti-BrdU) moving from non-apoptotic population (G0/
G1 and G2 phase) to the apoptotic population (S phase).
3.6. Intracellular Ca2+ level in the DBA-treated promastigotes
To assess the variation of cytosolic Ca2+ levels in both treated and
untreated L. donovani promastigotes, Fluo4 was used as a calcium
fluorescent probe. The data showed a significant increase in fluores-
cence attributable to the cytosolic Ca2+ ion content in promastigotes
with increasing concentrations of DBA (MFI: 45.52 ± 1.48 at 20 μg/ml
and 67.86 ± 1.79 at 40 μg/ml) as compared to control (MFI:
30.07 ± 0.15) (Fig. 4D). However, the low concentration of DBA
(10 μg/ml) failed to change the Ca2+ level in promastigotes (MFI:
32.50 ± 1.78).
3.7. Measurement of mitochondrial membrane potential
TEM study revealed that DBA induced significant changes and da-
mage in promastigote mitochondria. Based on this, membrane potential
of treated promastigotes was determined by flow cytometry using a
cell-permeable dye, MitoTracker deep red. Treatment with different
concentrations (0–40 μg/ml) of DBA induced decrease in the MFI
(158.36 ± 1.60, 139.02 ± 0.07 and 137.14 ± 0.13 respectively) re-
lative to the untreated promastigotes (224.97 ± 1.44) (Fig. 5A). Other
investigator has shown that mitochondrial membrane dissipation may
be caused due to increase in cytosolic Ca2+ level [54]. Since depolar-
ization of mitochondrial membrane potential is an important event in
apoptosis, we investigated the increase in cytosolic Ca2+ level, which is
upstream event of mitochondrial membrane dissipation, to confirm
apoptosis.
3.8. Measurement of intracellular non-protein thiols
Reduced glutathione (GSH) is an important cellular component for
protecting kinetoplastids, such as Leishmania from ROS and it is a
component of trypanothione, a major antioxidant of this parasite [55,
56]. Accordingly, the effect of DBA on the level of this reduced in-
tracellular non-protein thiol was assessed using fluorescent probe,
CMFDA. Promastigotes treated with DBA at different concentrations
(0–40 μg/ml) showed remarkable decrease in the level of GSH from
untreated (MFI: 278.54 ± 1.02) to treated cells (MFI: 60.52 ± 0.40,
48.82 ± 0.12 and 52.45 ± 0.31) (Fig. 5B).
From the above-mentioned results, we can conclude that DBA
caused depolarization of the mitochondrion and the depletion of re-
duced glutathione, triggering an apoptotic response in L. donovani
promastigotes.
4. Discussion
Cytotoxic and antiparasitic effects of curcumin on protozoan para-
sites have been demonstrated earlier in cultures against Trypanosoma,
Giardia, and Plasmodium falciparum [57–59]. It has been reported to be
cytotoxic to several strains of Leishmania including L. major, L. tropica L.
infantum [29] and L. donovani [21]. Limited investigations have been
carried out to decipher the mode of cell death using curcumin as a
leishmaniacidal agent [21]. There is only one reported study estab-
lishing apoptosis as the main mechanism of cell death in Leishmania
parasites in response to curcumin [13].
Among the curcumin structural modifications evaluated, elimina-
tion of the hydrolysis-prone diketo functionality and incorporating a
range of alternate substituents on the terminal phenyl rings have re-
sulted in new synthetic curcuminoids with improved structural stability
and increased tissue uptake under both in vitro and in vivo conditions
resulting in enhanced bioavailability [24, 25]. In the present study, we
have investigated the leishmanicidal activity of DBA, an easily
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Parasitology International 67 (2018) 627–636
synthesizable, simple monoketone analog of curcumin without the
highly reactive β-diketone moiety and with unsubstituted terminal
phenyl rings in its structure to provide stability essential for clinically
desirable pharmacokinetic profile.
We for the first time report the antiproliferative effect of this cur-
cumin analog (DBA) in the intracellular amastigotes of L. donovani. The
observed antiproliferative effects indicated that DBA was more potent
against intracellular amastigotes, the clinically more relevant stage of
the parasite than its promastigotes stage. On comparison with reference
drug miltefosine, which is in clinical use in the endemic areas of India,
DBA showed IC50 value (7.43 μg/ml) closer to that reported for milte-
fosine (0.01–10.9 μg/ml)(https://www.accessdata.fda.gov/drugsatfda_
docs/nda/2014/204684Orig1s000MicroR.pdf). DBA showed no cyto-
toxicity against the BALB/c mouse cell line J734A.1.
Alterations in morphology of the DBA-treated parasites were as-
sessed by SEM which showed remarkable changes when compared to
untreated cells. The main ultrastructural alteration was observed in the
mitochondrion-kinetoplast complex, which showed intense mitochon-
drial swelling with an increase in the number of cristae. DBA changed
the profile of the cell cycle with an increase in G0/G1 population as-
sociated with a decrease in S (synthesis) and G2/M phases when
compared to control. Although it has been established by Weingartner
et al. [60] that Leishmania promastigotes lack phosphatidylserine, An-
nexin-V binding as an early indicator of apoptosis has been used by
other investigators [61]. As shown in Fig. 4, L. donovani parasites upon
treatment with DBA do not indicate early apoptotic characteristics. If
they had shown early apoptotic features, then we would have obtained
Annexin+/PI−. Instead, we obtained PI+ and then Annexin+\PI+,
which means that the parasites enter into late apoptotic stage without
going into early apoptotic phase and these cells have lost their mem-
brane integrity. DBA caused an increase in cytosolic Ca+2 level leading
to mitochondrial membrane potential dissipation [54]. We further
correlated loss of membrane potential with depletion of antioxidant
molecules such as intracellular non-protein thiol (GSH).
Our results demonstrate that DBA has potent anti-prolific activity
against L. donovani. It alters the general ultrastructure and the mi-
tochondrial physiology of the parasite and triggers apoptotic cell death.
In conclusion, based on its potent in vitro antiparasitic activity against L.
donovani, this study has identified DBA, a simple curcumin analog as a
potential candidate for further investigations on leishmaniasis. With its
low toxicity profile, DBA compares favorably, at least in vitro, with
existing drug miltefosine and is a suitable candidate for further in vivo
investigations against VL.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgments
The authors are grateful to Mr. A.L. Vishwakarma for assistance
with flow cytometry. This work was supported by Council of Scientific
and Industrial Research (CSIR) (BSC0114) funded network project
“Host Interactome analysis: Understanding the Role of Host molecules
in Parasitic Infection (HOPE).” This is CDRI communication no. 196/
2015/NS. While this manuscript was under preparation, Prof. Dr.
Govind J. Kapadia passed away on August 23, 2016. He will be re-
membered by students, faculty and co-workers as an accomplished
scientist, dedicated teacher, inspiring mentor and loyal friend.
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