Biomedicine & Pharmacotherapy 111 (2019) 224–235

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Biomedicine & Pharmacotherapy

journal homepage: www.elsevier.com/locate/biopha

Serine protease inhibitors rich Coccinia grandis (L.) Voigt leaf extract induces T
protective immune responses in murine visceral leishmaniasis
⁎
Asmita Pramanik, Dibyendu Paik, Pijush Kanti Pramanik, Tapati Chakraborti
Department of Biochemistry and Biophysics, University of Kalyani, Kalyani, 741235, West Bengal, India

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

Keywords: Leishmaniasis is a parasite-mediated tropical disease affecting millions of individuals worldwide. The available
Antileishmanial activity antileishmanial chemotherapeutic modalities exhibit adverse toxicity, exorbitant price and advent of drug-re-
Coccinia grandis (L.) Voigt sistant parasites. Hence, plant-derived products are an alternative preference for the emergence of novel and
Immunomodulation effective antileishmanial agents that rejuvenate the host immunity with limited toxicity. The present work is
Leishmania donovani
complementary to our previous report that revealed the in vitro antileishmanial and immunomodulatory activity
Serine protease inhibitor
Visceral leishmaniasis
of Coccinia grandis (L.) Voigt leaf extract (Cg-Ex) rich in serine protease inhibitors. Thus, preliminary objectives
of the study were to elucidate the leishmanicidal activity and host effector mechanism in Leishmania donovani
infected BALB/c mice treated with Cg-Ex. Oral administration of Cg-Ex significantly reduced the spleen and liver
parasite burden at dose-dependently. The parasite elimination was associated with generation of ROS and NO
that are interrelated with up-regulation of disease-suppressing Th1 cytokines and down-regulation of disease-
promoting Th2 cytokines at both protein and mRNA level. Moreover, Cg-Ex augmented the delayed-type hy-
persensitivity (DTH) response and serum IgG2a level which are correlated with the diminution of parasite
burden with no hepatic and renal toxicity. Additionally, histological analysis of spleen depicted the improvement
of structural disorganization of white and red pulp after Cg-Ex treatment. Therefore, our intriguing findings have
presented the first indication of in vivo antileishmanial efficacy through activation of pro-inflammatory immune
responses of the host by a natural plant leaf extract (Cg-Ex) containing serine protease inhibitors which could
have a role as a potential immunomodulator against visceral leishmaniasis.

1. Introduction leishmaniasis (PKDL) remain as major problems to eliminate the VL

Leishmania parasite belongs to the order of Kinetoplastid and resides
in the phagolysosome of phagocytic cells by subverting the mammalian
host immune system. The parasite develops a wide array of clinical
manifestations ranging from the asymptomatic cutaneous leishmaniasis
(CL) to the fatal visceral leishmaniasis (VL), which affects around 350
million individuals in 98 endemic countries and territories [1]. Until
now, treatment strategies of VL have been primarily devoted to che-
motherapy due to the deficiency of commercially available effective
vaccines. However, the limited option of antileishmanial drugs, devel-
opment of multiple drug-resistant parasite strains, relapse of the disease
even with HIV-VL co-infection and the onset of post-kala-azar dermal
from India [2]. The emergence of novel and promising antileishmanial
medicine has a paramount preference on the ground that prevention of
the disease is very challenging. It is well studied that Leishmania spp.
deactivate the defence machinery of the host macrophage cells and
propagate disease-associated symptoms. Thus, development of the new
chemotherapeutic approach against VL especially focuses on the direct
leishmanicidal effect and concurrent stimulation of the host’s protective
immune system with minimal toxicity [3].
Proteases play an essential role in the various biological processes of
human as well as protozoan parasites. In Leishmania parasites, proteases
exhibit a putative function for their proliferation, life cycle, and also act
as a very important virulence factor in the propagation of infection.

Abbreviations: AST, aspartate transaminase; ALT, alanine transaminase; Cg-Ex, Coccinia grandis (L.) Voigt leaf extract; CL, cutaneous leishmaniasis; DTH, delayed-
type hypersensitivity; FCS, fetal calf serum; IFN-γ, interferon-gamma; IgG1, immunoglobulin G1; IgG2a, immunoglobulin G2a; IL-10, interleukine-10; IL-12, in-
terleukine-12; iNOS, inducible nitric oxide synthase; LD-SP, Leishmania donovani serine protease; NO, nitric oxide; PBS, phosphate buffer saline; RNS, reactive
nitrogen species; ROS, reactive oxygen species; SAG, sodium stibogluconate; SLA, soluble leishmanial antigen; TGF-β, transforming growth factor-beta; TNF-α,
tumour necrosis factor-alpha; VL, visceral leishmaniasis
⁎
Corresponding author.
E-mail address: tcbiochem@gmail.com (T. Chakraborti).

https://doi.org/10.1016/j.biopha.2018.12.053
Received 4 September 2018; Received in revised form 10 December 2018; Accepted 14 December 2018
0753-3322/ © 2018 Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
A. Pramanik et al.
Additionally, Leishmania proteases (metallo, cysteine and serine) par-
ticipate in the establishment of the infection by modulating various
processes of the host systems among them the deactivation of host
immune system is one of the most important events. So, the use of
protease inhibitors targeting these proteases which are automatically
affects the protease mediated immune mechanism and block the pro-
pagation of infection [4,5]. Our previous findings of a virulence factor
from the Indian strain of Leishmania donovani is an intracellular serine
protease (58 kDa) which makes provision of an additional objective for
drug targeting aspect [6–8]. Thus, investigation of protease inhibitor
from natural sources could be beneficial to develop a unique protease-
based treatment strategy for combating visceral leishmaniasis.
Plant, is a precious wealth of nature, have a gigantic reservoir of
novel bioactive materials and these are alternative and attractive op-
tions for the drug discovery. Hence, plant-derived products can be
considered as a glimmer of hope for advancement of the chemother-
apeutic approach in different diseases. Currently, many medicinal plant
derived natural-products are used as the drug candidates for various
diseases [9]. Several scientific studies in recent years have suggested
the efficacy of different plant extracts and purified components against
fungus, protozoan parasites, helminths and other diseases [10]. As
noted that plant kingdom represents an excellent source of endogenous
protease inhibitors along with numerous secondary metabolites and
plant-based protease inhibitors are recently found to be efficient for
cancer, cardiovascular, and inflammatory diseases. In addition, plant
serine protease inhibitors particularly isolated from leaves and seeds
have also been studied in the search of alternative therapeutic agents of
these diseases [11].
Coccinia grandis (L.) Voigt belongs to the Cucurbitaceae plant family,
a climbing perennial tropical medicinal plant [12]. In the Indian tra-
ditional medicine, leaves of this plant have been used to treat diabetes
and also prevent gonorrhoea over a century. Furthermore, serine pro-
tease inhibitor from C. grandis leaf shows the evidence of antibacterial
and antifungal activity [13]. In our earlier reports, serine protease in-
hibitors enriched C. grandis leaf extract (Cg-Ex) exhibited anti-
proteolytic activity against Leishmania serine protease (SP-Ld) and has
also shown in vitro antileishmanial activity against L. donovani with a
negligible cytotoxic effect on macrophages [14,15]. Thus, consequences
of in vivo study with Cg-Ex might acquire significant importance for its
identity as a potent antileishmanial agent. Our primary objective of
present study was to exploit the antileishmanial efficacy and im-
munomodulatory role of Cg-Ex on L. donovani infected BALB/c mice for
the anticipation of developing an innovative antileishmanial agent for
visceral leishmaniasis.
2. Materials & methods
2.1. Preparation of Coccinia grandis (L.) Voigt leaf extract (Cg-Ex)
Fresh leaves from Coccinia grandis (L.) Voigt were collected from
University garden and extracted by physical method in 100 mM phos-
phate buffer, pH = 7.6 (1:5 w/v) at 4 °C, followed by centrifugation at
8000×g for 30 min and supernatant (crude extract) was collected. Then
crude extract was fractionated by ammonium sulfate precipitation
(20–85% saturation) and the precipitate was recovered by centrifuga-
tion at 10,000×g for 20 min at 4 °C. The precipitate was resuspended in
10 mM phosphate buffer, pH = 7.6. Then dialysed against 10 mM
phosphate buffer saline (PBS), pH = 7.6 for 48 h at 4 °C and after that
lyophilized. Subsequently, lyophilized sample was dissolved in 10 mM
phosphate buffer saline, pH = 7.6 and was stored at −20 °C until used
[14].
2.2. Parasite cultures
Indian strain of pentavalent antimony-responsive Leishmania dono-
vani promastigotes (MHOM/IN/1983/AG83) was used for this work.
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Biomedicine & Pharmacotherapy 111 (2019) 224–235
The virulence of promastigotes was maintained in BALB/c mice. They
were infected with the late stationary phase of second passage pro-
mastigotes, and virulent promastigotes were obtained from infected
spleen after transforming amastigotes. Subculture of promastigotes
were passed after 72 h at 22 °C in medium 199 (M199, Gibco) with
Hank’s salt containing HEPES (Sigma-Aldrich), 100 U/ml penicillin
(Gibco), and 100 mg/ml streptomycin (Gibco) supplemented with 10%
(v/v) heat-inactivated fetal calf serum (FCS, Gibco) [16].
2.3. Ethical clearance
To perform various experiments on the BALB/c mouse model, the
ethical clearance was obtained from Institutional Animal Ethics
Committee (IAEC), Department of Zoology (Registration number:892/
GO/Re/S/01/CPCSBA, Pr. 20.04.2014-28.04.2019), University of
Kalyani, Kalyani, West Bengal, India.
2.4. Animals
Adult female BALB/c mice 4–6 weeks (20–25 g body weight) were
selected as in vivo model and maintained in the animal house of
Department of Zoology, University of Kalyani, Kalyani, West Bengal,
India, according to the animal ethics committee guidelines. They were
kept at a standard temperature 20 ± 5 °C on a 12 h day and night cycle
and were provided standard food pallet diet and water ad libitam.
BALB/c mice were used for the isolation of splenocytes and main-
tenance of the virulent parasites as well as for the determination of in
vivo antileishmanial efficacy of Cg-Ex [16].
2.5. In vivo infection and treatment regiment
Each female BALB/c mouse was infected through intracardiac route
with 1 × 107 virulent stationary-phase L. donovani promastigotes [17].
The experimental sets were divided into eight groups (n = 3 mice/
group) such as uninfected control, normal mice with Cg-Ex, infected
control, vehicle control, positive control, Group I, Group II, Group III.
After 30days of post-infection, infected animals were assigned into
three categories Group I (12 mg/kg body weight/day), Group II (24 mg/
kg body weight/day), Group III (48 mg/kg body weight/day) and re-
ceived Cg-Ex by oral gavage for consecutive 15days. Another group of
normal mice were orally received 48 mg/kg body weight/day of Cg-Ex
for consecutive 15days. Efficacy of Cg-Ex will be evaluated at 1day and
15days of post-treatment by measuring different parameters. Vehicle
control mice were orally received sterile PBS during the treatment
schedule. Sodium stibogluconate (SAG, 20 mg/kg body weight/day for
15days) was used as a positive control (Table 1).
Table 1
Description of groups used in the in vivo experiment.
Serial No. Group Name Dosage
1 Uninfected Control Age-match control
2 Normal control+†Cg-Ex 48mg/kg body weight †Cg-Ex for 15days oral
administration in normal mice
3 Infected Control 1×107 L. donovani promastigotes by cardiac puncture
4 Vehicle Control Sterile #PBS treated
5 Positive Control 20mg/kg body weight §SAG for 15days intravenous administration
6 Group I 12mg/kg body weight †Cg-Ex for 15days oral administration in infected
mice
7 Group II 24mg/kg body weight †Cg-Ex for 15days oral administration in infected
mice
8 Group III 48mg/kg body weight †Cg-Ex for 15days oral administration in
infected mice
†
Cg-Ex=Coccinia grandis (L.) Voigt leaf extract, PBS = Phosphate buffer saline,
SAG = Sodium stibogluconate.
A. Pramanik et al.
2.6. Soluble leishmanial antigen (SLA) preparation
For the measurement of nitrite, superoxide and cytokines, soluble
leishmanial antigen (SLA) was prepared from stationary phase L. do-
novani promastigotes according to the standard protocol [18]. Briefly,
promastigotes (1 × 109/ml) were washed twice with sterile PBS. Then
the lysate was prepared by six cycles of freezing (−70 °C) and thawing
(37 °C) followed by the 5 min incubation on the ice. The partially lysed
promastigotes were fully disrupted by a sonicator thrice for 30 s each
and centrifuged at 10,000 rpm for 30 min at 4 °C. The supernatant
containing soluble antigen was collected and the protein concentration
was determined by BCA assay method (Pierce, Thermo Fisher Scien-
tific). SLA was stored at −20 °C until further use.
2.7. Serum collection
Blood was collected by cardiac puncture after anaesthetization of all
groups of BALB/c mice. Then serum was collected by centrifugation at
1000 rpm for 5 min at room temperature and stored at −80 °C to per-
form the quantitative analysis of IgG1, IgG2a and various biochemical
markers [19].
2.8. Enumeration of in vivo parasite burden
BALB/c mice from different groups were sacrificed for enumeration
of parasites in liver and spleen at 1day and 15days of post-treatment.
Spleen and liver of each animal from respective groups were excised
and weighted. Thereafter, spleen and liver parasite burden was mi-
croscopically determined from Giemsa (Sigma-Aldrich) stained spleen
and liver imprints respectively and expressed as Leishman Donovan
Units (LDU) of Stauber by the following formula [20]:
LDU = (No. of amastigotes/1000 cell nuclei) × Weight of organ (in
mg)
2.9. Measurement of intracellular reactive oxygen species (ROS) production
Intracellular ROS generation induced by Cg-Ex was measured using
cell-permeable probe H2DCFDA in different groups of mice. Splenocytes
were isolated from the individual animal of all experimental groups as
per the standard protocol [17] and plated in phenol red-free RPMI
medium and incubated with (50 μg/ml) SLA for 72 h at 37 °C CO2 in-
cubator (Thermo Electron Corporation, Forma II series). After that,
splenocytes were incubated with 10μM H2DCFDA (Sigma-Aldrich) for
20 min at 37 °C in dark and production of ROS was measured in terms of
geometric mean fluorescence intensity by FACS Calibur flow cytometer
(Becton Dickinson, USA) with FL-1 filter and data were analysed by
CellQuestPro software.
2.10. Determination of intracellular nitric oxide (NO) generation
The level of intracellular nitric oxide (NO) in the splenocytes was
measured using cell permeable dye DAF-2DA. Isolated splenocytes from
different experiment groups of animal were stimulated with SLA
(50 μg/ml) for 72 h at 37 °C CO2 incubator (Thermo Electron
Table 2
Forward and reverse primer sequence of different genes.
Gene Sequence of forward primer
IL-12 5′-GGA AGC ACG GCA GCA GAA TA-3′
TNF-α 5′-CAT CTT CTC AAA ATT CGA GTG ACA A-3′
IFN-γ 5′-TCA AGT GGC ATA GAT GTG GAA GAA-3′
IL-10 5′-GGT TGC CAA GCC TTA TCG GA-3′
TGF-β 5′-TGA CGT CAC TGG AGT TGT ACG G-3′
β-actin 5′-CGC CAT GGA TGA TGA TAT TGC-3′
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Biomedicine & Pharmacotherapy 111 (2019) 224–235
Corporation, Forma II series). Then, splenocytes were incubated with
5μM DAF-2DA (Sigma-Aldrich) for 20 min at 37 °C in dark. The flow
cytometer (FACS Calibur, Becton Dickinson, USA) was used for mea-
surement of NO and fluorescence intensity was quantified by using FL-1
green filter. Here, data analysis was done by CellQuestPro software
[21].
2.11. Quantification of Th1/Th2 cytokines by ELISA
Production of Th1 and Th2 cytokines from splenocytes of the dif-
ferent sets of BALB/c mice were quantitatively measured by sandwich
ELISA. Briefly, isolated splenocytes (1 × 106 /ml) from all mice were
seeded in 96-well plates and stimulated with SLA (50 μg/ml) for 72 h at
37 °C CO2 incubator (Thermo Electron Corporation, Forma II series).
The released pro-inflammatory and anti-inflammatory cytokines from
the splenocytes were measured in the supernatant as per manufacturer’s
instruction using commercial ELISA kits (BD Bioscience, USA). The
detection limit of these assays was 2.0, 2.5, 5.1, 1.5 and 4.6 pg/ml for
IL-12, TNF-α, IFN-γ, IL-10 and TGF-β respectively [17].
2.12. RNA isolation and semi-quantitative RT-PCR analysis of cytokine
genes
Total RNA from the isolated splenocytes of different groups of
BALB/c mice was extracted according to the standard Ribozol method
(Life Science Amresco). The first strand cDNA synthesis was done as per
the protocol provided by qScript cDNA synthesis kit (Quanta
Biosciences) and aliquots of 1 μg total RNA were used in 20 μl reaction
volume. Then PCR amplification was performed in a Thermal Cycler
(Bio-Rad, USA) using iProof High-Fidelity Master Mix (Bio-Rad, USA) of
50 μl reaction mixture. PCR products were subjected on 1.5% agarose
gels and visualized under UV illumination after staining with ethidium
bromide (Bio-Rad Gel-Doc, USA). The forward and reverse primer se-
quence of different genes are given in Table 2.
2.13. Assessment of delayed-type hypersensitivity (DTH) response
Delayed-type hypersensitivity (DTH) response is a salient feature of
cell-mediated immunity (CMI) in animal model. According to Kaur et al
DTH response was measured in every group of mice [19]. In brief, all
the groups of mice were subcutaneously injected with SLA in the right
footpad and PBS in their left footpads, 48 h prior to the euthanisation.
Before sacrificing mice, the thickness of both footpads was measured
with a Vernier calliper. Results were expressed in diameter of the swell
region of the right and left footpad.
2.14. Quantitative analysis of IgG1 and IgG2a isotypes
The Leishmania parasite-specific serum immunoglobulin G (IgG)
isotype antibody response was measured by a commercially available
ELISA kit (Abcam). Serum samples from each group of animals were
added after two-fold dilutions into an antibody-coated 96-well ELISA
plate. After washing the plate HRP-labelled-secondary detector anti-
body (rabbit anti-mouse IgG1 or IgG2a) was added in each well. The
chromogenic substrate was added and after 30 min of incubation stop
Sequence of reverse primer
5′-AAC TTG AGG GAG AAG TAG GAA TGG-3′
5′-TGG GAG TAG ACA AGG TAC AAC CC-3′
5′-TGG CTC TGC AGG ATT TTC ATG-3′
5′-ACC TGC TCC ACT GCC TTG CT-3′
5′-GGT TCA TGT CAT GGA TGG TGC-3′
5′-AAG CCG GCC TTG CAC AT-3′
A. Pramanik et al. Biomedicine & Pharmacotherapy 111 (2019) 224–235

Fig. 1. Determination of in vivo antileishmanial efficacy of Coccinia grandis (L.) Voigt leaf extract (Cg-Ex) on Leishmania donovani infected BALB/c mice. Estimation of
(a) splenic parasite burden and (b) hepatic parasite burden in terms of LDU in infected BALB/c mice. Cg-Ex was orally administered on L. donovani infected BALB/c
mice for consecutive 15days. Efficacy of Cg-Ex on Group I (12 mg/kg body weight), Group II (24 mg/kg body weight), Group III (48 mg/kg body weight) and SAG
treated animals (20 mg/kg body weight) was evaluated on 1day and 15days post-treatment by enumeration of intracellular parasites after Giemsa staining. (c)
Measurement of spleen weight of each group of mice. SAG (20 mg/kg body weight) was used as a positive control. Data was represented as mean ± SD (n = 3) for
three animals per group. Statistical significance was analysed between infected control and treated groups using Two-way ANOVA followed by Dunnett's multiple
comparison test (*p < 0.05, **p < 0.001, ***p < 0.002, ****p < 0.0001).

solution was added to each well. Then immediately measured the ab- 2.17. Protein estimation
sorbance on ELISA plate reader (iMark ELISA plate reader, Bio-Rad) at
450 nm with the wavelength correction at 570 nm [22]. The protein concentration of different samples was estimated by
BCA method (Pierce, Thermo Fisher Scientific).
2.15. Evaluation of biochemical markers
2.18. Densitometry analysis
For evaluation of toxicity of Cg-Ex on hepatic and renal function,
serum levels of alanine transaminase (ALT), aspartate transaminase
Densitometric analyses of semi-quantitative PCR were performed
(AST), urea and creatinine from different groups of animals were as-
with Quantity One software (Bio-Rad Laboratories, USA) and expressed
sayed using commercially available kits (Span Diagnostics Ltd., India)
as arbitrary densitometric units (ADU) in the bar plot.
[23].

2.16. Histopathological analysis 2.19. Statistical analysis

Spleen tissue fragment from each experimental set of animals was
fixed in 10% neutral buffered formalin solution (Sigma-Aldrich).
Histological sections were cut into 5 μm thick after paraffin embedding
and mounted on microscope slides. The tissue sections were stained
with haematoxylin and eosin (H&E stain, Sigma-Aldrich) and subse-
quently examined structural changes of spleen lymphoid tissue, the cell
population in white pulp and red pulp under the light microscope (Axio
skop2 Plus, Zeiss) [24].
All the data were expressed as mean ± SD. Two-way ANOVA fol-
lowed by Dunnett's multiple comparison test was performed for statis-
tical analysis by GraphPad Prism version 8.00 (Windows, GraphPad
Software, San Diego, USA). The difference between mean of various
groups were assessed for statistical significance, and p values < 0.05
were considered as statistically significant.

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A. Pramanik et al.
Fig. 2. Quantification of intracellular reactive oxygen species (ROS) and nitric oxides (NO) in the activated splenocytes. (a) Intracellular ROS level was measured in
SLA activated splenocytes of different groups after 1day and 15days post-treatment by cell-permeable H2DCFDA dye using FACS analysis. (b) Intracellular NO was
determined in the splenocytes isolated from each group of mice after 1day and 15days post-treatment by DAF-2DA dye using FACS analysis. SAG was used as a
positive control for measurement of ROS and NO. Values are represented as the mean ± SD (n = 3), and statistical significance was examined between infected
control and treated groups by Two-way ANOVA followed by Dunnett's multiple comparison test (*p < 0.05, **p < 0.001, ***p < 0.002, ****p < 0.0001).
3. Results
3.1. Cg-Ex confers in vivo protection by reducing parasitemia
To validate the in vivo antileishmanial efficacy of the Cg-Ex, spleen
and liver parasite burden of experimental groups of mice were de-
termined in terms of LDU. Accordingly, the observation was made after
oral administration of Cg-Ex for 15days in infected mice and the
parasite burden was measured on 1day and 15days post-treatment. In
case of infected control group the LDU of spleen was 755.5 ± 14.8 at
1day post-treatment and 834.0 ± 11.3 at 15days post-treatment. At
lowest dose of Cg-Ex (Group I, 12 mg/kg body weight) parasite burden
were decreased 29.6 ± 2.97% (LDU = 531.5 ± 16.3) and
44.1 ± 1.92% (LDU = 476.0 ± 9.9) from spleen at 1day and 15days
of post-treatment respectively. In Group II (24 mg/kg body weight)
mice similarly the splenic parasite load was reduced by 46.2 ± 3.87%
(LDU = 406.0 ± 12.0) and 70.6 ± 5.91% (LDU = 245 ± 7.1) at
1day and 15days of post-treatment. At the highest dose of Cg-Ex (Group
III, 48 mg/kg body weight) considerable reduction of parasite burden in
the spleen has appeared with 52.2 ± 4.04% (LDU = 361.0 ± 8.5) and
81.9 ± 3.84% (LDU = 151.0 ± 9.8) respectively at the same time
points. Likewise, in the positive control, i.e, SAG treated mice have
shown 69.0 ± 2.67% (LDU = 234.0 ± 8.5) and 89.2 ± 1.99%
(LDU = 90.0 ± 7.4) reduction in splenic parasites at 1day and 15days
of post-treatment respectively (Fig. 1a). This observation clearly depicts
that the diminution of splenic parasite burden upon Cg-Ex treatment
occurs in dose-dependent manner.
Additionally, determination of liver parasite burden represents a
key parameter for monitoring Leishmania infection in a mouse model. In
the infected group of mice the liver parasite burden in terms of LDU was
4139.0 ± 29.7 at 1day post-treatment and 4052.5 ± 38.9 at 15days
post treatment. Therefore, upon Cg-Ex treatment in Group I mice the
diminution of liver parasite load was observed by 31.7 ± 1.01%
(LDU = 2851.0 ± 26.9) and 50.6 ± 0.5% (LDU = 2039.0 ± 24.0) at
1day and 15days respectively. In the same duration of time Group II
BALB/c mice have exhibited 43.7 ± 0.32% (LDU = 2630.0 ± 31.1)
and 65.2 ± 1.47% (LDU = 1415.0 ± 37.5) decrease of parasite load
in the liver. Correspondingly, in case of Group III treated mice liver
parasite burden was declined about 68.5 ± 1.10%
(LDU = 1269.5 ± 34.6) and 78.0 ± 2.92% (LDU = 891.5 ± 33.2) in
the same time. A parallel observation was made within SAG treated
mice where the reduction of parasitemia from liver was about
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Biomedicine & Pharmacotherapy 111 (2019) 224–235
65.0 ± 1.12% (LDU = 1148.5 ± 40.3) and 84.0 ± 2.1%
(LDU = 643.0 ± 21.2) with respect to the infected control at equal
time span (Fig. 1b).
Splenomegaly is one of the major hallmarks of chronic VL due to the
parasitic load and cellular infiltration in the spleen [25]. With the
progression of infection spleen weight considerably increased 2.5 fold
and 2.7 fold at 1day and 15days post-treatment. However, the sig-
nificant decrease of 1.8 fold and 2.4 fold in weight of spleen was ob-
served in the Group III BALB/c mice at 1day and 15days post-treatment
with respect to the infected control mice (Fig. 1c). A similar pattern of
reduction in weight of spleen was noted in SAG treated mice by 1.9 fold
and 2.5 fold at 1day and 15days post-treatment respectively. Values
obtained from these experiments are statistically significant
(*p < 0.05, **p < 0.001, ***p < 0.002, ****p < 0.0001) compared
to respective control.
3.2. Cg-Ex restores intracellular oxidative burst
Intracellular Leishmania parasites deactivate the macrophages and
survived by scavenging oxygen free radicals such as ROS and NO within
the host. Nitric oxide generation depends on the activation of iNOS
expression in Leishmania infected macrophages [26]. In contrast, gen-
eration of ROS and NO by macrophages upon treatment with antil-
eishmanial agent facilitates the elimination of parasites from the host
[27]. Therefore, intracellular ROS and NO were measured in spleno-
cytes of all groups of BALB/c mice and presented by GMFC (Geometric
Mean Fluorescence Channel). Splenocytes from different groups of mice
exhibited a significant elevation of intracellular ROS in response to Cg-
Ex at dose-dependent manner on both time points. Splenocytes from
Group III mice displayed exaggeration of ROS generation at 5.1 fold and
4.4 fold at 1day and 15days of post-treatment respectively when com-
pared to infected control. In case of other two groups (Group I and
Group II) ROS was also generated in dose-dependently (Fig. 2a).
The present study was also demonstrated that NO production was
inhibited by 2.2 fold and 3 fold in infected control compared to the
uninfected animals at 1day and 15days post-treatment. Conversely,
splenocytes from Group III BALB/c mice augmented NO generation by
7.8 fold and 10.7 fold at 1day and 15days post-treatment in addition to
the generation of ROS (Fig. 2b). Other two groups of Cg-Ex treated
animals (Group I and Group II) showed the elevation of intracellular
nitric oxide (NO) level in a dose-dependent manner. All data in these
two experiments are statistically significant (*p < 0.05, **p < 0.001,
A. Pramanik et al.
Fig. 3. Analysis of pro-inflammatory and anti-inflammatory cytokines secreted by stimulated splenocytes. Th1 cytokines (a) IL-12 (pg/ml), (b) TNF-α (pg/ml), (c)
IFN-γ (pg/ml) and Th2 cytokines (d) IL-10 (pg/ml), (e) TGF-β (pg/ml) were quantified in culture supernatant of splenocytes isolated from different sets of animals
after 72 h of SLA stimulation by sandwich ELISA. Results were expressed as mean ± SD (n = 3) from three replicate experiments and statistical significance was
checked using Two-way ANOVA followed by Dunnett's multiple comparison test (*p < 0.05, **p < 0.001, ***p < 0.002, ****p < 0.0001).
***p < 0.002, ****p < 0.0001) compared to the infected control.
3.3. Cg-Ex induces secretion of pro-inflammatory cytokines at protein and
mRNA level
It is fully established that Leishmania infection critically skews from
pro-inflammatory response to anti-inflammatory response for sus-
taining the intracellular parasites in phagocytic cells [28]. Thus, the
Th1 immune response was assessed by the quantification of IL-12, TNF-
α, and IFN-γ cytokine in splenocytes culture supernatant. Levels of IL-
10 and TGF-β were also used as indicators of Th2 immune response.
The anti-inflammatory cytokines stimulate the progression of infection
within host. Hence, we examined whether the Cg-Ex treatment could
modulate pro-inflammatory cytokine (IL-12, TNF-α, and IFN-γ) and
anti-inflammatory cytokine (IL-10, TGF-β) release and expression at the
mRNA level in infected groups of animals. Level of Th2 cytokines were
found to be maximum in infected mice both at protein and mRNA level,
whereas their concentrations were reduced significantly in all treated
groups of animals in all post-treatment days. In Group III mice, there
was a considerable amount of increment in IL-12 (2.4 fold), TNF-α (5.6
fold) and IFN-γ (5.8 fold) level (Fig. 3a–c) in culture supernatant of
activated splenocytes with concomitant reduction of IL-10 (3 fold) and
TGF-β (2.7 fold) in protein level (Fig. 3d, e). A similar effect of SAG was
observed on Th1 and Th2 cytokines in the culture supernatant at
15days of post-treatment.
The quantitative data of cytokine expression as observed by the
ELISA method was confirmed by mRNA expression of the aforemen-
tioned cytokines using semi-quantitative PCR analysis. The results re-
vealed that there was a considerable inhibition of IL-12, TNF-α, and
IFN-γ expression in the infected splenocytes compared with uninfected
splenocytes. In Cg-Ex treated infected splenocytes, 3.1, 4.0, and 3.5-fold
increment was shown in the expression of IL-12, TNF-α, and IFN-γ re-
spectively compared with infected splenocytes (Fig. 4a–d). On the other
hand, mRNA profile exhibits a significant induction of IL-10 and TGF-β
in infected splenocytes with respect to the uninfected splenocytes. In-
triguingly, when infected splenocytes were treated with Cg-Ex, there
was 3.45 and 3.62 -fold reduction in the expression of IL-10 and TGF-β
respectively considering with infected control splenocytes (Fig. 4a, e,
229
Biomedicine & Pharmacotherapy 111 (2019) 224–235
and f). Treatment with PBS in animals as vehicle control did not showed
any effect on the release of IL-12, TNF-α, and IFN-γ that proves im-
munological inertness of PBS in the in vivo model (data not shown).
Data are statistically significant (*p < 0.05, **p < 0.001,
***p < 0.002, ****p < 0.0001) compared to the respective control.
3.4. Cg-Ex enhances leishmanial antigen-specific DTH response
DTH response predicts the peripheral blood T-cell immunity as well
as it correlates the clearance of parasites in animal model [29]. Con-
sequently, DTH response was measured in the infected mice at 1day and
15days post-treatment. A slight increase (1.4 fold) in footpad thickness
was observed in infected mice as compared to normal mice. But a sig-
nificant rise (2.28 fold) of DTH response was observed in Group III mice
footpad at 15days post-treatment (Fig. 5). Each value is statistically
significant (*p < 0.05, **p < 0.001, ***p < 0.002, ****p < 0.0001)
in comparison to the infective control.
3.5. Cg-Ex develops humoral immune responses
Healing of leishmaniasis is connected with the development of
strong humoral immune responses like IgG2a production while estab-
lishment of infection is associated with higher IgG1 production [30].
The IgG2a antibody response is a marker of protective Th1 type of
immune response. Therefore, Cg-Ex treated BALB/c mice produced
significant level of IgG2a antibody when compared with the infected
control. The serum IgG2a was increased about 2 fold in Group III mice
(Fig. 6a). Again, IgG1 antibody response points towards Th2 type of
immune response. Therefore, it was found that IgG1 level was higher in
infected BALB/c mice as compared to uninfected ones whereas IgG1
level was significantly reduced (2.9 fold) in the Group III mice at
15days post-treatment as compared to infected controls (Fig. 6b).
However, in SAG treated infected animals the increase in IgG2a level
and decrease in level of IgG1 was lower compared with higher dose of
Cg-Ex treated animals (Group III). All values are statistically significant
(*p < 0.05, **p < 0.001, ***p < 0.002, ****p < 0.0001) compared
to the infective control.
A. Pramanik et al. Biomedicine & Pharmacotherapy 111 (2019) 224–235

Fig. 4. Evaluation of Th1 and Th2 cytokines in activated splenocytes using semi-quantitative PCR. The mRNA was isolated from each sample and PCR amplification
was done. PCR product of Th1 and Th2 cytokines was run on the 1.5% agarose gel (a) Lane no. 1–5 in agarose gel are represented the normal control, infected control,
Group I, Group II, and Group III of 15 days post-treatment respectively. Here, β-actin was used as control housekeeping gene. (b) The band intensities of IL-12, TNF-α,
IFN-γ, IL-10, and TGF-β were evaluated and plotted as the bar diagram. Data are represented as the mean ± SD (n = 3) and statistical significance was analysed
between infected control and treated groups by Two-way ANOVA followed by Dunnett's multiple comparison test (*p < 0.05, **p < 0.001, ***p < 0.002,
****p < 0.0001).

Fig. 5. Delayed-type hypersensitivity response induced by Cg-Ex in infected
BALB/c mice. DTH response was measured in various groups of BALB/c mice at
1day and 15days of post-treatment. Statistical significance was analysed in all
groups by Two-way ANOVA followed by.
Dunnett’s multiple comparison test (*p < 0.05, **p < 0.001, ***p < 0.002,
****p < 0.0001) and expressed as mean ± SD (n=3).
were done by measuring the activity of serum AST, ALT and also the
level of urea and creatinine. The activity of liver enzymes in infected
control animals have been found in higher level with respect to unin-
fected control, which was reduced in a dose dependent manner in Cg-Ex
treated infected BALB/c mice at 1day and 15days post-treatment
(Table 3). Same pattern of increment was observed in liver transami-
nase enzymes of SAG treated mice. Elevation of transaminase activity in
SAG treated mice indicates the hepatotoxicity of this conventional drug,
while reduction of these enzymes in Cg-Ex treated mice to the normal
level implies that Cg-Ex has no hepato-toxicity in mice. Values are
presented as mean ± SD and differences between means were statis-
tically significant (*p < 0.05, **p < 0.001, ***p < 0.002,
****p < 0.0001) in comparison to the infected control.
Similarly, kidney function tests also showed the normal level of urea
and creatinine in serum of uninfected mice whereas infected control
mice have higher level of urea and creatinine. Although Cg-Ex treated
mice demonstrated the significant reduction of urea and creatinine level
in the serum to normal level but during SAG treatment this level was
increased. So, these observations signify the non-nephrotoxic effect of
Cg-Ex in BALB/c mice (Table 4). Vehicle control animals showed no
toxic effect on the liver and kidney function. Normal animals treated
with the highest dose of Cg-Ex (48 mg/kg body weight) did not show
any hepatic and renal toxicity after 1day and 15days post-treatment
(Tables 3 and 4). Data are represented as mean ± SD and differences
between means were statistically significant (*p < 0.05, **p < 0.001,
***p < 0.002, ****p < 0.0001) with respect to the infected control.

3.6. Non-toxic effect of Cg-Ex in liver and kidney function 3.7. Improvement of structural organization after Cg-Ex treatment

Hepato- and renal toxicity are common in currently available drugs Structural disorganization of white pulp and red pulp remains a
for VL [31]. In order to examine any hepatotoxicity or nephrotoxicity characteristic property of the murine model of VL [32]. Thus, histo-
exerted by Cg-Ex in the BALB/c mice, the liver and kidney function tests pathological analysis of spleen was carried out to examine the tissue
remodelling in the infected BALB/c mice. In this experiment, it was

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A. Pramanik et al. Biomedicine & Pharmacotherapy 111 (2019) 224–235

Fig. 6. Humoral immune response augmented by Cg-Ex in Leishmania donovani infected BALB/c mice. Leishmania-specific antigen (a) IgG2a and (b) IgG1 in the serum
of uninfected control, infected control and different treated groups of BALB/c mice at 1day and 15days post-treatment were quantified by the ELISA method.
Statistical significance was determined compared with infected control group (p* < 0.05) by Two-way ANOVA followed by Dunnett's multiple comparison test
(*p < 0.05, **p < 0.001, ***p < 0.002, ****p < 0.0001) and expressed as mean ± SD (n=3).

observed that the spleen from all the groups contained an intact capsule
but there was some architectural abnormality in white pulp and red
pulp. Herein, the non-infected group of animals was shown a compact
structure of white and red pulp (Fig. 7a) whereas the untreated infected
group has possessed extensive cellular disorganization in white and red
pulp architecture (Fig. 7b). Interestingly, at 1day post-treatment of
Group III mice have showed a modest level of regeneration of white
pulp from the widespread distorted structure (Fig. 7c). At 15days of
post-treatment, the structural integrity of white pulp and red pulp of
spleen from Group III animals seems more organized (Fig. 7d).
4. Discussion
The pathogenesis of visceral leishmaniasis concerns about the sub-
version and modulation of both innate and adaptive immunity of the
host leading to immunosuppression in individuals [33]. Hence, the
prevention of VL is challenging and the initiation of an appropriate
immune response is a necessary criterion to cure the disease. In fact,
conventional drugs like sodium stibogluconate and miltefosine act as an
immunomodulator that elicits the Th1-biased response and activates
the oxidative burst in macrophages to eliminate Leishmania parasites
[34,35]. However, the disease management is limited due to the small
therapeutic index and the emergence of resistance against available
drugs for VL [36].
In this perspective, searching for innovative antileishmanial agents
from the natural sources is an alternative approach. Various plant ex-
tracts and plant-derived products represent leishmanicidal activity by
targeting different biochemical pathways [37]. Plants provide an ex-
cellent source of protease inhibitors protecting from many diseases,
pests, insects and herbivorous animals. Very recently scientists devoted
their attention to the development of plant protease inhibitor-based
novel therapeutic agents against cardiovascular diseases, inflammatory
disease, bacterial and fungal diseases [11]. Clemente et al. have re-
ported that Bowman-Birk inhibitor, a serine protease inhibitor from
Pisum sativum, inhibits the in vitro growth of human colorectal adeno-
carcinoma cells [38]. Again, the serine protease inhibitor of Coccinia
grandis demonstrated the highest inhibitory activity against trypsin and
chymotrypsin as well as exhibiting the antimicrobial and antifungal
activity [13]. It is also noted that some plant-derived protease inhibitor
has shown antileishmanial activity with non-toxic effect on the mac-
rophages [5]. In another report, epoxy-α-lapachone, a plant-derived
serine protease inhibitor of oligopeptidase B, was also found to reduce

Table 3
Effect of Cg-Ex on liver enzymes activity of different groups of BALB/c mice.
Enzymes Post Normal control Normal Infected control Vehicle Group I Group II Group III (48mg/ Sodium
(Normal treatment control+ control (12mg/kg §b.w (24mg/kg §b.w kg §b.w †Cg-Ex) stibogluconate
† †
range) days 48mg/kg §b.w Cg-Ex) Cg-Ex) (20mg/kg §b.w)
†
Cg-Ex
¶
AST 1 22.36 ± 3.11 23.17 ± 2.34 50.97 ± 1.72 51.26 ± 1.45 38.51 ± 0.88 33.06 ± 2.27 27.61 ± 2.46 61.90 ± 2.36
(5-40 (*p˂0.05) (****p˂0.0001) (****p˂0.0001) (****p˂0.0001)
IU/L)
15 26.92 ± 2.32 24.56 ± 2.78 59.43 ± 2.72 60.14 ± 2.56 31.79 ± 1.99 27.64 ± 2.08 24.96 ± 3.98 67.20 ± 2.94
(****p˂0.0001) (****p˂0.0001) (****p˂0.0001) (****p˂0.0001)

#
ALT 1 24.31 ± 2.61 24.11 ± 1.31 60.61 ± 2.44 59.97 ± 2.26 54.69 ± 2.99 33.21 ± 1.32 30.95 ± 2.42 76.96 ± 1.86
(5-35 (****p˂0.0001) (****p˂0.0001) (****p˂0.0001) (****p˂0.0001)
IU/L)

15 26.24 ± 2.81 25.67 ± 2.45 75.31 ± 2.94 77.37 ± 1.82 46.56 ± 2.05 31.27 ± 1.69 27.05 ± 1.40 82.96 ± 2.57
(****p˂0.0001) (****p˂0.0001) (****p˂0.0001) (****p˂0.0001)

#
ALT = Alanine Transaminase; AST = Aspartate Transaminase, b.w = body weight, Cg-Ex=Coccinia grandis (L.) Voigt leaf extract, SAG = Sodium stibogluconate.
†BALB/c mice (n = 3) received Cg-Ex for consecutive 15days and activity of liver enzymes were estimated after 1day and 15days post-treatment (mean ± SD) using
commercial kits. All values are statistically significant (*p < 0.05, **p < 0.001, ***p < 0.002, ****p < 0.0001) and statistical significance was analysed with
respect to the infected control using Two-way ANOVA followed by Dunnett's multiple comparison test.

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A. Pramanik et al. Biomedicine & Pharmacotherapy 111 (2019) 224–235

Table 4
Consequence of Cg-Ex on the renal function of various groups of BALB/c mice.
Metabolites Post Normal control (Normal Infected control Vehicle Group I Group II (24mg/ Group III (48mg/ Sodium
(Normal treatment control+ control (12mg/kg kg b.w†Cg-Ex) kg b.w†Cg-Ex) stibogluconate
range) days 48mg/ b.w†Cg-Ex) (20mg/kg b.w)
kg§b.w†Cg-Ex)

Urea 1 32.98 ± 1.74 33.21 ± 2.08 57.63 ± 3.32 55.12 ± 2.67 49.06 ± 1.34 40.57 ± 1.87 33.86 ± 1.97 68.58 ± 1.29
(10-45 (**p˂0.001) (****p˂0.0001) (****p˂0.0001) (****p˂0.0001)
mg/dl)
15 36.57 ± 2.18 35.56 ± 2.64 65.12 ± 1.49 66.78 ± 2.23 43.33 ± 1.43 34.09 ± 1.72 31.84 ± 1.69 80.46 ± 2.05
(****p˂0.0001) (****p˂0.0001) (****p˂0.0001) (****p˂0.0001)

Creatinine 1 0.87 ± 0.03 0.86 ± 0.02 1.45 ± 0.06 1.49 ± 0.02 1.35 ± 0.04 1.20 ± 0.01 0.92 ± 0.04 1.54 ± 0.03
(0.6-1.2 (*p˂0.05) (****p˂0.0001) (****p˂0.0001) (****p˂0.0001)
mg%)
15 0.85 ± 0.03 0.85 ± 0.04 1.68 ± 0.04 1.72 ± 0.07 1.27 ± 0.01 1.14 ± 0.04 0.82 ± 0.02 1.88 ± 0.02
(****p˂0.0001) (****p˂0.0001) (****p˂0.0001) (****p˂0.0001)

#
b.w = body weight, Cg-Ex=Coccinia grandis (L.) Voigt leaf extract, SAG = Sodium stibogluconate.
†Cg-Ex was orally administered in BALB/c mice (n = 3) for consecutive 15days and metabolites were estimated after 1day and 15days post-treatment (mean ± SD)
by commercial kits. All values are statistically significant (*p < 0.05, **p < 0.001, ***p < 0.002, ****p < 0.0001) and statistical significance of different groups
were examined with respect to the infected control group using Two-way ANOVA followed by Dunnett's multiple comparison test.

Fig. 7. Assessment of histopathological alterations in the spleen after treatment of Cg-Ex.

Histology of spleen of BALB/c mice (a) uninfected control (b) infected control (c) Cg-Ex treated Group III mice (48 mg/kg body weight) at 1day post-treatment (d) Cg-
Ex treated Group III mice (48 mg/kg body weight) at 15days post-treatment. Tissue sections were stained with H&E stain and scale bar is 20 μm (20X magnification).
Capsule, white pulp, and red pulp are indicated by the black arrow in the histological section of spleen. (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)

232
A. Pramanik et al.
lesion size in Leishmania amazonensis infected BALB/c mice [39]. Ad-
ditionally, our previous reports have shown the antileishmanial activity
of Coccinia grandis (L.) Voigt leaf extract (Cg-Ex) that is enriched with
three serine protease inhibitors (64.8, 55.8 and 15.3 kDa). The Leish-
manial intracellular serine protease inhibitor (58 kDa, LD-SP) was in-
hibited by Cg-Ex without displaying any toxicity on murine macro-
phages [14,15]. The present study revealed that Cg-Ex exerts
antileishmanial activity on mouse model of VL by reducing the parasite
growth. Another investigation stated that the leaf extract of Croton
caudatus eliminates in vivo parasitic burden by up regulation of Th1
cells of the host [40]. Thus, other reports support our present findings
of antileishmanial activity of Cg-Ex on murine model of VL.
For the establishment of infection within host macrophages,
Leishmania parasites hinder the production of parasiticidal molecules
from macrophages such as reactive oxygen species (ROS) and reactive
nitrogen species (RNS). Therefore, macrophage-derived reactive inter-
mediates are involved in the innate host defence mechanism as integral
components for controlling Leishmania infection within the host cell
[27]. It is familiar that IFN-γ stimulates the expression of iNOS with
substantial production of nitric oxide in macrophages [41]. The present
study demonstrated that Cg-Ex exerts antileishmanial activity through
the production of ROS and NO in infected BALB/c mice. Our observa-
tions very clearly depicted that Cg-Ex could effectively mediate NO
generation as it significantly induced iNOS expression at the protein
level in L. donovani-infected animals. The similar effect was shown by
cystatin, a natural cysteine protease inhibitor, which exhibits in vivo
leishmanicidal effect by switching Th2 to Th1 cytokine responses and
also produced intracellular NO via the activation of JAK/STAT pathway
[42].
The chemotherapeutic efficacy of a drug for VL includes restoration
of T-cell response as this response is impaired during the progression of
the disease. Macrophages and T-cell subsets secret IL-12 that helps in
the development of Th1 cells and the effectiveness of antileishmanial
therapy is related with the reestablishment of IFN-γ and IL-12 secretion
[33].
Moreover, Leishmania proteases particularly cysteine proteases and
gp63 play a crucial role in the immune evasion process by degradation
of various proteins of host, such as the complement factors (C3b),
transcription factor NF-κB and the MHC class II molecules which
modify the immune response of the macrophages and facilitates per-
sistence of infection within the host. The pieces of literature also re-
ported that some natural protease inhibitors have shown leishmanicidal
activity by inhibiting the leishmania proteases and also stimulates the
Th1 responses in macrophages [4,5,26]. The oral administration of Cg-
Ex has also enhanced Th1 response especially IL-12, TNF-α, and IFN-γ
with simultaneous diminish of IL-10 and TGF-β in the infected BALB/c
mice. Several reports suggested that various plant-derived products
exert antileishmanial activity via modulation of T-cell response [43].
Delayed-type hypersensitivity (DTH) or type-IV cell-mediated hy-
persensitivity represents a localized inflammatory reaction where Th1
cells play a pivotal role in this response. DTH response induces the
clearance of parasites from infected host cells by activation of T-cell
that leads to the release of Th1 cytokines resulting in the infiltration of
lymphocytes and macrophages into the infected tissue [44]. It is re-
ported that plant-derived immunomodulator, eugenol exhibits DTH
response in BALB/c mice. This observation suggested that higher the
DTH response lower the parasitic load and greater the efficacy of im-
munomodulatory plant-derived compound [23]. The current study re-
ported that DTH response was induced by Cg-Ex in BALB/c mice.
During the course of Leishmania infection, the host IgG1 increases
which is responsible for secretion of disease-promoting IL-10 cytokine
whereas IgG2a antibody decreases after resolution of infection that is
linked with IFN-γ production [30]. Thus, the detail immunological
study demonstrated that Cg-Ex treatment induces production of IgG2a
level due to the up-regulation of IFN-γ. Azadirachta indica bioactive
fraction significantly increases serum IgG2a level but simultaneously
233
Biomedicine & Pharmacotherapy 111 (2019) 224–235
decreases IgG1 level in treated BALB/c mice [45]. Therefore, con-
sidering these observations it can be concluded that Cg-Ex activates
both cell-mediated immunity and humoral immunity to clear the
parasitic burden from infected mice.
Hepatomegaly is a characteristic property of the VL and renal dys-
functions have been well documented that comprises interstitial and
glomerular defect [46]. Furthermore, the renal and hepatic dysfunction
are major side effects of the currently available drugs used for treatment
of VL [31]. A significant decrease of transaminase enzyme activity and
serum urea and creatinine level indicate the non-toxic effect of Cg-Ex on
liver and kidney. Similarly, the plant derived bioactive fraction of Piper
nigum has demonstrated the in vivo nontoxic effect on liver and kidney
function with antileishmanial property [47]. Here, the SAG treated
animals showed antileishmanial activity with extensive toxicity in liver
and kidney whereas Cg-Ex does not express any hepatotoxicity and
nephrotoxicity in the animal model of VL.
During visceral leishmaniasis, extensive damage of tissue occurs in
the spleen due to the disruption of both fibroblastic reticular cell net-
work and follicular dendritic cell network [28]. Upon oral adminis-
tration of Cg-Ex, the cellular disorganization in white pulp and red pulp
of spleen was restored by preventing tissue disruption in the infected
BALB/c mice. Dalton et al. demonstrated that antimonial drug along
with sunitinib maleate (Sm), an inhibitor of receptor tyrosine kinase,
enhanced leishmanicidal activity as well as block tissue remodelling
and splenomegaly in experimental VL [48]. In the similar fashion, ur-
solic acid, a plant-derived compound, showed the reduction of parasitic
burden in liver and spleen as well as changed the histological archi-
tecture of spleen white pulp [49].
Our overall findings indicate that Cg-Ex possesses a protective role
in the L. donovani infected BALB/c mice model without nephrotoxicity
and hepatotoxicity after oral administration. The potential antil-
eishmanial activity of Cg-Ex was augmented by both cell mediated and
humoral immunity of the host as described in Fig. 8 and Cg-Ex also
prevents the tissue damage in the spleen. Consequently, Cg-Ex can be a
deliberate candidate for achieving a new identity as therapeutic agent
against visceral leishmaniasis.
5. Conclusions
Altogether, our study illustrates the in vivo leishmanicidal potency
of Cg-Ex that occurs by means of the up regulation of Th1 immune
response and simultaneous production of ROS and NO exerting non-
toxic effect on the mouse model. Combining results of our studies as
observed previously in vitro and currently in vivo systems, it is suggested
that serine protease inhibitor enriched leaf extract (Cg-Ex) can inhibit
the Leishmania donovani serine protease and could be a potential option
for VL chemotherapy. The current study accredits further investigations
to identify the Cg-Ex-derived highly active protease inhibitor against
Leishmania serine protease. This endeavour might facilitate the ex-
ploration of the exact mechanisms behind the antileishmanial activity
of a novel natural plant derived lead component in the treatment of VL.
Conflict of interest
The authors declare that they have no conflict of interest.
Acknowledgements
AP is recipient of ICMR-SRF and this experimental work was fi-
nancially supported by the grants from the Indian Council of Medical
Research (ICMR, Government of India), New Delhi (sanction No.
Fellowship/18/2018-ECD-II), Government of India. Authors are
thankful to the DST-FIST phase-III (Project no. FIST Sl. NO. 185),
Government of India and UGC-SAP phase-II (Project No. F 5-3/2018/
DRS-II) and University of Kalyani.
A. Pramanik et al.
Fig. 8. Summary of immunological responses during establishment of infection and elimination of infection from Leishmania donovani infected BALB/c mice upon
oral administration of Cg-Ex. The figure illustrates that during L. donovani infection in BALB/c mice, the Th2 type immune response was predominant with sup-
pression of ROS and NO production. In contrast, Th1 type immune response is up-regulated following treatment with Cg-Ex. The generation of ROS and NO with
positive DTH response facilitates reduction of the parasite burden in the infected BALB/c mice.
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