Gallic acid induces reactive oxygen species generation and apoptosis in Leishmania donovani in vitro: Probable antileishmanial cellular mechanism

DOI: https://doi.org/10.21203/rs.3.rs-1791137/v1

Abstract

Leishmaniasis is one of the important neglected tropical diseases affecting more than 12 million worldwide with emergence of 3 million cases every year. Leishmaniasis manifests into different clinical outcomes and the most fatal being Visceral Leishmaniasis. Emerging cases of parasite resistance to the limited chemotherapeutic drugs necessitates novel strategies and drugs with antileishmanial potential which can replace the current drugs. Bioactive phytomolecules like polyphenols and tannins have been reported for their antileishmanial activities. Amongst such compounds, Gallic acid is a naturally occurring plant phenolic compound which is a well-known immunomodulator, anti-inflammatory, antioxidant was tested for anti-leishmanial activity on the Leishmania donovani Dd8 promastigotes, in vitro. The IC90 of Gallic acid (1mM) on Leishmania donovani promastigotes was evaluated by MTT assay and the treatment of Gallic acid to Leishmania promastigotes demonstrated cell shrinkage, cytotoxicity and triggered apoptosis like events - chromatin condensation, release of reactive oxygen species (ROS), loss of mitochondrial membrane potential, showed DNA fragmentation, cell cycle arrest at the sub-G0/G1 phase (94.4%) and membrane permeability. After Gallic acid and MIL treatment, 89% and 63.78% promastigotes showed apoptotic death for 24 h respectively. After 6 h incubation with four-fold of IC90, only 12.94 ± 0.12% hemolysis was observed with gallic acid treatment indicating its non-toxic nature. Our data indicates Gallic acid to be a promising anti-leishmanial compound that mediates its activity on the promastigotes, in vitro; via a caspase 3 independent programmed cell death and accordingly, deserves consideration and further investigation as a therapeutic option for the treatment of leishmaniasis.

Introduction

The World Health Organization (WHO) considers Leishmaniasis as one of the six major infectious diseases which affects more than 12 million people and 3 million new cases are reported worldwide annually(Mitropoulos et al. 2010). Leishmaniasis has several clinical manifestations capable of severe deformities and the most fatal being the Visceral Leishmaniasis (VL). According to the World Health Organization, VL which is caused by the protozoan parasite Leishmania donovani has an incidence and prevalence rate of 0.5 million and 2–5 million VL cases per year respectively, accounting for high DALYs (disability-adjusted life years)(WHO Report, Geneva 2010) Presently, the limited chemotherapeutic drugs against leishmaniasis in clinics include Sodium Antimony Gluconate (SAG), Sodium Stibo Gluconate (SSG), Amphotericin B and Miltefosine (Frézard et al. 2009). However, these therapeutic drugs are expensive, have severe side effects and are proving ineffective against resistant strains of Leishmania (Croft et al. 2006). Thus, the severe toxicity of current anti-leishmanials to normal cells and the high resistance of the causative agent, Leishmania donovani have necessitated the need for discovery of new alternative drugs (Murray et al., 2005; Rosenthal and Marty, 2003). In the absence of an effective vaccine, chemotherapy remains the sole weapon in the arsenal against leishmaniasis (Rosenthal and Marty 2003). In the ongoing search for efficient anti-leishmanial compounds, plant-derived products are being evaluated for low toxicity and high efficacy features (Kayser et al. 2003). Previously a number of tannins and phenols have been tested and evaluated against Leishmania spp. (Herbert Kolodziej and Kiderlen, 2005). Amongst such compounds, Gallic acid is a naturally occurring plant phenolic compound which is also known as 3,4,5-trihydroxybenzoic acid occurring both in free state and as a constituent of tannins namely: Gallo tannin(Fitzpatrick and Woldemariam 2017). It commonly found in Indian gooseberry: Emblica officinalis (Amla), is a well-known antioxidant and has been tested for its efficacy against L. major amastigotes in vitro (Radtke et al. 2004). Several indigenous pharmacological properties like anti-inflammatory, antioxidant (Yang et al. 2015), antimutagenic (Lee et al. 2003; Lu et al. 2010), anticancer (Paolini et al. 2015), antimicrobial(Sarjit et al. 2015) has been attributed to gallic acid (GA). In addition, recently GA and its derivatives are reported to be potential novel treatment agents for several gastrointestinal diseases through its interaction with the gut microbiota and immunomodulatory responses. In vitro and animal model studies indicate its therapeutic interventions making it a potential candidate for treating several high morbidity diseases (Yang et al. 2020). Leishmania spp. upon infection harbors in the macrophages where, it evades the host immune system by multiple mechanisms like reduction in iNOS (inducible Nitric Oxide synthase) activity, increase in Th2 response by stimulating the production of anti-inflammatory cytokines (IL4, IL10) (Shahi et al., 2013). Earlier studies indicated an increase in Nitric oxide (NO) and cytokine levels in L. major amastigote infected RAW 264.7 cells when treated with GA by studying their gene expression profiles (Radtke et al. 2004). The immunomodulatory effects of GA were reinforced when GA induced an increase in phagocytosis ability of murine macrophages, nitrite release, intracellular calcium levels and lysosomal volume against L. major (Alves et al. 2017). Though the effect of Gallic acid on L. major amastigotes has been tested, the probable antileishmanial cellular effects on L. donovani promastigote forms has not been yet elucidated. The metacyclic promastigote form of the parasite is the infective stage that is transmitted from the sandfly to the mammalian host. This led us to investigate the effect of Gallic acid on promastigotes in vitro. It was previously reported tolerance of up to 128 mg/kg/day Gallic acid administration in F344 rats (Niho et al. 2001). This is the first attempt to check the effect of Gallic acid on L. donovani promastigotes within administrative doses and determine the cellular mode of action within the promastigotes. In this study, an attempt was made to evaluate the cellular mechanism(s) mediating the anti-leishmanial properties of Gallic Acid.

Materials And Methods

Reagents

RPMI-1640 and fetal calf serum (FCS) were purchased from GIBCO; In Situ Cell Death Detection Kit (TUNEL Kit) from Roche Applied Science, USA; Miltefosine from Zentaris, GmbH and Ethanol from Merck. Caspase -3 Assay Kit, Gallic acid, Sodium Dodecyl Sulphate (SDS), MTT, 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide, 2'-7'-Dichlorodihydrofluorescein diacetate (H2DCFDA), JC-1 (5,5’,6,6’-tetrachloro-1,1’,3,3’ tetraethylbenzimidazolylcarbocyanine iodide), Acridine Orange, Ethidium Bromide, Propidium Iodide (PI) and RNase A were purchased from Sigma (St. Louis, MO, USA). All other reagents used were of the highest analytical grade possible.

In vitro culture of parasites

Promastigotes of the WHO reference strain Leishmania donovani Dd8 (MHOM/IN/80/Dd8) was kindly provided byDr. Patole (NCCS, India). They were cultured in RPMI-1640 medium supplemented with 10%heat-inactivated fetal calf serum at 23°C. For all experiments carried out, promastigotes in the latelogarithmic phase of growth were used.

In vitro cytotoxicity measurement

Late log-phase promastigotes (5 × 105 per well) were resuspended in fresh RPMI medium and incubated in 96-well culture plates with increasing concentrations of Gallic Acid (0, 0.01, 0.05, 0.1, 1 and 2 mM) or Miltefosine (0, 0.01, 0.025, 0.06, 0.125 and 0.25 mM) for 24 h and promastigote viability was evaluated by the quantitative colorimetric MTT assay (Pai et al. 1997). Briefly, after drug treatment, MTT (0.4 mg/ml) was added to each well and plates were incubated for 4 h at 23°C. The purple formazan crystals were solubilized in 10% SDS (W/V) and absorbance was measured in a microplate reader at 550 nm. The concentration of drug that decreases cell growth by 90% (IC90) was calculated by the formula given: 

% Inhibition = 100 – [Absorbance of Drug treated cells / Absorbance of Untreated cells]. 

The results were expressed as the means of three independent experiments and their standard errors.

Cell size analysis

Promastigotes were treated with the antileishmanial drugs for 24 h, washed with 1×PBS and fixed in 2% (V/V) formaldehyde. Control included promastigotes without any drug treatment. The difference in the cell size of 1 mM Gallic acid or 0.2 mM Miltefosine treated and untreated cells was analyzed on a Partec Flow Cytometer (Sysmex Partec, GmbH, Münster, Germany) where, a forward scatter Vs side scatter was plotted for 20,000 cells (Paris et al. 2004). 

Analysis of cellular morphology

The morphology of promastigotes after Gallic acid or Miltefosine treatment for 24 h was determined under a phase contract microscope (Carl Zeiss, Axioskop 40, 100X/ 1, 30 Oil immersion/ 0, 17 Plan - NEOFLUAR with an ocular W-PI 10X/ 23) and images were captured using AxioCam MRC camera and analyzed by AxioVision LE Rel. 4.2 software. 

Measurement of Reactive Oxygen Species (ROS)

ROS generation in Leishmania promastigotes was measured by fluorescent probe H2DCFDA. 1 × 106 Promastigotes were incubated with or without 1 mM Gallic acid or 0.2 mM Miltefosine for 0-6 h. After each time point, live cells were washed and then probed with H2DCFDA (100 μM) for 30 min at 23°C. Intracellular ROS was measured fluorometrically (Dutta et al. 2007) on Chameleon fluorescence plate reader at excitation 485 nm and emission 535 nm and analyzed by Micro Win software. 

In situ detection of DNA fragmentation by terminal deoxynucleotidyltransferase (TdT)-mediated dUTP end labeling (TUNEL)

DNA fragmentation with the untreated and drug treated promastigotes was analyzed in situ by TUNEL (Roche Applied Science, Indianapolis, IN, USA) assay, according to the manufacturer’s instructions with some modifications. Briefly, after 24 h of 0.2 mM Miltefosine or 1 mM Gallic acid treatment, promastigotes were fixed in 4% formaldehyde solution for at least 30 min, washed and permeabilized by a mixture of 0.1% Sodium citrate and 0.2% Triton X-100 in PBS for 10 min. Permeabilized cells were washed and fragmented DNA was tagged with TUNEL Reaction Mixture for 1 h. At least 20,000 cells were washed and acquired on a Partec flow cytometer (Sysmex Partec, GmbH, Münster, Germany) by a blue laser at an excitation of 488 nm and emission detection at 530 nm. The data was analyzed by FloMax software. 

Mitochondrial membrane potential determination (ΔΨm)

Difference in the mitochondrial membrane potential in untreated and drug treated promastigotes was detected by JC-1 (Mukherjee et al. 2009). Briefly, after individual drug treatments, promastigotes were washed and incubated with JC-1 (1μM) for 30 min at 37°C. Subsequently, after washing 20,000 cells were acquired on Partec flow cytometer (Sysmex Partec, GmbH, Münster, Germany) and ratio of J-aggregate forms of JC-1 (excitation at 535 nm and emission at 600 nm) against the monomeric form (excitation at 485 nm and emission at 535 nm) was plotted using the FloMax software for analysis. 

Acridine Orange/ Ethidium Bromide staining for Apoptosis detection

Treated or untreated promastigotes were washed and suspended in Acridine Orange and Ethidium Bromide (100 μg/ml) (Kern and Kehrer, 2002; Liegler et al., 1995). At least 20,000 cells were acquired on a Partec flow cytometer (Sysmex Partec, GmbH, Münster, Germany) and analyzed by FloMax software. A ratio of green fluorescence (excitation at 485 nm and emission at 535 nm) Vs red fluorescence (excitation at 535 nm and emission at 630 nm) was plotted. 

Flow cytometry analysis of cell cycle

After drug treatments, promastigotes were washed and incubated at -20°C overnight in 70% (V/V) ethanol. Cells were washed and resuspended in 100 μg/ml Propidium Iodide (PI) in 1×PBS containing RNase A [0.1 mg/ml] ( Sen et al. 2004; Sen et al. 2007). The fluorescence intensity of PI incorporated in 20,000 promastigotes was analyzed on a FACS Partec flow cytometer (Sysmex Partec, GmbH, Münster, Germany) and FloMax software upon excitation at 535 nm and emission at 630 nm.

Determination of Caspase 3 activity

To determine the role of caspase 3 in Gallic acid induced Leishmania cytotoxicity, Caspase 3 Assay Kit (colorimetric) was used according to the manufacturer’s protocol. Briefly, 5 × 106 Leishmania promastigotes were treated with 1 mM Gallic acid or 0.2 mM Miltefosine for 24 h at 23°C. Promastigotes were washed and lysed in 50 μl of lysis buffer on ice for 20 min. Cell lysates were collected after centrifugation at 600 g for 20 min at 4°C. 5 μl of cell lysate or positive control (5 μg/ml Caspase 3, supplied by the kit) was mixed with 85 μl Assay buffer in a 96-well microtiter plate. 10 μl Caspase 3 substrate (acetyl-Asp-Glu-Val-Asp p-nitroanilide) was added to each well and incubated at 37°C for 2 h. Plate was read at 405 nm in an ELISA plate reader. 

 Estimation of hemolysis (%) for Gallic acid-induced toxicity by Human Red Blood Cells (RBC) lysis assay

Drugs, their metabolites, or excipients used in formulation can cause toxic hemolysis. Therefore, the FDA recommends testing for hemolytic potential (FDA guidance for Industry). Any drug administered needs to be checked for its cytotoxicity, as high concentration (IC90 1mM) of gallic acid was used against parasites in the present study so RBC hemolysis assay was carried out to evaluate the toxicity of compounds or drugs.    The collection of human blood and ex vivo assessment of toxicity of gallic acid by RBC lysis assay was carried out after pre-approval by the Institutional Ethical Committee (IEC) (SPPU/IEC/2019/53 dated: 4/10/2019). Briefly, 5 ml blood was drawn from a healthy donor directly into K2-EDTA-coated Vacutainer tubes to prevent coagulation. This was followed by centrifugation of the blood at 500×g for 5 min and removal of the upper yellowish plasma which was discarded into biohazard waste after bleach treatment. The RBCs were washed with 1×PBS and centrifuged at 500×g for 5 min and this was carried out twice. Human RBC suspension was prepared in 10mM PBS, pH-7.4. RBC suspension was incubated with different concentrations of drug ranging from 1×IC90 up to 32×IC90 for different time intervals such as 1, 3, 6, 12 and 24 h. RBC suspension treated with 2% Triton X-100 was taken as positive control while, RBC suspension containing 1×PBS was negative control. After incubation, cells were centrifuged at 2000 rpm for 5 min and supernatant was taken in microtiter plate. Absorbance was measured at 541 nm for the estimation of hemolysis as a function of hemoglobin absorbance. Percent hemolysis was calculated using the following formula –

Statistical analysis

Data are expressed as mean ±S.E.M. unless mentioned. Comparisons were made between treatments and untreated controls using unpaired Student’s t-test. P-values <0.05 were considered significant.

Results

The inhibitory effect of Gallic acid and Miltefosine on L. donovani promastigotes was verified by the MTT assay, where an increase in the amount of formazan crystals indicates cell viability and the decrease represents cell inhibition. The observed IC90 values of Gallic acid and Miltefosine for L. donovani Dd8 promastigotes were 1 mM and 0.2 mM, respectively (Fig. 1). Cell cytotoxicity of promastigotes to Gallic acid and Miltefosine was confirmed by Trypan blue stain exclusion (data not shown). The confirmation of cell cytotoxicity to Gallic acid and Miltefosine as observed by Trypan blue staining (data not shown) showed a marked change in the Leishmania promastigotes morphology. The effect of Gallic Acid on the promastigotes was further monitored microscopically wherein, the promastigotes demonstrated fragmented and rounded bodies with a reduction in cell size (Fig. 2A). The morphological changes as observed by microscopy were confirmed by flow cytometry analysis of FSC versus SSC (Fig. 2B). The results of DNA fragmentation in promastigotes treated with Gallic acid and MIL drugs expressed threefold apoptotic death (Fig. 3A) than the untreated (Fig. 3B). Thus, Gallic acid caused DNA fragmentation, a characteristic of apoptosis in L. donovani Dd8 promastigotes. Upon interaction with the reactive oxygen species like H2O2, hydroxyl ions and superoxide ions generated within the cell, the dye DCFDA gets converted to a non-permeable fluorescent derivative H2DCFDA. The fluorescence of H2DCFDA is proportional to the amount of ROS generated within the cells (Dutta et al. 2007). With an increase in time an increase in ROS generation was observed in Gallic acid treated Leishmania promastigotes up to 6 h (Fig. 4). However, the ROS measured after 24 h was not detected (data not shown). Gallic acid is known to possess pro and antioxidant properties (Sakagami, 1997). The fluorescent dye JC-1 signals the loss of the mitochondrial membrane potential. In healthy cells, the dye accumulates to form J-aggregates that stain the mitochondria bright red. While in apoptotic cells, the mitochondrial membrane potential decreases and the dye remains in monomeric form, thus giving the apoptotic cells a green fluorescence. The flow cytometry data (Fig. 5) indicates a distinguishable loss in the mitochondrial membrane potential in the Gallic acid treated promastigotes as compared to the untreated cells. Chromatin condensation and nuclear fragmentation remain the hallmarks of apoptotic cells. The Acridine orange / Ethidium bromide staining assay was performed to assess the apoptotic index and cell membrane integrity simultaneously. Acridine orange (AO) permeates all cells and makes the nuclei appear green. Ethidium bromide (EB) is only taken up by cells when cytoplasmic membrane integrity is lost and stains the nucleus red. Thus, live cells have a normal green nucleus; early apoptotic cells have bright green nucleus with condensed or fragmented chromatin; late apoptotic cells display condensed and fragmented orange chromatin; cells that have died from direct necrosis have a structurally normal orange nucleus (Kern and Kehrer 2002). After Gallic acid treatment, 89% and 63.78% promastigotes showed apoptotic death (Fig. 6B) and Miltefosine (Fig. 6A) treatment for 24 h respectively. Internucleosomal DNA fragmentation is one of the typical nuclear features of apoptosis. Previous experiments to determine the DNA fragmentation by TUNEL showed DNA fragmentation in promastigotes. Promastigotes were permeabilized and stained with propidium iodide to measure the amount of DNA fragmentation by flow cytometry. The amount of bound PI correlates with the DNA content in a given cell; DNA fragmentation in apoptotic cells translates into a fluorescence intensity lower than that of G1 cells (sub-G1 peak) as represented by the RN2 region in Fig. 7. After 24 h treatment with Gallic Acid, 94% promastigotes (Fig. 7B2) were found in the sub-G1 peak, as compared to 75% in Miltefosine treated promastigotes (Fig. 7B3). This was extremely high compared to 2% found in untreated (Fig. 7B1) promastigotes. This confirmed that Gallic acid induces DNA degradation and cell shrinkage in L. donovani Dd8 promastigotes. Cell lysates of Gallic acid treated promastigotes did not show any detectable amount of caspase 3 activity when compared to the positive control. The anti-leishmanial activity of Gallic Acid could be via a Caspase 3 independent pathway (Dutta et al. 2007). RBC when treated with gallic acid (1 mM) for 1 h (standard time for hemolysis assay) showed no significant hemolysis with concentration corresponding to IC90 value. Even after a prolonged incubation at higher concentrations, minor hemolysis was found as compared to positive control. After 6 h incubation with four-fold of IC90, only 12.94 ± 0.12 % hemolysis was observed with gallic acid treatment. After 24 h of incuation with 32-fold IC90, 30.746 ± 0.10% hemolysis was observed (Table 1).

Table 1

The values given in the table are mean ± standard deviation and represent three independent experiments done in triplicate. Gallic acid treated RBC showed significantly less hemolysis versus positive control (p < 0.00001).

Concentration (×IC90)

% Hemolysis

Time (h)

1

3

6

12

24

Positive Control (0.2% Triton-X-100)

100

100

100

100

100

Medium Control

0

0.13 ± 0.02

0.16 ± 0.02

0.27 ± 0.01

0.29 ± 0.02

0.36 ± 0.12

Gallic Acid (mM)

1

0.181 ± 0.15

0.448 ± 0.05

0.757 ± 0.09

1.758 ± 0.02

4.476 ± 0.13

2

1.281 ± 0.15

1.548 ± 0.05

1.857 ± 0.09

4.858 ± 0.02

9.476 ± 0.13

4

10.09 ± 0.18

11.94 ± 0.02

12.94 ± 0.12

15.824 ± 0.03

15.426 ± 0.09

8

12.519 ± 0.11

12.78 ± 0.22

14.66 ± 0.08

18.77 ± 0.05

20.23 ± 0.02

16

22.70 ± 0.13

23.20 ± 0.11

24.58 ± 0.12

26.02 ± 0.08

26.75 ± 0.11

32

25.05 ± 0.12

25.48 ± 0.12

26.86 ± 0.12

28.07 ± 0.11

30.74 ± 0.10

Discussion

Previous studies on antileishmanials have focused on natural products and their application for treatment has been promising especially from plant sources. Several natural compounds isolated from plant origin have been studied in the last decade for their usage as alternatives for antileishmanial drugs. Amongst the bioactive molecules derived from plant sources, polyphenols have demonstrated antileishmanial activity in vitro and in vivo (Kayser et al. 2003; Kolodziej et al. 2001). Novel plant based antileishmanials with high specificity towards Leishmania spp. and low toxicity towards macrophages or the host system is the need of the hour owing to the increase in resistant strains against the limited chemotherapeutic drugs. In this regard, toxicological studies of the drug become imperative due to the fact that Leishmania is an intracellular obligate parasite (de Medeiros et al. 2011; Islamuddin et al. 2015). Among the various natural compounds tested, Gallic acid is an important immunomodulator found in many plants that are a part of the Indian medicine system: Ayurveda. It is a major constituent of the Indian Gooseberry, Emblica officinalis. Gallic acid is a water-soluble compound and is highly absorbed in humans (Manach et al. 2005; You and Park 2010). Various biological activities of Gallic Acid have been reported including anti-viral, anti-tumor (Dalla Pellegrina et al. 2005; Faried et al. 2007). Earlier studies (Kolodziej et al., 2001; Herbert Kolodziej and Kiderlen, 2005) have tested Gallic acid and its derivatives for immunomodulatory properties on RAW murine macrophage cell line, while its anti-leishmanial (anti-amastigote) potential in vitro has also been evaluated (Kolodziej et al. 2001). As reported by Zhao (Zhao et al. 2007), an oral dose of 5000 mg/kg Gallic acid was permissible to rabbits while, Niho (Niho et al. 2001) estimated intra-peritoneal delivery of 119 and 128 mg/kg/day Gallic acid for male and female F344 rats respectively. Thus, on the basis of the literature available, we evaluated the effects of Gallic acid on Leishmania promastigotes up to 1 mM concentration. In this study, our objective was to evaluate the putative mechanism(s) underlying the antileishmanial property of Gallic Acid. Among various mechanisms for mediating parasiticidal activity, programmed cell death (PCD) or apoptosis appears to be the most preferred. This mechanism does not damage the host cells while, effectively eliminating the pathogen (Dutta et al. 2007). The mode of death of the promastigotes was determined by different apoptosis/ necrosis detection assays. A number of reports have shown apoptosis-like death in Leishmania promastigotes treated with different drugs (Alzate et al., 2008; Das M, 2001; Dutta et al., 2007; Mukherjee et al., 2009; Paris et al., 2004; N. Sen et al., 2004; R. Sen et al., 2007). Microscopic examinations of Gallic acid treated promastigotes showed a marked effect on the promastigote morphology (Fig. 2). To confirm the effects of Gallic acid on the cellular morphology, the FSC versus SSC scatter plot was analyzed and a reduction in the scatter typically indicated the reduction in cell size (Fig. 2). The hallmark of apoptosis being DNA fragmentation; the fragments were assessed by TUNEL assay to measure the number of TUNEL positive cells. The effect of Gallic acid on the nuclear DNA fragmentation was further confirmed by flow cytometry analysis (Fig. 3). These results were also validated by the PI binding to the DNA where, 94.4% population was arrested in the sub-G0/G1 peak in case of Gallic acid treated promastigotes as compared to 2.67% of untreated cells (Fig. 7). Thus, Gallic acid had a pronounced effect on the nuclear material of Leishmania promastigotes. The other major events in apoptosis are loss of mitochondrial membrane potential and membrane permeability. In apoptotic cells, the membrane permeability was intact while, there is a loss in mitochondrial membrane potential. The difference in the mitochondrial membrane potential was measured by the dye JC-1 and its ability to form red aggregates at a higher membrane potential. JC-1 remains as green monomers in the cytoplasm at a lower membrane potential (Sen et al. 2007). Gallic acid treatment decreased the membrane potential at least 4-fold when compared with that of the untreated cells (Fig. 5). Another agent, Licochalcone A has been shown to interfere with the function of parasite mitochondria (Zhai et al. 1995). The membrane permeability of the untreated and Gallic acid treated promastigotes was verified by the dual staining employing a cell permeable stain acridine orange and selectively permeable stain ethidium bromide that enters cells with a compromised cell membrane, found in case of late apoptotic and necrotic cells. Acridine orange fluorescence green in apoptotic cells and bright green in live cells (Kern and Kehrer 2002). This differential staining confirmed the death of promastigotes by Gallic acid via an apoptotic pathway (Fig. 6). ROS generation and caspase release in cells during apoptosis is a known phenomenon. A comparable ROS generation was found in promastigotes treated with Gallic acid up to 6 h of treatment (Fig. 5); however, after 24 h ROS could not be detected. An initial increase in the ROS levels and a decrease after 24 h could be due to the pro-oxidant and antioxidant properties of Gallic acid (You and Park 2010). As observed in previous findings, Caspase 3 activity was absent in the Leishmania promastigotes (Mukherjee et al. 2009). Thus, Gallic acid mediated death in Leishmania donovani promastigotes is apoptotic, caspase independent and could be due to ROS generation. Previous reports indicate activation of macrophages and subsequent reduction in Leishmanial infection due to the fact that GA is capable of activating several signaling pathways which may be responsible for making the intracellular unfavorable along with its inherent antiparasitic activity (Herbert Kolodziej and Kiderlen, 2005; Macedo-Silva et al., 2011) The dichotomy between Th-1 and Th-2 immune responses in the host during infection plays a crucial role in determining the outcome of the disease. This favors natural therapeutic agents which not only possess antileishmanial activity but also immunomodulatory properties to induce Th-1 type immune response in the host (Islamuddin et al. 2015). Several studies indicate immunomodulatory activities of phenolic compounds including GA which include production of various proinflammatory cytokines like TNF-α, INF-γ, increased NO synthesis, etc.(Alves et al., 2017; Herbert Kolodziej and Kiderlen, 2005; Yadav et al., 2012). Immunomodulatory properties of GA may also be attributed to the IP3/PLC/PKC pathway as in the case of MIL (Verma and Dey 2004) Since Gallic acid is water soluble and an important dietary constituent, so it merits further studies and serious consideration as an antileishmanial compound. Assessment of percent RBC hemolysis suggested this compound to be safe as insignificant hemolysis was found even at 4-fold IC90 concentration of gallic acid with prolonged incubation time (Table 1). Research work is underway on the efficacy of Gallic acid (1mM) on Leishmania clearance in an intra-macrophage and mouse models.

Declarations

Funding

This work was supported by Indian Council of Medical Research (ICMR) (ICMR-58/4/2011-BMS 26/7/2012), Board of Research in Nuclear Sciences–Department of Atomic Energy (BRNS/DAE), Government of India (GOI), Centre for Advanced Studies (CAS), Department of Zoology, Savitribai Phule Pune University and Board of College and University Development (BCUD). Nutan Jadhav is grateful for the Bhabha Atomic Research Centre (BARC/DAE) - UoP PhD collaboration program fellowship. 

Competing Interests

“The authors have no relevant financial or non-financial interests to disclose.”

Authors Contribution

Material preparation, data collection and analysis were performed by Nutan Jadhav. Data review and editing were carried out by Divya Prakash, Kalpana Pai and T B Poduval. The final draft of the manuscript was written by Divya Prakash and all authors read and approved the final manuscript.

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