IN VITRO ANTILEISHMANIAL ACTIVITIES OF THREE MEDICINAL PLANTS: ARGEMONE MEXICANA, MURRAYA KOENIGII AND CINNAMOMUM TAMALA AGAINST MILTEFOSINE RESISTANT PROMASTIGOTES OF LEISHMANIA DONOVANI PARASITES

Objective: Leishmaniasis is one of the neglected tropical diseases in terms of drug development and discovery. Non-responsiveness and resistance to the drug in Leishmania species need to develop new antileishmanial potentials; herbal medicines could be the alternative one.
Methods: In the present study, semi-purified fractions were prepared from the traditionally used three medicinal plants of India: Argemone mexicana (aerial shoot), Murraya koenigii (stem), and Cinnamomum tamala (bark) by using multiple solvent systems (non-polar to polar, beginning with petroleum ether followed by n-hexane, benzene, and chloroform) and an effort was given to assess the leishmanicidal activities against Leishmania donovani miltefosine resistant HePC-R (Ld/MIL-30) promastigotes in vitro and the IC50 concentrations were estimated.
Results: The study revealed that the semi-purified fractions of A. mexicana, M. koenigii, and C. tamala have effective antileishmanial activities and the 50% inhibitory concentrations (IC50) are 50 μg/ml, 98 μg/ml, and 200 μg/ml, respectively. At these (IC50) concentrations, these plant semi-purified fractions were found to interfere in lipid and protein biosynthesis, alter cell morphology, DNA content, mitochondrial membrane potential, generating ROS, and apoptosis in promastigotes. The semi-purified fractions were also found noticeably non-toxic towards host splenocytes.
Conclusion: These results could suggest that A. mexicana, M. koenigii, and C. tamala could carry potential novel compounds for the development of new drugs against Leishmaniasis.


Leishmaniasis
The native medicinal plants and plant-derived drugs are the potential source of alternative medicine and are extensively used for the treatment of various health ailments [5,6]. About 25 % of prescribed drugs were obtained from plants with or without further modification [7,8]. Use of the medicinal plants is a core component at the primary health care level due to cheaper and easier availability, acceptability, compatibility, and affordability and with less or no side effects. A broad range of available plant species was reported with potentially active leishmanicidal activities [7,9,10].
is one of the neglected tropical diseases in terms of drug discovery and development and is endemic in our country (India) [1]. The chemotherapy used to treat this disease has been proved to be highly toxic and has persistence of resistance issues. Miltefosine (MIL) or Hexadecylphosphocholine, is the first orally administrable anti-leishmanial drug but due to its long half-life, it is highly susceptible to resistance. Parasites with decreased drug vulnerability have been associated with treatment failure [2][3][4]. The emergence of drug resistance is the biggest threat to the successful treatment of leishmaniasis. As a consequence, the need for ideal leishmanicidal molecules to overcome resistance issues has notably increased in recent years.
In the present work, an attempt had been made to evaluate the leishmanicidal activities of semi-purified fractions of three different plants Argemone mexicana (Papaveraceae), Murraya koenigii (Rutaceae), and Cinnamomum tamala (Lauraceae), which are also used as traditional medicine in India. Argemone mexicana is an annual herb with an extremely prickly branched stem and showy yellow flowers [11]. In Homeopathy, the medicine prepared from this herb is used for the treatment against tapeworm [11]. Murraya koenigii is a deciduous shrub with a short trunk and smooth, greyish, or brown bark and has a dense shady crown [12]. It is widely used for culinary purposes and as traditional medicine. Also, it has multiple scientific reports of its medicinal properties [13,14]. Cinnamomum tamala is a medium-sized evergreen tropical tree with small, yellowish flowers [15]. The leaves of C. tamala have been used for flavoring food and it has been used in traditional medicines as an astringent, stimulant, diuretic, carminative, and cardiac disorders [15]. This present study aimed to find out the active antileishmanial potentials against miltefosine resistant Leishmania donovani parasites from medicinal plants of India.

Collection and identification of plant material
The plant materials of Argemone mexicana (Papaveraceae) were collected from the village Gobindapur of Malda district in April 2015; Murraya koenigii (Rutaceae) plant was collected from the village Gazole of Malda district in July 2015 and the plant materials of Cinnamomum tamala (Lauraceae) were collected from the village Checkpost of Malda district in September 2015. The samples were identified in the Animal behavior and Natural product research laboratory, West Bengal State University, WB, India, with the plant identification numbers (SSS23413), (SSS3414), (SSS3416), respectively.

Phytochemical work-up procedures
The dried coarsely ground plant materials were extracted following the protocol for the enrichment of compounds. The isolated aerial part of A. mexicana, the stem of M. koenigii, and the bark of C. tamala were dried at a hot air oven (50-55 °C) for 48 h and crushed with a mixture grinder. The dried powder materials of the three plants were mixed with petroleum ether (1:5 W/V) individually, extracted in Soxhlet apparatus for 2 d at room temperatures, and then filtered separately with Whatman No. 1 filter paper. The filtrated phytochemicals were concentrated from solvent petroleum ether (1:5 W/V) using the rotary evaporator (Buchi, model no. B 100). The leftover materials were dried again at room temperature and were sequentially partitioned into n-hexane (1:5 W/V), benzene (1:5 W/V) and finally chloroform (1:5 W/V) for 3 d (each step) [16,17]. Each fraction was concentrated to dryness by evaporation with the Buchi rotary evaporator (Model B 100) of the solvent. Activated charcoal was used to remove the unnecessary pigments from chloroform-derived phytochemicals for bioassay-guided activity studies.

Sample preparation
The semi-purified fractions were dissolved in sterile dimethyl sulfoxide (DMSO). Appropriate stock solutions (20 mg/ml) were stored at-20 °C until use (antileishmanial activity studies).

Parasite maintenance
Leishmania donovani drug-resistant parasites HePC-R (Ld/MIL-30) strain was used for experimental purposes. L. donovani miltefosineresistant strain (named HePC-R) was developed in our laboratory from the bone marrow aspirates of a relapsed miltefosine-treated visceral leishmaniasis (VL) patient from West Bengal, India, by transforming and maintaining in continuous drug pressure with stepwise increasing doses of miltefosine as per the approval (reference number WBSU-IEC/06, dated 24 April 2012) described earlier [18]. Parasites were maintained [19], in vitro in complete M199 medium, supplemented with 1% penicillin-streptomycin and 10% fetal calf serum at requisite temperature (22 °C). For experimental purposes, log-phase promastigotes were sub-cultured every 72-96 h, inoculums being 5×10 5 cells/well) without (control) or with 50 μg/ml concentration of all three semipurified fractions in vitro, using 7-amino actinomycin D (7-AAD) as described earlier [19] in FACS. Samples were run on a flow cytometer (BD FACSVerse™, BD Biosciences, USA) and analyzed by Flowing software, version 2.5 [20]. The unstained population was taken as a reference.

Promastigote viability assay by 7AAD staining in FACS
The 50% inhibitory concentration (IC50) on L. donovani HePC-R promastigotes and cytotoxic effect on murine splenocytes without or with increasing concentration of semi-purified fractions of A. mexicana, M. koenigii, and C. tamala was estimated by MTT [3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] method with the doses started from 50 μg/ml to 400 μg/ml described earlier [19,21]. Three independent experiments were performed in triplicate for each set. The 50% inhibitory concentrations of semipurified fractions have been determined from the graph of percent inhibition against increasing concentrations. Statistical analyses for all experiments were performed by one-way ANOVA in R-3.4.1 software followed by Tukey's test.

Effect on protein content in promastigotes
The protein contents of promastigotes treated without (control) or with IC50 concentrations of these three semi-purified fractions were assessed by staining with fluorescein isothiocyanate (FITC), an acidic dye that covalently binds with the positively charged proteins as described earlier [20] in FACS. The acquisition was done in a flow cytometer (BD FACSVerse™, BD Biosciences, CA, USA) and was analyzed by Flowing 2.5 version software19. The unstained population was taken as a reference.

Effect on neutral hydrophobic lipids in promastigotes
Neutral lipid contents were assessed as described earlier [19]. Neutral lipids were assessed in promastigotes (1×10 6 Analysis of cell cycle phases cells/ml) treated without or with IC50 concentrations of these three semi-purified fractions for 48 h by staining with Nile red (Sigma) and acquired in a flow cytometer (BD FACSAria™, BD Biosciences, USA) and analyzed by Flowing 2.5 version software [20].

Measurement of reactive oxygen species (ROS)
) were incubated without or with IC50 concentrations of semi-purified fractions and were analyzed for cell cycle progression by fluorescent dye propidium iodide [19] that binds to the DNA. The acquisition was performed on a flow cytometer (BD FACSVerse, BD Biosciences) and data analyzed using Flowing software 2.5 [20].
The changes in cellular ROS, in plants semi-purified fraction treated promastigotes have been compared with cells pre-incubated with NAC and without any treatment (control) using 2',7'dichlorodihydrofluorescein diacetate (H2DCFDA) (Sigma-Aldrich) (20 μM) and analyzed by flow cytometer (BD FACSVerse, BD Biosciences) as described previously [19,21] and analyzed by Flowing 2.5 version software [20]. The induction of ROS in promastigotes has been compared with positive inducer H2O2

Variation of the mitochondrial membrane potential (ΔΨm)
. The variation of the accumulation of rhodamine 123 in promastigotes which is directly related to the mitochondrial membrane potential, was assessed in promastigotes after treatment with IC50 concentration of semi-purified fractions of A. mexicana (50 μg/ml), M. koenigii (98 μg/ml) and C. tamala (200 μg/ml) at different time points (1 h, 3 h, 6 h, and 12 h) under standard conditions, as described previously [21]. Rhodamine 123 accumulation was monitored in a flow cytometer (BD FACSVerse, BD Biosciences, USA) and analyzed by Flowing 2.5 version software. The changes in mitochondrial ΔΨm in promastigotes have been compared in cells, preincubated with NAC and without any treatment (control).

Detection of chromatin condensation
The nuclear condensation in semi-purified fractions of all three plant treated promastigotes have been detected under FACS (BD FACSAria™, BD Biosciences) after staining with DAPI (4',6diamidino-2-phenylindole) by a slight modification of the methods described earlier [19] and cells were analyzed by Flowing 2.5 version software [20].

Acridine orange/ethidium bromide staining method
Promastigotes were seeded in 6-well tissue culture plates (1×10 6 cells/well), treated without or with IC50 concentrations of the three semi-purified fractions individually for 24 h, and the cells were washed with 1XPBS (Phosphate Buffered Saline). Then, the cells were stained with a mixture of acridine orange (3 μg/ml) and ethidium bromide (10 μg/ml). Place 10 μl of cell suspension onto a microscopic slide, cover with a glass coverslip, and observed immediately in a fluorescence microscope (Carl Zeiss, Germany) using a fluorescein filter and a 40X objective. Higher or lower magnification could also be desired, counting on DNA fragmentation assay cell type. Each sample should be mixed just before microscopy and must be evaluated immediately [21].
DNA fragmentation assay of L. donovani HePC-R promastigotes (10 7 cells) without (control) or with IC50 concentrations of plants semipurified fraction were done by agarose gel electrophoresis to work out the fragments of DNA generated during apoptosis

Detection of the change in morphology by scanning electron microscopy (SEM)
, total cellular DNA was isolated by a previously described method [22] and analyzed by agarose gel electrophoresis. Total DNA was mixed with tracking dye and loaded on 1% agarose gel containing ethidium bromide. The gel was run at 50 V for 2.5 h.
L. donovani HePC-R promastigotes (2×10 6 cells) in log phase were incubated at 22 °C in complete M199 medium, incubated with the three semi-purified fractions (IC50 against promastigotes) for 24 h and 48 h respectively. After incubation, promastigotes were centrifuged and the pellet was washed in 1X cold PBS (×2) and finally resuspended in 1X PBS. Promastigotes were then fixed with 2.5% Gluteraldehyde (Sigma Aldrich), dehydrated in ethanol, critical point dried in CO2, mounted on stubs, sputtered with a thin platinum layer, and observed under a scanning electron microscope [19] (Model: ZEISS EVO-MA 10).

The semi-purified fractions of A. mexicana; M. koenigii and C. tamala inhibited the proliferation of L. donovani miltefosine resistant (HePC-R) promastigotes in vitro
Anti-proliferative effects of all three semi-purified fractions were evaluated on L. donovani HePC-R promastigotes, the causative agent of visceral leishmaniasis. It was found that semi-purified fractions of A. mexicana, M. koenigii, and C. tamala inhibited the proliferation of L. donovani HePC-R promastigotes in vitro. The 50% inhibitory concentration for A. mexicana, M. koenigii, and C. tamala against HePC-R promastigotes at 48 h were detected as 50±2.56 μg/ml, 98±1.81 μg/ml, and 200±1.57 μg/ml, respectively ( fig. 1A), which could restrict the proliferation of murine splenocytes only by 4.4±2.51%, 3.51±1% and 3.4±1.59% respectively at 96 h ( fig. 1B). Our data indicated that the three compounds have noteworthy antiproliferative effects on L. donovani HePC-R promastigotes as well as non-toxic to normal murine primary cells. Data represent three independent experiments' mean±standard error. Statistical significance was determined by one-way ANOVA in R software followed by Tukey's test. Differences were considered to be statistically significant if the p-value is less than 0.05 (*p<0.05 vs control).

The semi-purified fractions (IC50) of A. mexicana; M. koenigii and C. tamala caused a decrease in total protein content in HePC-R promastigotes
The total cellular protein content of treated promastigotes was determined and compared with control promastigotes taking the unstained group as reference. It has been found that the treatment with semi-purified fractions could reduce the percentage of promastigotes with positively charged groups of proteins and were estimated as 11.71±5.04% (A. mexicana), 16 2B).

The semi-purified fractions (IC50) of A. mexicana; M. koenigii and C. tamala caused the accumulation of lipids in HePC-R promastigotes
As semi-purified fractions of all three plants exhibited substantial anti promastigote activity in vitro, and reduced the total protein, we were interested to examine its effect on lipid metabolism in promastigotes. Nile red was used for studying lipid metabolism. At 48 h treated promastigotes were estimated with high neutral lipid content in comparison to the control promastigotes culture. The semi-purified fractions were also found to increase the MFI when treated and estimated for the neutral lipid droplets content (control vs treated) in promastigotes. The MFI was found to increase to 2109.69 (A.  The study of mitochondrial membrane potential has become a focus of apoptosis regulation as many investigations demonstrated a major functional impact of mitochondrial alterations on apoptosis. Mitochondria are critical for the survival of any cell as they act as the warehouse of ATP. The proton gradient across the inner mitochondrial membrane is crucial during oxidative phosphorylation as the source for ATP production. Thus, the maintenance of mitochondrial membrane potential (ΔΨm) is essential for this chemical transformation as well as for cell survival [23]. The incorporation of rhodamine 123 has been used to determine the changes in mitochondrial membrane potential. All the three semi-purified fractions of A. mexicana, M. koenigii, and C. tamala caused loss of mitochondrial membrane potential and were found to be continued from 1 h to 12 h of treatment. The alteration of ΔΨm in the semi-purified fraction of A. mexicana treated promastigotes was found to be started gradually from 1 h (2.31-fold decrease in comparison to control promastigotes) continued to the extent of decrease by 2.4-fold after 3 h, 3.83-fold after 6 h and even 5.2-fold loss after 12 h of treatment. The alteration of ΔΨm in the semi-purified fraction of M. koenigii treated promastigotes decreased by 2.44-fold after 1 h, 3.44-fold after 3 h, 3.76-fold after 6 h, and even 5.32-fold loss after 12 h of treatment in comparison to control promastigotes. The alteration of ΔΨm in the semi-purified fraction of C. tamala treated promastigotes was also found started from 1 h (1.2-fold decrease in comparison to control promastigotes) continued to the extent of decrease by 1.94-fold after 3 h, 2.53-fold after 6 h and even 3.79-fold loss after 12 h of treatment. Interestingly, pre-incubation with NAC prevented the alteration of plant semi-purified fractions-induced mitochondrial membrane potential, which could be further linked with the prospect of ROSmediated inhibition of L. donovani promastigotes by the semipurified fractions (fig. 3B).   Fig. 3: The IC50 concentrations of semi-purified fractions of (a) A. mexicana, (b) M. koenigii, and (c) C. tamala (A)  The treated promastigotes were stained with acridine orange/ethidium bromide to verify the apoptosis in treated promastigotes. After the treatment with IC50 concentrations of semi-purified fractions of A. mexicana, M. koenigii, and C. tamala individually for 24 h, the majority of cells exhibited diffused orangecolored promastigotes or orange fluorescence, while in control promastigotes, green fluorescence was observed. Treated promastigotes developed orange and orange-red fluorescence, indicating membrane disruption (fig. 4B). These results support that all three semi-purified fractions could induce apoptosis at IC50 concentrations.

HePC-R promastigotes
Scanning electron microscopy exposed specific morphological alterations in promastigotes after treatment with the IC50 concentration of semi-purified fraction of A. mexicana, M. koenigii, and C. tamala plants for 24 h and 48 h. Promastigotes appeared irregularly shaped with gradual loss of cell membrane and flagella concerning the control cultured flagellated and slender promastigotes ( fig. 4C).

The semi-purified fractions (IC50) of A. mexicana; M. koenigii and C. tamala treatment resulted in the fragmentation of DNA
Tests for an additional hallmark of apoptosis is the internucleosomal genomic DNA degradation, shown by the experimental group which had nucleosome-sized DNA fragments, giving a DNA ladder-like pattern identified by agarose gel electrophoresis of DNA from the cells treated with IC50 concentration of semi-purified fractions of A. mexicana, M. koenigii and C. tamala plants ( fig. 4D).   fig. 2A). The IC50 concentrations of these three semi-purified fractions caused a decrease in total protein content ( fig. 2B) and accumulation of lipids in cells as analyzed by FACS, staining with Nile red (fig. 2C). Accumulation of lipids in cells could be related to the alterations of plasma membrane biophysical properties and also the degradation of abnormal lipids. Thus, the accumulation of lipid in cells as the consequence of the treatment with these three semi-purified fractions could hamper the plasma membrane integrity, resulting in cell death. This result discriminates live promastigotes from dead one on a membrane integrity basis. Reactive oxygen species (ROS) have been reported as the key modulator against the Leishmania parasites [24]. Interestingly, the IC50 concentration of these three semi-purified fractions against the promastigotes was found to induce oxidative stress by producing the highest amount of (ROS) measured in FACS, ( fig. 3B) and this result was also found correlated with the alteration in mitochondrial membrane potential in treated promastigotes ( fig. 3B). Treatment with IC50 of these three semi-purified fractions was found to alter the cell cycle phases caused promastigotes to remain resting in G0/G1 cells and inhibited their entry into the S phase time-dependently ( fig. 1C) which was a clear indication of apoptosis in promastigotes which was also supported by acridine orange/EtBr staining experiment ( fig. 4B). Fragmentation of the nucleus is considered the hallmark of apoptosis [23,25]. IC50 of these three semi-purified fractions caused DNA condensation, observed by DAPI staining (fig. 4A) and DNA fragmentation giving the DNA a ladder-like pattern identified by agarose gel electrophoresis ( fig. 4D) and also the morphological alterations were observed by SEM in L. donovani HePc-R promastigotes ( fig. 4C). This data substantially indicated that these three semi-purified fractions were proficient in arresting the proliferation of L. donovani promastigotes by inducing apoptosis (fig. 4). These three active semi-purified fractions against promastigotes were also found non-toxic to normal murine splenocytes ( fig. 1B). However, investigation on bioactive molecules from Argemone mexicana, Murraya koenigii, and Cinnamomum tamala has not been done extensively. So, further phytochemical and pharmacological investigations are necessary for the search of active anti-leishmanial constituents from Argemone mexicana, Murraya koenigii, and Cinnamomum tamala. The consequences of this study can be used as a reference for further phytochemical and pharmacological investigations within the CONCLUSION effort for the search of novel anti-leishmanial leads.
The results of the present study established that plant materials are potential sources of novel and selective agents which contribute a lot to primary health care and most likely are promising agents for the treatment of leishmaniasis. Semi-purified fractions of A. mexicana, M. koenigii, and C. tamala showed potential activities against drugresistant (Miltefosine) Leishmania species and also no toxicity on splenocytes authorized its specific anti-promastigote effect which is induced by reactive oxygen species (ROS). The study will help to develop potent, non-toxic plant-derived anti-leishmanial drugs. Further investigations are needed for the identification and isolation of active plant constituents.
ACKNOWLEDGEMENT I like to acknowledge Prof. Narayan Ghorai, WBSU for sharing his expertise on natural product research. We are also thankful to the Director, Centre for Research in Nanoscience and Nanotechnology (CRNN), the University of Calcutta for access to the scanning electron microscopy facility. I am very thankful to UGC/MANF, Govt.

AUTHOR CONTRIBUTION
The author confirms sole responsibility for the following: study conception and design, data collection, experimentation, analysis and interpretation of results, manuscript preparation, critical revision of the article, and final approval of the version to be published.