Bioassay-based Corchorus capsularis L. leaf-derived β-sitosterol exerts antileishmanial effects against Leishmania donovani by targeting trypanothione reductase

Leishmaniasis, a major neglected tropical disease, affects millions of individuals worldwide. Among the various clinical forms, visceral leishmaniasis (VL) is the deadliest. Current antileishmanial drugs exhibit toxicity- and resistance-related issues. Therefore, advanced chemotherapeutic alternatives are in demand, and currently, plant sources are considered preferable choices. Our previous report has shown that the chloroform extract of Corchorus capsularis L. leaves exhibits a significant effect against Leishmania donovani promastigotes. In the current study, bioassay-guided fractionation results for Corchorus capsularis L. leaf-derived β-sitosterol (β-sitosterolCCL) were observed by spectroscopic analysis (FTIR, 1H NMR, 13C NMR and GC–MS). The inhibitory efficacy of this β-sitosterolCCL against L. donovani promastigotes was measured (IC50 = 17.7 ± 0.43 µg/ml). β-SitosterolCCL significantly disrupts the redox balance via intracellular ROS production, which triggers various apoptotic events, such as structural alteration, increased storage of lipid bodies, mitochondrial membrane depolarization, externalization of phosphatidylserine and non-protein thiol depletion, in promastigotes. Additionally, the antileishmanial activity of β-sitosterolCCL was validated by enzyme inhibition and an in silico study in which β-sitosterolCCL was found to inhibit Leishmania donovani trypanothione reductase (LdTryR). Overall, β-sitosterolCCL appears to be a novel inhibitor of LdTryR and might represent a successful approach for treatment of VL in the future.


Corchorus capsularis L. leaf-derived β-sitosterol (β-sitosterol CCL ): bioassay-guided isolation and characterization.
Major fractions (F1 to F13) were obtained according to the schematic presentation shown in Fig. 1A. The antipromastigote activity of all the fractions at doses ranging from 0 to 200 µg/ml was determined by calculating the half-maximal inhibitory concentration (IC 50 ) using the MTT assay method ( Fig. 1B and Supplementary Table S1). The dose response curve (Fig. 1B) demonstrated that F4 is the most active fraction, having the maximum inhibitory effect with an IC 50 value of 17.7 ± 0.43 µg/ml. Then, the lead fraction F4 (ethyl acetate:hexane = 7:93) was fully characterized on the basis of spectroscopic analysis (FTIR, 1 H NMR, 13 C NMR and GC-MS), the results of which are shown below, and the obtained lead phytocompound (F4) was found to be β-sitosterol.
β-Sitosterol CCL affected 7.04 ± 0.38% of host cells at a high dose (200 µg/ml), but commercial β-sitosterol exhibited greater toxicity at a high dose, exhibiting 18.04 ± 0.75% killing of normal host cells (Fig. 3B). Thus, β-sitosterol CCL displays antileishmanial properties with negligible cytotoxicity even at a high doses. Therefore, β-sitosterol CCL seems to be more reliable than commercial β-sitosterol for subsequent evaluation of antileishmanial potency against L. donovani. β-Sitosterol CCL induces intracellular ROS production in promastigotes. Intracellular production of ROS is the central factor involved in triggering apoptosis in promastigotes 22 . Hence, we initially monitored the generation of intracellular ROS in β-sitosterol CCL -treated L. donovani promastigotes by using H 2 DCFDA, a cellpermeable dye. H 2 DCFDA is a nonfluorescent molecule, but once it enters cells, it is ultimately converted to the fluorescent DCF (2′,7′-dichlorofluorescein) by exposure to proper oxidants present within the cells. Therefore, the detected fluorescence of DCF is considered to be an indicator of the intracellular ROS level. Flow cytometry demonstrated that in the early hours of β-sitosterol CCL treatment, there was a gradual time-dependent increase in ROS production for up to 12 h, which triggered oxidative stress in β-sitosterol CCL -treated parasites compared www.nature.com/scientificreports/ to the untreated control. Maximum production of ROS was found at 12 h, after which, ROS generation drastically decreased, reaching close to the level in control cells at 24 h. However, pre-treatment of promastigotes with the ROS quencher NAC (N-acetyl-l-cysteine) restrained the level of ROS generation in β-sitosterol CCL -treated cells to the level in control parasites at the same time points. A comparison of the mean fluorescence intensity (MFI) of the treated and control cells is shown in a bar graph (Fig. 4A), and shifting of the fluorescence intensity (FL1-H channel) in β-sitosterol CCL -treated parasites compared to the control is also shown by a histogram (Fig. 4B).
β-Sitosterol CCL alters the morphological structure of promastigotes. Changes in the morphological structure of promastigotes are the key indication of an apoptosis-like mode of cell death 23 . Henceforth, morphological deformities in β-sitosterol CCL -treated promastigotes compared to the untreated cells were detected. For comparison, miltefosine (10 µM) was used as a positive control, showing distinct morphological alterations at both 24 h and 48 h. A snapshot captured by phase contrast microscopy (Carl Zeiss, Germany) showed that untreated parasites were structurally organized and elongated in shape with intact flagella after 24 h and 48 h. Similarly, β-sitosterol CCL -treated parasites were morphologically atypical, exhibiting features such as roundness with shrinkage and shortening of flagella, compared to the untreated control parasites. This phase contrast microscopic data was also confirmed with SEM imaging of treated and untreated parasites at the same time points (Fig. 5A).

β-Sitosterol CCL causes ultrastructural changes in promastigotes.
Regarding ultrastructural alterations, β-sitosterol CCL -treated parasites exhibited notable deformities of internal cellular organelles, such as vacuolated nuclei, distortion of flagellar pockets, and disorganization of mitochondria and kinetoplasts at 24 h and 48 h. However, untreated control parasites displayed normal morphological structures with well-organized intracellular organelles consisting of intact flagellar pockets with flagella, nuclei at central positions and kinetoplasts at the proper location (Fig. 5B). In parallel, miltefosine (10 µm) was used as a positive control for comparison of ultrastructural deformities as observed in the β-sitosterol CCL -treated parasites at 24 h as well as 48 h.

β-Sitosterol CCL triggers lipid body accumulation in promastigotes.
Excess accumulation of intracellular lipid droplets is a key characteristic of cellular stress and triggers apoptosis in parasites 24 . Therefore, while searching for apoptotic events in parasites caused by β-sitosterol CCL , we were greatly concerned with evaluating the enhanced accumulation of lipid bodies in treated L. donovani promastigotes by using the common fluorescent marker Nile Red, which generally stains intracellular lipid droplets. Upon β-sitosterol CCL treatment for 24 h and 48 h, superfluous lipid droplets were observed to be randomly distributed throughout the cytoplasm of promastigotes, in contrast to the untreated control parasites (Fig. 6A).   www.nature.com/scientificreports/ β-Sitosterol CCL causes mitochondrial membrane depolarization in promastigotes. Elevated production of intracellular ROS causes oxidative stress inside promastigotes and subsequently induces mitochondrial membrane depolarization, a major indication of apoptosis 25 . Therefore, the significant effect of β-sitosterol CCL on ROS production made us curious to observe its simultaneous effect on mitochondrial transmembrane potential. Therefore, measurement of mitochondrial membrane depolarization was carried out in promastigotes using the dye JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′ tetraethylbenzimidazolcarbocyanine iodide). The dye usually forms aggregates in mitochondria after entering cells. However, in apoptotic cells, aggregated JC-1 is released from mitochondria to the cytoplasm as a monomer. Consequently, aggregated JC-1 at higher potential emits red fluorescence, whereas at lower membrane potentials, JC-1 remains as a monomer within the cytoplasm and emits green fluorescence 26 . Thus, the shift in the cell population towards green fluorescence, as observed by the FACS study, indicates mitochondrial membrane potential depolarization. In the JC-1 assay, FACS data showed that β-sitosterol CCL treatment induced the loss of mitochondrial membrane potential in promastigotes by 26.53% and 56.16% at 24 h and 48 h, respectively (Fig. 6B). Correspondingly, in miltefosine (10 µm)-treated promastigotes, loss of mitochondrial membrane potential was perceived by 35.99% (24 h) and 61.01% (48 h) of the promastigotes.

β-Sitosterol CCL induces externalization of phosphatidylserine in promastigotes.
Externalization of phosphatidylserine to the outer surface of the plasma membrane is the core indication of apoptosis in unicellular eukaryotic cells 27 . Phosphatidylserine exposure is usually analysed by flow cytometry through a dual staining process with annexin V-fluorescein isothiocyanate (annexin V-FITC) and propidium iodide (PI) 27 . In the early stage of cellular apoptosis, when the plasma membrane loses its symmetry, membrane phospholipids are eventually translocated from the inner to outer plasma membrane, and annexin V-FITC rapidly binds with high affinity to the phosphatidylserine exposed to apoptotic cells (early and late). PI selectively binds to DNA in necrotic cells in which membrane integrity is already interrupted, and PI eventually discriminates apoptotic cells from necrotic cells 28 . Thus, the different labelling patterns of annexin V-FITC/PI in the dot plot describe various conditions of cells, which are as follows: annexin V-FITC-negative and PI-negative cells are considered viable, After 48 h of miltefosine treatment, 25.88% and 39.42% parasites were detected in the early and late apoptotic stages, respectively, compared to the untreated control parasites (Fig. 6C). Similarly, β-sitosterol CCL treatment for 24 h resulted in 23.62% and 11.78% early and late apoptotic cells, respectively. Increasing the exposure time of β-sitosterol CCL to 48 h increased the amounts of early and late apoptotic cells by 31.45% and 38.51%, respectively (Fig. 6C). Therefore, taken together, these flow cytometry data undoubtedly indicate β-sitosterol CCL to be a potent apoptosis-triggering agent in L. donovani promastigotes.
β-Sitosterol CCL decreases non-protein thiol levels and trypanothione reductase (TryR) activity in promastigotes. Thiols play a key role in protecting parasites against ROS-mediated oxidative stress 29 , and thus, depletion of non-protein thiols could be considered an appropriate focus for drug targeting. In addition, trypanothione reductase (TryR), an indispensable enzyme of kinetoplastid parasites, participates in their unique thiol-redox metabolism 20 . Therefore, the ROS-mediated induction of apoptosis by β-sitosterol CCL made us interested in examining the trypanothione reductase activity and intracellular levels of non-protein thiols. The level of the thiols was monitored flow cytometrically in β-sitosterol CCL -treated promastigotes by using the well-known dye 5-chloromethylfluorescein-diacetate (CMFDA). The dye penetrates the cells and usually binds to non-protein thiols and is ultimately converted to a fluorescent thioether 30 . Therefore, the detected fluorescence reflects the level of total non-protein thiols present within the parasites. In β-sitosterol CCL -treated promastigotes, a gradual time-dependent decrease in fluorescence intensity was noticed by flow cytometry for up to 24 h compared to the intensity in the control. A comparison of the MFI values of β-sitosterol CCL -treated and control parasites is shown in a bar graph (Fig. 7A) and histogram (Fig. 7B).
Since β-sitosterol CCL causes a reduction in the levels of thiols in Leishmania promastigotes, TryR inhibition by this component was initially performed in soluble extracts of L. donovani promastigotes. The percentage of inhibition of this enzyme in parasite extracts was calculated in terms of the decrease in absorbance at 340 nm, indicating NADPH oxidation. Thus, the optical density itself indicates the consumption of NADPH by TryR. The control lacking any inhibitor was considered as having 100% TryR activity. However, the presence of β-sitosterol CCL (IC 50 dose) inhibited NADPH consumption by TryR by 52.77 ± 1.04% in parasite extracts compared to the control ( Supplementary Fig. S4). To validate the observation, commercial β-sitosterol (Abcam, USA) was also used, which showed a similar inhibitory effect as that of β-sitosterol CCL (Supplementary Fig. S4).

β-Sitosterol CCL competitively inhibits Leishmania donovani trypanothione reductase (LdTryR).
To determine the type of inhibition of L. donovani trypanothione reductase (LdTryR) exhibited by β-sitosterol CCL , an enzyme kinetics study was performed on recombinant LdTryR. The Lineweaver-Burk www.nature.com/scientificreports/ plot obtained from the kinetics study showed a gradual increase in apparent K m with increasing β-sitosterol CCL concentration without any effect on Vmax. As a result, the inhibition of LdTryR by β-sitosterol CCL appeared to be competitive, and the inhibition constant (Ki) of β-sitosterol was found to be 3.5 µg/ml (8.43 µM) (Fig. 8).

Molecular docking-based binding interactions of β-sitosterol CCL with Leishmania donovani
trypanothione reductase (LdTryR). Trypanothione reductase (TryR), an NADPH-dependent enzyme, is unique to kinetoplastid parasites, including Leishmania. The key role of TryR in redox metabolism has made it an interesting option for the design of advanced antileishmanial drugs 20,31 . To better understand the binding interactions of the ligand β-sitosterol CCL (Corchorus capsularis L. leaf-derived β-sitosterol), molecular docking simulations were performed using the three-dimensional structure of the ligand with L. donovani trypanothione reductase (LdTryR). Due to the lack of a crystal structure for LdTryR, homology modelling was performed to generate a three-dimensional model of the protein LdTryR. The homology model of LdTryR was built using the crystallographic structure of TryR from Leishmania infantum in complex with TRL156 (PDB Code: 6I7N, chain B), which was used as the template because it shared 98% sequence identity with the LdTryR protein. The modelled structure of LdTryR was superimposed on the crystal structure of the template, and the RMSD of the superimposition was found to be 0.25 Å (Supplementary Fig. S5A). The validation of stereo-chemical qualities of the modelled structure of LdTryR was estimated by the PROCHECK program, and 90.4% of the amino acid residues were in the most favoured region of the Ramachandran plot (Fig. 9A). The structural qualities of the modelled protein were again analysed by Verify3D, and the model passed the criteria of Verify3D; thus, the constructed LdTryR structure was considered a good quality model. Then, the modelled protein ( Supplementary Fig. S5B) was docked to the ligand to obtain the three-dimensional (3D) docking conformation with a close-up view of the binding interactions of LdTryR and the ligand, as shown by the circle in Fig. 9B Fig. S5C). The amino acids of LdTryR involved in the binding interactions with the ligand β-sitosterol CCL are shown as two-dimensional (2D) representations (Fig. 9C)

Discussion
Plant-derived natural products possess diverse pharmacological activities and consequently are an attractive resource for the development of advanced chemotherapeutics to treat a wide range of disease conditions, including microbial infections 32 . For the last few years, plant sources have also been reported to be very important in Leishmania research 8 . Existing drugs against visceral leishmaniasis exhibit several limitations, such as the emergence of resistance, toxicity and high cost 33,34 . Therefore, in the search for economically feasible antileishmanial agents with better efficacy and low toxicity, plant sources are highly prioritized. In this regard, our previous work on the antileishmanial activity of C. capsularis L. leaf extract revealed its efficacy against L. donovani promastigotes 9 ; therefore, isolation of the natural lead component seems to be significant for exploration of its therapeutic utility in the treatment of VL. Thus, based on the antileishmanial property 9 and relatively low cytotoxicity of C. capsularis L. leaf extract (see Supplementary Method, Results, Fig. S6), the current work was undertaken with the aim of identifying bioactivity-based lead compounds that may play a role in killing parasites. The subsequent bioassay-guided fractionation process resulted in the isolation of a major phytosterol, β-sitosterol, from Corchorus capsularis L. leaves (β-sitosterol CCL ). In many earlier reports, the medicinal value of plant-derived β-sitosterol has been demonstrated, and the role of β-sitosterol as an antimicrobial agent has also been revealed in the treatment of various infectious diseases 13,14 . β-Sitosterol from a variety of plant sources is applied in the treatment of many parasitic diseases 15,16 . Plantderived β-sitosterol has also been studied against different forms of leishmaniasis; for example, phytosterols (stigmasterol + β-sitosterol) isolated from Musa paradisiaca fruit peel have been tested for antileishmanial properties in vitro by studying growth inhibition of L. infantum chagasi promastigotes and amastigotes 19 . Similarly, β-sitosterol isolated with other components from Sassafras albidum stem bark was studied against Leishmania amazonensis, where the β-sitosterol was found to be least effective against the promastigotes, which was evident from its highest IC 50 value in comparison to that of the other isolated products 17 . Furthermore, although the leishmanicidal effect of β-sitosterol from Ifloga spicata showed apoptosis-type killing of L. tropica promastigotes, the detailed mechanism of apoptosis is unclear 18 . Previously, β-sitosterol isolated from Thalia geniculata was also tested against L. donovani amastigotes (strain MHOM/ET/67/L82), but no significant activity of β-sitosterol was documented 16 . Therefore, despite the many studies on the effect of plant-derived β-sitosterol on various forms of leishmaniasis, the mechanisms by which the compound kills the parasites remain unclear. On the basis of previous reports, it could be suggested that although the effect of different plant-derived β-sitosterols has been examined on various Leishmania spp., the effectiveness varies among different Leishmania species and strains [16][17][18][19]35 . The discrepancy in the efficacy of drugs depends on the phenotypic variability of different species or strains, clinical appearances and geographical origin 36,37. Certain antileishmanial drugs have also been reported to exhibit species-and strain-specific efficiency 37-40. Additionally, various plant-derived secondary metabolites have been shown to exhibit target-specific activity against parasites 15 . Therefore, considering these aspects, the present investigation of Corchorus capsularis L. leaf-derived β-sitosterol (β-sitosterol CCL ) against the Indian strain of L. donovani promastigotes [(MHOM/IN/1983/AG83)] seems very reasonable and unique.
As β-sitosterol is available commercially, during our investigation of the antileishmanial properties of β-sitosterol CCL , it was also of interest to evaluate the efficacy of commercial β-sitosterol on L. donovani www.nature.com/scientificreports/ promastigotes. Therefore, commercial β-sitosterol was initially compared herein with β-sitosterol CCL in terms of its antileishmanial properties and cytotoxicity. Although both β-sitosterol CCL and commercial β-sitosterol exhibited potent antileishmanial activity, β-sitosterol CCL was less toxic than commercial β-sitosterol. Similar observations have also been reported previously, showing that plant extracts or plant-derived components are safer and more efficient than any synthetic drugs against parasitic diseases 18 . As a result, the current investigation on the natural lead component β-sitosterol from the commonly available edible plant C. capsularis L. focused on β-sitosterol CCL as a novel agent for elucidating the cell death mechanism in L. donovani.
In the present study, β-sitosterol CCL initially led to the appearance of major features of apoptosis, such as the formation of intracellular ROS and, subsequently, an atypical morphology of L. donovani promastigotes with alterations in internal organelles. Similar morphological and ultrastructural changes were observed during apoptotic death of L. donovani promastigotes treated with clerodane diterpene (K-09) obtained from Polyalthia longifolia leaves 24 . Lipid droplets are very special, dynamic and complex organelles that play a key role in regulating lipid metabolism in unicellular protozoan parasites 41 . Surplus cytoplasmic accumulation of these lipid droplets is also regarded as a symbolic feature of apoptotic cells 42 . A report suggests that the dibenzylideneacetone A3K2A3 exerts leishmanicidal activity through the excess accumulation of lipid bodies within the parasites 43 . An elevated number of lipid droplets upon clerodane diterpene K-09 exposure was also noted in L. donovani parasites 24 . Generally, impeding lipid metabolism causes excess production of lipid precursors that accumulate in the form of lipid bodies inside cells and provoke cell death 24,42 . Correspondingly, in the current study, the antiparasitic nature of β-sitosterol CCL was also observed as enhanced accumulation of lipid bodies in β-sitosterol CCL -treated parasites, which ultimately led to parasite killing due to probable alteration of lipid metabolism.
Leishmania parasites possess a single mitochondrion that plays an essential role in the survival of parasites by maintaining homeostasis; thus, loss of mitochondrial membrane potential could be considered a very striking characteristic of cell death by means of apoptosis 22,23 . Interestingly, an appreciable time-dependent decrease in the mitochondrial membrane potential was noticed in β-sitosterol CCL -treated parasites and consequently provided an excellent indication of apoptosis. Subsequently, depolarization of the mitochondrial membrane potential allowed us to explore the apoptosis promotion capability of this β-sitosterol CCL in L. donovani promastigotes. Apoptosis is a common physiological phenomenon that leads cells towards death 27 . In higher eukaryotic unicellular organisms, apoptosis is represented by externalization of phosphatidylserine from the inner leaflet to the outer surface of the plasma membrane. Interestingly, increased exposure of phosphatidylserine was similarly noticed in L. donovani promastigotes after β-sitosterol CCL treatment, which further confirms the mode of parasite killing exhibited by this natural lead component.
Furthermore, the ROS-mediated apoptosis-like programmed cell death events in promastigotes caused by β-sitosterol CCL exposure inspired us to explore the effect of the compound on non-protein thiols, which play an important role in protecting parasites under oxidative stress condition 29,30 . Fascinatingly, in the current study, significant depletion of non-protein thiol levels was also noticed in L. donovani promastigotes upon β-sitosterol CCL treatment. Thus, β-sitosterol CCL causes redox imbalance situations with increased ROS production and decreased antioxidant-like thiol levels in L. donovani promastigotes followed by apoptosis. Moreover, the viability and infectivity of Leishmania parasites generally depend on some key enzymes, the major enzyme among which is a redox-maintaining enzyme, trypanothione reductase (TryR). In general, the redox balance in Leishmania is regulated by TryR, which provokes a cascade of events via the reduction of trypanothione disulphide [T(S) 2 ] to the dithiol form [T(SH) 2 ] by neutralization of ROS 20 . Thus, the antileishmanial function of β-sitosterol CCL targeting the enzyme TryR appears very imperative and relevant in the current study. Consequently, an enzymatic assay was performed, which showed that β-sitosterol CCL significantly inhibits TryR activity in soluble extracts of L. donovani promastigotes. Therefore, a subsequent enzyme kinetics study on recombinant LdTryR was performed with β-sitosterol CCL and clearly demonstrated that β-sitosterol CCL is an efficient competitive inhibitor of LdTryR. This further confirms that β-sitosterol CCL acts as a potent antileishmanial agent by competitively inhibiting the enzyme LdTryR. Although many synthetic compounds are also reported to inhibit the parasitic trypanothione reductase enzyme in a competitive manner 21,44,45 , the inhibition of LdTryR by β-sitosterol obtained from C. capsularis L. leaves is the first report of an efficient antileishmanial agent. Therefore, the significant inhibitory effect of β-sitosterol CCL against LdTryR led us to check the binding affinity of the compound with the enzyme with the help of molecular docking simulation.
Molecular modelling, a computer-assisted tool, has emerged as an attractive platform for better understanding drug design and is widely used to predict the preferred binding orientation of pharmacologically active molecules (ligands) with biological macromolecules 46 . Trypanothione reductase (TryR) is an NADPH-dependent homodimer that comprises the cofactor FAD bound to each subunit and helps in electron transfer from NADPH to oxidized trypanothione through the prosthetic group, FAD and a redox-active cysteine disulphide residue 20,47 . Previously, many phytocompounds and synthetic compounds have been reported to dock with TryR from different Leishmania spp. 18,20,21 . Docking studies have been performed between TryR from L. infantum (PDB ID 4APN) and β-sitosterol 18 . However, the efficiency of plant-derived β-sitosterols in the binding of L. donovani trypanothione reductase has yet to be studied. Hence, we used molecular docking to investigate the binding capacity of β-sitosterol (β-sitosterol CCL ) with trypanothione reductase of L. donovani. The X-ray crystallographic structure of L. donovani trypanothione reductase (LdTryR) is not well documented 21 . Therefore, we employed homology modelling to generate the three-dimensional structure of LdTryR using the X-ray crystallographic structure of TryR from Leishmania infantum in complex with TRL156 (PDB Code: 6I7N, chain B) as a template. The sequence identity between LdTryR and the template was 98%. Then, we made a structural comparison between the modelled structure of LdTryR with the template 6I7N, chain B by structurally aligning their C-α backbone atoms. The root mean squared deviation (RMSD) between the backbones of the proteins was found to be 0.25 Å, which indicates a significant structural similarity between the modelled LdTryR and the template 6I7N, chain B. Ultimately, the modelled structure of LdTryR was used to dock with the ligand β-sitosterol (β-sitosterol CCL ) in www.nature.com/scientificreports/ the presence of FAD (cofactor) and NADPH; the stability of the molecular interaction was also established from the ΔG value. Next, we tried to compare the distribution of the active site amino acid residues of LdTryR with the same proteins from other members of the Leishmania and Trypanosoma families. For that purpose, we used the amino acid residues of the following sets of proteins: • LiTryR from Leishmania infantum TryR.
We compared the amino acid sequences of the above mentioned proteins by a multiple sequence alignment method and identified the conserved active site amino acid residues in the proteins (Supplementary Fig. S7). These residues are conserved in all species. In our model, all these residues were also found to be involved in binding interactions, which take place in and around the active sites. Therefore, the in silico interaction study again validates the observations of the inhibitory activity of β-sitosterol CCL targeting TyrR of L. donovani promastigotes.
Overall, the antileishmanial activity of β-sitosterol CCL against L. donovani promastigotes exhibits very specific apoptotic features by targeting LdTryR, as depicted in the proposed model (Fig. 10). Thus, the present work might provide an efficient way to use Corchorus capsularis L. leaf-derived β-sitosterol (β-sitosterol CCL ) as an alternative novel therapeutic agent against visceral leishmaniasis in the future.

Conclusion
The present investigation introduces Corchorus capsularis L. leaf-derived β-sitosterol (β-sitosterol CCL ) as a novel antileishmanial agent that competitively inhibits Leishmania donovani trypanothione reductase. Overall, the antiparasitic efficiency and the ability of this phytosterol to block one of the most crucial parasitic enzymes emphasize its proficiency as a strong candidate for treatment of visceral leishmaniasis. The fractions (F1-F13) were screened against L. donovani promastigotes, testing for viability, by the MTT assay 9 . Log-phase promastigotes (1 × 10 7 /ml) were seeded in 96-well plates (BD falcon) and treated separately with these fractions (0-200 µg/ml) for 48 h. Next, MTT was added, and the plate was incubated for 6 h at 37 °C. Then, viable cells were estimated by conversion of MTT to formazan at 570 nm in an iMark Microplate Reader (Bio-Rad). The IC 50 value of each fraction was calculated by GraphPad Prism software (version 5).

Materials and methods
Characterization of Corchorus capsularis L. leaf-derived β-sitosterol (β-sitosterol CCL ). FTIR analysis of the isolated lead compound was carried out to identify the existing functional groups. A thin disc www.nature.com/scientificreports/ of the sample was prepared by using a KBr pellet, and the spectral data were recorded by FTIR spectrometry (Perkin Elmer) 49 . 1 H and 13 C NMR spectra were scanned with a Bruker Avance spectrometer at 400 MHz and 100 MHz, respectively, by using CDCl 3 as the solvent system 49 . GC-MS analysis was performed with a gas chromatograph (Agilent Technologies 7980A) equipped with a mass spectrometric system (7000, GC/MS triple quad). An HP-5MS column (30 m length, 0.25 mm I.D., film thickness 0.25 μm) was employed 50 . Agilent Mass Hunter software (Version B.50.00) was used for instrument control and data analysis.
Surface morphology analysis of β-sitosterol CCL . The compound was dried under vacuum for scanning electron microscopy (SEM) imaging. Images of the surface morphology of the compound were then obtained by SEM (ZEISS EVO LS 10).
Antipromastigote activity and cytotoxicity assay of β-sitosterol CCL and commercial β-sitosterol. To check the inhibitory effect of commercial β-sitosterol on the growth of L. donovani promastigotes, an MTT assay was performed as described previously 9 . Promastigotes (1 × 10 7 /well) were treated individually with β-sitosterol CCL and commercial β-sitosterol (Abcam, USA) (0-200 µg/ml) for 48 h. The IC 50 value was determined from a graphical representation (GraphPad Prism software, version 5).
Similarly, cytotoxicity was tested on RAW 264.7 macrophages (1 × 10 5 /ml) by an MTT assay with β-sitosterol CCL and commercial β-sitosterol. Macrophages were treated with both the β-sitosterols at a dose of 0 to 200 µg/ml for 48 h. The percentage of host cells affected by the compounds was calculated through graphical exploitation by using GraphPad Prism software (version 5). Parasite morphology analysis of β-sitosterol CCL -treated promastigotes. Promastigotes were treated with β-sitosterol CCL (IC 50 dose) for 24 h and 48 h. Then, treated and untreated cells were fixed in methanol and stained with Giemsa (Sigma-Aldrich). Subsequently, images were obtained under a Meiji (ML 2955) light microscope (100× objective). For scanning electron microscopy (SEM), fixation of cells was performed in 2.5% glutaraldehyde and 2% paraformaldehyde for 3 h at room temperature, and the cells were left overnight at 4 °C 53 . Then, the samples were dehydrated with an increasing gradient of ethanol washing solution and imaged under a scanning electron microscope (ZEISS EVO LS 10). Herein, miltefosine (10 µm) was used as a positive control.

Measurement of ROS in β-sitosterol
Ultrastructural study of β-sitosterol CCL -treated promastigotes. Promastigotes were treated with β-sitosterol CCL (IC 50 dose) for 24 h and 48 h. Cells were then fixed with 2.5% glutaraldehyde and 2% paraformaldehyde in sodium cacodylate buffer (pH 7.2). Then, samples were prepared for TEM, and ultrastructural imaging was performed using a Tecnai G2 20 S-Twin transmission electron microscope at SAIF, AIIMS, New Delhi 54 .

Scientific Reports
| (2020) 10:20440 | https://doi.org/10.1038/s41598-020-77066-2 www.nature.com/scientificreports/ Trypanothione reductase assay. Soluble extract of L. donovani promastigotes was obtained by resuspending the washed pellet in buffer containing 40 mM HEPES (pH 7.4) and 1 mM EDTA 57 . The cell suspension was then lysed in a Dounce homogenizer and centrifuged at 12,000×g for 15 min. The supernatant collected was considered the soluble fraction containing trypanothione reductase. Then, the assay was performed by incubating the soluble protein fraction (1 mg/ml) for 10 min with β-sitosterol CCL (IC 50 dose). Then, the reaction was initiated by the addition of NADPH (0.1 mM) in HEPES (40 mM) and EDTA 1 (mM) plus 100 mM substrate (trypanothione disulphide, Sigma-Aldrich). Herein, commercial β-sitosterol (Abcam, USA) was also tested in parallel, and a positive control set was made by incubation with clomipramine (10 µM), a known TryR inhibitor 31 . TryR activity was measured in a spectrophotometer (Hitachi, Japan) by measuring NADPH consumption at 340 nm. The percentage of inhibition was eventually calculated based on a decrease in optical density 20,57 . Enzyme kinetics study of the effect of β-sitosterol CCL on recombinant Leishmania donovani trypanothione reductase (LdTryR). Recombinant L. donovani trypanothione reductase (LdTryR) 47 was kindly provided by Dr. Neena Goyal, The CSIR-Central Drug Research Institute, Lucknow, India. An enzyme inhibition assay was performed spectrophotometrically 58 . The enzyme inhibition kinetics were determined in an assay mixture (40 mM HEPES, 1 mM EDTA at pH 7.5) containing LdTryR and the substrate trypanothione disulphide T(S) 2 (Sigma-Aldrich) at varying concentrations (25, 50, 100 and 200 µM), where β-sitosterol CCL as an inhibitor was added at concentrations of 0, 3.75, 7.5, and 15 µg/ml. The reactions were monitored by the addition of 100 µM NADPH (Sigma-Aldrich), and changes in absorbance were measured at 340 nm 44 . In the presence of β-sitosterol CCL , the mode of inhibition was detected by a Lineweaver-Burk plot, and the value of the inhibition constant (Ki) was calculated.

Molecular docking simulation.
The three-dimensional coordinates of the atoms of the ligand β-sitosterol were obtained from Pubchem 59 . The amino acid sequence of the protein LdTryR from L. donovani was retrieved from UniProt with the accession number P39050. We used homology modelling to build the three-dimensional structure of LdTryR using 6I7N and chain B as templates. The stereo-chemical qualities of the modelled structure were validated through a Ramachandran plot by using the PROCHECK program. The structural qualities of the modelled protein were again analysed by Verify3D. A molecular docking study between the modelled structure of LdTryR and ligand (β-sitosterol CCL ) was performed using the tool GOLD, and the best docking poses were chosen as per the GOLD scores 60 . The significance of differences was calculated by using Student's t-test (two-group comparison) and one-way ANOVA with Dunnett's multiple comparison test (multiple-group comparison), where *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 were considered statistically significant.