Identification of novel inhibitors from Rubus ellipticus as anti-leishmanial agents targeting DDX3-DEAD box RNA helicase of Leishmania donovani

Abstract

Visceral leishmaniasis (VL), caused by Leishmania donovani, remains challenging to treat due to severe side effects and increasing drug resistance associated with current chemotherapies. Our study investigates the anti-leishmanial potential of Rubus ellipticus from Uttarakhand, India, with extracts prepared from leaves and stems using ethanol and hexane. Advanced GC–MS analysis identified over 100 bioactive compounds, which were screened using molecular docking to assess their binding to LdHEL-67, a DDX3-DEAD box RNA helicase of L. donovani. Our results spotlighted nine major compounds with high binding energy, which were then further analyzed for ADMET properties and toxicity predictions, demonstrating their promising pharmacokinetic profiles. Among these, clionasterol emerged as the standout compound, displaying superior results in all in silico analyses compared to Amphotericin B (the control). Notably, clionasterol was present in significant proportions across all the mentioned extracts. Subsequent treatment with these extracts led to a remarkable reduction in the intracellular amastigote and axenic amastigote, and promastigote forms of L. donovani and non-toxic to THP-1-derived macrophages. Moreover, the extracts induced apoptotic effects, as evidenced by the fragmentation of parasitic genomic DNA. This study marks a significant leap in developing herbal-based, target-specific inhibitors against VL. Hence, our findings highlight the immense potential of R. ellipticus as a natural treatment for VL.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13205-024-04183-4.

Introduction

Leishmania donovani, characterized as a protozoan parasite, is the causative pathogen of visceral leishmaniasis (VL). It is a severe systemic infection in humans transmitted by female Phlebotomine sandflies (Ridley 1988; Freitas-Junior et al. 2012; Siddiqui et al. 2012). Despite its significant global impact, only a fraction of VL, roughly 25% to 45%, cases are reported to the World Health Organization (WHO) per annum, with an estimated 50,000 to 90,000 new cases arising worldwide each year. Current control measures for VL are limited by the absence of effective vaccines, relying heavily on synthetic drugs that often necessitate prolonged treatment durations and are associated with severe side effects, including drug resistance, toxicity, etc. (Maltezou 2009; Saint-Pierre-Chazalet et al. 2009; Chakravarty and Sundar 2010; Dorlo et al. 2012; Singh et al. 2016). Previous research highlights various potential drug targets within Leishmania, including DDX3-DEAD BOX RNA helicase (LdHEL-67) (Chawla and Madhubala 2010; Raj et al. 2019; Gouri et al. 2021; Brar et al. 2022; Kanojia et al. 2024). Separate studies have reported that LdHEL-67 in L. donovani plays a crucial role in the parasite survival (Linder and Jankowsky 2011; Padmanabhan et al. 2016b). LdHEL-67 possesses both ATPase and RNA helicase activities and is essential for RNA metabolism, cellular survival, maintaining mitochondrial proteostasis, and stress response (Padmanabhan et al. 2016a, b). Further, LdHEL-67 is also required for initiating promastigote to amastigote differentiation (Pandey et al. 2019; Gouri et al. 2022). In this study, a computational approach was employed as an initial step to explore the bioactive compounds of Rubus ellipticus. Previous studies have demonstrated that molecular docking is an effective tool in the screening process within drug discovery, highlighting its utility in identifying potential therapeutic candidates (Siddiqui et al. 2021). R. ellipticus, a plant of the Uttarakhand Himalayan commonly known as Yellow Himalayan Raspberry, has been essential in traditional medicine for centuries and is well-known for its therapeutic properties (Saini et al. 2014; Pandey and Bhatt 2016; Pandey et al. 2016). Hence, these reported medicinal properties of R. ellipticus gave us the direction to also explore the anti-leishmanial properties of this plant, which have not been reported yet. Therefore, to proceed in this direction, we collected R. ellipticus from Uttarakhand, India. We then prepared leaf and stem extracts using polar (ethanol) and non-polar (hexane) solvents through the Soxhlet extraction technique. The extracts were labeled as follows: RLEE (Rubus leaf ethanol extract), RSEE (Rubus stem ethanol extract), RLHE (Rubus leaf hexane extract), and RSHE (Rubus stem hexane extract). The gas chromatography–mass spectrometry (GC–MS) analysis of these extracts identified over 100 compounds. We selected 45 compounds based on their high percentage composition and used a molecular docking approach to examine their interaction with the LdHEL-67 protein. We found that around nine compounds exhibited superior binding energy, some of which are near that of the synthetic anti-leishmanial drug Amphotericin B. Specially, clionasterol was occurred at RLEE, RSEE, RLHE, and RSHE in major % composition, this compound was displaying high binding energy as same as Amphotericin B (the control). These top nine hits were then subjected to ADMET and drug-likeness studies, which revealed significant pharmacokinetic characteristics, indicating their potential therapeutic nature.

Furthermore, all the prepared R. ellipticus extracts demonstrated significant anti-leishmanial activity, effectively inhibiting both the promastigote and amastigote forms of L. donovani in a concentration-dependent manner. Notably, we observed a substantial decrease in intracellular amastigote counts in infected THP-1 cells treated with RLEE, RSEE, RLHE, and RSHE extracts. At specific concentrations, these extracts achieved complete parasitic reduction in THP-1-derived macrophages while maintaining low toxicity toward THP-1 mammalian cells. In addition, the inhibition of intracellular amastigotes was confirmed by the dose-dependent downregulation of JW11 mRNA expression level, quantified by real-time PCR, following treatment with all the extracts. The effectiveness of these extracts extended to the promastigote genomic DNA, as evidenced by fragmentation observed after post-treatment with R. ellipticus extracts which is clear indication toward apoptosis in parasite. Consequently, the strategy utilized in our study will aid in comprehending the action mechanism of these herbal compounds, specifically targeting LdHEL-67. This approach provides a clear pathway for further exploration and evaluation of these compounds, potentially leading to the development of targeted, effective, and low-toxicity treatments for visceral leishmaniasis. Based on our findings, we underscore the potent anti-leishmanial properties of R. ellipticus, which targets the LdHEL-67 protein of the parasite and controls its propagation in all forms.

Material and methods

Plant collection and identification

The healthy plant of R. ellipticus was collected from Ranikhet, Uttarakhand, India. This plant specimen was identified and authenticated by the Regional Ayurveda Research Institute, (Ministry of Ayush, Government of India), Ganiadholi (Ranikhet), District Almora, Uttarakhand, India. Collection of R. ellipticus leaves and stem samples was done from March to April.

Initial preparation

Collected R. ellipticus plant sample (leaves and stems) was washed thoroughly with tap water and then with distilled water to eliminate dust and other contaminants. Later, these leaves and stems samples were cut into small pieces and dried in the shade until the moisture was removed, followed by grinding of these samples into powder form.

Soxhlet extraction

The extraction process was conducted using the Soxhlet extraction technique (Kasiramar and Gopalasatheeskumar 2019), aiming to obtain extracts suitable for compound identification and screening of anti-leishmanial activity. The extraction was performed using ethanol (a polar solvent) and hexane (a non-polar solvent) for 9–10 h, following the method described by Murugan and Parimelazhagan (2014) and Kasiramar and Gopalasatheeskumar (2019). Fractions were then subjected to evaporation in a water bath and subsequent lyophilization at temperatures ranging from 40 to 45 °C. All experiments were conducted using filtered extracts.

GC–MS profiling and ligands (phytocompounds) preparation

The phytocompounds present in the prepared extracts of R. ellipticus were then identified by GC–MS analysis. The process was done at the Advanced Instrumental Research Facility (AIRF), Jawahar Lal Nehru University (JNU), New Delhi. The GC–MS profiling provided a catalog of natural compounds present in R. ellipticus. However, the 3D structure of these compounds was retrieved from the PubChem compound database (https://pubchem.ncbi.nlm.nih.gov). The structures of these compounds were downloaded in SDF format and were converted into PDB format using PyMOL software.

Sequence retrieval and analysis of target protein

The amino acid sequences of the HEL-67 in FASTA format were retrieved from the National Centre for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov/). ExPASy ProtParam Server (https://web.expasy.org/protparam/) was used for structure analysis like molecular weight, theoretical isoelectric point (pI), amino acid composition, atomic composition, aliphatic index, instability index, and grand average of hydropathicity index (GRAVY) (Singh and Chaube 2014). The ExPASy ProtParam tool is used to analyze protein sequences, structures, and physicochemical properties of the protein model (Gasteiger et al. 2005). Moreover, PROSITE is a protein database that identified the motifs present in LdHEL-67 (https://prosite.expasy.org/) (Sigrist et al. 2009).

Secondary structure prediction

SOPMA and GORIV (https://npsa.lyon.inserm.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_sopma.html and https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_gor4.html) were used for secondary structure prediction of LdHEL-67 (Singh and Chaube 2014).

3D structure prediction via homology modeling approach

Because of the inaccessibility of LdHel-67 in any of the protein databases, a predictive model of L. donovani (LdHel-67) was generated by a homology modeling approach. Modeling of the three-dimensional structure of the LdHel-67 was done by a Swiss model web-based program (Bordoli and Schwede 2012; Prasanna et al. 2023). The Swiss model template library was utilized to search for suitable templates. Templates of highest quality were selected for generating the model which was built based on the alignment model quality of the target template. The newly generated LdHel-67 model quality was evaluated by the QMEAN score and validated by Ramachandran plot generation (Prasanna et al. 2023).

Molecular docking

The binding energy between the generated LdHel-67 model and phytocompounds was calculated using CB-Dock2 (https://cadd.labshare.cn/cb-dock2/php/blinddock). CB-Dock2 is an upgraded form of AutoDock Vina for blind molecular docking (Dodia et al. 2024). Moreover, the binding efficiency was also evaluated using the AutoDock Tool (Rizvi et al. 2013). The interactions between compounds and the target protein were analyzed using Discovery Studio Visualizer and PyMOL (Shaweta et al. 2021).

ADMET study of selected phytocompounds

To predict the ADMET study, compounds were identified via GC–MS, and the structures were drawn using Chem draw (de Paula et al. 2019). To start the prediction, first, the molecular formula of the compounds was transformed into SMILES format using PubChem (https://pubchem.ncbi.nlm.nih.gov). The pharmacokinetic properties of the compounds were forecasted using Swiss ADMET software, as well as toxicity profiling was done using the ProTox-II webserver (https://comptox.charite.de/protox3/). Toxicity in selected compounds was forecasted in the sense of different criteria, i.e., oral toxicity, organ toxicity (hepatotoxicity), and taxological endpoints (mutagenicity, cytotoxicity, immunotoxicity, and cardiotoxicity) (Bhat and Chatterjee 2021).

Leishmania cell culture condition

The Bob strain (LdBob/strain/MHOM/SD/62/1SCL2D) of L. donovani promastigotes taken from Dr. Stephen Beverly (Washington University, St. Louis, MO) were grown in M199 medium (Sigma-Aldrich, USA), supplemented with 100 µg/ml streptomycin, 100 units/ml penicillin (Sigma-Aldrich, USA), (Sigma-Aldrich, USA), and 10% heat-inactivated FBS (fetal bovine serum-Biowest) at 22 °C. Sub-culture was done every 4th–5th day accordingly.

Anti-promastigote and anti-amastigote activity

The logarithmic stage of L. donovani promastigotes was used to evaluate the inhibitory efficacy by MTT assay (Das et al. 2017; Pandey et al. 2019). First, 10⁵ cells density of L. donovani were seeded on 96-well plates and incubated with R. ellipticus leaves and stem extract samples (0 μg/ml to 1000 µg/ml conc.) for 72 h. After this incubation period, MTT (20 µl of 5mg/ml stock solution; Himedia) was added to each well of respective plates followed by incubation for 4 h. Then 100 μl of dimethyl sulfoxide (DMSO, Merck) was added and mixed properly. The O.D. was measured at 540nm by a microplate reader (Bio-Rad). The parasite inhibition was calculated by comparing the %inhibition with the untreated control (Pandey et al. 2019; Tandon et al. 2023). Amphotericin was used as a standard control; each experiment was done in triplicates.

Healthy promastigotes were differentiated into axenic amastigotes in different environmental cues, such as variations in temperature (ranging from 34 to 37 °C), pH (acidic, pH 4), and slight modifications in the composition of the culture media (Alves et al. 2005; Zilberstein 2020). This differentiation process typically requires 5–6 days to reach completion. The anti-axenic amastigote activity of the R. ellipticus extracts was also evaluated using the MTT assay, following the previously described protocol (Pandey et al. 2019; Tandon et al. 2023).

THP-1 cell culture condition

THP-1 (202 TIB; American Type Culture Collection, Rockville, MD) is a non-adherent, acute monocytic leukemia-derived human cell line cultured in RPMI-1640 (Sigma-Aldrich, USA) medium which is supplemented with 100 units/ml penicillin and 100 µg/ml streptomycin and 10% heat-inactivated FBS (Biowest, UK) at 37 °C with 5% CO₂. For the healthy growth of THP-1, proper sub-culturing was performed accordingly.

THP-1 cell culture and cytotoxicity

Healthy THP-1 cell lines were used to induce differentiation into macrophage-like cells, and THP-1 cells were treated with 50 ng/ml conc. of PMA for 48 h (phorbol-12-myristate-13-acetate; Sigma-Aldrich, USA). To evaluate the effect of plant extracts on THP-1-derived cell viability, 6 × 10³ cells were seeded in 96-well plates and supplemented with plant extract containing RPMI media for 72 h and an MTT assay was done, following the previously described protocol (Pandey et al. 2019; Tandon et al. 2023).

Anti-intracellular amastigote activity

5 × 10⁵ THP-1 were seeded on a glass coverslip in six-well plates and incubated with 50 ng/ml PMA for differentiating into macrophage-like cells. For infection, THP-1 was incubated with late log-phase healthy promastigote (multiplicity of infection of 20:1) for 6 h. After this, washing with PBS (phosphate-buffered saline) removed the excess non-adherent promastigote from host cells. Then differentiated THP-1 was maintained in RPMI medium with different conc. of R. ellipticus extracts for 72 h. All the steps of incubation have been done at 37 °C with 5% CO₂ supply. Intracellular L. donovani load was visualized by PI (propidium iodide) staining (Roy et al. 2020).

RNA extraction and quantitative RT-PCR to determine kDNA minicircle-specific JW11 mRNA expression levels

Total RNA (TRIZOL reagent; Sigma-Aldrich, USA) was extracted from infected macrophages that had been treated with plant extracts at various concentrations. The RNA underwent precipitation through phenol–chloroform protocol, and quantification of the RNA was carried out via spectrophotometric analysis. Following RNA extraction, cDNA synthesis was performed using 2 μg of total RNA and the First Strand cDNA Synthesis Kit as per the manufacturer’s instructions (Thermo Fisher Scientific, USA), utilizing random hexamer primers. The resulting cDNA was then subjected to quantitative real-time PCR (qRT-PCR) experiments employing SYBR Green PCR Master Mix (Thermo Fisher Scientific) and gene-specific primers. The qRT-PCR reactions were conducted on the Applied Biosystems 7500 Fast Real-Time PCR System (CA, USA) (Roy et al. 2020).

DNA fragmentation

Genomic DNA was extracted from both untreated and R. ellipticus-treated L. donovani promastigotes in different concentrations after 72 h using the phenol–chloroform protocol. An equal concentration of DNA samples (15 ng each) was loaded onto 1% agarose gel and subjected to electrophoresis for 1 h at 80V. Subsequently, the DNA bands were visualized under UV light (Ebrahimisadr et al. 2013; Das et al. 2017). As a positive control, promastigotes treated with Amphotericin B (50 µg/ml) was included.

Statistical analysis

The experiments were performed in triplicates, and compiled as mean±SD. Further analysis of the results was done by one-way ANOVA and T test, and GraphPad Prism (version 9) software was utilized for all the analyses.

Results

Yield percentage

Collected healthy leaves and stems from Ranikhet, Uttarakhand, India used for Soxhlet extraction technique. After the successful extraction procedure, four distinct extracts were prepared: Rubus leaves ethanol extract (RLEE), Rubus stem ethanol extract (RSEE), Rubus leaves hexane extract (RLHE), and Rubus stem hexane extract (RSHE) (Table 1).

Table 1.

Percentage yield of different extracts from R. ellipticus

S. No	R. ellipticus	Weight (g)	Yield	% Yield
1	RLEE	50	9.9	19.8
2	RSEE	50	9.1	18.2
3	RLHE	50	6.3	12.6
4	RSHE	50	3.9	7.8

Identification and preparation of compounds

The identification of compounds in the above-mentioned extracts of R. ellipticus was analyzed by GC–MS technique. In this analysis, all the extracts (RLEE, RSEE, RLHE, and RSHE) were found to contain over 120 compounds. Of these compounds, 45 were selected based on their percentage composition or peak area in gas chromatogram. (Table S1). Among the identified compounds, β-D-glucopyranoside methyl was notably abundant, constituting 31.91% and 49.0% of the total extract of RLEE and RSEE, respectively. In addition, 5-hydroxymethylfurfural was present at 7.40% and 14.69% of the total extract of RLEE and RSEE, respectively. Phytol was detected at 5.14% and 7.08% in RLEE and RLHE, respectively. Clionasterol was a common compound found in all extracts, with varying concentrations: 10.57%, 4.48%, 13.82%, and 5.72% in RLEE, RSEE, RLHE, and RSHE, respectively. Moreover, other significant compounds included eicosane and pentacosane, with respective proportions of 8.92% and 9.22% in RLHE. D-Allose was present at 6.32% in RSEE, while a compound 1,3,4,5-Tetrahydroxy-Cyclohexanecarboxy has 48% of the extracted area. Moreover, vitamin E was present at 4.40% and 1.46% in RLEE and RLHE, respectively. Xanthosine was present at 2.06% in RSEE. 4,8,12,16-Tetramethylheptadecan-4-olide was present at 1.18% in RLHE. Dihydroactinidiolide was present at 1.17% in RLHE. Squalene and 1,6-Anhydro, β- D-Glucopyranose were present at 2.43 and 4.23%, respectively, in RLEE. α-Tocospiro B was present at 1.27 and 7.4 in RLEE and RLHE, respectively. Methyl linolenate is present at 0.52%, 1.80%, and 0.28% in RLEE, RLHE, and RSHE, respectively, in the total extract (Table S1).

Based on their percentage composition, the 3D structures of the 45 identified compounds were retrieved from the PubChem database server in SDF format. These structures were subsequently converted into PDB format using PyMOL software, enabling further evaluation of molecular docking interaction between the identified compounds and the target protein.

Sequence analysis and preparation of protein

The ProtParam analysis of LdHEL-67 shows it is composed of 612 amino acids with a theoretical isoelectric point (pI) of 9.09, which is the pH at which the molecule has no net charge. The analysis also reveals that LdHEL-67 contains 61 negatively charged amino acids (Asp + Glu) and 72 positively charged ones (Arg + Lys). The atomic composition of LdHEL-67 is presented in Table 2. Its protein instability index (II) is 34.67, indicating stability, as proteins with an instability index below 40 are considered stable. The aliphatic index of 62.76 reflects the relative volume of aliphatic side chains (alanine, valine, leucine, isoleucine), and a higher index generally increases the thermostability of globular proteins. The grand average of hydropathicity (GRAVY) value for this protein is −0.622, calculated by averaging the hydropathy values of all amino acids in the sequence. A higher positive GRAVY score suggests greater hydrophobicity but does not consider the protein’s three-dimensional structure or the proportion of residues in the hydrophobic core. In addition, PROSITE identified three sequence motifs (Q-Motif, Helicase-ATP-BIND, and HELICASE-CTER) in the protein (Fig. 1).

Table 2.

The atomic composition of LdHEL-67

Atom	Total
Carbon (C)	2908
Hydrogen (H)	4508
Nitrogen (N)	880
Oxygen (O)	884
Sulfur (S)	20

Fig. 1.

Three sequence motifs including Q-Motif, Helicase-ATP-BIND, and HELICASE-CTER identified in LdHEL-67 sequence analysis using Expasy-PROSITE Tool

Secondary structure analysis

Using SOPMA and GORIV tools for secondary structure prediction, various regions of secondary structure in the protein sequence were identified. The analysis showed that the protein’s secondary structure includes alpha helices and beta sheets. Comparative analysis using both SOPMA and GORIV indicated that alpha helices are the most predominant structure, followed by extended strands. Detailed secondary structural annotation revealed that the protein comprises 45.75% random coils, 29.25% alpha helices, 15.52% extended strands, and 9.48% beta turns. Other secondary structures, such as 310 helices, pi helices, beta bridges, bend regions, ambiguous states, and other states, were completely absent. The result is graphically represented in Fig. 2.

Fig. 2.

Secondary structure annotations of LdHEL-67 using GORIV (A) and SOPMA (B). Both GORIV and SOPMA tool represent the percent composition in LdHEL-67, in target protein alpha helix (29.25 and 23.04), extended strand (15.52 and 20.75) and random coil (45.75 and 56.21), respectively

3D structure prediction via homology modeling approaches

The amino acid sequences of LdHEL-67 in FASTA format were retrieved from the National Centre for Biotechnology Information (NCBI). The Swiss model, an online tool, was used for 3D structure prediction based on homology modeling (Bordoli and Schwede 2012; Prasanna et al. 2023). The Swiss Model Template Library was searched in parallel both with BLAST, a basic local alignment search tool (Camacho et al. 2009), and HHblits, a protein sequence search tool (Steinegger et al. 2019) to identify templates and obtain target-template alignments. Based on sequence identity, we selected one template to build the 3D model. The predicted model showed 54.31% sequence identity, 0.44 sequence similarity, 0.68 coverage, and template sequence has been solved by X-ray, 3.20 Å diffraction and biounit oligo state is monomer. The model quality was estimated based on the QMEAN (Fig. 3) and validated by Ramachandran plot analysis, LdHEL-67 favored 93.06% Ramachandran plot and 6.8% of residues present in the allowed region (Fig. 3).

Fig. 3.

Homology modeling of LdHEL-67. A 3D structure generated by Swiss model tool, B V = validation by Ramachandran plot, C and D QMEAN Z-scores of LdHEL-67 and the red star represents the Z-score position of the LdHEL-67 protein model

Molecular docking

Next, we assessed whether the identified compounds from R. ellipticus by GC–MS analysis could bind to the LdHEL-67 protein using a molecular docking approach to determine the binding energy of the docked structures. Following CB-Dock2 analysis, we found that most of the compounds from R. ellipticus successfully docked with the target protein (Table S1). The top nine compounds were selected including xanthosine, clionasterol, vitamin E, 4,8,12,16-tetramethylheptadecan-4-olide, dihydroactinidiolide, squalene, α-tocospiro B, methyl linolenate, and 1,6-anhydro-β-d-glucopyranose based on high binding energy (Table S2 and Fig. 4).

Fig. 4.

Clionasterol exhibited the high binding energy (− 10.0 kcal/mol) among the best nine compounds, with the generated LdHEL-67 protein out of ten runs using blind docking with the AutoDock Tool. The figure illustrates the protein–ligand interaction, showing A the docked complex, and (B, C) both 3D and 2D interactions including protein receptor, ligand, interacting atoms and pocket atoms. Discovery Studio was used for interaction analysis

The interaction efficiency of these top nine compounds with LdHEL-67 was further validated using the AutoDock tool. In the AutoDock analysis (Center Grid Box: X-center: 19.221, Y-center: −32.823, Z-center: 10.345), clionasterol (Fig. 5) and 1,6-anhydro-β-D-glucopyranose exhibited binding energies of −10.0 kcal/mol and −9.89 kcal/mol, respectively, which were close to that of the anti-leishmanial drug Amphotericin B (−10.97 kcal/mol). In addition, the other seven compounds also demonstrated favorable binding energies, as detailed in Tables 3, 4 and Figs. 6 or 7.

Fig. 5.

Best hits using blind docking with the AutoDock Tool: A xanthosine, B vitamin E C 4,8,12,16-tetramethylheptadecan-4-olide, D dihydroactinidiolide. Discovery Studio was used for interaction analysis

Table 3.

Binding energies of best 9 hits by using AutoDock tool

S. No	Compounds	Binding energy
1	Xanthosine	− 6.28 kcal/mol
2	Clionasterol	− 10.0 kcal/mol
3	Vitamin E	− 6.53 kcal/mol
4	4,8,12,16-Tetramethylheptadecan-4-olide	− 4.82 kcal/mol
5	Dihydroactinidiolide	− 6.47 kcal/mol
6	Squalene	− 5.48 kcal/mol
7	α-Tocospiro B	− 5.79 kcal/mol
8	Methyl linolenate	− 4.42 kcal/mol
9	1,6-Anhydro, β-D-glucopyranose	− 9.89 kcal/mol
*	Amphotericin B	− 10.97 kcal/mol

Table 4.

Different parameters of pharmacological properties of selected compounds by using Swiss ADMET analysis

Compounds	TPSA (A2)	Consensus Log Po/w	nRB	nOHA	nOHD	Water solubility	GI absorption	BBB permeant	P-gp substrate	CYP1A2 inhibitor	CYP2C19 inhibitor	CYP2C9 inhibitor
Xanthosine	153.46	− 1.92	2	7	5	Very soluble	Low	No	No	No	No	No
Clionasterol	20.23	7.19	6	1	1	Poorly soluble	Low	No	No	No	No	No
Vitamin E	29.46	4.32	2	2	1	Moderately soluble	High	Yes	No	Yes	Yes	Yes
4,8,12,16-Tetramethylheptadecan-4-olide	26.3	6.1	12	2	0	Poorly soluble	Low	No	No	Yes	No	Yes
Dihydroactinidiolide	26.3	2.41	0	2	0	Soluble	High	Yes	No	No	No	No
Squalene	0	9.38	15	0	0	Insoluble	Low	No	No	No	No	No
α-Tocospiro B	63.6	6.06	13	4	1	Poorly soluble	High	No	No	No	No	No
Methyl linolenate	26.3	5.55	14	2	0	Poorly soluble	High	Yes	No	Yes	No	Yes
1,6-Anhydro, β-D-glucopyranose	110.38	− 2.26	1	6	5	Highly soluble	Low	No	Yes	No	No	No

TPSA (A2) topological surface area, Consensus Log Po/w: lipophilicity, nRB number of rotatable bonds, nOHA number of H-bond acceptors, nOHD number of H-bond bonds donor, GI (gastrointestinal) absorption, BBB (blood–brain barrier) permeant, P-gp (P-glycoprotein) substrate, isozymes of the cytochrome group (CYP1A2, CYP2C19 and CYP2C9) inhibitor

Fig. 6.

Best hits using blind docking with the AutoDock Tool: (E) squalene (F) α-tocospiro B (G) methyl linolenate. Discovery Studio was used for interaction analysis

Fig. 7.

In vitro anti-leishmanial activity at different conc. of R. ellipticus extracts (RLEE, RSEE, RLHE, and RSHE) on promastigotes. The bar diagrams show the % inhibition of promastigotes after 72 h. The results are mean ± SD of three biological replicates. The statistically significant p value was calculated using the one-way ANOVA. The difference was considered significant at p < 0.05

In silico ADMET study

ADMET analysis characterizes the essential features of an ideal drug, focusing on absorption, distribution, metabolism, excretion, and toxicity. These factors play crucial roles in drug discovery and development. The drug-likeliness action of compounds was assessed using Swiss ADME online software. To examine the drug properties of the following compounds clionasterol, vitamin E, and 4,8,12,16-tetramethylheptadecan-4-olide Lipinski’s, Egan’s, and Veber’s rules were followed. As per Lipinski’s rule, the compound must have a TPSA (topological surface area) < 140, nOHD (number of H-bond bonds) ≤ 5, nOHA (number of H-bond acceptors) ≤ 5, and nRB (number of rotatable bonds≤ 10 for the oral drugs. The present study depicts eight compounds that exhibit potent drug-like properties and subsequently follow Lipinski’s rule. A good brain penetration potential was observed with TPSA values less than 120 Å2. The effective bioactive nature was confirmed by a bioavailability score of 0.55 observed in all compounds. P-glycoprotein (P-gp) substrate was found in 1,6-anhydro-β-D-glucopyranose, recommending poor fine intestinal absorption of compounds. The consensus Log Po/w (0.84−4.56) value predicts good lipophilicity, which is observed in vitamin E and dihydroactinidiolide. Vitamin E, dihydroactinidiolide, α-tocospiro B, and methyl linolenate demonstrated high gastrointestinal absorption power. In addition, vitamin E, dihydroactinidiolide, and methyl linolenate cross the blood–brain barrier (BBB). The selected compounds interact with four isozymes of the cytochrome group, namely CYP1A2, CYP2C19, and CYP2C9 attributing their potency and possessing the least toxicity. The compounds and their drug-like properties are mentioned in Table 4. Moreover, toxicity prediction of selected compounds was done using Protox II as mentioned in Table 5. Vitamin E, 4,8,12,16-tetramethylheptadecan-4-olide, squalene, 1,6-anhydro-β-D-glucopyranose were predicted as inactive for all toxicity parameters, while xanthosine and dihydroactinidiolide were predicted as slightly inactive for hepatotoxicity and carcinogenicity. In addition, α-tocospiro B was predicted slightly inactive for carcinogenicity and cytotoxicity, while clionasterol was predicted to be slightly inactive for carcinogenicity and active for immunotoxicity with 0.99 probability (Table 5). Both ADMET analysis and toxicity prediction confirmed that these six compounds possess drug-like properties and may be further evaluated as potential drug agents.

Table 5.

Toxicity prediction and their probability of selected compounds by using Protox-II

S. No	Compounds	Toxicity report (probability/prediction)
		Hepatotoxicity	Carcinogenicity	Immunotoxicity	Mutagenicity	Cytotoxicity	LD50 (mol/kg)
1	Xanthosine	0.57 Slightly inactive	0.68 Slightly inactive	0.99 Inactive	0.88 Inactive	0.90 Inactive	15,000
2	Clionasterol	0.87 Inactive	0.60 Inactive	0.91 Active	0.98 Inactive	0.94 Inactive	890
3	Vitamin E	0.93 Inactive	0.79 Inactive	0.95 Inactive	0.95 Inactive	0.89 Inactive	5000
4	4,8,12,16-Tetramethylheptadecan-4-olide	0.70 Inactive	0.69 Inactive	0.99 Inactive	0.88 Inactive	0.83 Inactive	4400
5	Dihydroactinidiolide	0.61 Slightly inactive	0.52 Slightly inactive	0.91 Inactive	0.88 Inactive	0.94 Inactive	34
6	Squalene	0.79 Inactive	0.76 Inactive	0.99 Inactive	0.98 Inactive	0.81 Inactive	5000
7	α-Tocospiro B	0.97 Inactive	0.57 Slightly inactive	0.99 Inactive	0.85 Inactive	0.78 Slightly inactive	300
8	Methyl linolenate	0.63 Slightly inactive	0.58 Slightly inactive	0.97 Inactive	0.83 Inactive	0.73 Inactive	20,000
9	1,6-Anhydro, β- D-glucopyranose	0.81 Inactive	0.64 Inactive	0.99 Inactive	0.54 Inactive	0.79 Inactive	8000
*	Amphotericin B	0.97 Inactive	0.73 Inactive	0.99 Active	0.89 Active	0.83 Inactive	100

Effect of R. ellipticus on promastigote and axenic amastigote form of L. donovani

The anti-promastigote activity of R. ellipticus was calculated with doses ranging from 0 μg/ml to 1000 µg/ml in a dose-dependent manner. The inhibition of the parasite was determined using the MTT assay. Treatment with R. ellipticus extracts led to decrease in the growth of promastigotes in the log phase (Fig. 7). At a concentration of 50 µg/ml, RLEE, RSEE, RLHE, and RSHE significantly decreased promastigote growth to 50.5% ± 2.267, 49.26% ± 1.847, 51.05% ± 4.017, and 69.13% ± 3.067, with p value <0.0001, <0.0001, 0.0026, and 0.7264, respectively, at 72 h. At 1000 µg/ml for 72 h, only RSHE and RSEE significantly inhibited promastigote growth by 93.08% ± 0.6866 and 88.14% ± 1.448, with p values of 0.0047 and <0.0001, respectively (Fig. 7).

In addition, all four extracts of R. ellipticus were also examined against the axenic amastigotes form of L. donovani in the range between 0–1000 µg/ml. After 72 h of treatment with 50 µg/ml of RLEE, RSEE, RLHE RSHE, axenic amastigote growth was reduced to 41.91% ± 1.234, 58.22% ± 2.848, 51.61% ± 2.446, and 53.53% ± 4.159, with p value <0.0001, 0.0011, <0.0001, and 0.0039, respectively. Further, we found that RLHE and RSHE at a concentration of 1000 µg/ml inhibited axenic amastigotes growth to 92.86% ± 2.422 and 90.41% ± 1.059, respectively, with p values of 0.0006 and 0.0482 (Fig. 8). Amphotericin B was used as a positive control and this drug exhibited 80% inhibition of both promastigote and axenic amastigote forms of L. donovani multiplication.

Fig. 8.

In vitro anti-leishmanial activity at different conc. of R. ellipticus extracts (RLEE, RSEE, RLHE, and RSHE) on axenic amastigotes. The bar diagrams show the % inhibition of axenic amastigotes after 72 h. The results are mean ± SD of three biological replicates. The statistically significant p value was calculated using the one-way ANOVA. The difference was considered significant at p < 0.05

Cytotoxicity of R. ellipticus on THP-1 cell

Next, we examined the effect of these extracts on THP-1 cells by treating the cells with differing concentrations for 72 h. The THP-1 cells remained viable up to a concentration of 250 µg/ml indicating the extracts are not toxic to the mammalian cells (Fig. 9).

Fig. 9.

Effect of different concentrations of R. ellipticus extracts (RLEE, RSEE, RLHE, and RSHE) on the survival of THP-1-derived macrophages. The bar diagrams show the percentage viability of THP-1 of after 72 h treatment of extracts. The results are mean ± SD of three biological replicates

Effect of R. ellipticus on intracellular amastigotes in THP-1 derived macrophage

To assess the effectiveness of these extracts on intracellular amastigotes, L. donovani-infected THP-1 macrophages were treated with extracts at concentrations of 50 µg/ml and 70 µg/ml. Treatment with R. ellipticus at 50 µg/ml significantly decreased the load of intracellular amastigotes (Figs. 10 and 11A), while at a concentration of 70 µg/ml, it completely reduced the parasite load (Fig. 10). The number of amastigotes in both treated and untreated THP-1 derived cells was counted microscopically, and the results found were presented as a percentage decrease of amastigotes (Fig. 11A). RLEE, RSEE, RLHE, and RSHE were found to lessen the intracellular amastigote burden in infected THP-1-derived macrophages with p value 5.11992E−06, 7.09491E−07, 1.37733E−06 and 3.01914E−07, respectively. The fact that these extracts were toxic to intracellular amastigotes and non-toxic or less toxic to the host cell confirmed their selectivity to the parasite as compared to the host cell.

Fig. 10.

Microscopic analysis shows the effects of R. ellipticus extracts (RLEE, RSEE, RLHE, and RSHE) on intracellular amastigotes at conc. of 50 µg/ml and 70 µg/ml conc. At 70 µg/ml conc. of extracts reduces the complete parasitic load in THP-1 host after 72-h treatment

Fig. 11.

A Graphical representation of intracellular amastigotes inhibition assay at 50 µg/ml results extracts significantly reduces the parasite load without causing significant harm to the host after 72 h, while at 70 µg/ml, extracts eliminate the complete parasitic load. B1 and B2 Graphical representation confirmed the significant reduction in intracellular amastigote by real-time PCR targeting the kDNA minicircle-specific JW11 mRNA expression after the treatment of R. ellipticus extracts in a dose-dependent manner at 50 µg/ml and 70 µg/ml conc. for 72 h. The results are mean ± SD of two biological replicates. The statistically significant p value was calculated using the paired t-test. The difference was considered significant at p < 0.05

Inhibition of intracellular amastigotes by R. ellipticus results in a reduction of kDNA minicircle-specific JW11 mRNA expression levels

The kinetoplast of Leishmania contains a concatenated network of circular DNA molecules that includes the kDNA minicircle-specific jw11 gene (Prasanna et al. 2023). Detection of intracellular amastigotes was performed using SYBR Green-Based Real-Time PCR targeting the kDNA minicircle-specific jw11 gene post-treatment with R. ellipticus extracts. For this experiment, THP-1 cells were first infected with L. donovani followed by treatment with the extracts at concentrations of 50 µg/ml and 70 µg/ml for 72 h. RLEE, RSEE, RLHE, and RSHE at 50 µg/ml conc. (p value 0.00019, 9.88701E−07, 2.64618E−05 and 3.04851E−07) and 70 µg/ml conc. (4.30179E−05, 4.60789E−07, 3.0524E−06 and 2.83446E−08) significantly decreased the mRNA expression of JW11 after 72-h post-treatment in a dose-dependent manner compared to the untreated sample (Figs. 11B1 and B2).

DNA fragmentation assay

DNA fragmentation is a characteristic feature of apoptosis (Shadab et al. 2017). Therefore, we next assessed whether the R. ellipticus extracts induced apoptosis in L. donovani. A clear smear (DNA fragmentation) was observed in parasitic DNA treated with RLHE and RSHE at a concentration of 500 µg/ml (Fig. 12), while the remaining extract samples showed slight fragmentation at concentrations of 500 µg/ml, 250 µg/ml, and 100 µg/ml. The untreated DNA sample was used as a negative control, and AmB was used as a positive control (Fig. 12).

Fig. 12.

DNA fragmentation analysis in L. donovani by agarose gel electrophoresis. The DNA of promastigotes treated with R. ellipticus extracts (RLEE, RSEE, RLHE, and RSHE) at 500, 250, and 100 conc. for 72 h (lanes 2–13) show fragmentation which is a DNA characteristic feature of apoptosis. DNA ladder (lane 1); RLEE at 500 µg/ml, 250 µg /ml, and 100 µg /ml represent lanes 2, 6, and 10, respectively; RLHE at 500 µg /ml, 250 µg /ml, and 100 µg /ml represent lanes 3, 7, and 11, respectively; RSEE at 500 µg /ml, 250 µg /ml, and 100 µg /ml represent lanes 4, 8, and 12, respectively; and RSHE at 500 µg /ml, 250 µg /ml, and 100 µg /ml represent lanes 5, 9, and 13, respectively. Lane 14 represents negative control or untreated DNA; lane 15 represents positive control or AmB-treated DNA at 25 µg /ml DNA (lane 14)

Discussion

Leishmaniasis presents a significant challenge due to the identification of acquired resistance mechanisms in the human host (Moncada-Diaz et al. 2024). While chemotherapeutics remain the primary treatment for VL, their efficacy is compromised by the widespread appearance of synthetic drug resistance and adverse side effects (Saini et al. 2014; Singh et al. 2016; Kumar et al. 2018). Accordingly, the exploration of alternative, safer target-based drug options is needed. Plant-based therapeutics offer a promising path, potentially overcoming the limitations associated with synthetic drugs (Rodrigues et al. 2015; Ghodsian et al. 2020; Gouri et al. 2021, 2022; Tandon et al. 2023). The World Health Organization (WHO) report approves the progress and utilization of natural products as leishmanicidal agents (WHO 2010). Previous research has identified natural compounds targeting specific proteins of Leishmania, such as Luteolin and Niranthin (topoisomerase I and II), Betulin (trypanothione enzymes), Piperine (pteridine reductase1), Genistein (protein kinase), Licochalcone A, Berberine (mitochondrial protein), Carvacrol, vanillin, and p-coumaric (LdHEL-67) (Gouri et al. 2022). In line with these previous reports and to further elucidate the target-based approach of our study, we selected LdHEL-67 as the target protein based on its relevance to Leishmania biology (Pandey et al. 2020). LdHEL-67 plays a crucial role in initiating promastigote to amastigote differentiation and is actively involved in RNA metabolism, specifically RNA splicing (Fig. 14). Furthermore, it functions to prevent RNA degradation facilitated by an antisense rRNA-mediated pathway (Padmanabhan et al. 2016b) (Fig. 14). Moreover, previous studies have highlighted the potential of plant-based treatments. For example, the methanol extract of Sterculia villosa has been reported for its anti-leishmanicidal action against L. donovani, with an IC₅₀ at 17.5 µg/ml, and it is considered safer for the host macrophage and animal model (Das et al. 2017). Extracts derived from plants such as Nyctanthes arbortristis, Tinospora cordifolia, Alstonia scholaris, Tibouchina semidecandra, and Swertia chirayita exhibited 75% inhibition of L. donovani in in vivo experiments (Singha et al. 1992; Rocha et al. 2005; Iqbal et al. 2016; Wafula 2023; Gouri et al. 2022). Similarly, the plant's Khaya senegalensis and Anthostema senegalense showed inhibitory activity against L. donovani with IC₅₀ values of 9.8 and 9.1 mg/ml, respectively (Abreu et al. 1999). Moreover, other plants, including Azadirachta indica (ethanolic fraction and ethyl extract), Allium sativum (methanolic extract), Annona crassiflora (exanolic and ethanolic extract), Cocos nucifera (aqueous extract), Croton caudatus (hexanic extract), etc., have been evaluated against L. donovani (Gouri et al. 2021).

Fig. 14.

A Phytocompounds derived from R. ellipticus (identified by GC–MS analysis) were docked with the LdHEL-67 protein. Hence, based on the present in silico study, it can be predicted that selected compounds inhibit the LdHEL-67 protein and reduce the parasitic load in the host cell. B1 and B2 In Leishmania, LdHEL-67 is involved in RNA metabolism, specifically RNA splicing and it functions to prevent RNA degradation facilitated by an antisense rRNA-mediated pathway that leads to parasitic survival. However, after the treatment with phytocompounds of R. ellipticus, inhibition processes take place. C1 and C2 LdHEL-67 regulates the mitochondrial stress response in the parasitic Leishmania, while inactivation of LdHEL-67 by phytocompounds leads to the accumulation of mitochondrial reactive oxygen species (ROS), mitochondrial membrane potential loss, mitochondrial fragmentation, inhibit central role in mitochondrial proteostasis and cell death. D1 and D2 LdHEL-67 plays an essential role in promastigote to amastigote differentiation, which leads to infection in the host cell. However, phytocompounds of R. ellipticus interrupt the processes and reduce the parasitic burden in host macrophages. Thus, the overall figure highlights the role of target-based phytocompounds in the treatment of VL

In our study, we focused on R. ellipticus due to its well-documented biological properties and abundance in nature (Pandey and Bhatt 2016; Karn et al. 2022). Employing Soxhlet extraction techniques, we prepared leaf and stem extracts using ethanol and hexane solvents. After GC–MS profiling, we found number of compounds present in extracts. To determined compounds, potential molecular docking was performed and then we observed that compounds, namely xanthosine, clionasterol, vitamin E, 4,8,12,16-tetramethylheptadecan-4-olide, dihydroactinidiolide, squalene, α-tocospiro B, methyl linolenate, and 1,6-anhydro, β-D-glucopyranose demonstrated notable binding energy which is nearby Amphotericin B interaction with LdHEL-67, thereby impeding the survival of L. donovani (Fig. 13). These notable results admired to performed next in silico ADMET analysis which predicts that these phytocompounds possess drug-like properties. Furthermore, the majority of the compounds were noted to be inactive in toxicity prediction assessments. These selected compounds present in R. ellipticus extracts in major percentage composition. Among all of the compounds, clionasterol showed high binding energy as compared to Amphotericin B and this particular compound occur in all the extract RLEE, RSEE, RLHE, and RSHE in major quantity such as 10.57%, 4.48%, 13.82%, and 5.72%, respectively. The research article by Cerqueira et al. (2003) demonstrated the potential of clionasterol as a therapeutic drug for modifying the complement system by elucidating its inhibitory effects on complement component C1 in this study. Clionasterol provides a novel method of regulating complement activation by targeting C1, which may lessen the harmful consequences of excessive complement activity (Cerqueira et al. 2003) which support our study. In addition, most of the selected compounds were found in RLEE and RLHE. However, after the in silico study, we concluded that R. ellipticus extracts contain compounds that are effective against the target protein (LdHEL-67) of L. donovani. In addition, in in vitro examinations, all the extracts were effective against various forms of L. donovani (Table 6). Among all extracts, RSHE and RSEE exhibited greater effectiveness against promastigotes, while RLHE and RSHE showed higher efficacy against axenic amastigotes. The IC₅₀ values of RLEE were 50 µg/ml and 243 µg/ml, RSEE demonstrated 50 µg/ml and 42 µg/ml, and RSHE displayed 36 µg/ml and 47 µg/ml against promastigotes and amastigotes, respectively. RLHE had consistent values of 49 µg/ml for both forms; these results suggest differential efficacy, with RSHE showing the highest potency against promastigotes and RSEE performing best against amastigotes. Importantly, all extracts exhibited low toxicity toward host THP-1 cells while effectively reducing the intracellular amastigote load. Furthermore, our findings indicate a distinct impact of the extracts on parasite JW11 mRNA expression levels. The extracts downregulated JW11 mRNA expression in a dose-dependent manner, with RSHE and RLHE showing the strongest effects, followed by RSEE and RLEE. Post-treatment with extracts resulted in the fragmentation of promastigote DNA, indicating a potential mechanism of action at the genomic level and suggesting parasite apoptosis. RLHE and RSHE extracts showed particularly high fragmentation of L. donovani promastigote DNA. All these findings underscore the promising therapeutic potential of R. ellipticus against L. donovani. In summary, our study highlights the identification of novel inhibitors from R. ellipticus that have the potential to inhibit LdHEL-67 through molecular interactions. Extracts from this plant have shown promising anti-leishmanial activity in various in vitro experiments. Therefore, we hypothesized that the components of R. ellipticus may effectively target LdHEL-67 and its associated pathways (Fig. 14). These findings contribute to the growing field of research supporting the exploration of plant-based therapeutics as alternative treatments for VL. Further investigations are needed to authorize the mechanism of action of these compounds and to evaluate their safety in animal models and clinical trials. This success could catalyze the development of novel and effective drugs against Leishmania infections. This suggests their potential as safe and effective candidates for further drug development.

Fig. 13.

Chemical structure of best nine selected compounds derived from R. ellipticus

Table 6.

Top 9 selected hits identified via GC–MS analysis along with their % Composition in extracts and % inhibition at 50 µg/ml, 100 µg/ml & 250 µg/ml concentration

S. No	Extracts from R. ellipticus	Compounds	% Composition in extracts	Dose of extracts	Promastigote mean ± SD	Axenic amastigote mean ± SD
1	RLEE	Clionasterol	10.57	50 µg/ml 100 µg/ml 250 µg/ml	50.5 ± 2.267 53.89 ± 1.525 58.67 ± 0.511	41.91 ± 1.23 43.17 ± 4.38 51.3 ± 2.49
		Vitamin E	4.40
		Squalene	2.43
		α-Tocospiro B	1.27
		Methyl linolenate	0.52
		1,6-Anhydro, β- D-glucopyranose	4.23
2	RSEE	Clionasterol	4.48	50 µg/ml 100 µg/ml 250 µg/ml	49.26 ± 1.847 55.93 ± 4.405 68.07 ± 1.274	58.22 ± 2.848 58.23 ± 4.64 64.32 ± 3.34
		Xanthosine	2.06
3	RLHE	Clionasterol	13.82	50 µg/ml 100 µg/ml 250 µg/ml	51.05 ± 4.017 55.94 ± 6.206 58.17 ± 5.707	51.61 ± 2.44 52.78 ± 2.13 72.62 ± 3.66
		Vitamin E	1.46
		4,8,12,16-Tetramethylheptadecan-4-olide	1.18
		Dihydroactinidiolide	1.17
		α-Tocospiro B	7.4
		Methyl linolenate	1.80
4	RSHE	Clionasterol	5.72	50 µg/ml 100 µg/ml 250 µg/ml	69.13 = 3.067 86.28 ± 1.35 93.35 ± 0.570	53.53 ± 4.159 57.02 ± 5.168 61.44 ± 3.213
		Methyl linolenate	0.28
*	Control	–	–	50 µg/ml	74.96 ± 2.672	75.12 ± 2.198

Conclusion

This study underscores the potential of R. ellipticus as a promising therapeutic agent against leishmaniasis. In in-silico analyses, compounds derived from R. ellipticus exhibited strong binding interactions and favorable pharmacokinetic profiles, highlighting their suitability for further drug development. Notably, clionasterol major component present in all plant extracts demonstrated the most robust binding affinity with the target LdHEL-67 protein, suggesting that it plays a crucial role in the plant’s anti-leishmanial activity. This compound’s prevalence across extracts in substantial percentages reinforces its significance as a primary bioactive constituent responsible for the observed activity. Moreover, in vitro assays further validated the efficacy of R. ellipticus extracts, revealing potent leishmanicidal activity with a preferential selectivity toward intracellular amastigotes, while showing minimal cytotoxicity to host cells. This selectivity is particularly advantageous, as it minimizes potential side effects and underscores the safety profile of these extracts for therapeutic use. Consequently, the findings of this study contribute valuable insights into the development of herbal-based inhibitors that are both target-specific and effective, paving the way for novel anti-leishmanial drug candidates with a natural origin. These results collectively suggest that R. ellipticus could serve as a foundation for new, plant-based treatment options against leishmaniasis. Hence, they can serve as a platform for further in vivo studies, which could potentially accelerate the leap forward in natural drug development to address leishmaniasis, especially against the existing drug arsenal, in light of the emerging resistance.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

VL

Visceral leishmaniasis

WHO

World Health Organization

LdHEL-67

DDX3-DEAD BOX RNA helicase

NCBI

National Centre for Biotechnology Information

pI

Isoelectric point

GRAVY

Grand average of hydropathicity index

QMEAN

Qualitative model energy analysis

GC–MS

Gas chromatography–mass spectrometry

ADMET

Absorption, distribution, metabolism, excretion, and toxicity

TPSA

Topological surface area

nOHD

Number of H-bond bonds

nOHA

Number of H-bond acceptors

nRB

Number of rotatable bonds

P-gp

P-glycoprotein

MTT

3-(4, 5-Dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide

PMA

Phorbol-12-myristate-13-acetate

RPMI

Roswell Park Memorial Institute

THP-1

Acute monocytic leukemia-derived human cell

PI

Propidium iodide

DMSO

Dimethyl sulfoxide

RT-PCR

Reverse transcription polymerase chain reaction

Author contributions

Vinita Gouri: investigation, methodology, visualization, writing—original draft preparation, writing—review and amp; editing. Gargi Roy: investigation, reviewed, and edited the manuscript. Akanksha Kanojia: investigation, reviewed, and edited the manuscript. Sumeet Singh: data curation, reviewed and edited the manuscript. Rohini Muthuswami: supervised, formal analysis, resources, funding acquisition, reviewed, edited, and finalized the manuscript. Mukesh Samant: supervised, investigated, project administration, resources, funding acquisition, conceptualized, validated the data, reviewed, edited, and finalized the manuscript.

Funding

This work is supported by the DST-FIST grant SR/FST/LSI/2018/131 to the Department of Zoology. S.S.’s financial support provided by the UGC, India, in the form of a JRF (Ref. No. 211610156180). R.M. was supported by DST-DPRP funding (VI-D&P/569/2016-17/TDT). R.M. also acknowledges the facilities/laboratories supported by DBT BUILDER (BT/INF/22/SP45382/2022) and DST FIST-II (SR/FST/LSII-046/2016(C)). A.K. is supported by the ICMR Senior Research Fellowship.

Data availability

The data supporting the conclusions of this article are all included within the article.

Declarations

Conflict of interests

On behalf of all authors, the corresponding author states that there is no conflict of interest.

Research involving human participants and/or animals

Not applicable.

Informed consent

Not applicable.