GC–MS analysis of phytoconstituents of Acacia nilotica and its molecular docking with drug-targets of  Leishmania donovani to combat leishmanaisis


 Acacia nilotica is an important medicinal plant, found in Africa, Middle East, and Indian subcontinent. Every part of the plant possesses a wide array of biologically-active and therapeutically important compounds and have been used in traditional-system of medicine. We reported the antileishmanial activity of Acacia nilotica (A. nilotica) bark methanolic extract through in vitro assays and dissected the mechanism of its action through in silico studies. Bark methanolic extract exhibited anti-promastigote and anti-amastigote potency with IC50 value of 19.6 + 0.9037 µg/ml and 77.52 + 5.167 µg/ml respectively in time and dose dependent manner. It showed very low cytotoxicity having CC50 value of 432.7 + 7.71 µg/ml on human-macrophage cell line, THP-1. The major constituents identified by GC-MS analysis are 13-docosenoic acid (34.06%), lupeol(20.15 %), 9,12-octadecadienoic acid (9.92 %) and 6-octadecanoic acid (8.43 %) bind effectively with the potential drug-targets of Leishmania donovani (L. donovani ) including sterol 24-c-methytransferase (SMT), trypanothione reductase (TR), pteridine reductase (PTR1) and adenine phosphorybosyl transferase (APRT); suggest the possible mechanism of its antileishmanial action. The highest affinity with all these targets was shown by lupeol. The pharmacokinetic studies, predicted bioactivity scores and acute toxicity studies of major extract constituents support safe antileishmanial drug candidate. This study proved the antileishmanial potential of bark-methanolic extract A. nilotica and its mechanism of action through the inhibition of potential drug targets of L. donovani.

among the neglected tropical diseases 4,5 . It is a significant problem for the economically weaker section of the society. Due to their unhygienic living environment, they are more vulnerable to the disease. Illiteracy is another factor which is directly proportional to the lack of awareness which leads to major morbidity and mortality. The available chemotherapy of visceral leishmaniasis is limited and undermined by drug resistance. Currently, in general the drug used in the Indian subcontinent sodium antimony gluconate (SAG) showed no response in more than 64 % of the patients due to development of resistance against the parasites 6 . Alternate drugs, miltefosine, amphotericin B and its lipid formulations have several limitations because of high toxicity, cost and unavailability, limit its use. The present scenario of disease and its limited treatment options demand an urgent need to develop a promising and cost-effective operational drug to overcome the disease. In case of parasitic disease, directly targeting parasites is found to be significant in disease recovery. Till date, large numbers of medicinal plants and their extracts had been studied for antileishmanial activity and proved to be potential therapeuticoptions 7,8 .
We also tried to dissect the mechanism of its antileishmanial action through different in silico approaches. SMT, TR, PTR1 and APRT are prerequisite enzymes for survival, pathogenicity and transmission of L. donovani. Therefore, we selected these potential drug-targets for molecular docking study of major constituents of bark-extract identified by GC-MS, with these mentioned essential enzymes of Leishmania.

Materials and method
2.1 Chemicals: M199 media, Roswell park memorial institute (RPMI) 1640 media, penicillin streptomycin antibiotic cocktail, fetal bovine serum (FBS) were purchased from Gibco. HEPES, sodium bicarbonate and paraformaldehyde were purchased from Sigma Aldrich. Miltefosine, MTT assay reagents, DMSO and different solvents were procured from Merck. Propidium iodide and Annexin V apoptosis kit were procured from Thermo scientific. All the other chemicals and reagents were purchased from Sigma Aldrich or Merck unless stated otherwise.

GC-MS analysis of extract:
GC-MS analysis was performed to identify the secondary metabolites that may be responsible for the antileishmanial efficacy of A nilotica,. Bark was crushed, powdered, and extracted in methanol and then analyzed on Shimadzu QP2010 armed with a DB-5MS column. The mass spectrums of the sample were produced in an electron impact ionization mode of 70 eV and the phytochemicals were identified after correlation of the recorded mass spectrum with the reference library WILEY8.LIB and NIST14.LIB supplied with the software of the GC-MS system.

Molecular docking studies:
To begin with structure-based virtual screening and docking, we used various bioinformatic tools, such as PyRx 25  2JK6_A) and X-ray diffracted crystal structure 1.34 Å resolution (PDB id: 5WP4_A) were used as template structures to model the 3D structures of trypanothione reductase and sterol 24-cmethyltransferase, respectively. PDB was used to retrieve the template structure. Homology modelling was carried out using Modeller 9.24 32 and PyMol was used for the visualization of the 3D structures. The energy minimization was performed using Discovery studio. The PROCHECK program, Ramachandran plots was also used for the assessment of the model 33 .

Pharmacokinetics studies
The selected ligands were evaluated for their pharmacological profiles by analyzing for (https://molinspiration.com/cgi-bin/properties). The successfully screened ligands were further evaluated for ADMET (absorption, distribution, metabolism, excretion and toxicity) properties by GUSAR 36 and SwissADME database 37 .

Growth reversibility assay after extract treatment:
A. nilotica, treated and untreated parasites were washed with PBS after 7 days and old media was

Molecular docking of A. nilotica methanolic extract of major constituents with potential drug-targets of L. donovani
The TR and SMT enzymes were modelled using Modeller 9.24 and the energy minimization was carried out by BIOVIA Discovery studio. The three-dimensional cartoon representation of TR and SMT enzymes are shown in Supplementary Fig 1A & 2A. The models were selected by analysing its stereochemical quality using PROCHECK program. The generated models of TR and SMT show a good quality structure having 99.8 % and 99% residues in the allowed regions of Ramachandran plot respectively (Supplementary Fig. 1B & 2B). PDBsum tool was used to analyze and found that the 3D structure of the enzyme is composed of mixed α-helices and βstrands (α+β) secondary structures 33 (Table 2).

Pharmacokinetics studies of A. nilotica bark-methanolic extract constituents:
The pharmacological studies was done for the selected ligands against Adenine phosphoribosyl transferase, Pteridine reductase, trypanothione reductase and sterol 24-c-methyltransferase proteins for a good oral administration established through the Lipinski rule of five 38 Table 3.
The bioactivity prediction of the major constituents of A. nilotica bark-methanolic extract was analysis through molinspiration. The activity was calculated against G-protein coupled receptorligand, ion channel modulator, a kinase inhibitor, nuclear receptor ligand, protease inhibitor and enzyme inhibitor 40 . The interpreted values for bioactivity were as: active (bioactivity score ≥ 0), moderately active (bioactivity score: between −5.0 to 0.0) and inactive (bioactivity score ≤ −5.0) 41 . Lupeol, 9,12-Octadecadienoic acid, 6-Octadecenoic acid, and 13-Docosenoic acid were evaluated as active enzyme inhibitors with values 0.52, 0.23, 0.12 and 0.10, respectively. Lupeol and 9,12-Octadecadienoic acid were evaluated as active protease inhibitor as well as ion channel modulator. (Table 4) The principal aim of predicting the acute toxicity is to evaluate undesirable side effects of a compound after single or multiple exposures to an organism via a known administration route (oral, inhalation, subcutaneous, intravenous or intraperitoneal). GUSAR was used to determine the acute toxicity of the successfully docked compounds. The parameters used by GUSAR to probe compounds based on the prediction of activity spectra for substances algorithm and quantitative neighborhoods of atoms descriptors. The obtained results were compared with SYMYX MDL Toxicity Database to further categories them on the basis of Organisation for Economic Co-operation and Development (OECD) chemical classification manual 36 . The criteria used for these compounds to elicit toxicity based upon the administration route when the compound dose is more than 7000 mg/kg for intravenous route, more than 500,000 mg/kg in case of the oral route, and more than 20,000 mg/kg for intraperitoneal route and subcutaneous database as shown in Table 5. As per the OECD chemical classification 9,12-Octadecadienoic acid, 6-Octadecenoic acid, 13-Docosenoic acid found to be non-toxic and Lupeol is a Class 5 chemical.  Table 2.

Discussion
Pharmacological studies of these selected inhibitors for Lipinski rule of 5 indicated violation of only one Lipinski parameter, as shown in Table 3.The pharmacokinetic properties and acute toxicity of lupeol; 9,12-Octadecadienoic acid;6-Octadecenoic acid and 13-Docosenoic acidhave shown relatively low toxicity profile, which meansrequire high doses to evoke toxic response.
The majority of the compounds are non-toxic chemicals whereas lupeol is a Class 5 chemical with very low toxic effects (31,54).

Conflict of interest
The author showed no conflict of interest.

Author Contribution
Study was conceptualized by AR, SB and AABD, ASA; data acquisition and data analysis were