Identification of phytoconstituents from Dicliptera paniculata and study of antibacterial activity guided by molecular docking

Abstract

According to WHO, antibiotic resistance is one of the biggest healthcare challenges to the global community. Therefore, it is absolutely essential to discover new antibiotics to address the challenge. Dicliptera paniculata (ForssK.) I. Darbysh, a rare medicinal herb of Acanthaceae, is known for its noteworthy uses as a flavoring, spicing, and antibacterial agent. The primary goal of the study is to identify novel antibacterials from D. paniculata. The petroleum ether fraction of the methanol extract of D. paniculata was subjected to GC–MS and identified 14 compounds. Several bacterial target proteins were used for molecular docking. The antibacterial activity of petroleum-ether fraction was evaluated on bacteria whose target protein interacts most strongly with identified molecules. The molecules DP_02, DP_06, and DP_14 exhibited the highest docking scores with Staphylococcus aureus dihydrofolate reductase, which were − 6.283, − 7.705, and − 6.364 kcal/mol, respectively. The MM-GBSA binding energy of compounds DP_02, DP_06, and DP_14 were − 46.736, − 42.366, and − 35.734 kcal/mol, respectively. The MM-GBSA binding energy and decent docking score of the compounds DP_02 and DP_06 were both encouraging, and both of the compounds are drug-like. The finding was validated through studies on antibacterial effectiveness against S. aureus and showed encouraging results. These two molecules might serve as the building blocks for the future development of potent antibiotics.

Introduction

Medicinal plants have been used as therapeutic agents since ancient times and are as old as 4000–5000 BC. (Tewari et al. 2012). Natural compounds from medicinal plants might be used to combat the current challenge of multi-drug resistance in bacteria. The biological activities of plants unique to particular species or groups are consistent with the concept that the combination of secondary metabolites for a specific plant is taxonomically distinct (Wink et al. 1999). Screening of such secondary metabolites as active compounds from plants has led to the invention in the drug discovery process in pharmaceutical science for isolating new natural drugs that have efficient protection and treatment roles against various diseases (Sheeja et al. 2007).

The North-Eastern hilly states of India are affluent in their floristic composition. It is also represented by various forest dwellers and local ethnic tribal communities who play an important role in the country's social, cultural, historical, economic, and industrial development and in maintaining the region's ecological balance. Traditionally, the ethnic group uses the vegetation of their ambient environment to get food, fodder, fuel, fiber, medicine, etc. (Sharma et al. 2011). In developing countries, up to 80% of the population relies on medicinal plants for primary health care (Ekor 2014). Ethno-pharmacological studies have shown the implications of plants as a remedy for various diseases. Several researchers identified antibacterial plant fractions using the bioassay-guided method (Mehta et al. 2021; Rolta et al. 2020); some of them targeted phytoconstituents based on the rational drug design approach (Mehta et al. 2022; Salaria et al. 2021).

Dicliptera paniculata (Forssk) I. Darbysh, a perennial herb with pink flowers, belongs to the family Acanthaceae. It is distributed through Tropical Africa, Myanmar, and India (Tamilnadu, Kerala, and Tripura). Generally found along the roadside and desolate regions. The plant is used as fodder, and the water decoction of the leaves is taken orally to cure anemia in India. The leaves are used to prepare various soups in India as a flavoring and spicing agent (Adeniyi and Odufowora 2000). Leaf paste is applied in cutting wounds, and paste of the whole plant is applied in bone fractures (Debnath et al. 2016). A yellowish-brown essential oil extracted by steam distillation shows anti-tuberculosis activity in vitro and inhibits the growth of multiple Mycobacterium tuberculosis strains. The crude ethanolic extract of fresh leaves exhibits extensive antibacterial activity. The antibacterial potency of crude ethanolic extract of D. paniculata against Aspergillus niger, Aspergillus clavatus, and Rhizopus stolonifer was also reported (Giwa 2010). The herb shows anti-inflammatory, analgesic, expectorant, and antipyretic activities (Sawadogo et al. 2006; Satyanarayana 1993).

S. aureus is a bacterium widely found on human and animal skin or in nares (Al-Kharabsheh and Ahmad 2022). When it penetrates the human body, or its population explodes, it can cause a wide range of infections like pneumonia, bone and joint infection, endocarditis, and more (Clark and Hicks 2023). We can efficiently suppress bacterial DNA synthesis by inhibiting the S. aureus enzyme dihydrofolate reductase, which inhibits bacterial growth and proliferation. Mycobacterium tuberculosis is the bacterial species that causes tuberculosis (TB) in humans. Tuberculosis is a common infection that typically affects the lungs but may also affect other organs. 7,8-dihydropteroate synthase is a crucial enzyme in the folate biosynthesis pathway, and inhibiting it using inhibitors has proven to be an effective therapy for tuberculosis infections. NDM-1 is a β-lactamase enzyme that has been identified in various bacterial species, including Escherichia coli and Klebsiella pneumoniae, which are common causes of healthcare-associated disorders. β-lactamases are enzymes generated by bacteria that can degrade β-lactam antibiotics, which include penicillins, cephalosporins, and carbapenems. MAO-B inhibitors are medications that suppress MAO-B activity. These inhibitors are used in the treatment of Parkinson's disease. The present work focuses on identifying various phytoconstituents and finding the antibacterial properties of petroleum ether fraction of crude methanol extract from D. paniculata.

Materials and methods

Plant material and sample preparation

The plant sample, D. paniculata, was collected from the roadside at Suryamaninagar, near the Tripura University campus (23º45ʹ48.53ʺ N 91º15ʹ58.18ʺ). The whole plant was air-dried in the absence of sunlight. The dried plant was powdered (500 gm) in a grinding machine and used for solvent extraction.

The powdered plant material was cold macerated in an extracting jar in methanol for 48 h, filtered at room temperature using filter paper, and the process was repeated five times. The filtrate was then evaporated to dryness using a rotary evaporator to obtain a greenish residue. The dried crude methanol extract was dissolved in 200 ml of distilled water and extracted with petroleum ether and ethylacetate using a separating funnel. The oily petroleum ether fraction was subjected to GC–MS as nonpolar, volatile, and small molecular weight compounds are available in petroleum ether fraction (Lynch 2017).

GC–MS analysis of the extract

The petroleum ether fraction was analyzed by using the Agilent model 6890N. A gas chromatograph fitted with DB-5 (30 m × 0.25 mm × 0.50 µm) column and HED-EM detector. Helium was used as carrier gas at 1 ml/min flow rate. GC. oven temperature was held at 70 °C for 3 min and 4 °C/min heated to 320 °C for 5 min. The GC–MS analyses were performed on an Agilent Model 5957C GC-MSD system with a m/Z 30-1000 mass range. Relative percentages of separated compounds were calculated from scan chromatograms, and 14 compounds were identified by using the NIST database (https://chemdata.nist.gov). Then, the in-silico binding affinity towards bacterial target proteins of S. aureus, M. tuberculosis, and New Delhi Metallo-β-lactamase-1 with the compounds was studied by molecular docking.

Data collection

The compounds identified by GC–MS further searched for their target selection (http://www.swisstargetprediction.ch/). Based on traditional knowledge of plant use from the literature, the binding affinity of compounds determined by molecular docking with enzyme dihydrofolate reductase of Staphylococcus aureus, 7,8-dihydropteroate synthase (DHPS) of Mycobacterium tuberculosis, and New Delhi Metallo-β-lactamase 1 (NDM-1). The X-ray crystal structures of Human Monoamine Oxidase B (Binda et al. 2001) (PDB ID: 1GOS, Resolution: 3.00 Å), Dihydrofolate reductase (PDB ID: 2W9G, Resolution: 1.95 Å) (Souter and Miller 2009; Heaslet et al. 2009) and enzyme-7,8-dihydropteroate synthase (DHPS) (PDB ID: 1EYE, Resolution: 1.70 Å) were retrieved from the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank (PDB). The methanol extracts of D. paniculata were analyzed using GC–MS, and 14 compounds were detected. The molecular docking studies of the compounds were carried out using a Windows 10 64-bit system with a Core 2 Duo CPU and 4 GB RAM. On the basis of binding affinity, the antibacterial activity was studied against S. aureus.

Ligand preparation

The compounds of D. paniculata were sketched using Chem Draw Professional 15.1 and saved in sdf format, then imported these structures in Maestro (Maestro, Schrödinger, LLC, New York, NY, 2021). These structures were prepared using the LigPrep module of Schrodinger 2022-3 (LigPrep, Schrödinger, LLC, New York, NY, 2021). The OPLS_2005 force field was applied during ligand preparation, and LigPrep generates accurate, energy-minimized 3D molecular structures, keeping the original ionization states.

Protein preparation, receptor grid generation, and molecular docking

The proteins were prepared by using the "Protein Preparation Wizard" (Protein Preparation Wizard; Epik, Schrödinger, LLC, New York, NY, 2021; Impact, Schrödinger, LLC, New York, NY; Prime, Schrödinger, LLC, New York, NY, 2021). The enzyme human monoamine oxidase B has two chains, A and B. The residues of chain B were identical to A up to residue ILE496, but chain A has four extra residues GLY497, LEU498, THR-499, and THR-500. As chain A possesses four more residues, it was preserved throughout protein preparation and chain B was removed while chain A and the co-ligand flavin-adenine dinucleotide were kept in. The energy of protein was optimized, and finally, restraint energy was minimized using the OPLS_2005 force field. The enzyme dihydrofolate reductase (PDB ID: 2W9G) of Staphylococcus aureus, 7,8-dihydropteroate synthase (1EYE) of Mycobacterium tuberculosis, and New Delhi Metallo-β-lactamase 1 (6LIP) were prepared. The receptor grids were generated in all cases by selecting the co-ligands, and the grid dimension was 15.0 Å from the center of the co-ligand. The molecular docking of 14 compounds of D. paniculata was performed with the prepared receptor of bacterial target grids using XP Glide (Glide, Schrödinger, LLC, New York, NY, 2021). The molecular docking of all compounds was performed using Extra Precision Glide (XP) (Glide Schrödinger 2021; Sastry et al. 2013; Friesner et al. 2006, 2004; Halgren et al. 2004).

Validation of docking protocol

The validation of the docking protocol is essential for molecular docking reliability. The root means square deviation (RMSD) was used to determine the reliability of the docking protocol. To determine the RMSD, the co-ligand of the receptor was docked with the corresponding receptor grid using Extra Precision (XP) Glide docking and superimposed the best-docked pose on the original crystallographic bound conformation of the co-ligand and calculated the RMSD. The lower the value of RMSD, the higher the accuracy of the docking protocol. The allowed RMSD limit is 3.0 Å. (Hevener et al. 2009). The RMSD of co-ligands of dihydrofolate reductase (PDB ID: 2W9G) and 7,8-dihydropteroate synthase (1EYE) were previously determined, and the values were 0.8490 and 0.7462, respectively (Debnath et al. 2022).

Generalized-born surface area (MM-GBSA) binding free energy calculation

A well-liked end-point free energy method is MM-GBSA, which calculates ∆G using the implicit generalized Born (GB) solvent model (Kollman et al. 2000). It is a key parameter that provides crucial information about the ligands' affinity for the receptor. The MM-GBSA binding energy was predicted using protein–ligand complexes using Prime (Prime, Schrödinger, LLC, New York, NY, 2021).

ADME prediction

The potential compounds' ADME (absorption, distribution, metabolism, excretion, and toxicity) properties were predicted by the open-source server SwissADME (http://www.swissadme.ch/index.php). The drug-like properties of these compounds were evaluated by looking at Lipinski's Rule of Five, blood–brain barrier permeability, and central nervous system permeability property.

Antibacterial activity

The bacterial cultures used for this study were S. aureus (MTCC 96) and M. smegmatis (MTCC 06). Bacterial strains were cultured overnight at 35 °C in Luria–Bertani agar (LBA). The Agar disc-diffusion method is used (Bauer et al. 1966) to evaluate antibacterial activity. Petroleum ether fractions (PEF- 500, 250, 50, 25, 5, and 2.5 µg/ml) and Ethyl acetate fractions (EAF-300, 150, 30, 15, 3, 1.5 µg/ml) were dissolved in DMSO. Five mm filter paper discs were soaked in different dilutions, allowed to air dry at room temperature, and placed on the surface of the agar (LB Agar). Commercial antibiotic Amphicilin solution (100 µg/ml) and DMSO were used as control and kept in a BOD incubator at 35 °C for 16 h. Tests were performed in duplicate.

All plates were observed for growth inhibition zones, and these zones' diameter was measured in mm. The MIC value was recorded as the lowest essential fraction concentration to inhibit microbial growth. The results were expressed in µg/ml.

Results

The GC–MS analysis of the petroleum ether fraction of D. paniculata extract identifies 14 compounds with quantification (Table 1). Among these compounds, Neophytadiene (DP_01, 24.254%) is the most abundant, followed by 4-(Diethylaminomethyl)-2,5-dimethylphenol (DP_11, 23.031%), 9,12,15-Octadecatrienoic acid, methyl ester, (Z,Z,Z)-(DP_09, 15.715%), 1,3-butanedione, 1-(2,3-dihydro-2-oxo-1H-indol-1-yl)-(DP_06, 9.150%) others were present in lower concentration ( < 7%).

Table 1.

Chemical components of extract from D. paniculata (Forssk) I. Darbysh

RT, retention time of the compound

The validity of the docking method was determined using the RMSD calculation, and the values for the co-ligands of 2W9G were 0.8490 Å. The RMSD of co-ligand of PDB ID: 1GOS and 6LIP were 2.20 and 1.59 Å, respectively (Fig. 1). The RMSD of the co-ligand was within the acceptable range. The XP glide score of the top three hits DP_02, DP_06, and DP_14 were − 7.993, − 7.265, and − 7.021 kcal/mol, respectively, with Human Monoamine Oxidase B. The MMGBSA energy of the top three hits, DP_02, DP_06, and DP_14, were − 39.228, − 48.55, and − 36.82 kcal/mol, respectively. The binding energy of protein ligand complexes with amino acids are displayed in Table 2. The 2D and 3D ligand interaction of best hits DP_02, DP_06, and DP_14 with S. aureus, are shown in Fig. 2.

Fig. 1.

Superposition of the docked co-ligands (sky colored) on its crystallographic bound conformation of A Coligand of human monoamine oxidase B: Flavin-adenine dinucleotide and B Coligand of New Delhi metallo-β-lactamase 1 (NDM-1): (2R)-1-[3-sulfanyl-2-(sulfanylmethyl)propanoyl]pyrrolidine-2-carboxylic acid

Table 2.

The table exhibits important phytoconstituents of D. paniculata with their XP docking score, MMGBSA dG binding energy with Dihydrofolate reductase (PDB ID: 2W9G) and interactive amino acid residues

Compounds	Receptor	XPGS	MMGBSA dGbind	2D ligand interaction
DP_02	Dihydrofolate reductase	− 6.283	− 46.736	Π-π interaction with PHE-92
				Hydrophobic: LEU-5, VAL-6, ALA-7
				ILE-14, LEU-28, VAL-31, PHE-98
DP_06	Dihydrofolate reductase	− 7.705	− 42.366	Hydrogen Bond Acceptor: ALA-7
				Hydrophobic: LEU-5, VAL-6, ILE-14
				LEU-20, PRO-21, LEU-28, VAL-31
DP_14	Dihydrofolate reductase	− 6.364	− 35.734	Hydrogen Bond Acceptor: ALA-7
				Hydrophobic: VAL-6, ILE-14, LEU-20, PHE-92

Fig. 2.

2D, and 3D ligand interaction, of best hits DP_02, DP_06 and DP_14 with S. aureus

The XP docking score of the top three hits DP_02, DP_06, and DP_14 were − 6.283, − 7.705, and − 6.364 kcal/mol, respectively, with dihydrofolate reductase. The MMGBSA binding energy of hits DP_02, DP_06, and DP_14 were − 46.736, − 42.366, and − 35.734 kcal/mol, respectively. The highest docking score for enzyme 7,8-dihydropteroate synthase (DHPS) (PDB ID: 1EYE) of M. tuberculosis and New Delhi Metallo-β-lactamase 1(PDB ID: 6LIP) was − 3.822 (for compound DP_11) and − 5.752 (DP_02) kcal/mole, respectively. The XP glide score of all phytoconstituents with different target proteins is summarized in Table 3. The binding affinity and MMGBSA dG binding energy of compounds of D. paniculata for dihydrofolate reductase were high. Therefore antibacterial activities of the petroleum ether extract containing the potential compounds were evaluated against S. aureus.

Table 3.

XP Glide score of phytoconstituents with different target proteins

Compound	XP glide score (kcal/mol)
	2W9G	1EYE	1GOS	6LIP
DP_01	− 4.318	− 0.767	− 6.358	ND
DP_02	− 6.283	− 2.505	− 7.993	− 5.752
DP_03	− 6.358	− 3.104	− 5.502	− 4.668
DP_04	− 1.886	ND	− 2.975	ND
DP_05	− 5.341	− 2.743	− 4.732	− 2.853
DP_06	− 7.705	− 3.357	− 7.265	− 5.619
DP_07	− 3.385	− 0.949	− 5.262	− 5.004
DP_08	ND	− 3.400	− 4.154	− 4.976
DP_09	− 4.264	− 3.224	− 5.438	− 3.370
DP_10	− 4.044	− 1.803	− 3.407	− 3.996
DP_11	− 5.218	− 3.822	− 6.195	− 4.780
DP_12	ND	ND	ND	ND
DP_13	− 4.610	− 2.439	− 1.678	− 4.502
DP_14	− 6.364	− 3.188	− 7.021	− 5.382

ND, not detected

Several physicochemical parameters, such as the number of rotatable bonds, hydrogen bond donors (HBD), acceptors (HBA), molecular weight, and logP value, influence the drug-like properties of molecules, i.e., Lipinski's Rule of Five (Lipinski 2016). The HBD and HBA must be in the range of 5 and 10, respectively, for an excellent drug-like molecule. The molecular weight must be less than 500 Da, and the logP value must be under 5. All the compounds in the current study satisfied the criteria for drug-like properties (Table 4, Fig. 3). All the potential inhibitors have the required drug-like properties and positive ADME characteristics according to the findings of the DP_02, DP_06, and DP_14 physicochemical and pharmacological ADME criteria. The results of physicochemical and pharmacological ADME properties of DP_02, DP_06, and DP_14 indicated that all the compounds have suitable drug-likeness properties and good ADME values.

Table 4.

Physiochemical parameters of selected inhibitors predicted by SwissADME

Parameters	DP_02	DP_06	DP_14
MW	206.28	217.22	205.25
NHA	15	16	15
NAHA	6	6	6
NRB	4	3	2
NHBA	2	3	2
NHBD	1	0	1
MR	62.18	61.46	61.72
TPSA (Å2)	37.30	54.45	38.33
iLOGp	2.17	1.77	2.02
Log S (ESOL)	− 3.36	− 1.64	− 2.94
MLOGP	3.13	1.24	1.99
GI	High	High	High
BBBP	Yes	Yes	Yes
vLROF	0	0	0
vGR	0	0	0
vVR	0	0	0
BS	0.85	0.55	0.55
SA	1.92	1.77	2.73

MW, Molecular weight; NHA, Num. heavy atoms; NAHA, Num. arom. heavy atoms; NRB, Num. rotatable bonds; NHBA, Num. H-bond acceptors; NHBA, Num. H-bond donors; MR, Molar Refractivity; TPSA, Topological Polar Surface Area; Log S, Solubility class; SC, Solubility class, 1.34e-02 mg/ml; 3.27e-05 mol/l Class; GI, Gastrointestinal absorption; BBBP, Blood Brain Barrier Penetration; vLROF, Violation of Lipinski’s rule of five; vGR, Violation of Ghose rule; vVR, Violation of Veber rule; BS, Bioavailability Score; SA, Synthetic accessibility

Fig. 3.

The bioavailability radar plot of selected inhibitors DP_02, DP_06, and DP_14 are depicted schematically (INSTU: unsaturation; INSOLU: Insolubility, LIPO: Hydrophobicity, FLEXI: Rotatable bonds, SIZE: Molecular weight; POLAR: Polar surface area)

The antibacterial activity of the petroleum ether fraction of methanol extract of D. paniculata against S. aureus revealed the minimum inhibitory concentration (MIC) value of 2.5 µg/ml (Fig. 4). In the case of M. smegmatis, only the ethylacetate fraction shows inhibition at 300 µg/ml. Keeping the main skeleton of the best 2W9G inhibitors DP_02 and DP_06, four compounds were designed, DP_01, DP_02, DP_03, and DP_04, with XP glide scores greater than DP_02 and DP_06. The XP glide score of DP_02 and DP_06 with 2W9G were − 6.283 and − 7.705 kcal/mol, respectively. On the other hand, the XP glide scores of DP_01, DP_02, DP_03, and DP_04 were − 9.412, − 9.253, − 8.091, and − 8.067, respectively. The details of 2D interactions and XP glide score of designed hits are depicted in Fig. 5.

Fig. 4.

Antibacterial activity of petroleum ether fraction of methanol extract on S. aureus (A, B) and Growth inhibition Plot at different concentrations of petroleum ether fraction (C)

Fig. 5.

2D ligand interaction diagram of designed hits DES_01, DES_02, DES_03 and DES_04 against 2W9G

Discussion

D. paniculata family (Acanthaceae) plays a significant role in the traditional treatment and the discovery of novel bioactive molecules for the treatment of many lethal diseases. Many members of this family harbor numerous secondary metabolites with multiple therapeutic uses. Previous workers (Renju Krishna and Drisya 2018) reported the presence of alkaloids, glycosides, saponin, phytosterol, tannin, and flavonoid groups of compounds from D. paniculata In the present study, fourteen active compounds were identified through GC–MS analysis from the petroleum ether fraction of the methanol extract of D. paniculata. Based on molecular docking results, antibacterial studies carried out with the petroleum ether fraction showed potential inhibition against S. aureus at a minimum concentration of 2.5 µg/ml, while no inhibition against M. smegmatis. This finding is interestingly supported by the traditional treatment of cutting/wounds using leaf paste, which acts as an antibiotic agent (Debnath et al. 2016). This study also corroborates with the previous findings of Akbar et al. 2021, Mbopi et al. 2021, Prasad et al. 2023. Akbar et al. (2021) showed that the insilico study of Vanillic acid from D. bupleuroides has good affinity (− 24.26 kcal/mol) towards DNA gyrase of Streptococcus pneumoniae. Prasad et al. 2023 reported sixty-nine compounds from the essential oil of Dicliptera roxburghiana analyzed by GC-FID and GC–MS and this essential oil is highly potent against some gram-positive and gram-negative bacteria. Mbopi et al. 2021 stated that the EtOH extract of D. verticillate showed bactericidal effect against S. aureus, Klebsiella pneumonia and E. coli. This result clearly indicates that the genus Dicliptera and specially D. paniculata is significantly potent against bacteria. The present study shows that extraction with organic solvent has better activity. While there are no previous records of the analysis of phytoconstituents of D. paniculata, this finding will be a new contribution to the researcher working in the field of pharmaceutical science and Ayurveda. The XP glide score of four newly designed molecules DES_01, DES_02, DES_03, and DES_04 based on the main skeleton of the best 2W9G inhibitors DP_02 and DP_06 were significantly higher than the DP_02 and DP_06. Using this technique, more potential inhibitors can be identified in the future.

Conclusion

Finding novel antibacterials from natural sources is a useful option in the era of drug resistance, and natural compounds showed little side effects as compared to synthetic compounds. In conclusion, this study highlights the challenges of antibiotics and the evaluation of in-silico-guided antibacterial activity against S. aureus. The petroleum ether extract of D. paniculata can be considered an effective healing agent for bacterial-borne diseases. The extract contains compounds DP_02 and DP_06, which showed potential binding affinity against dihydrofolate reductase of S. aureus. Both the compounds are drug-like and could be developed as selective drugs against S. aureus.

Author contributions

AS: Extraction, GC–MS analysis, the study of antibacterial activity, Resource collection, Methodology Validation; SD: Docking, and Editing; BDC: Extraction, Resource collection, Formal analysis; RG: Schrödinger software support; BD: Conceptualization, Writing the original draft, Final editing of the manuscript.

Funding

This research work is done under the Project support of the Department of Biotechnology, New Delhi, Govt. of India (Project No. BT/PR16867/NER/95/327/2015 Dated: 13/01/2017).

Declarations

Conflict of interest

The authors declare no conflict of interest.