Multi-target Inhibitory Potency of Active Metabolites Dictates the Antimicrobial Activity of Indigenous Medicinal Plant Leucas biflora: GC-MS Analysis, Biological Evaluations, and Molecular Docking Studies

ABSTRACT The metabolites present in the crude methanolic extract of Leucas biflora were identified and characterized to obtain new leads for compounds with antibacterial effects. The plant extract showed antibacterial effects against both the gram-positive and gram-negative bacteria in disc-diffusion assay. Gas chromatography-mass spectrometry (GC-MS) results revealed nine types of high and low molecular weight chemical entities with varying quantities in the extract. An in silico target-fishing approach identified two compounds with multi-target-directed activities. Two metabolites, 3-Oxo-18-Nor-ent-ros-4-ene-15.alpha.,16-acetonide and 4-Dehydroxy-N-(4,5-methylenedioxy-2-nitrobenzylidene)-tyramine, inhibited two promising bacterial targets, FabH and DNA gyrase B.


Introduction
Around 25% of marketed pharmaceutical products are of indigenous origin and represent a 2000 billion dollar share in the global market. A bioprospecting approach is highly crucial in identifying novel leads with medicinal properties from natural resources. [1] Two of the plants of the genus Leucas (Lamiaceae) have been used as medicinal plants to treat many diseases. The genus Leucas comprises approximately 80 species [2] and its highest species diversity is observed in East Africa. [3] Forty-three species are available in India. [4] Mature leaves of L. biflora with Centella asiatica (whole plant) are taken in 1:2 proportion, cut into pieces and ground to extract the juice and used to treat nasal bleeding. The mixture of five leaves of L. biflora and one leaf of Piper beetle helps women suffering from white discharge. [5] L. aspera extracts elicited antioxidant and anticancer activity in HeLa cell lines. [6] Leucas species are rich in flavonoids, lignans, coumarins, steroids, terpenes, fatty acids, and long aliphatic chain compounds. These metabolites exhibit a wide range of biological properties as antioxidant, antidiabetic, hypoglycemic, antimicrobial, anticarcinogenic, antimalarial, anti-inflammatory, anticholinergic, and antileprosy. [7] Flavonoids and phenolic acids are secondary metabolites found in plants [8] that play an important role in protecting biological systems against harmful effects of oxidative stress. [9][10][11][12][13] The genus Leucas is nontoxic when used in mice at therapeutic doses. [14] Hence, the active metabolites of L. biflora plant extracts may be nontoxic and possess diverse medicinal properties.
Gas chromatography-mass spectrometry (GC-MS) is a system that unites the characteristics of gas-liquid chromatography and mass spectrometry to determine the different substances present in a given test sample. [15,16] GC-MS was used to identify the phytoconstituents present in the methanolic extracts of L. biflora. The antimicrobial potency of the methanolic extract of L. biflora was evaluated using the disc-diffusion assay assessing the antimicrobial efficiency. [17] Molecular modeling studies were further carried out to provide mechanistic insights into the possible mode of action of the active metabolites identified from the plant extract.

Plant Collection
The whole plant of L. biflora was collected from Bidhan Chandra Krishi Vishwavidyalaya, Nadia, West Bengal, India, identified at the Central National Herbarium, Botanical Survey of India, Howrah, India, and a specimen deposited (AU/AC-01).

Preparation of plant sample and extraction
Plant sample preparation and extraction was done as described with modifications. [18] The whole plant was dried at room temperature for 7 d, ground to get a powder form. Then, 10 g of the powder was dissolved in 100 mL of 80% methanol and kept at room temperature for 7 d. The extract was then filtered using Whatman No. 1 filter paper to produce a clear filtrate.

GC-MS analysis
GC-MS analysis of the extracts was carried out as described with some modifications [19] using the Thermo Scientific™ ISQ™ 7000 Single Quadrupole GC-MS system equipped with DB5 capillary column (0.25 mm thickness and 30 m in length). About 0.1 mL of concentrated extract was diluted to 1 mL by adding the solvent (methanol) and transferred to standard GC-MS sample tubes. The ion source was 230, and the start time (solvent delay) = 2 min (solvent cutting). The temperature was maintained at 40°C for 4 min and gradually increased to 280°C at 20°C min −1 . The total run time was 30 min. The GC and trans-line section temperature was higher (280°C) than the MS section (250°C) to ensure the complete shifting of ions to the MS section. Helium was used as the carrier gas with a constant flow of 1 mL min −1 . The electron impact ionization was 70 eV. The compounds were evaluated using total ion count (TIC) for constituent identification after comparison with the database of the known component available in the computer library attached to the GC-MS instrument. NIST mass spectral search program software (Version 2.0) was used for metabolite identification.
Lipinski's "Rule of 5" predicts the drug likeness of a biologically active compound designed for oral route of administration. It stated that an ideal drug molecule will comply with the physicochemical properties like having a molecular weight (MW) of <500 g mol −1 , hydrophobicity of <5, <5 hydrogen bond donors (HBDs), <10 hydrogen bond acceptor (HBA) sites. [20,21] After obtaining the phytochemical profile, an investigation of the biologically active compounds and their compliance with the Lipinski's rule of 5 following the data available in PubChem database was done.

Antimicrobial test (Disc diffusion assay)
The anti-microbial assay was performed as described with modifications. [22] Glassware and Muller-Hinton agar were autoclaved, and culture plates were prepared and solidified. Bacterial cultures of Escherichia coli (Gram-negative) and Bacillus subtilis (Gram-positive) were swabbed on two different plates. Methanolic extract of L. biflora was diluted to three concentrations: 1% weight/volume (w/v), 0.1% w/v and 0.01% w/v. Four sterile discs were taken. One was dipped in methanol, another dipped in pure methanolic plant extract (undiluted), and the rest of the two discs were dipped in two dilutions 1% w/v and 0.1% w/v of the extract and all the discs were placed on the plate denoted with different quadrants. The dilution 0.01% w/v showed no zone of inhibition in the initial phase of the experiment for both the microorganisms considered, thereby making 0.1% (w/v) to be the minimum inhibitory concentration (MIC) of the plant extract. The plates were then incubated at 37°C for 24 h.
All the ligand structures were prepared for docking. Nonpolar hydrogens were merged, and then charges were computed. Rotatable bonds were defined. The macromolecular sites were also prepared for docking. Similarly, nonpolar hydrogens were merged, and atomic charges were calculated. Finally, docking of each compound to the binding regions of both the proteins was carried out using AutoDock Vin [30] implemented in the PyRx virtual screening tool.

Results
GC-MS analysis of L. biflora methanol extract fraction contained nine major bioactive compounds (Table 1). From the typical chromatogram of crude methanolic extract of L. biflora ( Figure. 1), all the active metabolites were identified. GC-MS spectrum of each of the active metabolites was documented (Fig. S1-S9).
The zone of inhibition of extracts with different dilutions was measured in the plates cultured with E. coli and B. subtilis, separately (Figures 2 and 3). The MIC for E. Coli and B. Subtilis was 0.1 (%W/V) and the zone inhibition for the undiluted methanolic extracts was 9 mm and 7 mm for E. coli and B. subtilis, respectively (Table 2) The molecular basis behind the observed antibacterial activities of the extracts was further evaluated. Knowledgebase searching identified two potential antibacterial targets in E. coli: β-Ketoacyl acyl carrier protein synthase III (FabH) and DNA gyrase B. 3-D structures of E. coli FabH bound with the ligand, malonyl-coenzyme A was considered for docking. The entire ligandbinding site was considered in the molecular docking. The inhibitor bound structure of DNA gyrase was considered as the docking site in virtual screening The phytochemicals and their abidance by the Lipinski's rule of 5 were listed (Table 1). Among the nine metabolites, six metabolites were considered with a defined pharmacophoric feature: 2-Methoxy-4-vinylphenyl acetate, Eugenol, Acetylisoeugenol, 4-Dehydroxy-N-(4,5-methylenedioxy-2-nitrobenzylidene)tyramine, 3-Oxo-18-Nor-ent-ros-4-ene-15.alpha.,16-acetonide, and Alpha-Tocopherol (Table 3).

Discussions
The GC-MS analysis is widely used for the identification of the presence of biologically molecules in medicinal plants in order to draw a correlation among the biochemical compounds and describes the nature of the active compounds present that are presumed to cure certain ailments. [31,32] Molecular docking is performed extensively by the computational biologists to discover targeted drug therapies that will be useful in designing novel drugs from indigenous sources. [33,34] In the present study, the identified major metabolites through GC-MS are 2,3-Dimethylpentane, Methylcyclopentane, 2-Methyl-1-Pentene, 2-Methoxy-4-vinylphenyl acetate, and Eugenol. On the other hand, Acetylisoeugenol, 4-Dehydroxy-N-(4,5-methylenedioxy-2-nitrobenzylidene)tyramine, 3-Oxo-18-Nor-ent-ros-4-ene-15.alpha.,16-acetonide, Alpha-Tocopherol were present in relatively small amounts. Among these nine  bioactive compounds, three of them have been reported to play a crucial role in disease and metabolism in humans. Eugenol has anti-inflammatory, neuroprotective, antipyretic, antioxidant, antifungal, and analgesic (dental) properties. [35] Acetyl isoeugenol is widely used in food industries as a flavoring agent or food additive. Alpha-tocopherol, known for its antioxidant activities, is protective against cardiovascular disease and cancer. [36] The health-beneficial roles of the remaining active metabolites are yet to be reported. The methanolic extracts of L. biflora showed antibacterial activity against both the Gram-positive   (B. subtilis) and Gram-negative bacteria (E. coli), as evident from the disc diffusion assay results. Upon 10-time dilution, the extract still showed biological activities. β-Ketoacyl acyl carrier protein synthase III (FabH) [23 , 24 , 28] and DNA gyrase B [25][26][27] are considered to be potential anti-bacterial targets in E. coli. The former is essential for bacterial viability due to its catalyzing property in the fatty acid biosynthetic pathway. Hence, the ligand bound E. coli FabH targets were thought to be ideal for docking analysis. The other potential antibacterial target DNA gyrase is known to be involved in negative supercoiling of DNA. Thus, the protein is considered a promising target for antibacterial drug design. Recently, the inhibitor, 3-[[8-(methylamino)-2-oxidanylidene-1~{H}-quinolin-3-yl]carbonylamino]benzoic acid, bound structure of DNA Gyrase has been resolved using crystallography. Therefore, the inhibitor bound region was found to be appropriate as the docking site. Two metabolites, 3-Oxo-18-Nor-ent-ros-4-ene-15.alpha.,16-acetonide and 4-Dehydroxy-N-(4,5-methylenedioxy-2-nitrobenzylidene)-tyramine, showed high affinity with both the receptors. Thus, both the ligands are considered as multi-target directed ligands (MTDLs). While α-Tocopherol also shows higher binding affinity with the FabH, the remaining metabolites were low-affinity binders. Detailed interaction analysis of the two MTDL metabolites with both the FabH and DNA gyrase B ( Figure. 4) showed that both the metabolites bind close to the cognate inhibitors, as observed in the crystal structures (blue ligand in Figure 4). The 3-Oxo-18-Nor-ent-ros-4-ene-15.alpha.,16-acetonide and 4-Dehydroxy-N-(4,5-methylenedioxy-2-nitrobenzylidene)-tyramine bind to the active site crevices. The former formed a single hydrogen bond with the ASN210, while the latter formed two hydrogen bonds with the ASN247 and ASN274 of FabH ( Figure. 4A). On the other hand, both the ligands showed different modes of interactions with DNA gyrase B (Figure. 4B). 3-Oxo-18-Norent-ros-4-ene-15.alpha.,16-acetonide strongly fits within the binding site driven by surface complementarity without any hydrogen bonding contacts with the receptor. 4-Dehydroxy-N-(4,5-methylenedioxy-2-nitrobenzylidene)-tyramine binds to the receptor primarily driven by hydrogen bonds and van der Waals interactions. The ligand forms two hydrogen bonds with GLY77 and THR165 of DNA gyrase B.