Unravelling the antibacterial potential of hydroethanolic bark extract from Melia azedarach: In vitro and in silico investigation

Introduction

Antimicrobial resistance is one of the predominant global concerns, leading to the emergence of innumerable resistant bacterial species (Prestinaci et al., 2015) [23] . The main factor leading to the evolution of resistance bacteria is the indiscriminate use of antibiotics.

In this category of alternatives to antibiotics and sequential reduction in antibiotic use, new hope is generated from herbal medicine. Which has a very long history of treating many infectious and non-infectious diseases, a time when people have not heard the name of antibiotics. Hence, herbs or plants are hoped that provide a possible replacement for the antibiotics (Xie et al., 218) [38] . Under the category of traditional medicinal plants, Melia azedarach well well-known and popularly named as Chinaberry tree or Mahanimba in India. This plant is known for its multiple pharmacological activities. It possesses various properties anthelmintic, antilithic, diuretic, deobstruent, antimalarial, and antifungal (Maciel et al., 2006; Khan et al., 2011) [14,10] . Potential analgesic and antipyretic activities of M. azedarach L. leaf and M. root powder have been evaluated. Mild-to-moderate peripheral and central analgesic activity with antipyretic effect was reported (Vekariya et al., 2016) [36] . Seed extracts of M. azedarach were found to be effective in controlling grampositive and gram-negative human pathogenic infection agents under hospital conditions (Khan et al., 2011) [10] , while M. azedarach of flowers found effective against S. aures and E. coli (Muhammad et al., 2015) [18] . M. azedarach L. leaves contain huge quantities of limonoids of the nimbolinin type, which contribute to therapeutic effects, such as antimicrobial, antioxidant, and anticancer potential (Srivastava et al., 2020) [35] . Further, ZnO NPs biosynthesized using Melia azedarach leaf extra have substantial efficient biological activities regarding antioxidant and antibacterial potential have been reported (Dhandapani et al., 2020) [5] . Moreover, the broad-spectrum medicinal value of A. indica L. and M. azedarach L. leaf extracts have also been reported and recommended for the control of COVID-19 and other human microbes (Hemdan et al., 2023) [8] . In the search for an alternative to the antimicrobials and looking towards the potential properties of Melia azedarach, it was decided to explore the more antimicrobial potential of the bark-ethanolic extract against the mastitis-causing pathogen. In silico analytical tools will provide information about different molecular interactions through possible ligand-receptor interaction studies (Rizvi et al., 2013; Nabati et al., 2020) [27,20] . Hence, the advanced in-silico tool was used to explore the affinity of bioactive constituents of Melia azedarach against different targets of selected bacteria which establish the possible mechanism of action as an antibacterial agent.

Materials and Methods

Collection and processing of plant Materials

In the month of October 2022, the bark of Melia azedarach was collected from the Herbal Garden, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab, India. Thereafter, the bark chapped into 5-6 cm sizes, washed with plenty of water, and allowed to dry for several days in an Oven Dryer at 45 O C. After completion of drying, the powder was prepared in a grinding mill.

Extraction of plant material

Powder of bark from Melia azedarach was soaked in 60% alcohol (aqua-ethanol) and kept for 72 hours in a rotatory shaker. After that, soaked content was filtered with a fourfold of muslin cloth followed by filtration using Whatman

No.1 paper. The aqua-ethanolic bark extract (AEBE) was dried in oven dryer at 45±1 O C. Prepared AEBE was stored in air tied container until used. The percent recovery was calculated by taking the difference in the weight of the empty petri dish and the weight after drying and multiplying by 100 and expressed as percent extractability.

Antibacterial Assays Bacterial strain

Bacterial strains of Staphylococcus aureus (ATCC 25921) and Escherichia coli (clinical isolates) were obtained from the Department of Veterinary Microbiology, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana. Working bacterial cultures were prepared by transferring a colony to a fresh medium and then kept overnight at 37ºC for further growth. Turbidity of the bacterial solution inside normal saline was matched with 0.5 Mc Farland standard and also measured in a spectrophotometer for the optical density at 625 nm and then was serially diluted to achieve 10 7 CFU/mL with NSS.

Estimation of MIC

A minimum inhibitory concentration (MIC) of AEBE of Melia azedarach's against E. coli and S. aureus was measured by broth microdilution method as recommended by CLSI protocol (CLSI, 2014). A 96-well plate was taken to measure MIC of extract against each bacterium (1 plate for each bacterium). Different dilution of plant extract ranging from 128 mg/ml to 0.25 mg/ml was prepared in 1% DMSO. 100 µl of Mueller Hinton broth (MHB), different concentrations of extract and 20 µl of bacteria were added simultaneously in each of the wells. Afterward it was incubated at 37 ºC for 24 hrs. MHB + DMSO was used as a vehicle control. After completion of incubation period, 20 µl MTT was added and again incubated for 3 hrs. Change in colour from yellow to purple indicated as presence of bacteria. MIC was measured as the lowest concentration which do not change the colour.

GC-MS analysis

Gas Chromatography and Mass Spectrometer (GC-MS) analysis was carried out to find the phytochemical constituents present in the AEBE. GC-MS analysis data was obtained by proving the sample to Central Instrumentation Laboratory (CIL), Central University Punjab, Bhatinda (Shimadzu QP 2010 Ultra GC MS; Shimadzu Japan).

Molecular Docking Study

Phytochemical constituents identified by GC-MS analysis were selected as ligands for docking experiment. These constituents were first screened through PyRx (Dallakyan & Olson, 2015) [4] against protein of S. aureus (PDB ID-2ZCO, 4CJN, 1JIJ) and E. coli (PDB ID-1IEM, 7NAE, 1HNJ). Compounds with good binding energy were further selected for individual docking against both the proteins for validation by AutoDock 4.2. (Morris et al., 2009) [17] .

Protein preparation.

The crystal structures of receptor molecules of S. aureus (2ZCO, 4CJN, IJIJ) and E. coli (1IEM 7NAE, 1HNJ) were obtained from PDB database. Proteins were cleaned by removing the heteroatoms. Removal of water molecules and addition of hydrogen atoms were done by AutoDock tools 1.5.7. Afterwards, proteins were converted to PDBQT format. On the basis of the literature review, the amino acids involved in the active sites were selected and the grid box was placed covering active site. The grid-center for all receptor molecules is represented in tabulated form (Table 1).

Ligand preparation

All phytochemical constituents identified by GC-MS were retrieved in SDF format from the PubChem database. Discover studio visualizer was used to prepare a library of retrieved phytochemical constituents. PDB file of all constituents were prepared by entering the smile of all phytochemical constituents in CORINA CLASSIC.

Docking study

On the basis of the literature review, the amino acids involved in the active sites were selected (Table 2) and the grid box was placed to covering active site. The gridcenter and coordinates for XYZ of all receptor molecules are represented in tabulated form (Table 3). Before performing the docking, grid map file was generated for individual protein through AutoGrid. After that, docking parameter file was generated. Docking was performed through AutoDock 4.2. software. Analysis of docking was carried out on the basis of free binding energy and inhibition constant Ki.

Visualization of protein-ligand interaction was carried out by Discovery studio visualizer and Pymol, software. Drug-likeness studies: This prediction studies was carried out by using PreADMET (http://preadmet. bmdrc.org/).

Results and discussion

Effect on percent extractability and MIC AEBE from M. azedarach showed the percent extractability under standard laboratory conditions was 6.45±0.613 (g %).

MIC of AEBE from M. azedarach against S. aureus and E. coli were found to be 1mg/ml and 8 mg/ml, respectively. The AEBE showed very good antibacterial activity against both strains of bacteria (Fig. 1 and2).

Predicted bacterial targeted-protein & drug interaction

The results of in silico analysis after the screening of compounds through PyRx (drug computational software), a compound viz. CID 535221 and CID 500095 showed very good activity against targeted proteins of E. coli (PDB ID 1IEM, 7NAE, 1HNJ) and S. aureus (PDB ID 2ZCO, 4CJN, 1JIJ). These compounds were individually docked against respective proteins for further validation by AutoDock 4.2 software. They showed specificity and binding affinity with that of the receptors under 3D modeling system (Fig. 3 &4). The binding energy and inhibition constant of both compounds against targeted proteins are represented in tabulated form for comparison (Table 5). Drug-likeness (Table 6) was performed through PreADMET software.

In general, the cross-species transfer of bacteria has been made easier by the historical growth of agriculture as well as by the current industrialization and globalization of the livestock industry. This dissemination has most likely had a significant impact on the creation of diseases. S. aureus and E. coli both are opportunistic pathogens. These organisms are responsible for causing many diseases in animals. It is already known that S. aureus and E. coli are one of the predominant causes of mastitis which led to tremendous economic loss to the farmers. Along with this, S. aureus is responsible for causing different infections encompassing superficial to deep skin infections as well as septicemia (Mala et al., 2021) [15] . Also, E. coli infection is a substantial cause of neonatal mortality in calves due to septicemia or enteric infection (Peek et al., 2018) [21] Overuse use of antibiotics against these pathogens leads to the development of antibiotic-resistant strains. So, there is an urgent need to develop alternative compounds with higher potency, lower side effects, and sustainability. Melia azedarach plant comes under the family Meliaceae. It is commonly grown in areas of sub-Himalayan region 2000 m above sea level. This plant possesses a remarkable history of being used in Ayurveda. Bark of Melia azedarach is known to be used in diarrhoea, piles, fever, loss of appetite, rheumatic pain and ulcers (Sharma and Singla, 2013).

According to the present study, the minimum inhibitory concentration of Melia azedarach hydro-ethanolic bark extract was 1 mg/ml against S. aureus and 8 mg/ml against E. coli which depicts that hydro-ethanolic bark extract possesses significant antibacterial activity against both organisms. However, according to Hemdan et al. (2023) [8] MIC value of the methanolic leaf extract of Melia azedarach plant against S. aureus and E. coli was 75 mg/ml and 50 mg/ml corresponding to each organism. Which was much higher than the present reported value of MIC. This finding revealed that hydro-ethanolic bark extract possesses much higher antibacterial activity than methanolic leaf extract. Khatoon et al. (2014) [11] reported antibacterial potential of M. azedarach L. bark against human pathogens at fixed concentration 2mg/ml using n-hexan and methanolic extracts. They reported n-hexane extract exhibited highest zone of inhibition against S. flexneri, least against K. pneumoniae and resistant to S. mitis, S. aureus, B. subtilis. While methanolic extract showed highest zone of inhibition on S. mitis, least against K. pneumoniae and moderate against S. typhi, S. aureus and B. subtilis. It has been cleared that the solvent system influences the release of bioactive constituents that contributing to pharmacological activity against the bacterial strains. Another study, methanol extract of Melia azedarach leaves showed least MIC value (22.4 μg/ml) against B. cereus, while ethanol extract showed 37.6 μg/ml against P. aeroginosa. Against S. aureus and E. coli showed comparatively efficient MIC value 38.7 μg/ml and 39.6 μg/ml in methanol and ethanol extract, respectively (Sen and Batra, 2012) [32] . When compared these finding with present study suggested that the plant parts and type of solvent or fraction has influence over biochemical constituents. Their activity varies with the type of bacterial strains. When compared with report of Khatoon et al. (2014) [11] , present study hydro-ethanolic extracts showed comparatively better activity than the n-hexan and methanolic fractions. A previous study on GC-MS analysis of the flower of Melia azedarach showed the presence of fatty acids like hexadecenoic acid methyl ester and 9-octadecenoic acid (Z) which possess antibacterial activity against various bacteria (Muhammad et al., 2015) [18] . Another study showed that the presence of 6-Isopropen yl-4,8a-dimethyl-1,2,3,5,6,7,8,8aoctahydro-naphthalen-2-ol in essential oil from Ocimum gratissimum was one of the responsible constituents for antimicrobial activity (Salvi et al., 2022) [30] . The GC MS analysis of hydroalcoholic extract of bark from M. azedarach in this study also showed presence of abovementioned constituents. So, the antibacterial activity reported in this study may be due to presence of these bioactive compounds in hydroalcoholic bark extract from M. azedarach. In vitro assay along with in silico prediction showed that extract from Melia azedarach's bark possess a significant antibacterial activity against S. aureus and E. coli. The presence of bioactive compounds like organic acids, fatty acids and terpenoid identified by GC-MS may be given credit for exhibition of antimicrobial activity in the extract. In-silico study was carried out to screen the phytochemical constituents distinguished by the GC-MS analysis against various proteins of S. aureus and E. coli such as 2ZCO, 4CJN, 2FNP and 1IEM, 1HNJ, 7NAE, respectively. 2ZCO (Carotenoid dehydrosqualene synthase) is responsible for survival of S. aureus inside the host cell through the antioxidant activity of staphyloxanthin. Penicillin binding protein 2a (4CJN) is commonly involved in the synthesis of the bacterial cell wall by performing the role of transpeptidase activity (Shalaby et al., 2020) [33] . S. aureus tyrosyl-tRNA synthetase (1JIJ) possesses a central role in protein synthesis (Qiu et al., 2001) [24][25] AmpC betalactamase of E. coli (1IEM) is responsible for causing the inactivation of beta-lactam antibiotic (Power et al., 2011) [22] . beta-Ketoacyl-acyl carrier protein synthase-III (1HNJ) possesses key role in fatty acid biosynthesis which is essential part of bacterial membrane lipid and therefore important in viability of bacteria (Qiu et al., 2001) [24][25] . dihydrofolate reductase (7NAE) is responsible for trimethoprim resistance in gram-negative bacteria (Krucinska et al., 2022) [12] . So, it would be phenomenal to target these proteins to develop a new compound that possesses good efficacy and lower side effects. According to the in-silico results, it was found that phytochemical constituents identified by GC-MS had great activity against selected proteins of S. aureus (PDB ID 2ZCO, 4CJN, 1JIJ) and E. coli (1IEM, 7NAE, 1HNJ). Here, 6-Isopropenyl-4,8a-dimethyl-1,2,3,5,6, 7,8,8a-octahyd ronaphthalene-2,3diol and Isobenzofuran-1(3H)-one, 3-(3-furyll)-3a,4,5,6tetrahydr o-3a, 7-dimethyl showed best result against 2ZCO, 4CJN, 1JIJ, 7NAE, 1HNJ and 1IEM protein. Besides the good binding energies, these two best constituents also formed the hydrogen bond and alkyl bond with the active site amino acid residue which was presented in the binding pocket of the targeted protein. The interacting amino acids in a hydrogen bond, alkyl bond, and van der Waals interaction formed by ligand with the active site of targeted proteins was noticed (Table 7 and8). This finding supports that both constituents possess good interacting capacity against selected target proteins. So, in support of the above discussion, we can say that these compounds can be further studied and may be used for the development of an alternative to the currently used antibiotics. The present study based on the findings of the GC-MS analysis, in vitro antibacterial study, and in silico studies, suggests that AEBE from M. azedarach has potential antibacterial activity against both the organisms S. aureus and E. coli. The in-silico study showed that protein and ligand possess good binding activity. There is no effective cure without side effects predicted through ADME studies. So, these phytochemical constituents can be used as an initial compound for formulation development and/or a series of derivatizations may be done for improvement of antibacterial efficacy. Further, efficacy studies should be done based on individual compounds against different microbes to establish the mechanism of action and efficacy. Furthermore, in vivo antibacterial efficacy study may be carried out for proper dose determination against S. aureus and E. coli using a suitable animal model system.

Acknowledgement

AcknowledgmentsI would like to express my heartfelt gratitude to Dr. M.K Lonare, Associate Professor, Dept. of Veterinary Pharmacology & Toxicology, Rampuraphul, for his insightful guidance and technical counsel during the whole period of research. I am also thankful to Dr. S.P.S Saini Professor, Dept. of Veterinary Pharmacology & Toxicology, GADVASU, for their support and uplifting motivation. I am grateful to Dr. Sushree, Dr. Natasha and Dr. Sivaraman for their instructive feedback and unwavering support throughout the research work.

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