Abstract: Acanthus ilicifolius is lesser-known medicinal plant is used as medicine in traditional systems. Staphylococcus aureus is inducing many infections and it can infect tissues when the skin and mucosal barriers. The plant Acanthus ilicifolius GC-MS have reported the following phytochemicals, 26.27-Di (nor)-cholest-5,7,23-trien-22-ol, 3-methoxymethoxy, 9H-purin-6-amine, N, 9-bis (trimethylsilyl)-8-((trimethylsilyl) Oxy), Cyano colchicines and 3beta-methoxy-5-choleston-19-oic acid against a 12 essential protein and synthetic drug of S. aureus was collected through literature survey. S. aureus was studied zone of inhibition in different concentrations against microorganisms and essential proteins were used as target which were docked using phytochemicals (ligands) found in A. ilicifolius leaves. In the present study, to analyze the effect of A. ilicifolius against S. aureus through docking and inhibition studies, that A. ilicifolius has significant activity against S. aureus. This research was helpful attempt to the drug discovery in S. aureus.
INTRODUCTION
Acanthus ilicifolius Linn. (Acanthaceae), locally known as ‘sea holly’ is a spiny shrub found in the low lying coastal areas of southern districts in Bangladesh and the vast area of mangrove forest, the Sunderbans and it is also widely distributed. It is a viny shrub or tall herb and bushy with very dense growth (Lakshmi et al., 1997). Many phytochemicals are present in A. ilicifolius which is responsible for antimicrobial, chemo preventive, hepatoprotective, anti-osteoporotic activity and anti-inflammatory activities (Babu et al., 2001; Agshikar et al., 1979). Long back, the leaf juice of A. ilicifolius was commonly used both internally and externally for treating rheumatism, wounds, neuropathy and snake bites. The seeds were ground and their paste was administered for boils. In Ayurvedic medical systems, A. ilicifolius is as an astringent, nervine tonic, stimulant and expectorant.
Earlier GC-MS studies was done in our laboratory shown that the following major components present were in the A. ilicifolius leaves-26,27-di (nor)-cholest-5,7,23-trien-22-ol, 3-methoxymethoxy, 9H-purin-6-amine, N, 9-bis (trimethylsilyl)-8-((trimethylsilyl) Oxy), Cyano colchicines and 3beta-methoxy-5-cholesten-19-oic acid (Bala et al., 2011).
S. aureus has increased the risk of the infection development, both nosocomial and community-acquired (DeLeo et al., 2010; Rafee et al., 2012; Wang et al., 2010). In 1960 methicillin-resistant S. aureus. MRSA, has become one of the disease-producing agent for healthcare associated infections and it's become very difficult to treat (Bigos and Denys, 2008).
The present study aims to analyze the phytochemicals by GC-MS and evaluate the antimicrobial activity of most relevant compounds identified and quantified in A. ilicifolius. Furthermore, a Structure-activity Relationship (SAR) analysis and molecular docking studies against S. aureus were performed, in order to provide the mechanism of potential antimicrobial drugs for resistant microorganisms. Molecular docking is an in silico tool that predicts how a ligand interacts with a receptor and has been successfully applied in several therapeutic programme at the lead drug discovery (Ghosh et al., 2006).
MATERIALS AND METHODS
Antibacterial activity
Zone of inhibition: Acanthus ilicifolius were botanically identified by the Botanical survey of India, Tamil Nadu Agriculture University, Coimbatore. A voucher specimen of the plant has been deposited at the botanical survey of India herbarium (Voucher number-53582). Fresh leaves of A. ilicifolius were cleaned with deionized water and dried in the shade. The dried leaves were ground and sieved. Soxhlet extrtaction was carried out for the Acanthus ilicifolius leaf samples with solvent methanol (Okunade et al., 2007).
Microorganism was grown overnight at 37°C in nutrient broth (Perez et al., 1990). The antimicrobial activity of the crude leaf extract of A. ilicifolius was studied in different concentrations against bacterial strain, the antimicrobial potential of leaf extract was assessed in terms of zone of inhibition of bacterial growth.
Antibacterial activity
In silico analysis: The list of 12 genes involved in S. aureus infection pathway in human was collected from literature (Bartlett and Hulten, 2010). These were found to be essential for the survival of the organism as reported in the DEG database (www.deg.Org). They are Alpha hemolysin, Panton Valentine leukocidin, Enterotoxin, Toxic shock syndrome toxin-1, Staphylokinase, Aureolysin, Clumping factor A, Clumping factor B, Sec A protein translocase, Serine-aspartate repeat-containing protein C, 30S ribosomal protein S4, Serine-aspartate repeat-containing protein D (Table 1).
| Table 1: | Genes and proteins of S. aureus responsible for infection |
These proteins were taken as targets for docking with phytochemicals identified in A. ilicifolius. The structure of all target proteins for Staphylococcus aureus was retrieved from PDB (Protein Data Bank). The binding site of all target proteins was found using the Q site finder for grid generation in Schrodinger. Identifying the ligand binding sites on a protein is the functional importance of many applications including molecular docking, de novo drug design and structural identification and comparison of functional sites.
ISIS Draw 2.3 software was used to design the phytochemicals. Analogues were changed in MOL files and 3D optimization was done by ChemSketch 3D viewer of ACDLABS 8.0. The MOL files were converted into PDB format using VEG ZZ software. The docking studies have been used with Schrodinger Glide program version 4.0. The best 10 poses and corresponding scores have been evaluated using Glide in single precision mode (GlideSP) for each ligand. For each screened ligand, the pose with the lowest Glide SP score has been taken as the input for the Glide calculation in extra precision mode (Glide XP). The docking was carried out with Glide SP and Glide XP.
RESULTS AND DISCUSSION
A. ilicifolius methanolic extracts has shown significantly higher when compared to methanolic control (p>0.05). In order to verify the results, the experiment was triplicated and Mean±SD was reported (Table 2). The phytochemical reported in A. ilicifolius were considered as ligands for docking. These essential proteins in S. aureus were used as target proteins which were docked using phytochemicals (ligands) found in Acanthus ilicifolius leaves using Schrodinger software. (Table 3).
Although the phytochemicals exhibited bonding with proteins present in S. aureus, their interaction was found to be less when compared to common drug Penicillin G. So it is suggested that although the phytochemicals present in Acanthus ilicifolius leaves has individual insignificant activity against S. aureus (p>0.05), there may be a synergy effect of phytochemicals together in inhibiting the growth of S. aureus as seen in the zone of inhibition. This research was an attempt to take a one step towards the drug discovery for Staphylococcus aureus.
| Table 2: | Antibacterial activity of A. ilicifolius |
| Table 3: | Docking of phytochemicals present in the leaves of A. ilicifoilus with S. aureus target proteins |