Synthesis, In-Vitro and In-Silico Evaluation of Silver Nanoparticles with Root Extract of Withania somnifera for Antibacterial Activity via Binding of Penicillin-Binding Protein-4

Background: Metal Nanoparticles (NPs) have been widely used for various applications in biomedical sciences, including in drug delivery, and as therapeutic agents, but limited owing to their toxicity towards the healthy tissue. This warrants an alternative method, which can achieve the desired activity with much reduced or no toxicity. Being a biological product, Withania somnifera (W. somnifera) is environment friendly, besides being less toxic as compared to metal-based NPs. However, the exact mechanism of action of W. somnifera for its antibacterial activities has not been studied so far.

Objective: To develop "silver nanoparticles with root extract of W. somnifera (AgNPs-REWS)" for antimicrobial and anticancer activities. Furthermore, the analysis of their mechanism of action will be studied.

Methods: Using the in-silico approach, the molecular docking study was performed to evaluate the possible antibacterial mechanism of W. somnifera phytochemicals such as Anaferine, Somniferine, Stigmasterol, Withaferin A, Withanolide- A, G, M, and Withanone by the inhibition of Penicillin- Binding Protein 4 (PBP4). Next, we utilized a bottom-up approach for the green synthesis of AgNPs- REWS, performed an in-detail phytochemical analysis, confirmed the AgNPs-REWS by SEM, UVvisible spectroscopy, XRD, FT-IR, and HPLC. Eventually, we examined their antibacterial activity.

Results: The result of molecular docking suggests that WS phytochemicals (Somniferine, Withaferin A, Withanolide A, Withanolide G, Withanolide M, and Withanone) possess the higher binding affinity toward the active site of PBP4 as compared to the Ampicillin (-6.39 kcal/mol) reference molecule. These phytochemicals predicted as potent inhibitors of PBP4. Next, as a proof-of-concept, AgNPs- REWS showed significant antibacterial effect as compared to crude, and control; against Xanthomonas and Ralstonia species.

Conclusion: The in-silico and molecular docking analysis showed that active constituents of W. somnifera such as Somniferine, Withaferin A, Withanolide A, Withanolide G, Withanolide M, and Withanone possess inhibition potential for PBP4 and are responsible for the anti-bacterial property of W. somnifera extract. This study also establishes that AgNPs via the green synthesis with REWS showed enhanced antibacterial activity towards pathogenic bacteria.