Abstract

Background: The anti-inflammatory properties of Arak (Miswak, Salvadora persica) and its extracts have been firmly established through both in vitro and in silico studies. Nitric oxide (NO) plays a critical role as a signaling molecule in the pathogenesis of inflammation, which is a fundamental process in the development of various diseases, particularly carcinogenesis and the malignant transformation of cells. Herbal-derived compounds have demonstrated promising potential in inhibiting inflammatory diseases. This study aimed to investigate the inhibitory effects of Arak extract and its bioactive compounds on inducible nitric oxide synthase (iNOS) and NO production using vitro and computational methods and to identify potential phytocompounds with high binding affinity to iNOS.
Methods: An in vitro evaluation was performed using LPS-stimulated RAW macrophage cell lines to assess the inhibitory effect of Arak extract on NO production. Additionally, advanced in silico techniques, including all-atom molecular dynamic (MD) simulations, were used to model the interaction between six phytocompounds and iNOS, identify binding sites, and assess the stability and conformational shifts of the ligand-protein complexes.
Results: The in vitro analysis revealed strong inhibition of NO production by Arak extract. Computational studies confirmed that six bioactive compounds from the extract exhibited high binding affinity to iNOS, inducing conformational changes that enhanced ligand positioning within the active site. Among these, the compound 1/iNOS complex showed stable ligand interactions over the simulation period, suggesting a robust inhibitory effect.
Conclusions: This study highlights six promising phytocompounds from Arak extract as potent iNOS inhibitors. The results demonstrate the therapeutic potential of Arak in treating inflammation-related diseases. Further in vivo validation is warranted to confirm the clinical efficacy of these findings.

DOI: 10.7754/Clin.Lab.2025.250505