Flavonoids from Uvaria alba attenuate LPS-induced inflammatory responses by suppressing NF-κB activation in RAW 264.7 macrophages: In silico and in vitro perspectives

Hyperreactive inflammatory response occurs when there is excessive activation of NF-κB leading to a pathologic cascade of inflammatory reactions. Thus, we investigated the inflammatory modulating potentials of the Philippine medicinal endemic plant Uvaria alba. ELISA was initially performed to measure levels of proinflammatory mediators (NO and PGE 2 ) and cytokines (TNF-α, IL-1β and IL-6), followed by RT-PCR and western blotting to determine mRNA and protein expression, respectively. Using immunofluorescence staining combined with western blot analysis, the activation of NF-κB was further investigated. Putative flavonoids from the bioactive butanol subextract (UaB) were identified using high resolution LC-MS and subjected to in silico screening against COX-1/2, iNOS, TNF-α, TACE, and IKK via molecular docking and molecular dynamics simulations at 140 ns. UaB abrogated protein and mRNA expressions of iNOS, COX-2, TNF-α, IL-1β and IL-6 by suppressing the production of proinflammatory mediators and cytokines. Furthermore, UaB attenuated NF-κB activation by inhibiting the nuclear translocation of the transcription factor p65. LC-MS analysis with UaB revealed the presence of flavonol aglycones — quercetin (4) and kaempferol (6) — and their glycosylated derivatives — quercitrin (3), rutin (5), and kaempferol 3-O-rutinoside (7). Molecular docking analysis suggests that the major flavonol aglycones exhibited high affinity towards COX-2 NSAID-binding site, TNF-α and TACE, while the glycosylated flavonoids showed high affinity towards iNOS and IKK. The top protein-ligand complexes were found to be dynamically stable as shown by molecular dynamics simulations. This is the first report highlighting the mechanistic in silico and in vitro anti-inflammatory potential of U. alba.