Viola stocksii: A rich source of antioxidant and anti-inflammatory flavonoid glycosides with converged therapeutic potential against SARS-CoV-2 MPro, spike trimer, and surface glycoproteins

This research presents the first comprehensive investigation into the phytochemical and nutraceutical properties of Viola stocksii. The study aimed to identify the therapeutic efficacy and bioactive constituents of this medicinal herb, traditionally used as an herbal tea for managing respiratory and abdominal issues in remote areas of Pakistan. The therapeutic efficacy of the plant was assessed as an anti-oxidant-rich health tonic coupled with anti-inflammatory and immunomodulatory activities against COVID-19 and its evolving variants. The study involved sequential extraction, bio-assay guided fractionation, isolation, characterization, and biological evaluation. Preliminary in vitro studies have demonstrated that polar fractions of the plant possess significant antioxidant and antiinflammatory potential. A diverse combination of chromatographic techniques (Diaion resin HP-20, Sephadex LH-20, ODS-C18, and RP-HPLC) facilitated the purification of active constituents of the plant. Comprehensive analysis of NMR ( 1 H, 13 C, and 2D NMR) and mass (EIMS, HR-EIMS, and FAB-HRMS) spectral data lead to characterize the isolated compounds ( 1 – 4 ) as flavonoid glycosides. Results from in vitro and in-silico evaluations revealed that the isolated glycosides exhibit high antioxidant, antiinflammatory, and immunomodulatory activities, which synergistically show promising potential in combating SARS-CoV-2 variants. Besides M Pro , therapeutic potential against two unexplored proteins i.e. the spike trimer (7WZ1) and surface glycoproteins (7QTJ) was studied for the first time. Kaempferol ( KamGluαRh ) and quercetin ( QurGluαRh ) carrying rutinoside, were identified as the most active constituents of the plant, with the highest binding affinities in the range of −10 to −13 kcal/mol. Ten distinct active regions were explored on the spike trimer, including strong binding affinity with the essential RBD region, spike head, and tail of the protein. MD simulation validated the stability of M pro - KamGluαRh and M pro -QurGluαRh complexes evidenced by an average RMSD of 0.2 nm led to the mechanistic understanding of M pro inhibition. ADMET and pharmacokinetic studies authenticated the therapeutic ability and drug-likeness profiling of all extracted compounds. Results revealed the beneficial role of glycosides in boosting the medicinal effectiveness of flavonoids and the potential of Viola stocksii as an immunity booster against respiratory infections like COVID-19. • First comprehensive investigation into the potential of Viola stocksii as an immunity booster against Sars-CoV-2 variants • A systematic bioassay-guided approach, lead to isolation of active constituents using extensive chromatographic techniques. • These isolated compounds were characterized as flavonoid glycosides through advanced spectroscopic methods. • Detailed in-vitro and in-silico studies reveal significant antioxidant, anti-inflammatory, and immunomodulatory activities of the plant's constituents, which synergistically show promising potential in combating Sars-CoV-2 variants. • Kaempferol (KamGluαRh) and quercetin (QurGluαRh) carrying rutinoside, were identified as the most active constituents of the plant • Besides M Pro , therapeutic potential against two unexplored proteins i.e. the spike trimer (7WZ1) and surface glycoproteins (7QTJ) was studied for the first time. • Notably, beyond MPro, we report for the first time the therapeutic potential against two unexplored proteins: the spike trimer (7WZ1) and surface glycoproteins (7QTJ). • Cytotoxicity studies and drug profiling provide a scientific basis for the traditional use of Viola stocksii as a health tonic for respiratory infections.