Phytochemical Profiling and In Silico Molecular Interactions of the Bioactivity-Guided Fractions of Ocimum tenuiflorum L.: A Comprehensive Report of Antioxidant, Anti-Inflammatory, and Anti-Mutagenic Properties.

Ocimum tenuiflorum Linn. (Krishna Tulsi) holds significant traditional and ethnopharmacological relevance. This study aimed to identify the phytochemicals responsible for these properties through a bioactivity-guided fractionation approach. The aerial part extract was sequentially fractionated using solvents of varying polarity: n-hexane, ethyl acetate, and n-butanol. The fractions were screened for in vitro activities to determine the most bioactive one. High-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (HPLC-ESI-QTOF-MS) analysis was conducted on the most bioactive fraction, revealing high concentrations of flavonoid and anthocyanin compounds. The n-butanol fraction demonstrated superior antioxidant, enzyme inhibition, anti-inflammatory, and dose-dependent antimutagenic activities compared to other fractions. Cytotoxicity assays on HepG2 cells showed no toxicity at 50-500 µg/mL concentrations. In silico molecular docking studies further revealed that 17 bioactive compounds exhibited strong binding interactions, where apigenin 7,4-dimethyl ether (-8.96 kcal/mol), apigenin (-0.331 kcal/mol), and methyl gallate (-0.402 kcal/mol) had highest docking score with pro-oxidant enzymes (xanthine oxidase and NAD(P)H oxidase) and inflammatory mediators (C-reactive protein), validating the in vitro bioactivities. Molecular dynamics study of protein-ligand complex with the highest docking scores-maintained stability until 100 ns. In conclusion, this study outlines a systematic approach for developing phytopharmaceuticals for future in vivo studies.