Hepatoprotective Potential of <i>Boerhavia diffusa</i> L. Against Alcohol‐Induced Toxicity in HepG2 Cells: An In Vitro and In Silico Study

ABSTRACT Boerhavia diffusa Linn. (Nyctaginaceae) is popularly known as “Punarnava” in Ayurveda, is traditionally used for chronic alcoholism and liver dysfunction. However, associated molecular hepatoprotective mechanisms remain unexplored. In the present study, a rotenoid‐enriched fraction (REF) from B. diffusa roots was prepared and chemically characterized using ultra‐performance liquid chromatography (UPLC), ultra‐performance liquid chromatography high resolution mass spectrometry (UPLC‐HRMS). Further major rotenoid constituents viz. Boeravinone A and B were quantitatively analyzed by UPLC. REF was evaluated for its hepatoprotective potential against ethanol‐induced toxicity in HepG2 cells using in vitro assays and in silico docking. REF (100 and 150 µg/mL) significantly reduced reactive oxygen species (ROS) production [2′,7′‐dichlorodihydrofluorescein diacetate (DCFH‐DA assay)], lipid accumulation (Oil Red O assay), and nitric oxide production (NO assay) and enhanced activity of alcohol dehydrogenase and aldehyde dehydrogenase in HepG2 cells, compared to silymarin. In silico molecular docking and MD simulation studies revealed strong interactions of major rotenoids with key hepatoprotective targets, including cytochrome P450 2E1 (CYP2E1), nuclear factor erythroid 2–related factor 2 (NRF2), Sirtuin 1 (SIRT1), sterol regulatory element‐binding protein 1 (SREBP1), tumor necrosis factor alpha (TNF‐α), and toll‐like receptor 4 (TLR‐4), with binding affinities superior to silymarin by forming stable hydrogen bonds and hydrophobic interactions. This integrated chemical–biological approach supports further investigation of REF as a promising therapeutic agent for alcoholic liver disease (ALD).