Multi‐Target Anticancer Activity of Justicia betonica L.: Phytochemical Profiling, In Silico Screening, and Preclinical Evaluation

ABSTRACT Justicia betonica L. (Acanthaceae) is a traditionally used medicinal plant with limited evidence on its anticancer mechanisms. This study integrates phytochemical profiling, molecular docking, and in vitro/in vivo evaluations to elucidate its anticancer potential. Gas chromatography–mass spectrometry (GC–MS) and liquid chromatography–mass spectrometry (LC–MS) analyses identified a diverse phytochemical repertoire, including retronecine, maritimetin, hematopodin, mangiferin 6′‐gallate, and quercetin glycosides, many reported here for the first time in J. betonica L. In silico docking revealed high binding affinities of these constituents toward multiple oncogenic targets—CDK‐2, CDK‐6, B‐cell lymphoma 2 (Bcl‐2), vascular endothelial growth factor receptor‐2 (VEGFR‐2), and insulin‐like growth Factor 1 receptor (IGF‐1R)—suggesting multi‐target inhibitory potential. The ethanolic extract of J. betonica L. exhibited selective cytotoxicity against MCF‐7, A549, and HT29 cell lines, with IC 50 values of 42.11 ± 1.64, 35.39 ± 1.02, and 29.66 ± 1.34 µg/mL, respectively, while sparing normal human umbilical vein endothelial cells (HUVECs). Acute toxicity studies confirmed safety up to 2000 mg/kg. In the Ehrlich ascites carcinoma (EAC) model, oral administration of EEJB (400 mg/kg) significantly reduced tumor volume, packed cell volume, and viable cell count, extended mean survival time, and ameliorated tumor‐induced hematological and hepatic alterations. These findings position J. betonica L. as a promising plant‐derived, multi‐target anticancer candidate, meriting further isolation of active principles and mechanistic exploration.