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

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 IC₅₀ 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.