Evaluating the efficacy of Ocimum tenuiflorum as a therapeutic agent in colorectal cancer.

Colorectal cancer (CRC) remains a leading cause of cancer-related morbidity and mortality worldwide, with persistent challenges of systemic toxicity, chemoresistance, and limited efficacy of conventional therapies underscoring the need for safer therapeutic alternatives. Ocimum tenuiflorum (Holy Basil), a phytochemically rich medicinal herb with established antioxidant, anti-inflammato. An integrated in vitro and in silico approach was employed. The ethanolic extract of O. tenuiflorum leaves was prepared by Soxhlet extraction and evaluated for cytotoxicity (MTT, XTT, LDH release, Trypan Blue exclusion) against HCT116 colorectal cancer cells, antioxidant activity (DPPH, ABTS, FRAP assays), and anti-inflammatory activity (nitric oxide and cyclooxygenase inhibition assays) using RAW 264.7 macrophages. Computational analyses included protein-protein interaction (PPI) network construction via STRING, molecular docking (Glide SP/XP) of bioactive ligands against NF-κB (PDB: 1NFK) and JAK1 (PDB: 5XGJ), a 200 ns molecular dynamics (MD) simulation of the lead complex, and Density Functional Theory (DFT) calculations at the B3LYP/6-31G* level. The extract exhibited significant, concentration-dependent cytotoxicity against HCT116 cells (MTT IC50: 70.4 µg/mL), substantial antioxidant activity (DPPH IC50: 34.7 µg/mL), and notable inhibition of nitric oxide and COX activity, indicating modulation of oxidative stress and tumor-associated inflammation. PPI analysis revealed high-connectivity NFKB1 and PIK3CA signaling hubs central to CRC progression. Among all screened phytochemicals, rosmarinic acid demonstrated the highest binding affinity against NF-κB (docking score: -5.898 kcal/mol) and JAK1 (-6.812 kcal/mol), forming a stable complex over the 200 ns MD simulation (backbone RMSD: ±2.7 Å). DFT analysis revealed a moderate HOMO-LUMO gap (3.895 eV), indicating favorable electronic stability and chemical reactivity. The crude ethanolic extract of O. tenuiflorum demonstrated consistent, multi-target bioactivity, combining antioxidant, anti-inflammatory, and cytotoxic effects, supporting its potential as a chemopreventive and therapeutic agent against CRC. Within this extract, rosmarinic acid emerged as the principal bioactive contributor, exhibiting markedly higher binding affinity than all other screened phytochemicals and the reference standard doxorubicin at both NF-κB and JAK1 targets, with stable complex formation confirmed over 200 ns of MD simulation. These findings position O. tenuiflorum, and rosmarinic acid specifically, as a promising, safer alternative to conventional chemotherapy. Future studies should focus on in vivo validation, bioavailability-enhancing nanoformulation strategies, and clinical evaluation to translate these findings toward adjunctive cancer therapy.