Phytochemical Profiling, Cytotoxicity, and Molecular Docking of Juglans regia L. Leaf Extracts for Potential Breast Cancer Therapy

Background: Juglans regia leaves contain bioactive compounds with potential anticancer effects. This study evaluated their cytotoxic activity and interactions with cancer-related targets. Materials and Methods: Leaves of J. regia were subjected to Soxhlet and ultrasonic extraction using different solvents. Extract yields were calculated, and antioxidant activity was assessed using the DPPH• assay. Cytotoxicity was evaluated against MCF-7, HepG2, and Huvec cell lines. Apoptotic changes were visualized using Hoechst 33342 staining. Ethyl acetate extract was analyzed by GC-MS. SwissTargetPrediction and gene enrichment analyses were used to identify cancer-related targets. Molecular docking was performed using AutoDock Vina. Results: Soxhlet extraction with ethyl acetate yielded 1.64 g of extract compared to 0.262 g by ultrasonic extraction. The isopropanol Soxhlet extract showed the highest antioxidant activity (IC₅₀ = 0.68 µg/mL). The ethyl acetate extract exhibited cytotoxicity against MCF-7 (IC₅₀ = 100.5 µg/mL), HepG2 (IC₅₀ = 164.7 µg/mL), and Huvec cells (IC₅₀ = 244.0 µg/mL). Nuclear condensation and fragmentation were observed in Hoechst-stained cells. GC-MS analysis identified 30 compounds, including γ-tocopherol (17.43%), 1-heptacosanol (14.06%), and β-sitosterol (13.76%). SwissTargetPrediction identified 752 targets for eight major compounds; 434 were drug-related and 117 overlapped with breast cancer-associated genes. Ten hub targets were identified, including ERBB2, HIF1A, and MTOR. β-sitosterol showed strong docking scores with 4YAY (−9.9 kcal/mol) and 4JSV (−9.0 kcal/mol). Conclusion: The observed cytotoxic and pro-apoptotic effects of the J. regia ethyl acetate extract correlate with network pharmacology and docking results, where β-sitosterol and γ-tocopherol showed strong affinities for MTOR, ERBB2, HSP90AB1, and AGTR1. These interactions suggest modulation of key oncogenic pathways such as PI3K-Akt, MAPK, and ErbB, supporting the extract’s potential as a natural multi-target anticancer agent.