In silico and in vitro study of bioactive compounds of Nigella sativa for targeting neuropilins in breast cancer

Introduction: Breast cancer poses a significant global challenge, prompting researchers to explore novel approaches for potential treatments. Material and Methods: For in vitro study we used thin layer chromatography (TAC) for phytochemical screening, total antioxidant capacity (TLC) assay for antioxidant capacity, and hemolytic activity test for toxicity of Neuropilins (NRPs). We performed bioinformatic analyses to predict protein structures, molecular docking, pharmacophore modeling, and virtual screening to reveal interactions with oncogenes. We conducted 200 ns Molecular Dynamics (MD) simulations and MMGBSA calculations to assess the complex dynamics and stability. Results: We identified phytochemical constituents in Nigella sativa leaves, including tannins, saponins, steroids, and cardiac glycosides, while phlobatannins and terpenoids were absent. The leaves contained 9.4% ± 0.04% alkaloids and 1.9% ± 0.05% saponins. Methanol extract exhibited the highest yield and antioxidant capacity, with Total Flavonoid Content at 127.51 ± 0.76 mg/100 g and Total Phenolic Content at 134.39 ± 0.589 mg GAE/100 g. Hemolysis testing showed varying degrees of hemolysis for different extracts. In-silico analysis indicated stable Neuropilin complexes with key signaling pathways relevant for anti-cancer therapy. Molecular docking scores at different possesses (0, C-50, C −80, C-120,C −150, C −200 ns) revealed strong hydrogen bonding in the complexes and showed −12.9, −11.6, and −11.2 binding Affinities (kcal/mol) to support their stability. Our MD simulations analysis at 200ns confirmed the stability of Neuropilin complexes with the signaling pathways protein PI3K. The calculated binding free energies using MMGBSA provided valuable quantitative information on ligand potency on different time steps. These findings highlight the potential health benefits of N. sativa leaves and their possible role in anti-cancer treatments targeting angiogenesis. Conclusion: Nigella sativa leaves have shown significant medical potential due to their bioactive compounds, which exhibit strong properties in supporting organogenic processes related to cancer. Furthermore, studies have highlighted the promising role of neuropilins in anticancer treatment, demonstrating stable interactions and potential as targeted therapy specifically for breast cancer.