Integrated study of HR-LC/MS and network pharmacology to identify breast cancer-related molecular targets of Fritillaria cirrhosa D. Don active constituents in combination with molecular dynamic simulation and experimental evaluation

Fritillaria cirrhosa D. Don is a well-known medicinal plant in Kashmir Himalya. Traditionally, it has been used to treat several diseases, most notably in the treatment of various cancers particularly lung cancer. However, there is a significant gap between scientific research and its application in conventional medicine. The aim of the current work is to provide first-hand evidences both in-vitro and in silico showing that F. cirrhosa extracts exerts anti-cancer effects against breast cancer. Bulbs of F. cirrhosa was extracted with various solvents of increasing polarity. Compounds were identified by High resolution-liquid chromatography-mass spectrometry (HR-LC/MS) technique. Phytocompounds were studied for protein targets involved in pathogenesis of breast cancer using Binding 1DB (similarity index > 0.7). Later, the protein-protein interactions (PPI) network was studied using STRING programme and compound-protein interactions using Cytoscape. In addition, molecular docking was used to investigate intermolecular interactions between the compounds and the proteins software using Autodock tool. Molecular dynamics simulations studies were also used to explore the stability of the representative CDK2 + Peiminine complex. In addition, standard in-vitro biochemical assays were used to evaluate the in-vitro antiproliferative activity of active extracts of F. cirrhosa against several breast cancer cell lines. Bioactive components and potential targets in the treatment of breast cancer were validated through network pharmacology approach. HR-LC/MS detected the presence of several secondary metabolites. Afterward, molecular docking was used to verify the effective activity of the active ingredients against the prospective targets. Additionally, Peiminine showed the highest binding energy score against CDK2 (-12.99 kcal/mol). CDK2 + Peiminine was further explored for molecular dynamics simulations. During the MD simulation study at 100 nanoseconds (ns), a stable complex formation of CDK2 + Peiminine was observed. According to molecular docking results predicted, several key targets of breast cancer bind stably with the corresponding phytocompounds of F. cirrhosa. Lastly, F. cirrhosa extracts exhibited momentous anticancer activity through in vitro studies. Overall, the most important constituents were Imperialine-3-β-glucoside and Peiminine from the F. cirrhosa bulbs has effective anti-cancer efficacy by deactivating Akt1 on the PI3K-Akt signaling pathway. Therefore, these findings emphasized the momentous anti-breast cancer activity of F. cirrhosa extracts. This may open a new window and provide a theoretical foundation for further development and utilization of F. cirrhosa medicinal plant in the treatment of breast cancer.