HR-LCMS Metabolic Profiling from Kalanchoe pinnata for Tumor Suppression and Therapeutic Bioactivities by In-vitro and Integrated Network Pharmacology, Molecular Docking and Dynamics Simulations

• HRLC-MS analysis identified 16 bioactive phytocompounds, and FTIR analysis confirmed the presence of ketone, carboxylic, and phenolic groups in the methanolic extract of Kalanchoe pinnata. • K. pinnata showed significant, anti-oxidant, anti-inflammatory and anti-cancer activities in a dose-dependent manner. • The prominence of quercetin, quercetin-3-arbinoside, kaempferol, alpha amyrin and catechin gallate as potential therapeutic agents for breast cancer has been emphasized in network pharmacology. • Confirmation of binding affinity with MMP9, IGF1R, RELA, EGFR, IL2, TNF, NOX4, and CDK6 protein targets through molecular docking studies. • MDS refined docking predictions by revealing stable protein-ligand conformations, low RMSD fluctuations, and sustained interactions, confirming therapeutic complex viability. Breast carcinoma is the most prevalent cancer and leading cause of death among women. Phytochemical profiling of Kalanchoe pinnata leaves by Liquid Chromatography-Mass Spectroscopy revealed presence of quercetin derivatives, phenolics, flavonoids and terpenoids. Fourier Transform Infrared Spectroscopy revealed peaks of functional groups. Phytoconstituents demonstrated significant anti-oxidant, cytotoxic (IC 50 19.105±1.025 µg/mL), COX2 inhibition (IC 50 35.16±1.631 µg/mL) activities and improved glucose uptake in 3T3-L1 cell lines of mouse embryo fibroblasts (42.7% 2-NBDG expression). STRING and Kyoto Encyclopaedia of Genes and Genomes explored breast cancer protein targets. Interactive nodes in the network were built (Cytoscape). Quercetin, quercetin-3-arabinoside, kaempferol, catechin gallate and alpha amyrin are breast cancer therapeutics that modulate pathways and targets of breast cancer. Molecular docking of Matrix metallopeptidase 9 (MMP9), Insulin-like growth factor 1 receptor (IGF1R), v-rel Avian Reticuloendotheliosis Viral Oncogene Homolog A (RELA), Epidermal growth factor receptor (EGFR), Interleukin 2(IL2), Tumour necrosis factor (TNF), NADPH oxidase 4(NOX4) and Cyclin-dependent kinase 6 (CDK6) was performed. Catechin gallate showed significant binding ability to MMP9 (-9.6 Kcal/mol), IGF1R (-9.8 Kcal/mol), NOX4 (-9.7 Kcal/mol) and CDK6 (-9.6 Kcal/mol), and alpha amyrin revealed with TNF (-10.1 Kcal/mol). Molecular dynamics simulations further validated the stability of key protein-ligand complexes (2AZ5-APO,2AZ5-AMY and 2AZ5-CCL), revealing stable Root Mean square Deviation, Root Mean square Fluctuation, compact structural integrity, persistent hydrogen bond formation, flexibility reduction on both eigenvectors by principal component analysis and favourable free energy landscapes. This study provides insights into anti-cancer attributes through in-silico and in-vitro methods to provide a comprehensive bioactivity for therapeutic applications.