Proteomic profiling of Neem (Azadirachta indica) leaves

Modern scientific studies have contributed largely to creating well -validated research data for the holistically rooted use of the Neem (Azadirachta Indica) tree in disease management. Neem's astonishing historical and modern relevance reveal more evidence as to why Neem is the target of our study.Valuable studies have shown that the whole parts of the Neem tree are are markable natural source of more than 130 bioactive compounds, which are highly responsible for many therapeutic applications. Historically, Neem has been used as a source of complementary and traditional medicine Ayurvedic and Unani medicine for over 4,500 years. The mystical tree Neem originated from southern Asian countries. This research focuses on the proteomic profiling of Neem leaves and tests their anticancer potential on breast cancer cells. Recent Cancer studies have ranked breast cancer among the most rapidly increasing cancer types in women. Despite consistent advancements in therapies, breast cancer remains a concerning health issue and a deadly disease among women. Many of the existing and emerging Cancer therapies come with numerous drawbacks, increasing the thirst to find better the rapeutics from plant sources with safer and efficacious outcomes. Neem has shown a remarkable potential against cancer due to its wide range of biologically active compounds, including proteins. Studies on the proteins present in Neem remain largely unexplored. The reviewed SwissProt database in UniprotKB shows only three proteins reported from the Neem tree.Therefore, this study has great potential to explore the global proteomic profiling of Neem leaves. Further analysis has also evaluated the cytotoxic potential of proteins from Neem leaves against MDA-MB 231 breast cancer cell lines. The defatted Neem leaves were extracted in n -hexane, followed by extraction in a 20 mM Tris/HCl buffer, pH 8, and then precipitated using 80% ammonium sulfate. The crude extract was dialyzed in water using a 3.5MWCO dialysis tubing and then lyophilized. The anticancer assay of Neem leaves Crude protein at different concentrations was evaluated against MDA-MB-231 cells by using MTS assay, where cells were incubated for 24hours, followed by the treatment with Crude protein for 48 hours, and absorbance was measured at 490 nm. Concentration ranges (6.4- 0.2 mg/mL) showed inhibitory effects on MDA - MB 231 cells. Further, the downstream proteomics was achieved by purification of Crude using gel filtration chromatography first on Hiload Superdex-200 column. The electrophoretic profile was developed by SDS-PAGE electrophoresis using a 10% Tris/Glycine gel, confirmed the presence of proteins with MW ranging from 10 to 37 kDa. The Proteomic profiles were established by peptide mass fingerprinting using Q-tof LC-MS/MS. The bioinformatic analysis using PEAKS Studio X and the UniProtKB TrEMBL Viridiplantae database revealed 17 proteins in 11 groups at an FDR ≤ 1% with a minimum of two unique peptide matches.