Investigating the anticancer role of Calotropis procera leaf extract: LC-MS profiling and evidence for apoptosis and ROS induction

• Six local medicinal plants known for anticancer activity were characterised by FTIR. • The extracts were screened for their anticancer activity in human cell lines. • Calotropis procera methanol leaf extract (CPML) exhibited best anticancer activity. • CPML induced apoptosis, inhibited colony formation and increased ROS generation. • Treatment with CPML inhibited cell migration and induced cell cycle arrest. • LC-MS analysis revealed key components associated with anticancer activity. Aegle marmelos, Calotropis procera, Carica papaya, Morus alba, Tinospora cordifolia, and Trigonella foenum-graecum have been used for their antitumor properties. The present study aims to explore their anticancer activity in five cell lines viz. HeLa (cervical cancer), MCF-7 (breast cancer), T24 (urinary bladder carcinoma), SH-SY5Y (neuroblastoma), and LN229 (glioblastoma) to provide scientific evidence of their activity. Among the extracts, Calotropis procera methanol leaf extract (CPML) illustrated the best efficacy (IC 50 < 20 μg/mL) against all the cell lines except SH-SY5Y cells. CPML exhibited non-toxicity to HEK239T cells highlighting its specific anticancer activity. FTIR (Fourier Transform Infrared Spectroscopy) and LC-MS (Liquid Chromatography Mass Spectrometry) were performed for initial characterization of the extract. The mechanism of anticancer activity of CPML was through apoptosis induction as illustrated by AO/EB dual staining, ROS generation, and RT-qPCR. CPML treatment induced cell cycle arrest in the G2/M phase and inhibited colony formation and cell migration. LC-MS based phytochemical analysis led to the identification of Calotropin, Digitoxigenin, phenanthroline, hinokiresinol, Rhamnetin, 3-Methoxy Luteolin, Carveol, Lupulon, Azicitidine, Guaiacol and hydroxy-beta-cyclocitral in the extract. Presence of these active constituents might be responsible for the observed anticancer activity of CMPL. The study demonstrates the promising anticancer activity of CPML against HeLa, MCF-7 and T24 cells. Further studies can be carried out to explore the anticancer activity of the identified compounds alone and in combination therapy.