Acalypha indica is a medicinal plant widely used in traditional healthcare systems and is known for its diverse pharmacological properties. The present study aimed to evaluate the anticancer potential of its hexane-insoluble chloroform (HIC) fraction against cervical cancer cell lines, including HeLa (HPV-18 positive), SiHa (HPV-16 positive), and C33A (HPV-negative). Phytochemical characterization using GC–MS and LC–MS revealed the presence of various bioactive compounds such as alkaloids, phenolic, terpenoids, and fatty acids, including squalling, ruin, catching, and linoleic acid.

Biological assays demonstrated that the HIC fraction significantly induced cytotoxicity and apoptosis in all tested cell lines, as confirmed by Annexing V-FITC/PI staining and morphological changes. Cell cycle analysis revealed sub-G0 accumulation in HeLa and C33A cells, while G1/S phase arrest was observed in SiHa cells. Gene and protein expression studies indicated up regulation of pro-apoptotic markers (p53, Bax, p21, and cleaved caspase-3) and down regulation of anti-apoptotic and oncogenic markers (Bcl-2 and HPV E6).

Furthermore, the treatment induced reactive oxygen species (ROS) generation, mitochondrial membrane potential loss, and significant DNA damage, as evidenced by γH2AX incorporation and activation of AT Signalling pathways.