Antiproliferative activity, apoptotic induction, cell cycle arrest and p53 expression for dual herbal combination of W. somnifera with three Rasayana herbs: In vitro cytotoxic study against Jurkat cells

ANTICANCER POTENTIAL OF W. SOMNIFERA DUAL HERBAL COMBINATIONS AGAINST JURKAT CELLS

Abstract

The dual herbal methanolic formulations of Withania somnifera combined with Phyllanthus emblica (WP3), Bacopa monnieri (WB2), and Ocimum basilicum (WO3), prepared using our previously optimized and validated methodology for Ayurvedic phytochemical standardization, were investigated for their therapeutic relevance against leukemia. As phytotherapy emerges as a complementary strategy to reduce chemotoxicity and support long-term cancer management, evaluating rational botanical combinations is essential for advancing evidence-based integrative approaches. For the first time, this study delineates the collective anticancer effects of W. somnifera with three Rasayana co-herbs against Jurkat E6-1 T-cell leukemia cells. The extracts WP3 and WO3 were assayed for MTT cytotoxicity, exerting the strongest antiproliferative effects, with IC₅₀ values of 20.27 and 20.40 µg/mL, respectively. We utilised Annexin V/PI flow cytometry and AO/EB staining to validate both early and late stages of apoptotic progression following treatment. The WP3 results reflecteda substantial increase in apoptotic cell population for Sub-G0/G1 (18.37%) compared to (2.89%) control cells, while WO3 induced a lesser elevation of 6.53%, indicating DNA laddering resembling endonuclease fragmentation and apoptotic accumulation. Further cell cycle analysis showed reduced G0/G1phase population for WP3 (54.79%) and WO3 (56.82%) compared to the control (69.77%), interfering with cell cycle regulatory checkpoints for DNA synthesis.p53 protein expression was upregulated in WP3 treated cells (29.52%) relative to control (9.04%), suggesting stimulation of DNA tumor suppressor signaling pathways. The dual herbal combinations, treated with WP3, showed the strongest activity, suppressing leukemic cell growth by promoting apoptotic cell death, p53-mediated responses, and altering cell cycle progression. As a first-of-its-kind study, demonstrating phytochemical profiling, preclinical validation, and initiating a framework for Rasayana-derived combinatorial therapeutic strategies.