Hydroalcoholic Combination Extract of Withania somnifera (Ashwagandha)and Camellia sinensis (Green Tea) Induces Cytotoxicity, Apoptosis, andSuppresses Migration in MCF-7 Human Breast Cancer Cells via ReactiveOxygen Species Generation

Abstract

Background: Breast cancer remains one of the leading causes of cancer-related mortality among women worldwide, and the toxicity and drug resistance associated with conventional chemotherapy have renewed interest Ain plant-derived bioactive combinations. Withania somnifera (Ashwagandha; A) and Camellia sinensis (Green tea; G) are two widely studied medicinal plants individually reported to possess anticancer, pro-apoptotic and antioxidant-modulating properties. Objective: The present study was designed to evaluate the in vitro cytotoxic, apoptotic, pro-oxidant and anti-migratory potential of a hydroalcoholic combination extract of Ashwagandha and Green tea (designated A.G.) against the human breast adenocarcinoma cell line MCF-7. Methods: MCF-7 cells were treated with increasing concentrations of A.G. (10-400 µM) and cell viability was assessed by the MTT assay. Nuclear and membrane morphological changes associated with apoptosis were examined using acridine orange/propidium iodide (AO/PI) dual fluorescent staining. Intracellular reactive oxygen species (ROS) generation was measured using the 2',7' dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescent probe. Cell migratory capacity was evaluated using the in vitro wound-healing (scratch) assay at 0, 12 and 24 h. Results: A.G. produced a concentration-dependent decrease in MCF-7 cell viability (IC50 ≈ 100 µM at 24 h), with a non-significant effect at 10 µM and highly significant reduction in viability from 25 µMupward (p<0.05 to p<0.001). AO/PI staining of cells treated with A.G. (100 µM) revealed a marked shift from uniform green (viable) fluorescence in control cells to intense orange-red (PI positive) fluorescence, indicating apoptotic/late-apoptotic and membrane-compromised populations. A.G. treatment produced a significant increase in intracellular ROS level relative to untreated control (p<0.001), consistent with a pro-oxidant mechanism of cell killing. In the wound-healing assay, A.G. treated MCF-7 cells showed visibly delayed wound closure at 12 and 24 h compared with control, indicating suppression of cell migration. Conclusion: The hydroalcoholic Ashwagandha-Green tea combination extract (A.G.) exerts concentration-dependent cytotoxic and pro-apoptotic activity against MCF-7 breast cancer cells, mediated at least in part through excessive intracellular ROS generation, and also inhibits their migratory potential. These findings support further mechanistic and in vivo evaluation of A.G. as a candidate adjunct phytotherapeutic agent in breast cancer.