none 10.31254/phyto.2025.14407 BioMed Research Publishers BioMed Research Publishers 16195 184331103 316921 20251025020154379 10.31254 2025-10-25T02:02:00Z 2025-10-25T02:01:57Z 0 The Journal of Phytopharmacology J Phytopharmacol 2320480X 10.31254/phyto https://phytopharmajournal.com/ 09 30 2025 14 4 Preethy John https://orcid.org/0000-0002-7973-8205 AR Nisha Suresh N Nair Bibu John Kariyil Uma Radhakrishnan Background: Nanoparticle-mediated cancer therapies have gained increasing attention due to their targeted action and reduced side effects. Piperlongumine (PPLM), a natural alkaloid, has shown anticancer potential, and its combination with nanotechnology could enhance therapeutic outcomes. Dalton’s Lymphoma Ascites (DLA) cells serve as a reliable model for evaluating antineoplastic agents. Objective: To evaluate and compare the cytotoxic and apoptotic potential of biosynthesised silver nanoparticles (P-AgNPs), chemically synthesised silver nanoparticles (C-AgNPs), piperlongumine (PPLM), and the standard chemotherapeutic agent 5-fluorouracil (5-FU) against DLA cells, and to elucidate the probable underlying mechanisms of cell death. Materials and Methods: The cytotoxicity of test compounds was assessed using the MTT assay, and IC₅₀ values were calculated. Trypan blue dye exclusion assay was performed to evaluate cell viability after 3 hours of exposure. Apoptotic changes were observed through Acridine Orange/Ethidium Bromide (AO/EB) dual staining after 24-hour treatment with IC₅₀ concentrations. Mechanistic studies included DCF-DA assay for intracellular ROS levels, JC-1 staining for mitochondrial membrane potential, DNA fragmentation assay for DNA damage, and qRT-PCR for gene expression analysis of Caspase-3, p53, and Bcl-2, using GAPDH as reference. Results: P-AgNPs showed the highest cytotoxicity with an IC₅₀ of 1.25 µg/mL, outperforming PPLM (4.028 µg/mL), 5-FU (326.20 µg/mL), and C-AgNPs (743.60 µg/mL). Trypan blue assay indicated an average reduction in cell viability to 49.07% across all treatments. AO/EB staining revealed late apoptosis in P-AgNP- and PPLM-treated cells, early apoptosis with 5-FU, and minimal apoptotic effect from C-AgNPs. Mechanistic assays showed that P-AgNPs induced a marked increase in ROS and significant mitochondrial membrane depolarisation. DNA fragmentation confirmed apoptosis. Gene expression analysis showed upregulation of Caspase-3 and p53, and downregulation of Bcl-2 in P-AgNP-treated cells. Conclusion: Biosynthesised silver nanoparticles (P-AgNPs) demonstrated superior cytotoxic and apoptotic effects in DLA cells compared to chemically synthesised nanoparticles and conventional agents. The enhanced efficacy is attributed to ROS generation, mitochondrial dysfunction, and regulation of apoptotic genes, highlighting P-AgNPs as a promising candidate for anticancer therapy. 09 30 2025 274 280 1 10.31254/biomed-crossmark-policy www.biomedresearch.org false 10.31254/phyto.2025.14407 https://phytopharmajournal.com/articles/abstracts/835/year=2025&vol=14&issue=4