Maceration, Soxhlet Extraction, and Steam Hydrodistillation of Anethum graveolens Seeds: Phytochemical Profiles and Cytotoxic Effects

Dill, Anethum graveolens L., is a culinary plant used in traditional medicine to treat gastrointestinal conditions; its seeds have been previously reported to exhibit antimicrobial, anti-inflammatory, hypolipidemic, anticancer and antioxidant activities. This study aimed to assess dill seed extracts and essential oil in terms of chemical composition and cytotoxic effects, combined with an exploration of the potential underlying mechanisms. Four hydroethanolic seed extracts were prepared using Soxhlet extraction or maceration and the essential oil (AGEO) was prepared by steam distillation. Extracts were physicochemically analysed via LC-MS and spectrophotometry while the AGEO composition was determined through GC-MS. Their cytotoxic effects were assessed in A375, PANC-1 and SK-OV-3 cancer cells, while HaCaT keratinocytes were used as non-cancerous control. The mechanistic investigations included immunofluorescence assay, high-resolution respirometry, network pharmacology and molecular docking. Ferulic acid and fisetin were identified as major components of the hydroethanolic extracts, while D-limonene and carvotanacetone were the main compounds in the AGEO. All tested products were revealed to induce dose-dependent cytotoxicity (with IC50 values ranging between 619.8 and 810.2 μg/mL), diminished OXPHOS efficiency and a mitochondrial uncoupling effect. Morphological changes consistent with apoptosis were recorded, particularly in PANC-1 and A375 cells. Network pharmacology suggested a multitarget mechanism for EO that involved STAT3 and HSP90AA1, while the molecular docking calculations indicated that D-limonene and carvotanacetone could be accommodated within the ATP-binding pocket of Hsp90α.