Abstract
This study explores the bioactive potential of olive mill waste derived from cultivars grown in the Maltese Islands through various analytical approaches. Cell culture, cell staining, allelopathic assays, shrimp brine lethality assays, and HPLC analysis were conducted to assess the efficacy and bioactivity of the extracts using different treatments, including methanolic extraction, acid, and alkaline hydrolysis. Notably, the results from cell lines revealed that NB4r2 cells exhibited high susceptibility to the tested extracts, with the lowest IC50 recorded after 72 h of exposure. Notably, the 'Bajda' cultivar displayed the most effectiveness, particularly with acid hydrolysis. In allelopathic assays, higher concentrations of 'Malti', 'Bidni', and 'Bajda' extracts significantly inhibited lettuce seed germination. Similarly, in the brine shrimp lethality assay, higher concentrations led to increased mortality rates of Artemia salina, though rates decreased at lower concentrations. The identification of phenolic compounds found in olive mill waste was conducted using high-performance liquid chromatography (HPLC) with the use of internal standards. The identification revealed a variety of compounds, with 3-hydroxytyrosol and oleacein being present in high abundance in nearly all hydrolyzed and methanolic extracts, whereas gallic acid was found to be the least abundant. These findings highlight the rich bioactive potential of olive mill waste and provide insights into its applications in pharmaceuticals, nutraceuticals, and agriculture, emphasizing the importance of further research to fully exploit these valuable resources.