Formulation and Evaluation of Liposome-Encapsulated Phenolic Compounds from Olive Mill Waste: Insights into Encapsulation Efficiency, Antioxidant, and Cytotoxic Activities

Phenolic extracts from olive mill waste of the Maltese cultivars ‘Bidni’ and ‘Bajda’ were encapsulated in phosphatidylcholine (PC)/cholesterol liposomes using the thin-film hydration method. This study examines the impact of varying lipid composition and cholesterol content on the final encapsulation efficiency of the liposomal formulation. This study also compares two approaches to encapsulation of phenolic mixtures: co-dissolution of the extract within the lipids prior to hydration and introduction of the extract through the hydration medium during the hydration phase. Additionally, differences between the two cultivars were assessed. The study further evaluated in vitro cytotoxicity, focusing on the variation in cytotoxic potential between the free and encapsulated extracts. Total phenolic, flavonoid, orthodiphenolic content and CUPRAC were determined via spectrophotometric assays. Centrifree separation was employed to separate free extract from the extract-loaded liposomes and subsequently calculate encapsulation efficiency (%) with respect to all four assays. Encapsulation efficiencies ranged from 40% to 60% for TPC, 65% to 85% for TFC, 40% to 70% for TdOPC, and 25% to 65% for all CUPRAC related compounds. An increased percentage of cholesterol in the PC:Chol liposomal formulation correlated with higher encapsulation efficiency of phenolic compounds. Co-dissolution of the extract with lipids in chloroform before hydration resulted in preferential selectivity to flavonoids whereas the incorporation of the extract via the hydration medium during the hydration phase yielded a greater favouring of total phenolic compounds overall. MTT-Assay was used to determine cytotoxicity by IC50. Extracts from both cultivars exerted a proportional cytotoxic effect on human HL-60 cancer cells, applicable to both encapsulated and non-encapsulated forms. Logarithmic modelling of the IC50 indicated a rapid cytotoxic response (24 hours) for the free non-encapsulated extract, whereas the encapsulated extract demonstrated a more delayed yet more potent cytotoxic response at 48 hours.