Sustainable nanomedicine from olive leaf waste: biosynthesized ZnONPs with mechanistic validation against hepatocellular carcinoma

Abstract

Agri-food waste is an underutilized source of valuable bioactive compounds. In this study, olive leaf pruning residues (Olea europaea L. cv. Soury) were used for the green synthesis of zinc oxide nanoparticles (OL-ZnONPs) as a natural reducing and stabilizing agent. Physicochemical characterization showed that the OL-ZnONPs had a hexagonal wurtzite structure, a crystallite size of approximately 15.5 nm, 82.8% crystallinity, and a zeta potential of −23.7 mV. TEM and SEM-EDX analyses revealed aggregated spherical particles with sizes of 4.15 ± 1.23 nm and 0.192 ± 0.067 µm, respectively, with zinc and oxygen accounting for 75.29% and 24.71%, respectively. LC-ESI-MS/MS-MRM analysis identified and quantified 11 phenolic compounds, with rutin (354.15 µg g−1), 3,4-dihydroxybenzoic acid (78.21 µg g−1), and quercetin (45.75 µg g−1) being the most abundant. OL-ZnONPs showed cytotoxic activity against HepG-2, MDA-MB-231, and HCT-116 cells, with the greatest selectivity toward HepG-2 cells (IC50 = 0.344 µg mL−1; SI = 5.279), compared with crude extract and 5-FU. OL-ZnONPs treatment increased intracellular levels of total caspase-3 and caspase-9, reduced TNF-α and IL-6 levels, induced nuclear changes consistent with apoptosis, and reduced HepG-2 cell migration. Network pharmacology was performed using the selected phenolic compounds together with ZnO to explore potential molecular mechanisms associated with the anti-hepatocellular carcinoma (HCC). A total of 801 anti-HCC-HepG-2 targets were predicted including AKT1, TP53, TNF, and IL6, CASP3 as hub genes in protein–protein interaction. The compound-target-pathway network identified quercetin, 3,4-dihydroxybenzoic acid, and ferulic acid as hub compounds. KEGG and GO enrichment analyses highlighted pathways in cancer, apoptosis, PI3K-Akt signaling, and chemical carcinogenesis-reactive oxygen species, providing exploratory mechanistic context for the experimental findings. Molecular docking predicted favorable binding of rutin and quercetin to AKT1. Collectively, these findings highlight the potential of OL-ZnONPs as an anticancer nanomaterial, particularly against HepG-2 cells, while supporting the valorization of olive leaf pruning residues aligns with the 2030 UN Sustainable Development Goal 3 (Good Health), SDG-9 (Industry/Innovation), SDG-12 (Responsible Consumption/Production), and SDG-13 (Climate Action). Further studies are required to clarify the contribution of the ZnO core and associated phytochemicals using size-matched bare ZnONPs, alongside in vivo evaluation of efficacy, biosafety, and pharmacokinetics.