Nanoparticles made using olive-leaf extract, called OL-ZnONPs, showed the strongest reported sensitivity and selectivity in HepG-2 human hepatoma cells. Their IC50, the concentration measure used for a half-maximal effect, was 0.3440 µg/mL, and their reported selectivity index was 5.279. Activity was also reported in MDA-MB-231 cells, with an IC50 of 0.4199 ± 0.031 µg/mL and a selectivity index of 4.325, and HCT-116 cells, with an IC50 of 1.008 ± 0.08 µg/mL and a selectivity index of 1.802.
The comparison with the reference drug was limited to the same laboratory assays. The reported 5-FU selectivity index was below 1 in all three cancer lines: 0.943 in HepG-2, 0.849 in MDA-MB-231 and 0.888 in HCT-116. These numbers describe a pattern in cultured cells, not evidence that OL-ZnONPs would outperform 5-FU in patients.
Researchers exposed triplicate cultured cells to OL-ZnONPs, crude olive-leaf extract or 5-FU and assessed viability with the MTT assay. Results were summarized as mean ± standard deviation and analyzed with one-way ANOVA followed by Tukey's multiple-comparison test; results with p values below 0.05 were treated as statistically significant.
The normal-cell comparison matters
The normal-cell comparison adds an important caution. In HSF cells, the study's in-vitro normal-cell model, OL-ZnONPs had the lowest reported EC50, the concentration measure used for a half-maximal effect, at 1.816 ± 0.8244 µg/mL. The corresponding values were 3.19 ± 0.97 µg/mL for 5-FU and 20.02 ± 1.65 µg/mL for crude olive-leaf extract. The result shows measurable toxicity in the normal-cell model, and it does not establish a therapeutic window or organism-level biosafety.
From olive leaves to nanoparticles
The nanoparticles were produced by processing olive-leaf extract with zinc acetate and ammonium hydroxide, followed by centrifugation, washing and freeze-drying.
Characterization supported a hexagonal wurtzite structure, an approximately 15.5 nm crystallite size, 82.8% crystallinity and a surface charge of -23.7 mV. Zinc and oxygen accounted for 75.29% and 24.71% of the measured composition. The reported size depended on the technique: TEM gave an average of 4.15 ± 1.23 nm, while SEM-EDX gave 0.192 ± 0.067 µm. The analysis notes that these are different size measures and that the detailed crystallite-size values were internally inconsistent.
A targeted LC-MS/MS test detected 11 phenolic compounds in the 70% ethanolic olive-leaf extract. Concentrations ranged from 1.95 to 354.15 µg per g, with rutin highest at 354.15 µg per g, followed by 3,4-dihydroxybenzoic acid at 78.21 and quercetin at 45.75. The assay focused on phenolic acids and flavonoids, so it did not quantify the broader olive-leaf metabolome.
Signals, then predictions
Cell-level findings were consistent with apoptosis, a form of programmed cell death, and with changes in inflammation-related signals. The paper reports treatment-associated increases in total caspase-3 and caspase-9, reductions in TNF-alpha and IL-6, nuclear changes consistent with apoptosis and reduced HepG-2 migration. The migration result cannot by itself show a migration-specific effect, because reduced viability or proliferation may also reduce scratch closure.
To look for possible explanations, the computational analysis intersected 19,322 hepatocellular carcinoma-associated genes with 17,181 HepG-2-associated genes, leaving 16,756 common genes and 801 compound-overlapping targets. A protein-protein interaction network, a map of links among proteins, contained 801 nodes and 23,785 edges. Its reported top hubs included AKT1, TP53, TNF, IL6, EGFR, STAT3 and MYC.
Pathway analysis ranked pathways in cancer, chemical carcinogenesis involving reactive oxygen species, PI3K-Akt signaling and apoptosis among the strongest associations. Docking calculations predicted affinities of -11.381 kcal/mol for rutin with AKT1 and -9.278 kcal/mol for quercetin with AKT1. These scores are computational predictions, not proof that the compounds bind or control those targets in cells.
What the study still cannot answer
The main unresolved question is what part of the material drives the cell response. Because the study did not use a size-matched bare-ZnO comparator, it cannot separate the contribution of the ZnO core from the olive-leaf phytochemicals or determine whether the combination contributes to the effect. The work therefore offers laboratory evidence from cultured cells and computational leads, not a validated molecular mechanism or evidence of treatment benefit in people.
Paper data and sources
Original title: Sustainable nanomedicine from olive leaf waste: biosynthesized ZnONPs with mechanistic validation against hepatocellular carcinoma
Authors: Mohamaden Y, El-Hawary S, El-Fakharany E et al.
Journal/Repository: RSC advances
Status: Peer-reviewed
First online: 2026-10-07
DOI: Not available
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