Redox State Modulatory Activity and Cytotoxicity of Olea europaea L. (Oleaceae) Leaves Extract Enriched in Polyphenols Using Macroporous Resin

Abstract

The food products derived from Olea europaea are a fundamental part of the Mediterranean diet, and their health-promoting effects are well known. In this study, we analyzed the phytochemical characteristics, the redox state modulatory activity, and the cytotoxic effect of an olive leaf aqueous extract enriched by macroporous resin on different tumor and normal cell lines (LNCaP, PC3, HFF-1). HPLC-DAD analysis, the Folin-Ciocalteu and aluminum chloride methods confirmed the qualitatively and quantitatively high content of phenolic compounds (130.02 ± 2.3 mg GAE/g extract), and a DPPH assay (IC₅₀ = 100.00 ± 1.8 μg/mL), the related antioxidant activity. The biological investigation showed a significant cytotoxic effect, highlighted by an MTT test and the evident cellular morphological changes, on two prostate cancer cell lines. Remarkably, the extract was practically non-toxic on HFF-1 at the concentrations (100, 150, 300 µg/mL) and exposure times tested. Hence, the results are selective for tumor cells. The underlying cytotoxicity was associated with the decrease in ROS production (55% PC3, 42% LNCaP) and the increase in RSH levels (>50% PC3) and an LDH release assay (50% PC3, 40% LNCaP, established necrosis as the main cell death mechanism.

Keywords: ROS; glutathione; necrosis; oxidative stress; phytochemicals; plant extract; polyphenols; prostate cancer.

Figure 1

HPLC/DAD chromatograms, visualized at 280 nm (black line), 330 nm (blue line), and 350 nm (pink line) of the OLEE. See the text for further information and experimental details.

Figure 2

Cell viability in HFF-1, LNCaP, and PC3 cells untreated (Ctrl) and treated with vehicle or with OLEE for 48 h (A) and 72 h (B). Values are the mean ± s.e.m. of three different experiments with six wells assigned to each treatment. ^a Significant vs. untreated control cells: p < 0.05; ^b Significant vs. untreated control cells: p < 0.001, ^c Significant vs. untreated control cells: p < 0.0001; ^d Significant vs. treated cells: p < 0.05.

Figure 3

Optical microscope images of PC3 and LNCaP cells treated with vehicle or with OLEE for 24 h and 48 h. On the (left), PC-3 cells; on the (right), LNCaP cells. (A,B): Vehicle; (C,D): 100 µg/mL OLEE 24 h; (E,F): 150 µg/mL OLEE 24 h; (G,H): 100 µg/mL OLEE 48 h; (I,J): 150 µg/mL OLEE 48 h. Magnification 10× was used.

Figure 4

LDH release in untreated PC3 and LNCaP cells (Ctrl) and treated for 48 h with the extract (100–150 μg/mL). Values are the mean ± S.D. of five experiments in triplicate. ^a Significant vs. untreated control cells: p < 0.05; ^b Significant vs. treated cells: p < 0.05.

Figure 5

ROS levels in PC3 and LNCaP untreated cells (Ctrl) and treated for 48 h with extract (100–150 μg/mL). Values are the mean ± S.D. of five experiments in triplicate. ^a Significant vs. untreated control cells: p < 0.05; ^b Significant vs. treated cells: p < 0.05.

Figure 6

RSH levels in PC3 and LNCaP untreated cells (Ctrl) and treated for 48 h with extract (100–150 μg/mL). Values are the mean ± S.D. of five experiments in triplicate. ^a Significant vs. untreated control cells: p < 0.05.