Protective effects of Phyllanthus phillyreifolius extracts against hydrogen peroxide induced oxidative stress in HEK293 cells

Abstract

Phyllanthus phillyreifolius, a plant species indigenous to Reunion Island, is used in folk medicine for treating diarrhea and as a diuretic. In the present study acetone and hydroethanol extracts of P. phillyreifolius were evaluated for their cytotoxicity and antioxidant effects using in vitro (TPC, ABTS, DPPH, FRAP, ORAC) and in cellulo (MTT, DCFH-DA, RT-qPCR) assays. Major compounds were evaluated using UPLC-QTOF-MS. MTT cell viability assay showed low cytotoxicity of extracts towards human embryonic kidney 293 (HEK293) cell line. Both extracts were rich in polyphenols (mainly ellagitannins) and showed high antioxidant potential and intracellular ROS decreasing effect. Preconditioning of HEK293 cells with extracts influenced gene expression of antioxidant enzymes, however ROS level decreasing effect was more related to their capacity to scavenge free radicals and with their reducing power. Strong antioxidant activity of extracts as well as the presence of geraniin supports the use of P. phillyreifolius in traditional medicine.

Fig 1. Effect of P. phillyreifolius extracts on HEK293 cells viability analysed by mitochondrial metabolic activity (MTT) assay.

Cells were treated for 24 h with increased concentration of hydroethanolic (EH) or acetonic (AC) extracts of P. phillyreifolius. Data are represented as means ± standard deviations (n = 3).

Fig 2. Effect of P. phillyreifolius extracts on basal level of ROS.

HEK293 cells were treated with the noted concentrations of acetonic (AC) and hydroethanolic (EH) extracts for 24 h, then were washed and incubated with DCFH-DA (fluorescent probe as indicator of ROS) for 45 min. Data are represented as means ± standard deviations (n = 3) with one way ANOVA. The columns with different letters (a-d) differ significantly for Tukey’s test at p < 0.05.

Fig 3. Effect of P. phillyreifolius extracts on ROS generation induced by H₂O₂.

HEK293 cells were treated with extracts for A–3 h, B–24 h, then were washed, incubated with DCFH-DA for 45 min. Then supernatant was removed and cells were incubated for 1 h with H₂O₂ (100 μmol/L). Data are represented as means ± standard deviations (n = 3) with one way ANOVA. The columns with different letters (a-e) differ significantly for Tukey’s test at p < 0.05.

Fig 4

Effect of P. phillyreifolius extracts on A–SOD1, B–SOD2, C–CAT, D–GPx enzymes gene expression in unstressed and H₂O₂-stimulated HEK293. Cells were treated with extracts for 24 h, then were washed and incubated for 1 h with H₂O₂ (100 μmol/L). Data are represented as means ± standard deviations (n = 3) with one way ANOVA. The columns with different letters (a-d) differ significantly for Tukey’s test at p < 0.05.

Fig 5

Chromatograms of AC (a) and EH (b) extracts obtained by UPLC-Q-TOF and chemical structures of the main compounds, quantified in P. phillyreifolius: geraniin, elaeocarpusin, phyllanthusiin D and ellagic acid.