Cytoprotective Effect of Hydroalcoholic Extract of Pinus eldarica Bark against H2O2-Induced Oxidative Stress in Human Endothelial Cells

Abstract

Background: Pinus eldarica is a widely growing pine in Iran consisting of biologically active constituents with antioxidant properties. This study investigates the effect of hydroalcoholic extract of P. eldarica bark against oxidative damage induced by hydrogen peroxide (H2O2) in human umbilical vein endothelial cells (HUVECs).

Methods: The total phenolic content of P. eldarica extract was determined using Folin-Ciocalteu method. The cytotoxicity of P. eldarica extract (25-1000 µg/ml) on HUVECs was assessed using 3-(4,5- Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) method. Cytoprotective effect of P. eldarica extract (25-500 µg/ml) on H2O2-induced oxidative stress was also evaluated by MTT assay. The intra- and extra-cellular hydroperoxides concentration and ferric reducing antioxidant power (FRAP) were measured in pretreated cells.

Results: The total phenolic content of P. eldarica extract was estimated as 37.04±1.8% gallic acid equivalent. P. eldarica extract (25-1000 µg/ml) had no cytotoxic effect on HUVECs viability. The pretreatment of HUVECs with P. eldarica extract at the concentrations of 50-500 µg/ml significantly reduced the cytotoxicity of H2O2. P. eldarica extract decreased hydroperoxides concentration and increased FRAP value in intra-cellular fluid at the concentration range of 100-500 µg/ml and in extra-cellular fluid at the concentration range of 25-500 µg/ml.

Conclusions: This study revealed the antioxidant and cytoprotective effects of P. eldarica extract against H2O2-induced oxidative stress in HUVECs. Concerning the high content of phenolic compounds in P. eldarica, more research is needed to evaluate its clinical value in endothelial dysfunction and in other oxidative conditions.

Keywords: Antioxidants; Human umbilical vein endothelial cells; Oxidative stress; Pinus eldarica.

Fig. 1

The effect of P. eldarica extract on proliferation of HUVECs. Cells were incubated with different concentrations of P. eldarica extract (25-1000 µg/ml) or vitamin C (100 µg/ml) for 24 h. The cell viability was determined and compared with the control (untreated cells) by the MTT assay. Values are mean±SEM from three independent experiments in triplicate. ^**P<0.01 versus control (untreated cells

Fig. 2

The effect of P. eldarica extract on H₂O₂-induced oxidative stress in HUVECs. Cells were incubated with H₂O₂ (0.5 mM, 2 h) after pretreatment with different concentrations of P. eldarica extract (25-500 µg/ml) or vitamin C (100 µg/ml). The cell viability was determined by the MTT assay. Values are mean±SEM from three independent experiments in triplicate. ^###P<0.001 versus control (untreated cells), ^*P<0.05, ^**P<0.01 and ^***P<0.001 versus H₂O₂-stimulated cells

Fig. 3

The effect of P. eldarica extract on intra-cellular (A) and extra-cellular (B) hydroperoxides concentration in HUVECs. Cells were incubated with H₂O₂ (0.5 mM, 2 h) after pretreatment with different concentrations of P. eldarica extract (25-500 µg/ml) or vitamin C (100 µg/ml). The hydroperoxides concentration was determined by FOX1 method. Values are mean±SEM from three independent experiments in triplicate. ^###P<0.001 versus control (untreated cells), ^*P<0.05, ^**P<0.01 and ^***P<0.001 versus H₂O₂-stimulated cells

Fig. 4

Ferric reducing antioxidant power (FRAP) values of different concentrations of P. eldarica extract and vitamin C (25-500 µg/ml). Values are means±SEM from three independent experiments in triplicate. ^*P<0.05 and ^**P<0.01 versus vitamin C group at the same concentration

Fig. 5.

Effect of P. eldarica extract on intra-cellular (A) and extra-cellular (B) Ferric reducing antioxidant power (FRAP) value in HUVECs. Cells were incubated with H₂O₂ (0.5 mM, 2 h) after pretreatment with different concentrations of P. eldarica extract (25-500 µg/ml) or vitamin C (100 µg/ml). Values are means±SEM from three independent experiments in triplicate. ^***P<0.001 versus H₂O₂-stimulated cells