Oxidative stress prevention and anti-apoptosis activity of grape (Vitis vinifera L.) stems in human keratinocytes.

To date, grape stems have been partially assessed on their content in phenolics and their radical scavenging activity, whilst the potential to modulate oxidative stress in biological models remains underexplored. In the present work, the effect of grape stems' phenolics on redox unbalance was evaluated in human keratinocytes (HaCaT cells). Grape stems' extracts were assessed on their phenolic composition by high performance liquid chromatography coupled with photodiode array detection and electrospray ionization-mass spectrometry (HPLC-PAD-ESi-MSn), besides on radical scavenging capacity (ABTS and DPPH). In addition, their protective effect against oxidative stress induced by H2O2 by the determination of the level of glutathione, reactive oxygen species, lipid peroxidation, and overall oxidative stress in HaCaT cells by flow cytometry was evaluated. This characterization allowed to identify five flavonols, one cinnamic acid, and one stilbene. A close correlation between the concentration of these phenolics and the capacity to scavenge free radicals and with the potential to modulate the redox balance in vitro was observed. From the analysis of correlation, the activity of malvidin-3-O-glucoside, malvidin-3-O-(6-O-caffeoyl)-glucoside, and malvidin-3-O-rutinoside with respect to the prevention of basal oxidative stress and the capacity of isorhamnetin-3-O-(6-O-feruloyl)-glucoside and kaempferol-3-O-rutinoside to prevent H2O2-induced redox unbalance were stated. Furthermore, grape stems' phenolics also showed an efficient capacity to modulate apoptosis in HaCaT cells, reducing the frequency of annexin V/PI double positive apoptotic cells by up to 99.5% relative to controls, which was further confirmed by the determination of the appearance of the occurrence of apoptotic bodies and the expression of activated (cleaved) caspase-3 by flow cytometry and western-blot, respectively. These results supported the potential of individual phenolics from grape stems to modulate oxidative stress, allowing to envisage dedicated combinations of single compounds for the development of efficient formulations efficient against oxidative stress.