Cytoprotective effects of olive mill wastewater extract and its main constituent hydroxytyrosol in PC12 cells

Abstract

Evidence from epidemiological studies indicates that adherence to diets rich in secondary plant metabolites, such as polyphenols and phenolic acids, possibly reduces the risk of age-dependent neurodegeneration and subsequent cognitive decline. Olive mill wastewater is particularly rich in the ortho-diphenol hydroxytyrosol (HT) that can be recovered with ad hoc techniques. In a previous study, we showed that hydroxytyrosol-rich olive mill wastewater extract protects dissociated brain cells not only in vitro but also ex vivo after subchronic oral administration of the extract to mice. As plant extracts often contain constituents with potentially confounding activities, our present study aimed to test in vitro whether HT at concentrations present in olive mill wastewater extract is able to protect PC12 cells with similar efficiency. Following a short-term exposure (30 min) to the compounds of interest, cells were subjected to oxidative or nitrosative stress by adding either ferrous iron or sodium nitroprusside to the cell culture medium for 18 h, respectively. Cytotoxicity was assessed by measuring MTT reduction, cellular ATP levels and mitochondrial membrane potential in the absence and presence of HT or HT-rich olive mill wastewater extract. The results we obtained mainly confirm our previous observation of promising cytoprotection of brain cells by HT-rich olive mill wastewater extract in different stressor paradigms. Furthermore, correlation analyses revealed that the observed cytoprotective effects in PC12 cells are likely due to HT present in the extract.

Introduction

Extra virgin olive oil (EVOO) is a key constituent of the Mediterranean diet which is associated with lower mortality from coronary heart disease [1]. Recent evidence from epidemiological studies indicates that adherence to the Mediterranean diet might also reduce the risk of age-dependent neurodegeneration, particularly of Alzheimer's disease [2], [3], [4]. In contrast to the cardiovascular system [5], [6], little is know about the effect of EVOO and one of its main constituents, the ortho-diphenol hydroxytyrosol (HT), on the brain. Compared to other free radical scavengers such as vitamin E, BHT or resveratrol, HT showed a significantly higher antioxidant activity in test-tube experiments [7], [8], [9]. Uptake of HT into the brain has been demonstrated in rat studies [10], [11] and there is some evidence that HT exerts neuroprotection. For example, in brain slices obtained from HT-fed mice HT exerted a dose-dependent decrease in the efflux of lactate dehydrogenase, possibly indicating neuroprotective potential of HT in this rodent model [12]. Furthermore, supplementing human neuroblastoma cells with HT (10 μM) for 30 min resulted in significantly lower levels of hydrogen peroxide-induced DNA damage [13].

Olive mill wastewater is rich in organic matter and contains high amounts of polyphenols, including HT that can be recovered with ad hoc techniques [14]. In our previous work, we demonstrated that hydroxytyrosol-rich olive mill wastewater extract (HT-E) protected dissociated murine brain cells in vitro and ex vivo from deleterious impact of the stressors SNP (nitric oxide (NO) donor) and ferrous iron (Fe²⁺). Subchronic oral administration of HT-E in mice enhanced the resistance of dissociated brain cells, evident by reduced basal and stress-induced lipid peroxidation [15]. However, plant extracts, even after concentration of the active compound, often still contain constituents with potentially confounding biological activities [16], [17]. Hence, we decided to test in vitro whether HT at concentrations present in HT-E is able to protect PC12 cells, a commonly used neuronal-like cell culture model, from SNP- and Fe²⁺-induced cellular stress with similar efficiency.