Temperature-Dependent Olive Pomace Extraction for Obtaining Bioactive Compounds Preventing the Death of Murine Cortical Neurons.

High-pressure and temperature extraction (HPTE) can effectively recover bioactive compounds from olive pomace (OP). HPTE extract obtained by extracting OP with ethanol and water (50:50 v/v) at 180 °C for 90 min demonstrated a pronounced ability to preserve intracellular calcium homeostasis, shielding neurons from the harmful effects induced by N-methyl-d-aspartate (NMDA) receptor (NMDAR) overactivation, such as aberrant calpain activation. In this study, the extraction temperature was changed from 37 to 180 °C, and the extracts were evaluated for their antioxidant potency and ability to preserve crucial intracellular Ca2+-homeostasis necessary for neuronal survival. Additionally, to verify the temperature-induced activity of the extract, further extractions on the exhausted olive pomace were conducted, aiming to identify variations in the quality and quantity of extracted phenolic molecules through HPLC analysis. The results revealed a significant increase in bioactive compounds as a function of temperature variation, reaching 6.31 ± 0.09 mgCAE/mL extract for the extraction performed at 180 °C. Subsequent extraction of the exhausted residues yielded extracts that remained active in preventing calcium-induced cell death. Moreover, despite increased antiradical power, extracts re-treated at 180 °C did not display cell protection activity. Our results indicate that the molecules able to maintain physiological Ca2+-homeostasis in murine cortical neurons in conditions of cytotoxic stimulation of NMDAR are wholly recovered from olive pomace only following extraction performed at 180 °C.

A Bioactive Olive Pomace Extract Prevents the Death of Murine Cortical Neurons Triggered by NMDAR Over-Activation.

We have recently demonstrated that bioactive molecules, extracted by high pressure and temperature from olive pomace, counteract calcium-induced cell damage to different cell lines. Here, our aim was to study the effect of the same extract on murine cortical neurons, since the preservation of the intracellular Ca2+-homeostasis is essential for neuronal function and survival. Accordingly, we treated neurons with different stimuli in order to evoke cytotoxic glutamatergic activation. In these conditions, the high-pressure and temperature extract from olive pomace (HPTOPE) only abolished the effects of N-methyl-d-aspartate (NMDA). Particularly, we observed that HPTOPE was able to promote the neuron rescue from NMDA-induced cell death. Moreover, we demonstrated that HPTOPE is endowed with the ability to maintain the intracellular Ca2+-homeostasis following NMDA receptor overactivation, protecting neurons from Ca2+-induced adverse effects, including aberrant calpain proteolytic activity. Moreover, we highlight the importance of the extraction conditions used that, without producing toxic molecules, allow us to obtain protecting molecules belonging to proanthocyanidin derivatives like procyanidin B2. In conclusion, we can hypothesize that HPTOPE, due to its functional and nontoxic properties on neuronal primary culture, can be utilized for future therapeutic interventions for neurodegeneration.

Cell protection from Ca2+-overloading by bioactive molecules extracted from olive pomace.

We are reporting in the present study that molecules extracted from olive pomace prevent cell death induced by Ca2+-overloading in different cell types. Exposure of cells to these molecules counteracts the Ca2+-induced cell damages by reducing the activation of the Ca2+-dependent protease calpain, acting possibly through the modification of the permeability to Ca2+ of the plasma membrane. The purification step by RP-HPLC suggests that effective compound(s), differing from the main biophenols known to be present in the olive pomace extract, could be responsible for this effect. Our observations suggest that bioactive molecules present in the olive pomace could be potential candidates for therapeutic applications in pathologies characterised by alterations of intracellular Ca2+ homeostasis.

Characterization of a new p94-like calpain form in human lymphocytes.

Human circulating PBMC (peripheral blood mononuclear cells) contain three calpain isoforms distinguishable on the basis of their chromatographic properties. Two of these proteases belong to the ubiquitous calpain subfamily, corresponding to the classical mu- and m-calpain forms. The third, which shows peculiar activating and regulatory properties, is an alternatively spliced calpain 3 (p94) form. This new calpain differs from calpain 3 in that it has lost IS1 insertion and exon 15, a lysine-rich sequence regarded as a nuclear translocation signal. PBMC p94-calpain undergoes activation and inactivation without the accumulation of a low-Ca2+-requiring form that is typical of the classical activation processes of mu- and m-calpain. Furthermore, it differs from the ubiquitous forms in that it displays a lower sensitivity to calpastatin. On the basis of these selective properties, it can be postulated that PBMC p94-calpain can be activated in response to specific stimuli that are not effective on the other calpain isoenzymes. The enzyme is preferentially expressed in B- and T-lymphocytes, whereas it is poorly expressed in natural killer cells and almost undetectable in polymorphonuclear cells. This distribution might reflect the specific function of this protease, which is preferentially present in cells devoted to the production of the humoral, rather than to the cellular, immune response.