Green extraction of bioactive compounds from wine lees and their bio-responses on immune modulation using in vitro sheep model.

The objective of this study was to apply microwave-assisted extraction using green solvents starting from 3 different wine (white, rosé, and red) lees and to test their bio-response on sheep peripheral blood mononuclear cells proliferation, Bax/Bcl-2 ratio, and cytokines secretion. Wine lees (WL) of local organic farming from white wine, produced with Trebbiano cultivar, rosé and red wine, made with Nero di Troia cultivar, were collected. The WL were subjected to microwave-assisted extraction using 2 green solvents (water and ethanol) in 3 different combinations (water; water/ethanol 1:1 vol/vol; ethanol) with a dry matter-to-solvent ratio of 1:40 (wt/vol) at 4 temperature levels: 50, 100, 150, and 200°C. Sodium carbonate Na2CO3 (2 mmol/g of dry weight of lees) was used for increasing the polyphenol extraction yield. A total number of 6 extracts, 2 for each kind of WL investigated, according to their total phenolic content and in vitro antioxidant capacity, were selected to be tested on sheep peripheral blood mononuclear cells, as an animal model. All the WL extracts demonstrated a strong antiproliferative action. On the contrary, the cytokines' profile was mainly dependent on the different winemaking derived WL and the extraction solvent combination procedures. Red WL extract obtained by a combination of water/Na2CO3 and tested at 0.8 mg/mL, resulted in an increase of both IL-6 secretion and Bax/Bcl-2 ratio. Data from the present study demonstrated that WL extracts derived from different winemaking and solvent extraction could have a bimodal action on control of inflammatory mediated damage and highlighted the importance for further studies aimed at applying the biorefinery process on by-products to increase their economic value and exploit new derived bioactive compound.

Peripheral blood mononuclear cell proliferation and cytokine production in sheep as affected by cortisol level and duration of stress.

A large number of studies recognize glucocorticoids (Gc) as suppressors of inflammation; Gc exert an important role in coordinating the magnitude and duration of host immune responses. In the present in vitro investigation, we tested incremental levels of cortisol to verify the immunosuppressive or immunopermissive role of cortisol in sheep peripheral blood mononuclear cells (PBMC) after acute and chronic stress. Phytohemagglutinin (PHA)-stimulated PBMC were cultured for 24h and 96h at 37°C with 5% of CO2 and varying cortisol levels: 10 ng/mL (baseline), 100 ng/mL (physiological poststressor), and 1,000 ng/mL [hyperactivated hypothalamic-pituitary-adrenal (HPA) axis]. The cell-free supernatants were collected for determination of IL-6, IL-1ß, and IL-10 by ELISA, and the bromodeoxyuridine assay was performed on cells. Physiological cortisol concentration negatively affected the levels of IL-6 secreted by PBMC, resulting in increased cell proliferation after acute stress (24h of incubation). However, physiological cortisol concentration exhibited a reduction in cell proliferation induced by increased levels of IL-6 secreted by PBMC during chronic stress (96h of incubation). The cortisol concentration representing a hyperactivated HPA axis led to a reduction in cell proliferation after acute stress, which was probably induced by the elevated IL-10 production. Our results demonstrate that in sheep the effect of Gc on the immune system was related to the magnitude and the duration of stress. In particular, cortisol levels higher than physiological concentrations suppressed cell proliferation soon after acute stress. Instead, the physiological poststressor concentration of cortisol affected the immune responses in a bidirectional manner depending on the duration of the stressor.