The aim of this study was to determine the anti-inflammatory potential of pomegranate peel extracts (PPE) prepared from waste material of pomegranate juice production both in vitro on Caco-2 cells and ex vivo using porcine colonic tissue explants. Caco-2 cells were stimulated in vitro by TNF and colonic tissue explants were stimulated ex vivo with lipopolysaccharide (LPS). Both tissues were co-treated with PPE at 0, 1.0, 2.5, 5.0, 10 and 25 μg/mL. The secretion of CXCL8 in the supernatant of both experiments was determined by enzyme linked immunosorbent assay (ELISA) and the relative expression of inflammatory cytokines were evaluated in the colonic tissue by quantitative polymerase chain reaction (QPCR). The 2.5 to 25 μg/mL of PPE suppressed CXCL8 (p < 0.001) in the Caco-2 cells, whereas CXCL8 production was suppressed by only 5 and 25 μg/mL (p < 0.01) of PPE in the colonic explants. The 5 μg/mL of PPE also suppressed the expression of IL1A (p < 0.05), IL6 (p < 0.01) and CXCL8 (p < 0.05) in LPS challenged colonic tissues compared to controls. In conclusion, the 5 μg/mL of PPE consistently elicits strong anti-inflammatory activity. These results support the potential of bioactive compounds from the waste peel of pomegranate in terms of their anti-inflammatory activity in cells and tissues of the gastrointestinal tract.

Pomegranate peel extracts (PPE) were tested for the first time on BME-UV1, a valid cellular model to study the bovine mammary epithelial metabolism, to evaluate the effects on the oxidative stress and inflammatory status. Based on the statistical analysis of MTT data, PPE at 0.1, 1.0 and 10 μg/mL resulted not cytotoxic after 24 h, 48 h and 7 days of treatment. At the same concentrations, PPE induced a reduction of ROS production elicited by the addition of hydrogen peroxide or lipopolysaccharide evidencing an antioxidant effect confirmed also by a decrease of malondialdehyde. At 10 μg/mL, PPE reduced pro-inflammatory cytokines expressions showing an anti-inflammatory effect on BME-UV1 treated with lipopolysaccharide. Although in vivo experiments are necessary, the results of this study are promising for future applications of PPE as feed supplement for dairy cattle, in particular around calving, when the animals are more subject to oxidative stress and inflammatory diseases. Graphical Abstract

Fitzroya cupressoides (Molina) I. M. Johnst. and Austrocedrus chilensis (D. Don) Pic.Serm. & Bizzarri are two Chilean Cupressaceae that are naturally resistant to biodegradation. Secondary metabolites from these species display a variety of biological activities. To evaluate the antiproliferative activity of two lignans, a diterpene and a flavonol isolated from A. chilensis and F. cupressoides, to elucidate their cytological effects on P3X murine myeloma cells. The antiproliferative activity of yatein, isotaxiresinol, ferruginol, and isorhamnetin was evaluated in vitro using the MTT assay. The effect of yatein at the cellular level, due to its high antiproliferative activity was evaluated. P3X cells treated for 24 h with 12.5 and 25 µg/mL of yatein were also examined at the cytological level using immunofluorescence and scanning and transmission electron microscopy. Yatein, a lignan isolated from A. chilensis, potentially inhibited P3X murine myeloma cell proliferation, resulting in approximately 75% cell death in response to a 25 µg/mL treatment with the lignan. P3X cells lost membrane integrity at the nuclear and cytoplasmic levels, including organelles, in response to yatein treatment (12.5 µg/mL), and we observed changes in the cytoplasmic organization and distribution of microtubules. The other compounds tested had low activity. Yatein is a lignan precursor of podophyllotoxin, a key agent in anticancer drugs. Due to its structural similarities to podophyllotoxin, yatein could have similar cytoplasmic target(s), such as the microtubular apparatus. These findings suggest that yatein may be of potential pharmacological interest and warrants further investigation in human cell lines.

Pomegranate peel extracts prepared in our laboratories from a waste of juice fruit processing were tested on bovine peripheral blood mononuclear cells to evaluate the effects on viability, oxidative stress and proliferation. The (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay pointed out that the extracts were not cytotoxic at the tested concentrations (0.1, 1.0, and 10 µg/mL). A moderate protective effect against Reactive Oxygen Species production induced by hydrogen peroxide or lipopolysaccharide and a significant anti-proliferative activity against proliferation induced by concanavalin A were observed on cell lines treated with the extracts at 10 μg/mL. Based on these results, pomegranate peel extracts seem promising as feed supplement for dairy cattle, in particular around calving, when the animals are subjected to an increase of the metabolic activity, responsible for oxidative stress and diseases. However, in vivo studies are needed to investigate the stability of the extracts across the bovine gastrointestinal tract barrier. Graphical Abstract

Ethanolic extracts of Salvia officinalis leaves demonstrated antibacterial activity against Bacillus cereus. Fractionation of the extracts led to the isolation of the most active antibacterial compound, which, from spectroscopic and LC-MS evidence, was proved to be the diterpene, methyl carnosate.

In this in vitro study, for the first time was evaluated the antioxidant and anti-inflammatory effect of an Oleuropein-enriched extract (OleE) on bovine mammary epithelial cell line (BME-UV1). OleE was obtained from olives leaves and characterized by HPLC and NMR analysis. Cell viability test indicated that OleE at concentrations of 7.8 up to 250 μg/mL did not exert cytotoxic effect. At concentration of 31.2 up to 250 μg/mL, a dose dependent reduction of ROS production induced by hydrogen peroxide was observed. In addition, OleE at 62.5, 125 and 250 μg/mL showed a dose-dependent reduction in gene expression of TNF, IL1B, and IL10 after exposure to LPS. The downregulation of ROS production and cytokines expression in BME-UV1 by OleE confirmed the antioxidant and anti-inflammatory properties. In vivo experiments will be necessary for future applications of OleE as natural feed supplement in dairy cattle to reduce incidence of oxidative stress in peripartal period. Graphical Abstract Note: Visual Abstract Replacement Requested.