Antioxidant and anti-inflammatory effects of pomegranate peel extracts on bovine mammary epithelial cells BME-UV1

Abstract 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


Introduction
Pomegranate (Punica granatum L.) peel, a by-product of fruits processing, e.g. juice production, is a good source of bioactive phenolic compounds such as hydrolyzable tannins. Recent studies reported on the biological activity (Imperatori et al., 2018) and health benefits of both pomegranate peel extracts (PPE) and their components including the antioxidant and anti-inflammatory activities (El-Missiry, et al., 2015;Omar et al., 2016;Seok et al., 2018;Singh et al., 2018) which are relevant to prevent several serious diseases. However, to the best of our knowledge, the effects of PPE on bovine mammary cells have never been investigated so far.
Around calving, dairy cattle are subject to an intense metabolic activity, which could lead to an overproduction of Reactive Oxygen Species (ROS), harmful species responsible for the onset of the oxidative stress (Bernabucci et al., 2005). In addition, the animals are subject to endocrine and metabolic variations, which can cause inflammatory diseases (Lacetera et al., 2012) with both negative consequences on their health and economic losses for the farmers. In this scenario, natural compounds with antioxidant and anti-inflammatory activities are indeed promising candidates as feed supplement for dairy cattle in order to prevent and/or counteract periparturient related problems. This choice becomes even more attractive if bioactive compounds are recovered from agro-industrial by-products according to the recent "circular economy" strategy, which encourages the reuse and valorization of waste (Bernini et. al., 2018). Based on these considerations, the present work reported for the first time the evaluation of the activity of a standardized PPE on the oxidative stress and inflammatory status induced in bovine mammary epithelial cells BME-UV1.

Results and Discussion
PPE were obtained from fruits peel by an extractive process recently described by our group (Imperatori et al., 2018). The extract, characterized by HPLC/DAD/ESI-MS and 1 H-NMR analysis, resulted rich in hydrolyzable tannins, such as aand b-punicalagin, aand b-punicalin; minor components were granatin B, gallic acid, ellagic acid and derivatives ( Figure S1).
Cell viability was measured using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay on BME-UV1, a valid cellular model to study the bovine mammary epithelial metabolism (Ar evalo Turrubiarte et al., 2016). The statistical analysis of MTT data evidenced that PPE at 0.1 lg/mL, 1.0 lg/mL, 10 lg/mL for 24 h, 48 h and 7 days were not cytotoxic compared to the control. Only at high concentration (100 lg/mL), cell death was observed ( Figure S2). Subsequently, the antioxidant activity of PPE was evaluated on the bovine cell line by treatments at non-cytotoxic concentrations (0.1, 1.0, and 10 lg/mL); after 48 h hydrogen peroxide (H 2 O 2 ) was added to induce intracellular ROS production. Alternatively, cells were also treated with lipopolysaccharide (LPS) as indirect antioxidant agent. Experimental results evidenced that after 3 h, PPE at 1.0 lg/mL and 10 lg/mL showed a protective effect against ROS production induced by H 2 O 2 with a decrease of 14.3% and 42.2%, respectively, compared to the control. At the same time, PPE at 10 lg/mL produced a ROS decrease of 30% after the addition of LPS whereas at 1.0 lg/mL the extract resulted ineffective ( Figure 1A). In both experiments, PPE at 0.1 lg/mL did not produce ROS decrease. The antioxidant effect of PPE at 10 lg/mL was confirmed by the reduction of malondialdehyde (MDA) compared to the control (31.1%, p < 0.001, data not shown). In fact, MDA is one of the products deriving from lipid peroxidation induced by oxidative processes and thus widely used as biomarker (Ayala et al., 2014). Punicalagin, the main component of PPE, could be responsible of ROS and MDA decreases as demonstrated by both in vitro and in vivo experiments on Caco-2 intestine cell line and rats, respectively (Omar et al., 2016;El-Missiry, et al., 2015). Finally, the anti-inflammatory effect of PPE at 10.0 lg/mL was evaluated on BME-UV1. Bovine cells were treated with PPE for 48 h, then LPS was added to induce inflammation and after 3 h, cytokines mRNA expressions: TNF, IL1B, and IL10 were quantified. The experimental data evidenced that TNF, IL1B, and IL10 were significantly different compared to the control ( Figure 1B). In particular, TNF, IL1B and IL10 decreased of 18.0, 25.7 and 27.5%, respectively. The trend of these results for the pro-inflammatory cytokines TNF and IL1B is in accordance to already reported in the literature about their expression in RAW 264.7 macrophage murine cells treated with the same concentration of PPE (Du et al., 2018). In addition, the reduced anti-inflammatory cytokine gene transcription IL10 could be positively correlated with reduced transcription levels of pro-inflammatory cytokines during the inflammatory response (Dipasquale et al., 2018). Also the antiinflammatory property of PPE could be related to punicalagin as demonstrated on rats and primary human epidermal keratinocytes (El-Missiry et al., 2015;Seok et al., 2018).

Conclusions
This study has evidenced for the first time that standardized pomegranate peel extracts, obtained from a by-products of fruits processing, reduced the oxidative stress and inflammatory status induced in bovine mammary epithelial cells BME-UV1. These results are promising for a possible use of these extracts as feed supplement for dairy cattle, in particular in the periparturient period. However, further studies need to clarify the mechanisms of the observed effects and to investigate in vivo the fate of PPE across the gastrointestinal tract barrier after oral ingestion.

Disclosure statement
No potential conflict of interest was reported by the authors.