Anti-proliferative effect of pomegranate peel extracts on bovine peripheral blood mononuclear cells (PBMCs)

Abstract 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


Introduction
Plants are a precious source of valuable compounds exhibiting biological and pharmacological effects as antioxidant, antimicrobial and anti-inflammatory activities. For these relevant properties, plant extracts have been used in the past against numerous disorders and diseases, and currently they are object of intense studies to validate their potential as therapeutic agents in the promotion of health and disease prevention (D'Eliseo et al. 2019;Frezza et al. 2019;Mastrogiovanni et al. 2019).
Polyphenols are secondary metabolites variously distributed in the plant kingdom "derived from the shikimate/phenylpropanoid and/or the polyketide pathway, featuring more than one phenolic unit and deprived of nitrogen-based functions" (Yoshida et al. 2016). They exhibit many beneficial effects for human health benefits including the antioxidant, anti-inflammatory and anticancer activity (Barontini et al. 2014;Bernini et al. 2015Bernini et al. , 2018 and can be extracted from plants, fruits, and vegetables. When they were recovered from agro-industrial waste and by-products using green extractive technologies, a virtuous process of reuse and valorization process with economic and environmental benefits can be realized according to the "circular economy" strategy (Bianco et al. 2006;Romani et al. 2016;Bernini et al. 2017).
Pomegranate (Punica granatum L.) is a fruit crop brought in the Mediterranean area by the Phoenicians over 4000 years ago, which over the time has had a strong increase. Fruits, peel and seeds are considerable sources of polyphenols as hydroxycinnamic acids, hydroxybenzoic acids, flavonoids and hydrolyzable tannins (Singh et al. 2018;Russo et al. 2019).
Despite the wide number of in vitro and in vivo studies of pomegranate extracts to demonstrate the beneficial effects on human health, little is reported in the veterinary field. Based on this lack of literature, we recently planned to investigate the role of natural extracts to prevent the onset of the oxidative stress on dairy cattle during the calving period, when the animals are subject to an increase of the metabolic activity (Sordillo and Aitken 2009) and to endocrine variations, which can produce immunosuppression and serious diseases (Lacetera 2012).
In this context, we recently published a study on the activity of pomegranate peel extracts (PPE), rich of punicalagins on the oxidative stress and inflammatory status induced on bovine mammary epithelial cells, BME-UV1 (Mastrogiovanni et al. 2018). The experimental results have showed that, at non-cytotoxic concentrations (0.1, 1.0, and 10 lg/mL), PPE produced a reduction of the oxidative stress and at 10 lg/mL of the inflammatory status on BME-UV1 (Mastrogiovanni et al. 2018).
Based on these preliminary data, as a part of our research focused on the evaluation of of PPE as food supplement for dairy cattle, in this study we investigated the viability, the protective effect against Reactive Oxygen Species (ROS) production induced by hydrogen peroxide (H 2 O 2 ) or lipopolysaccharide (LPS) and the anti-proliferative effect on bovine peripheral blood mononuclear cells (PBMCs) induced by concanavalin A (ConA). To the best of our knowledge, this is the first study reporting the effects of PPE on bovine PBMCs.

Results and discussion
PEE were obtained by a green extraction procedure based on the use of water (Imperatori et al. 2018). As depicted in Figure S1, the HPLC analysis evidenced that PPE resulted rich of punicalagin (a-and b-isomer: 31.5 and 57.2% w/w, respectively) together to minor components as aand b-punicalin (3.10% w/w), granatin B (1.48% w/w), ellagic acid and derivatives (6.25% w/w), gallic acid (0.38% w/w).
Therefore, the effects of PPE at these concentrations were evaluated on PBMCs after ROS production induced by H 2 O 2 or LPS. After 48 h of treatment, PPE at 10 lg/mL showed a protective effect against ROS production induced by H 2 O 2 (À8.6%) or LPS (À17.5%); conversely, at the lowest concentrations, PPE did not show significant differences compared to the control (Figure 2A).
Finally, we investigated the effect of proliferation in PBMCs induced by ConA and treated with 0.1, 1.0, and 10 lg/mL of PPE. The Figure 2B evidenced a significant decrease of proliferation; in particular, PPE at 10 mg/mL induced a reduction of 26%. In accordance with the literature data, this activity be could be related to punicalagin found in PPE (88.8%); in fact, it is described that this hydrolizable tannin is able to downregulate the IL-2 expression and cell proliferation via inhibition of NFAT activation showing a potent immune-suppressive activity (Lee et al. 2008).

Conclusions
This study has evidenced for the first time that PPE rich of punicalagins, obtained from a by-product of the juice industry, exhibited biological effects on bovine PBMCs, reducing ROS production induced by H 2 O 2 or LPS and proliferation induced by ConA. The protective effect against ROS production was modest while the anti-proliferative activity induced by ConA was significant. These data, combined to those obtained in our previous study (Mastrogiovanni et al. 2018), make PPE a promising ingredient of feed supplement in dairy cattle to prevent and counteract various immune system derived pathologies, in particular during the calving period. However, in vivo studies needed to investigate the bioavailability of PPE in relation to the oral route of administration and the stability across the bovine gastrointestinal tract barrier.

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