In vitro antioxidant and anti-inflammatory activity of an oleuropein-enriched extract obtained from olives leaves on BME-UV1 cells

Abstract 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.


Introduction
In recent times, in all fields, the 'linear economy model', based on the traditional takemake-dispose linear production, is replacing with a 'circular economy model', focused on the 'zero waste concept' (Stahel 2016). Therefore, each productive process should be projected and realized in such a way that the waste derived from a primary production can become the raw materials of other processes. In this context, agroindustrial wastes are a valuable source of bioactive compounds that can be recovered and reused in different fields, including the feed industry (Mastrogiovanni et al. 2019;Mastrogiovanni et al. 2020).
Olea europaea L. is widely cultivated in the Mediterranean countries for the extraolive oil production. In the spring, pruning is a common management practice carried out on olive trees essentially to promote a healthy plant development, flowering, fruiting and to structure the trees into shapes suitable for an efficient harvesting. Due to this practice, olive leaves represent a waste. They constitute 25% in dry weight of the total pruning waste material that accounts for more than 1.5 Kg per each liter of olive oil produced. Olive leaves can be valorised, being rich in Oleuropein (Ole), a secoiridoid found in the Oleaceae family exhibiting antioxidant and anti-inflammatory properties .
Dairy cattle are likely to experience oxidative stress and inflammation during prepartum and early lactation that can have several detrimental effects on health status including an increased susceptibility to infections (Bernabucci et al. 2005). Consequently, the supplementation with natural antioxidant and anti-inflammatory compounds in dairy cattle nutrition could be beneficial, especially during periparturient period. For this reason, in this study an Oleuropein-enriched extract (OleE), obtained from olive leaves, was tested in bovine mammary epithelial cells (BME-UV1) for its potential to prevent or at least attenuate oxidative stress and inflammatory response induced by appropriate stimuli.
In vitro experiments were carried out using the bovine mammary epithelial cell line (BME-UV1), an effective cellular model for the study of bovine mammary epithelial metabolism. After OleE exposure, BME-UV1 cell viability was determined by MTT [3-(4,5-dimethylthiazol-2-ylÀ2,5-diphenyltetrazolium bromide] assay. Cells were treated with OleE at different concentrations (7.8, 15.6, 31.2, 62.5, 125, 250, 500, 1000 lg/mL) for 48 h. Cell viability was not significantly affected up to 250 lg/mL of OleE whereas cytotoxic effects were observed at concentrations of 500 lg/mL and 1000 lg/mL ( Figure S3). The non-cytotoxic concentrations of OleE were used for successive experiments. The effects of OleE on oxidative stress-induced damage of bovine mammary epithelial cells were addressed by treating in vitro cultured BME-UV1 cells with hydrogen peroxide (H 2 O 2 ). After a 48 h pre-treatment with OleE, H 2 O 2 (50 lM) was added to BME-UV1 cells to induce oxidative stress and stimulate the cellular response, which consists mainly in the production of reactive oxygen species (ROS). Experimental data demonstrated that OleE reduced of 9.8% ROS production already at the concentration of 31.2 lg/mL when compared with the control and further decreased of 16.2, 17.3 and 21.4% at concentrations of 62.5, 125 and 250 lg/mL, respectively (Figure 1). The OleE reduction of H 2 O 2 -induced ROS generation clearly suggests an antioxidant activity in BME-UV1 cells. In accordance with the literature data, this activity could be related to the high concentration (93.4%) of Ole found in OleE. In fact, Ole exerted a strong antioxidant activity on two human breast cancer cell lines MCF-7 and T-47D (Bulotta et al. 2011). Moreover, it reduced H 2 O 2 -induced DNA damage in human peripheral blood leukocytes and hepatocellular carcinoma (HepG2) cells suggesting that its effect might be attributed to its potential radical scavenging activity ( Cabarkapa et al. 2014).
In addition, OleE was accessed for anti-inflammatory potential by real time PCR evaluation of pro-and anti-inflammatory cytokines expression levels. The BME-UV1 cells pre-treated with OleE and then incubated for 3 h with lipopolysaccharides (LPS, from Escherichia coli 055: B5) to induce an inflammatory stimulus, showed a decrease in the mRNA expression of cytokines TNF, IL1B and IL10 compared with the control.  The mRNA levels of TNF, the main mediator of the pro-inflammatory response, are reduced by 70% using 62.5 lg/mL of OleE and IL1B mRNA levels of 40%, as well as IL10 mRNA, that normally acts by suppressing pro-inflammatory cytokines. At OleE concentration of 250 lg/mL, the transcript levels of TNF and IL10 were significantly lower than the control, confirming the potential of the OleE to prevent pro-inflammatory LPS-effect in BME-UV1 cells. OleE showed greater efficacy on the reduction of TNF expression levels compared to IL1B; indeed IL1B mRNA showed a reduction of 52% versus 90% of TNF mRNA compared to the control cells, at 125 lg/mL of OleE concentration. Overall, these results (Figure 2A, B and C) showed that OleE was able to reduce the TNF, IL1B and IL10 expression after LPS exposure in a dose-dependent manner and are indeed in line with the observed decrease in the expression of IL1B in LPS-stimulated macrophages Raw264.7 by Ole supplementation (Ryu et al. 2015). Moreover, the pro-inflammatory cytokine mRNA levels such as TNF and IL1B was suppressed by 10 lM Ole treatment in a time-dependent manner in LPS-treated BV-2 murine microglial cells (Park et al. 2017). Downregulation of TNF secretion in Polymorphonuclear cells culture in response to olive leaf extract treatment was observed (Qabaha et al. 2018). The authors indicated that this polyphenol-rich extract exhibited an anti-inflammatory effect, and Ole is the major component responsible for this property (Qabaha et al. 2018).

Conclusions
This communication described an example of valorization of olives wastes according to the 'circular economy model'. For the first time the in vitro antioxidant and antiinflammatory effect of an Ole-enriched extract (OleE), obtained from olive leaves through an eco-friendly procedure, was evaluated on BME-UV1 cells. The findings indicated that OleE reduced levels of ROS in cells and displayed anti-inflammatory effect by reducing the expression of pro-and anti-inflammatory cytokines in LPS-stimulated BME-UV1, demonstrating protection at multiple levels. Although in vivo experiments will be necessary, the experimental results described in the present study would suggest that OleE might be useful as feed supplement for dairy cattle, above all in the prevention of oxidant and inflammatory status during the periparturient period.

Experimental
All experimental data, Figures S1, S2, S3 and Table S1 are reported in the Supplemental Material.