Anti-oxidant and anti-inflammatory effect of polar extracts obtained from waste product of wine making

Abstract A lot of diseases are characterized by an increased inflammatory response with an exacerbated production of free radicals. The anti-inflammatory effect of different compounds with antioxidant capacity, as polyphenols present in grape is well known. Therefore, the objective is to evaluate the anti-oxidant and anti-inflammatory activity of waste product of wine production. Six different non-toxic-marc-polar extracts from Malbec and Syrah grape varieties were obtained, their total phenol and flavonoid content were evaluated, and their antioxidant and anti-inflammatory activity were determined. High content of total phenols and flavonoids were found mainly in extracts obtained from Syrah (80.51 ± 16.63 g equivalent to gallic acid/100 g and 25.47 ± 3.33 g equivalent to quercetin/100 g). In addition, they had a high antioxidant effect (above 88.5% of ABTS inhibition by Syrah extracts). Finally, all extracts decreased the nitric oxide (NO) production, but this was more accented when extract from Syrah obtained by infusion was used, which decreased NO levels to baseline (4.46 µM). Taking together, our results show the potential pharmaceutical use of waste product of wine making to prevent or to treat diseases which inflammatory response is exacerbated. Graphical Abstract


Introduction
The immune system protects of infections through an acute inflammatory response, characterized by an increase in pro-inflammatory cytokines and reactive oxygen and nitrogen species production (ROS and NOS) (Warnatsch et al. 2017). However, some diseases are characterized by a chronic inflammation response, and there is an exacerbated production of nitric oxide (NO) (Iwakiri and Kim 2015;Soufli et al. 2016;Torrisi et al. 2016). Mortality and morbidity caused by diseases characterized by chronic inflammatory response are on the rise, triggering increasingly human and economic losses (WHO 2018). For that, new therapies and preventive strategies are necessary.
There are a lot of studies about beneficial effect of consumption of foods on our health (Pejin et al. 2019). Specifically, food with large amount of anti-oxidant and antiinflammatory compounds could have positive effect against chronic inflammatory diseases (Pisoschi and Pop 2015;-Dordevi c, Todorovic et al. 2018). It has been widely demonstrated that grape and wine are rich in phenolic compounds with anti-oxidant capacity (Lingua et al. 2016 . During wine production, high amount of marc are thrown away; consequently, its use as pharmacological purpose could be a good option to diminish these waste products and give them added value.

Total phenolic and flavonoid content of grape marc extracts
Marc extracts from Syrah variety presented higher both total phenols and flavonoids content than extract from Malbec variety obtained using the same method of extraction. In respect of the methods of extraction used, methanol was which generated extracts with more phenols and flavonoids contents (Table S1).

Anti-oxidant activity of Grape-Marc extracts
Extracts from Syrah variety had the most anti-oxidant effect, reaching values near to 92% of inhibition of ABTS þÁ radical (Table 1).

Anti-inflammatory activity of Grape-Marc extracts
The effect of extracts on nitrite accumulation in the supernatant of RAW 264.7 cells stimulated with lipopolysaccharide (LPS) was used as a parameter of anti-inflammatory activity ( Figure S1). All of extracts decreased nitrite accumulation after 24 h of incubation using at least 1 mg/mL (p ˂ 0.001). Most of them also generated a statistically significant reduction when low concentrations were used. Syrah Aqueous Infusion (SAI) was the extract which generated the highest reduction, reaching basal levels of nitrites accumulation when cells were incubated with 1 mg/mL ( Figure S1A).
Our results show that extracts of grape marc from Syrah variety had the most total phenols and flavonoids contents, and anti-oxidant and anti-inflammatory activities, these results were more evident when SAI extract was used. Harbeoui found that grape seed extract from different varieties as Syrah exhibited a high total phenolic content, with significant anti-oxidant and anti-inflammatory effects on LPS-activated macrophages (Harbeoui et al. 2019). Accordingly, there are many studies that show biological effect of natural phenols and flavonoids present in grapes and wine due to their anti-oxidant and anti-inflammatory effects ( Moreover, there are a large number of reports related to NO production and modulation of molecular mechanisms that regulate inflammation vias (Lee et al. 2011;Van den Bossche et al. 2016). Hence, anti-inflammatory compounds that decrease NO production, could have a potential therapeutic effect to treat diseases which are characterized by a chronic inflammatory response. In addition, it has been demonstrated that these extract exhibited non toxic effect both in vitro and vivo (Germano et al. 2017).

Conclusions
Extracts obtained from waste natural products from wine making could be good candidates for pharmaceutical purposes due to their high contents of poly-phenols, flavoinoids and anti-oxidant and anti-inflammatory effects.

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