Vegetal By-products of Sicilian industry as high healthy molecules: Extraction and biochemical characterization

The valorisation of agro-industrial by-products is a crucial step in advancing sustainability and promoting the circular economy. In this context, grape pomace, a by-product of the winemaking industry, represents a valuable source of bioactive compounds, including secondary metabolites as polyphenols. These compounds exhibit different biological activities, such as anti-inflammatory, antioxidant, and anti-cancer properties.This doctoral research, funded by the PON & RI 2014–2020 scholarship focused on green theme, explores innovative methods for the extraction and application of bioactive compounds from agri-food by products, and specifically from Sicilian white grape pomace. The study is divided into two main parts, combining traditional and innovative extraction techniques.In the first part, a hydroalcoholic extract (HE) from grape pomace was prepared and characterized. HE was found to be rich in phenolic compounds, including gallic acid, catechin, and epicatechin, as identified by UPLC-MS analysis. The biological activity of the extract was evaluated on two cancer cell lines, HCT116 (colon cancer cells) and MDA MB-231 (triple-negative breast cancer cells). Cytotoxicity assays demonstrated that HCT116 cells were more sensitive to HE compared to MDA MB-231. The study also revealed that HE triggered apoptosis in HCT116 cells through the activation of the caspase-3/PARP1 pathway. In contrast, MDA MB-231 cells displayed resistance to apoptosis and underwent a G0-G1 phase arrest, which was accompanied by elevated levels of p21. These effects were linked to the modulation of reactive oxygen species (ROS) levels, which induced distinct cellular responses. In HCT116 cells, ROS levels peaked early and decreased over time due to the upregulation of antioxidant proteins such as Nrf-2, SOD2, and HO-1. In MDA MB-231 cells, ROS levels remained persistently high, leading to the induction of pro-survival autophagy. The protective role of autophagy in MDA MB-231 cells was confirmed by the enhanced cytotoxicity of HE following treatment with the autophagy inhibitor bafilomycin A1.The second part of the study focused on a greener extraction technology, specifically supercritical fluid extraction and subcritical water extraction. While the first one proved inefficient in extracting polyphenols from grape pomace, the subcritical water extraction emerged as a promising alternative. The technique employs water in a subcritical state, in which high temperature and pressure is employed, in order to extract bioactive compounds, offering an eco-friendly approach that avoids the use of toxic organic solvents. LC-MS analysis of Subcritical Water Extracts (SWEs) revealed a high content of phenolic compounds, particularly gallic acid and protocatechuic acid. The extract obtained at a temperature of 160 °C exhibited a significant antioxidant effect, as well as selective cytotoxicity against HCT116 and MDA MB-231 cancer cells, reducing cell viability of about 70% and 40%, respectively, while leaving normal human bronchial epithelial (HBE) cells unaffected. Given these results, the antioxidant activity of the SWE was further studied on normal HBE cells. The subcritical water extract was able to counteract both the basal levels of ROS and those induced following the treatment with the specific ROS inducer sodium arsenite. This effect was attributed not only to its scavenging activity but also to the ability to stimulate the expression of the main cellular antioxidant systems.The findings of this research underscore the potential of grape pomace as a sustainable source of bioactive compounds. By integrating hydroalcoholic extraction with innovative green methods like subcritical water extraction, this study not only maximizes the value of winemaking by-products but also contributes to the development of eco-friendly technologies to produce new functional ingredients. The dual focus on anticancer and antioxidant activities highlights the therapeutic potential of grape pomace-derived extracts, opening new prospective for future applications in nutraceuticals and biomedical fields.