STUDY OF THE BIOLOGICAL ACTIVITY OF PLANT EXTRACTS OBTAINED FROM BIOMASSES FROM WASTES OF THE AGRO-FOOD CHAIN WITH INNOVATIVE AND SUSTAINABLE METHODOLOGIES: RELEVANCE FOR CARDIOMETABOLIC HEALTH

Introduction. Metabolic dysfunction-associated steatotic liver disease (MASLD) is an emerging global health concern closely linked to obesity, type 2 diabetes mellitus, and metabolic syndrome. It is also associated with atherosclerotic cardiovascular disease (ASCVD), sharing critical pathogenic mechanisms such as insulin resistance, chronic inflammation, and oxidative stress. Despite extensive research, effective pharmacological treatments for MASLD remain limited, underscoring the need for sustainable and innovative alternatives. Nutraceuticals derived from agro-food waste, rich in bioactive compounds, present a promising strategy aligned with Circular Economy, One Health principles, and the United Nations’ Agenda 2030. Aim of the project. This project aims at developing a sustainable nutraceutical from agro-food waste targeting MASLD and ASCVD. The objectives include identifying sustainable agro-food waste rich in bioactive compounds, optimizing eco-friendly enzymatic extraction, chemically and biologically characterizing the extract, and validating its therapeutic potential using an in vitro steatotic liver cell model (HepG2-OA). A Prunus domestica extract will be employed to validate the model prior to testing the final extract from Malus domestica ‘Cripps Pink’ and subsequent nutraceutical formulation. Materials and Methods. A comprehensive literature review supported the nutraceutical design targeting MASLD and ASCVD. Agro-food waste consisting of Malus domestica ‘Cripps Pink’ apples, sourced from a certified sustainable supply chain, was selected. Enzymatic extraction was optimized via pilot trials using a blend of cellulase, xylanase, and glucosidase, followed by dynamic extraction with the Naviglio Estrattore® system. The extract underwent chemical (Folin–Ciocalteu assay, HPLC), microbiological, and nutritional characterization. The HepG2-OA steatotic model was obtained by treating HepG2 cells with 100 µM oleic acid for 7 days and characterized via lipid quantification (Oil Red O staining, spectrophotometry), glucose uptake, reactive oxygen species production (flow cytometry), mitochondrial function (Seahorse XF analysis), gene expression (RT-qPCR), GLUT1 trafficking (immunofluorescence), and metabolomic profiling. A Prunus domestica extract was used to validate the HepG2-OA dysfunctional model, and the bioactivity of the Malus domestica ‘Cripps Pink’ extract was assessed on both models (HepG2 and HepG2-OA) through MTT assay (cell viability), intracellular lipid accumulation (Oil Red O staining and quantification), ROS production (flow cytometry), glucose uptake (6-NBDG uptake by flow cytometry), and expression of genes involved in lipid and glucose metabolism, and oxidative stress (RT-qPCR). Results. A sustainable agro-food supply chain located in Ferrara, Italy, was identified based on environmental certifications and proximity to processing facilities. Among available waste, Malus domestica ‘Cripps Pink’ apples, discarded primarily for aesthetic defects, were selected due to their high content of bioactive compounds. Literature screening and phytochemical profiling confirmed their richness in bioactive compounds relevant to MASLD management. Green extraction was optimized through enzymatic pre-treatment using a blend of cellulase, xylanase, and glucosidase (25 U/g), followed by Naviglio Estrattore® dynamic extraction. This method significantly improved total polyphenol content (1.5 mg/g), and HPLC revealed a broad spectrum of active compounds including chlorogenic acid (70.9 µg/g) and quercetin (16.5 µg/g). The extract showed high microbiological quality and a nutritional profile rich in sugars and low in fats, fiber, and sodium. The HepG2-OA steatotic-like model was obtained by oleic acid treatment and showed increased lipid accumulation, altered lipid composition (via ¹H NMR), disrupted mitochondrial function, reduced glucose uptake, ROS accumulation, upregulated genes linked to lipid metabolism (DGAT1, and FASN) and oxidative stress (SOD, CAT, and Nrf2), and downregulated expression of genes linked to glucose metabolism (GLUT1, and GLUT2). In HepG2-OA cells, immunofluorescence analysis revealed a redistribution of GLUT1 from the plasma membrane to intracellular compartments. Moreover, staining with anti-actin and DAPI highlighted a remodeling of the actin cytoskeleton compared to HepG2 cells. Metabolomic profiling confirmed altered energy, amino acid, and lipid pathways. The suitability of the cell model in assessing natural compounds was validated by treatments with the Prunus domestica extract, which modulated ROS levels, glucose uptake, lipid accumulation, and gene expression in both HepG2 and HepG2-OA cells. Finally, the Malus domestica ‘Cripps Pink’ extract was tested in HepG2 and HepG2-OA, showing good cytocompatibility (MTT), and significantly reducing lipid accumulation and oxidative stress production, while improving glucose uptake and modulating expression of target genes involved in oxidative stress and lipid and glucose metabolism. Discussion and future prospective. This study established a human-relevant in vitro model of MASLD and demonstrated that a bioactive extract derived from enzymatically processed Malus domestica ‘Cripps Pink’ by-products significantly reduced lipid accumulation, oxidative stress, and altered gene expression linked to steatosis. The extract showed potential to restore metabolic balance in steatotic hepatocytes, highlighting its promise as a sustainable nutraceutical candidate. Future research will focus on investigating its bio accessibility, systemic effects via gut–liver models, mitochondrial activity, and formulation development. Clinical validation and intellectual property protection are also planned to support translation into industrial applications.