Bioactive Ingredients in the Mediterranean Diet for Hepatic Health: In Vitro Evaluation of Selected Extracts Against Non-Alcoholic Steatosis

Natural products derived from the Mediterranean diet have attracted considerable interest as potential drug leads owing to their antioxidant and anti-inflammatory properties, as well as their beneficial effects on metabolic disorders such as non-alcoholic fatty liver disease (NAFLD). This study investigated the individual and combined effects of carvacrol, thymol, basil essential oil, and lycopene in an in vitro HepG2 model of hepatic steatosis. Their antioxidant activity was first confirmed by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical-scavenging assays. Treatments significantly reduced intracellular lipid accumulation and lipid peroxidation, as demonstrated by Oil Red O staining, LipidSpot™ immunofluorescence, and the TBARS assay. In addition, the natural compounds restored lipid homeostasis by upregulating the β-oxidation- and lipolysis-related markers PPARα, CPT1α, and ATGL, while downregulating PPARγ, a lipogenic regulator, and the pro-inflammatory enzyme 5-lipoxygenase. The combined formulation consistently exhibited the strongest biological activity, showing enhanced efficacy in reducing lipid accumulation, oxidative stress, and oxidative DNA damage, as evidenced by decreased 8-oxo-7,8-dihydroguanine (8-oxo-dG) formation. Overall, these results demonstrate that Mediterranean-diet-derived bioactives, particularly when used in combination, exert multitarget antioxidant activity by reducing oxidative damage, restoring lipid homeostasis, and modulating inflammatory pathways, supporting their potential application in natural product-based strategies for the prevention and management of NAFLD.