Sequential Fractionation of Opuntia ficus-indica (L.) Reveals Isorhamnetin Glycosides Associated with Reduced Hepatic Lipid Accumulation and Oxidative Stress

Metabolic dysfunction-associated fatty liver disease (MAFLD) is the most prevalent chronic liver disease worldwide, affecting 37–52% of the population and potentially progressing to cirrhosis and hepatocellular carcinoma. Effective treatments remain limited, making nutraceuticals such as flavonoids promising therapeutic alternatives. Opuntia ficus-indica (L.) (OFI) has demonstrated beneficial effects against MAFLD, although its active compounds remain unclear. In this study, the complexity of the OFI sample was addressed through fractionation by sequential exhaustive extraction (SEE), resulting in six fractions enriched with different compound families, and metabolic markers related to steatosis were evaluated. Butanol (BT), ethanol (ET), and water (WA) extracts reduced lipid accumulation by 18–21%. BT and WA fractions also improved glucose uptake and decreased ketone body and reactive oxygen species production. UPLC-MS analysis showed that the WA extract was rich in piscidic acid and exhibited the highest antioxidant activity, whereas the BT displayed the strongest antisteatotic effect. Glycosylated flavonoids, representing 78% of the BT, were the predominant compounds. Isorhamnetin-glucosyl-rhamnosyl-rhamnoside and isorhamnetin-glucosyl-pentoside were inversely associated with triglyceride release, oxidative stress, and ketone body production. These findings identify isorhamnetin derivatives as key bioactive compounds underlying OFI’s beneficial effects against hepatic steatosis.