Optimization of Ultrasonic‐Assisted Extraction Process, Derivatives Preparation, Structural Characteristics, and Activities In Vitro of Polysaccharides From Olive ( <i>Olea europaea</i> L.) Leaves

Olive leaves, a major by-product of olive oil production, are an underutilized source of bioactive polysaccharides (olive leaves polysaccharide [OLP]). This study optimized the ultrasonic-assisted extraction (UAE) of OLP using response surface methodology, maximizing yield (4.28%) under conditions of a 1:30 g/mL solid-liquid ratio, 600 W power, and 3h extraction. The extracted polysaccharides (U-OLP) were chemically modified via phosphorylation, sulfation, and selenation. Structural analyses (Fourier transform infrared, UV, nuclear magnetic resonance) confirmed the successful introduction of functional groups. Monosaccharide composition analysis showed that both native and modified U-OLP contained the same eight monosaccharides (Man, GlcN, Rha, GlcA, GalA, Glc, Gal, and Ara), albeit in different proportions. Bioactivity assessments revealed that all modified derivatives, especially the sulfated form (U-OLP-S), exhibited significantly enhanced antioxidant (scavenging 1,1-diphenyl-2-picryl-hydrazyl, hydroxyl, and superoxide radicals) and hypoglycemic activities (inhibiting α-glucosidase and α-amylase) compared to the native U-OLP. Furthermore, in vitro cell assays demonstrated that U-OLP and its derivatives inhibited HepG2 cell proliferation and stimulated proliferation, phagocytosis, and nitric oxide release in RAW264.7 macrophages, with U-OLP-S showing the most potent immunomodulatory effects. The findings confirm that chemical modification, particularly sulfation, is a highly effective strategy for enhancing the bioactivity of olive leaf polysaccharides, broadening their application potential in functional foods and pharmaceuticals.