A Comparison Study on Polysaccharides Extracted from Citrus reticulata Blanco cv. Tankan Peel Using Five Different Methods: Structural Characterization and Immunological Competence

This is the first work to screen an optimal extraction method for <i>Citrus reticulata</i> Blanco cv. Tankan peel polysaccharides (CPP). The CPP was extracted using hot water extraction (HWE), acid extraction (AAE), enzyme extraction (EAE), high-pressure extraction (HPE), and ultrasound extraction (UAE), named CPP-W, CPP-A, CPP-E, CPP-P, and CPP-U, respectively. Results showed that CPP-A and CPP-P had higher extraction yields than other CPPs. The five CPPs varied chemically in molecular weight, monosaccharide composition, and microstructure, but shared similar IR spectra and core glycosidic linkages, indicating differential degradation while preserving core structures during extraction. Among these CPPs, CPP-A, CPP-E, and CPP-U exhibited stronger immunological activities, attributed to high galacturonic acid and low molecular weight. Moreover, CPPs significantly promoted secretion of cytokines (nitric oxide, NO; prostaglandin E₂, PGE₂; interleukin-6, IL-6; tumor necrosis factor-α, TNF-α) by activating downstream inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2)-related mitogen-activated protein kinases (MAPK) pathways. Overall, CPP-E possessed high extraction yield, low molecular weight, and strong immuno-stimulatory activity, suggesting that enzyme-assisted extraction was the optimal approach for extracting CPP.