Structure-function relationships of Alpinia oxyphylla Fruit polysaccharides via different extraction methods: IL-22 pathway activation mediates enhancement of intestinal barrier protection by ultrasound extraction

• AFP-U features rhamnogalacturonan-I and homogalacturonan domains alongside glucan motifs, distinct from those by enzymatic or hot water extraction. • TOPSIS analysis identified AFP-U (50 μg/mL) as the optimal treatment for intestinal barrier protection. • AFP-U enhances intestinal repair in colitis by activating the IL-22 pathway and restoring the mucus layer. • Extraction methods define polysaccharide structure and bioactivity, far beyond just determining the yield. The bioactivity of Alpinia oxyphylla fruit polysaccharides (AFP) is closely linked to their extraction-determined structural properties. This study systematically compared three extraction methods (ultrasound-assisted enzymatic (AFP-UE), pure ultrasound (AFP-U), and hot water (AFP-H)) for their efficiency in extracting AFP and influencing their structural features and intestinal barrier protective effects. Although AFP-UE yielded the highest extraction efficiency (4.75 %) and exhibited predominantly a neutral arabinogalactan, it was AFP-U (comprises mixed rhamnogalacturonan-I and homogalacturonan domains alongside notable glucan motifs) that demonstrated the most potent intestinal barrier protection in a dextran sulfate sodium (DSS)-induced zebrafish colitis model. At 50 μg/mL, AFP-U significantly reduced the pro-inflammatory cytokine TNF-α, while upregulating anti-inflammatory cytokine IL-4, tight junction protein ZO-1, and mucin 2. Mechanistically, AFP-U is very likely to mainly alleviated oxidative stress and promoted intestinal barrier repair primarily by activating the IL-22 pathway, as evidenced by enhanced IL-22/IL-10 expression and suppression of IL-22 Receptor Subunit Alpha-2 (IL-22RA2). These findings highlight the critical role of extraction methods in shaping polysaccharide structure and bioactivity, positioning pure ultrasound extraction as a green and efficient strategy for producing functional polysaccharides with enhanced gastrointestinal health benefits.