Isolation, purification, degradation of citrus pectin and correlation between molecular weight and their biological properties

Citrus pectin (CP) and two pectins with distinct molecular weights (CP-1 and CP-2) were prepared using ultrasound-assisted H₂O₂/ascorbic acid reaction, and their structural, anticancer, and immunostimulatory properties were investigated. Based on the methylation and nuclear magnetic resonance analyses, the structure of CP-2 was determined to be composed of an α-1,4-linked homogalacturonan backbone that exhibited high levels of O-6 methylation. In addition, both CP-1 and CP-2 induced apoptosis in various cancer cell lines, while CP exhibited minimal impact. All tested CPs activated RAW264.7 cells, promoting the production of nitric oxide (NO), tumor necrosis factor (TNF)-α, and interleukin (IL)-6 by upregulating the expression of TNF-α, IL-6, and inducible NO synthase genes. Among them, CP-2 exhibited the highest immunostimulatory activity closely linked with its molecular weight. Furthermore, we found that CP-2 could activate RAW264.7 macrophages through NOD-like receptor (NLR) signaling pathways involving nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways. In summary, the results of this investigation will enhance the comprehension of the structure-function correlation of CP, furthering its application within the functional food industry.