To promote the functional applications of lotus root polysaccharides (LRPs), the effects of noncovalent polyphenol binding on their physicochemical properties, as well as antioxidant and immunomodulatory activities, were investigated. Ferulic acid (FA) and chlorogenic acid (CHA) were spontaneously bound to the LRP to prepare the complexes LRP-FA₁, LRP-FA₂, LRP-FA₃, LRP-CHA₁, LRP-CHA₂ and LRP-CHA₃, and their mass ratios of polyphenol to LRP were, respectively, 121.57, 61.18, 34.79, 2359.58, 1276.71 and 545.08 mg/g. Using the physical mixture of the LRP and polyphenols as a control, the noncovalent interaction between them in the complexes was confirmed by ultraviolet and Fourier-transform infrared spectroscopy. The interaction increased their average molecular weights by 1.11~2.27 times compared to the LRP. The polyphenols enhanced the antioxidant capacity and macrophage-stimulating activity of the LRP depending on their binding amount. Particularly, the DPPH radical scavenging activity and FRAP antioxidant ability were positively related to the FA binding amount but negatively related to the CHA binding amount. The NO production of the macrophages stimulated by the LRP was inhibited by the co-incubation with free polyphenols; however, the inhibition was eliminated by the noncovalent binding. The complexes could stimulate the NO production and tumor necrosis factor-α secretion more effectively than the LRP. The noncovalent binding of polyphenols may be an innovative strategy for the structural and functional modification of natural polysaccharides.

Despite the interaction between polyphenols and polysaccharides in food products, their specific non-covalent interactions and effects on macrophage functions are not well understood. Therefore, the interaction and mechanism of purified lotus root polysaccharide (PLRP) with polyphenols, and the regulatory mechanisms of the PLRP-polyphenol complex on the macrophage functionals were studied. By combining ferulic acid (FA) and chlorogenic acid (CHA) with PLRP, the complexes PLRP-FA, PLRP-CHA and the physical mixtures PLRP&FA and PLRP&CHA were prepared, where their mass ratios of polyphenols to PLRP were 143.97 and 601.67 mg g⁻¹. Nuclear magnetic resonance (NMR), Fourier-transform infrared (FTIR), Ultraviolet (UV), and Transmission electron microscopy (TEM) analyses confirmed that PLRP and polyphenols may engage in non-covalent interactions via hydrogen bonds and hydrophobic interactions. We confirmed that non-covalent interactions led to high molecular weight, dense complexes. Both PLRP and its polyphenol complexes stimulated NO production by macrophages to varying degrees without exacerbating lipopolysaccharide-induced inflammatory responses. PLRP and PLRP-polyphenol complexes repaired cells with impaired antioxidant capacity, depending on doses. Those results indicated that after the combination of lotus root polysaccharide and polyphenol, the molecular weight and conformation changed significantly, which influenced the biological activity. RNA-seq analysis suggested that the regulatory mechanism of PLRP-polyphenol complex in macrophages may mainly involve oxidative phosphorylation, FoxO, TNF, IL-17, MAPK, NF-kappa B, and other signaling pathways. This study investigated the effects of polyphenol binding on the physicochemical characteristics and functional activities of polysaccharides, which provided references for the development of polysaccharide functional products and the control of nutritional quality.