Abstract
A purified polysaccharide with a galactose backbone (SPR-1, Mw 3,622 Da) was isolated from processed Polygonati Rhizoma with black beans (PRWB) and characterized its chemical properties. The backbone of SPR-1 consisted of [(4)-β-D-Galp-(1]9 → 4,6)-β-D-Galp-(1 → 4)-α-D-GalpA-(1 → 4)-α-D-GalpA-(1 → 4)-α-D-Glcp-(1 → 4,6)-α-D-Glcp-(1 → 4)-α/β-D-Glcp, with a branch chain of R1: β-D-Galp-(1 → 3)-β-D-Galp-(1→ connected to the →4,6)-β-D-Galp-(1→ via O-6, and a branch chain of R2: α-D-Glcp-(1 → 6)-α-D-Glcp-(1→ connected to the →4,6)-α-D-Glcp-(1→ via O-6. Immunomodulatory assays showed that the SPR-1 significantly activated macrophages, and increased secretion of NO and cytokines (i.e., IL-1β and TNF-α), as well as promoted the phagocytic activities of cells. Furthermore, isothermal titration calorimetry (ITC) analysis and molecular docking results indicated high-affinity binding between SPR-1 and MD2 with the equilibrium dissociation constant (K D) of 18.8 μM. It was suggested that SPR-1 activated the immune response through Toll-like receptor 4 (TLR4) signaling and downstream responses. Our research demonstrated that the SPR-1 has a promising candidate from PRWB for the TLR4 agonist to induce immune response, and also provided an easily accessible way that can be used for PR deep processing.