The Isolation, Structural Characterization and Anti-Inflammatory Potentials of Neutral Polysaccharides from the Roots of Isatis indigotica Fort.

Polysaccharides have been assessed as a potential natural active component in Chinese herbal medicine with anti-inflammatory properties. However, the complex and indefinite structures of polysaccharides limit their applications. This study explains the structures and anti-inflammatory potentials of three neutral polysaccharides, RIP-A1 (M-w 1.8 x 10(4) Da), RIP-B1 (M-w 7.4 x 10(4) Da) and RIP-B2 (M-w 9.3 x 10(4) Da), which were isolated from the roots of Isatis indigotica Fort. with sequenced ultrafiltration membrane columns, DEAE-52 and Sephadex G-100. The planar structures and microstructures of RIP-A1, RIP-B1 and RIP-B2 were further determined by HPGPC, GC-MS, methylation analysis, FT-IR, SEM and AFM, in which the structure of RIP-A1 was elucidated in detail using 1D/2D NMR. The Raw 264.7 cells were used for the anti-inflammatory activity in vitro. The results showed that RIP-A1, RIP-B1 and RIP-B2 are all neutral polysaccharides, with RIP-A1 having the smallest M-w and the simplest monosaccharide composition of the three. RIP-A1 is mainly composed of Ara and Gal, except for a small quantity of Rha. Its main structure is covered with glycosidic linkages of T-alpha-Araf, 1,2-alpha-Rhap, 1,5-alpha-Araf, T-beta-Galp, 1,2,4-alpha-Rhap, 1,3,5-alpha-Araf and 1,6-beta-Galp with 0.33:0.12:1.02:0.09:0.45:11.41:10.23. RIP-A1 significantly inhibited pro-inflammatory cytokines (NO, TNF-alpha, IL-6 and IL-1 beta) and increased anti-inflammatory cytokines (IL-4) in LPS-stimulated RAW 264.7 cells. Moreover, RIP-A1 could significantly inhibit the mRNA expression of TNF-alpha, IL-6 and L-1 beta. It could also activate IKK, p65 and I kappa B alpha (the components of the NF-kappa B signaling pathway). In conclusion, the above results show the structural characterization and anti-inflammatory potentials of RIP-A1 as an effective natural anti-inflammatory drug.