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 × 10⁴ Da), RIP-B1 (M_w 7.4 × 10⁴ Da) and RIP-B2 (M_w 9.3 × 10⁴ 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-α-Araf, 1,2-α-Rhap, 1,5-α-Araf, T-β-Galp, 1,2,4-α-Rhap, 1,3,5-α-Araf and 1,6-β-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-α, IL-6 and IL-1β) 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-α, IL-6 and L-1β. It could also activate IKK, p65 and IκBα (the components of the NF-κB 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.

Polysaccharides derived from Atractylodes chinensis (DC.) Koidz. (ACP), a traditional Chinese medicine, were extracted and analyzed for their structural characteristics and anti-gouty nephropathy (GN) activity. Sprague-Dawley (SD) rats were divided into six groups: control, model, positive control, and three treatment groups (ACP-60-L, ACP-60-M, and ACP-60-H). Treatment significantly reduced inflammatory responses and renal damage, as evidenced by decreased levels of uric acid (UA), creatinine (Cr), and blood urea nitrogen (BUN), alongside modulation of NOD-like receptor protein 3 (NLRP3) expression in renal tissues. ACP-60 was fractionated into three polysaccharides, including ACP-60-A (Mw 9.18 kDa), ACP-60-B (Mw 58.21 kDa), and ACP-60-C (Mw 109.01 kDa) using DEAE-52 cellulose column chromatography. Monosaccharide analysis revealed that ACP-60-A predominantly comprised fructose (Fru) and glucose (Glc), ACP-60-B contained rhamnose (Rha), galactose (Gal), Fru, and mannose (Man), and ACP-60-C included Man, Gal, Rha and xylose (Xyl). In vitro studies using HK-2 cells confirmed the anti-GN activity of all three fractions, with ACP-60-A demonstrating the highest efficacy. Structural elucidation of ACP-60-A identified its main glycosidic linkages as a →1)-β-Fruf-(2→ backbone with α-Glcp-(1→ and β-Fruf-(2→ branches. The underlying mechanism of ACP-60-A’s anti-GN activity is associated with inhibition of the NLRP3 inflammasome signaling pathway, suppression of downstream inflammatory factor release, and downregulation of NLRP3, ASC, and Caspase-1 protein expression. Further studies demonstrated that the superior activity of ACP-60-A is attributable to its lower molecular weight, specific monosaccharide composition, and unique glycosidic bond arrangement. ACP-60-A shows potential for increased anti-GN efficacy through purification or modification, laying the groundwork for developing novel therapeutic agents for GN.

The immunomodulatory potential of natural polysaccharides is often limited by their structural complexity and high molecular weight. In this study, DFIP-A3-1 (M_w = 8.68 × 10³ g/mol) was obtained from the leaves of Isatis indigotica Fort. by ultrafiltration, DEAE-650M, and Sephadex G-100 chromatography, followed by acid degradation. Fortunately, DFIP-A3-1 exhibited the most potent immunostimulatory activity in vitro. HPGPC, HPSEC-MALLS-RID, GC-MS, FT-IR, Congo red tests, SEM, and AFM were used to characterize their structure, and 1D/2D NMR was used for further investigation of DFIP-A3-1 for in-depth structural clarification. DFIP-A3-1 was primarily composed of Rha and Gal. Based on methylation and NMR analyses, the structure of DFIP-A3-1 was elucidated as follows: →1)-β-Galp-(4→1,4)-α-Rhap-(2→1,4)-α-Rhap-(2→1)-β-Galp-(6→1)-β-Galp-(6→1,6)-β-Galp-(3→1,6)-β-Galp-(3→. Furthermore, DFIP-A3-1 was found to exhibit a triple-helix conformation. DFIP-A3-1 markedly upregulated the secretion of NO, IL-6, and TNF-α and enhanced the mRNA expression levels of their related genes in RAW 264.7 cells. Moreover, DFIP-A3-1 activated p-IκBα, p-p65, and TLR4, while co-treatment with TAK-242 markedly suppressed the expression of these pathway-related proteins. All of the aforementioned findings suggested that DFIP-A3-1 is a promising natural immunomodulatory drug deserving of additional research and use.

Background: Hepatic fibrosis represents a critical pathological stage in the progression of chronic liver diseases, yet clinically effective and low-toxicity therapeutic options remain limited. Taraxacum mongolicum and Astragalus form a classic hepatoprotective herbal pair, and their derived polysaccharides- Taraxacum mongolicum polysaccharides (TMP) and Astragalus polysaccharides (APS)-both exhibit hepatoprotective activities. This study aimed to investigate the ameliorative effects of TMP combined with APS on CCl ₄ -induced hepatic fibrosis in mice and the gut-liver axis-associated changes. Methods: Mice were randomly assigned to the blank control, model, silybin, Lizhu Changle (live Bifidobacterium adolescentis capsules), TMP monotherapy, APS monotherapy, and combination groups. Following the establishment of fibrosis by CCl ₄ , a 4-week intervention was administered. Serum liver function parameters and fibrosis markers were measured, and hepatic histopathological changes were observed. Intestinal function was comprehensively evaluated. Gut microbiota composition was analyzed using 16S rRNA gene sequencing, and fecal short-chain fatty acid (SCFA) levels were quantified by high-performance liquid chromatography (HPLC). The protein expression of the hepatic TLR4/MyD88/NF-κB pathway was further assessed. Results: Compared with TMP or APS alone, the combination significantly improved multiple fibrosis-related, inflammatory, intestinal barrier, microbial diversity, and SCFA-related indices, although significant superiority was not observed for PC III and TNF-α. The combination also more strongly suppressed hepatic TLR4/MyD88/NF-κB pathway expression. Conclusion: TMP combined with APS exerts enhanced anti-hepatic fibrosis effects compared with either monotherapy. These effects are associated with changes in gut microbiota composition, elevated fecal short-chain fatty acid levels, reduced expression of hepatic TLR4/MyD88/NF-κB pathway-related molecules. These findings provide an experimental basis for further investigation of this herbal pair.