Insight into structure-immunostimulatory activity relationship of rhamnogalacturonan-I enriched pectic polysaccharides from Tartary buckwheat leaf in cyclophosphamide-induced immunosuppressed mice

Tartary buckwheat leaf is a promising sustainable source of rhamnogalacturonan-I-rich pectic polysaccharides (TBP) with marked in vitro immunostimulatory activity. However, their in vivo efficacy and structure-activity relationships remain unknown. Here, we investigated TBP fractions differing in esterification degree, molecular mass, and branching in a murine immunosuppression model. All TBP fractions attenuated immunosuppression, an effect associated with modulating gut microbial homeostasis, enhancing levels of short-chain fatty acids (SCFAs), reinforcing gut barrier integrity, and improving systemic immune markers. Structural modifications augmented bioactivity; low-esterified TBP-D and low-molecular-mass, low-branching TBP-F outperformed native TBP. TBP-F had the strongest effects, significantly improving immune organ indices, cytokines, immunoglobulins, SCFAs, and tight-junction proteins. Microbiota analysis revealed that all TBPs enriched Muribaculaceae and decreased dysbiosis-associated taxa, with TBP-F most strongly suppressing detrimental bacteria. These results imply that TBPs with lower molecular mass, less branching, or reduced esterification possess superior immunostimulatory potency, supporting their development as immune-enhancing functional foods.