Immunoprotective potential of steamed Panax notoginseng saponins: chemical characterization and mechanistic insights via the TLRs/NF-κB pathway

Steamed Panax notoginseng (SPN) has long been consumed as a functional food for immune support, yet its active constituents and mechanisms remain incompletely defined. This study investigated the immunoprotective effects of SPN and its saponin-enriched extract (SPNS) in cyclophosphamide-induced immunosuppressive mice and explored the underlying mechanisms. High-performance liquid chromatography identified and quantified major saponins, revealing SPNS contained over eightfold higher total ginsenosides than SPN. Both SPN and SPNS significantly improved peripheral blood cell counts, restored spleen and thymus indices, enhanced splenic lymphocyte proliferation, and increased serum cytokines and lysozyme activity. Network pharmacology and molecular docking identified 20( S )-Rh 2 , 20( S )-Rh 1 , Rh 4 , and Rk 3 as representative immunomodulatory saponins, and predicted their interactions with key targets in the Toll-like receptors/nuclear factor-kappa B (TLRs/NF-κB) pathway. Experimental validation in macrophages confirmed pathway activation and cytokine gene upregulation. These findings demonstrate that SPN and its saponins modulate immune responses via the TLRs/NF-κB pathway. By linking chemical characterization with mechanistic validation, the study provides new insight into the bioactive basis of SPN, supporting the translational potential of SPN saponins as functional food ingredients for immune restoration. • SPN and its saponins ameliorate cyclophosphamide-induced immunosuppression. • SPN saponin-enriched extract is effective at a dose tenfold lower than SPN. • Saponins S -Rh 2 , S -Rh 1 , Rh 4 , and Rk 3 are identified as key active components. • TLRs/NF-κB signaling pathway is involved in immune regulation of SPN.