Discovery of novel adjuvants: Identification of saponins from Hylomecon japonica (Thunb.) Prantl & Kündig and insights into their in vitro and in vivo activities.

Bioassay-guided approach resulted in the isolation of fourteen triterpenoid saponins were isolated from the herba of Hylomecon japonica (Thunb.) Prantl & Kündig including eight novel saponins (1–8) and six known saponins (9–14). Their chemical structures were unequivocally determined through comprehensive spectral data analysis. By screening all compounds to enhance the ability of RAW 264.7 cells to produce NO, the novel compound 1 (HA) was identified as a candidate for immune adjuvant. In vitro experiments demonstrated that HA promotes the proliferation of spleen lymphocytes, DC 2.4, and RAW 264.7 cells, while also enhancing the phagocytic activity of DC 2.4 and RAW 264.7 cells. HA also counteracts the LPS-induced decline in dendritic cell phagocytosis and works with LPS to further improve macrophage phagocytosis. The network pharmacology analysis identified a total of 46 targets and 20 pathways through which HA exerts its immune-enhancing effects. In vivo experiments, HA enhanced immune organ development, boosted specific serum antibodies, increased Th1 and Th2 cytokines, and amplified related gene expression. And HA could boost lymphocyte proliferation, increased CD3⁺ T cells and altered CD4⁺ and CD8⁺ levels. It also promoted dendritic cell maturation in lymph nodes, raised MHC II molecules and co-stimulatory factors. HA triggered the upregulation of TL4, MyD88, and IKK proteins and promoted the phosphorylation of NF-κB P65 and P-IkBα within the TLR4/NF-κB signaling pathway, while simultaneously suppressing IκBα protein expression. HA has the characteristics and functions of enhancing immunity and could be applied as an immune adjuvant. The findings presented herein establish a fundamental basis for investigating the immune adjuvant effect of triterpenoid saponins.