Novel hasubanan alkaloids and dimers from Stephania japonica var. discolor: phytochemical profile and anti-neuroinflammatory activity

Using a ¹H-NMR-guided isolation approach, twelve novel hasubanan alkaloids, bishernsubanins A-D (1—4) and hernsubanines G-N (5—12), along with eight known monomeric analogues (13—20), were isolated from Stephania japonica var. discolor. Notably, compounds 1—4 represent a new class of homologous dimeric hasubanan alkaloids. Their structures were elucidated by comprehensive spectroscopic data analysis, including NMR, ECD calculations, and single crystal X-ray diffraction. Their biosynthetic pathways are proposed to involve redox reactions, free radical coupling, isomerization, and condensation reactions. All isolates were evaluated for anti-neuroinflammatory activity in vitro. Alkaloids 1, 3, 4, 7, and 11—13 exhibited better inhibitory effects on nitric oxide production in lipopolysaccharide (LPS) induced BV-2 microglial cells than minocycline. Among them, the new compound bishernsubanin A (1) could inhibit Iba-1 expression, and suppress the release of inflammatory factors in LPS stimulated BV-2 microglial cells. Molecular docking, immunofluorescence, and drug affinity responsive target stability (DARTS) studies indicated that compound 1 modulates toll-like receptor 4 to exert its anti-neuroinflammatory effect, positioning it as a promising naturally derived candidate for neuroinflammatory inhibition.