Modulation of LPS-induced inflammation in RAW264.7 murine cells by novel isoflavonoids from Millettia pulchra

Millettia pulchra is a renowned anti-inflammatory herbal medicine in southeast provinces of China. However, the underlying anti-inflammation mechanism remained incompletely understood. Herein, four new isoflavones, pulvones A-D and eleven reported constituents were isolated from the stems of Millettia pulchra with their structures being elucidated by HRMS and NMR analysis. The anti-inflammatory activities of pulvones A and C were further evaluated due to the better inhibitory activity on nitric oxide production in LPS-stimulated RAW264.7 cells and no obvious cytotoxicity to RAW264.7 cells. Western blot showed that pulvones A significantly decreased the levels of iNOS and COX-2 proteins and pulvones C only decreased the level of iNOS protein. ELISA analysis demonstrated that pulvones A inhibited the production of both interleukin-6 (IL-6) and IL-1 beta while pulvones C showed better suppression effect on IL-1 beta production in LPS-stimulated RAW264.7 cells. Then, their potential inhibitory effects on NF-kappa B pathway were tested in LPS-stimulated RAW264.7 cells. Immunofluorescence and western blot assay showed that pulvones A and C reduced the nuclear translocation of NF-kappa B(p65) and interrupted I kappa B phosphorylation. The ADP-Glo (TM) kinase assay showed pulvones A and C could directedly inhibit the IKK13 kinase activity with the inhibitory rate of 40%, which were also verified by docking study. Collectively, these results suggested that pulvones A and C's anti-inflammatory effects were relevant to the interruption of NF-kappa B activation by inhibiting IKK beta kinase.