During the screening of natural anti-inflammatory agent, we identified some C21-steroidal pregnane sapogenins or the derivatives to inhibit TLR2, TLR3, and TLR4-initiated inflammatory responses respectively. Treatment with active compounds 10, 2j and 3p failed to impact tumor necrosis factor-ot (TNF-a) induced nucleus translocation of NF-kappa B p65 subunit. However, these compounds regulated distinct canonical or non-canonical NF-kappa B family members. Ectopic expression of TNF receptor associated factor 6 (TRAF6) abrogated the inhibitory activity of the compounds on production of pro-inflammatory cytokines downstream of TLR4. These results suggested that compounds 10, 2j, and 3p suppressed TLRinitiated innate immunity through TRAF6 with differential regulation of NF-KB family proteins. (C) 2017 Elsevier Ltd. All rights reserved.