Stramoniumlides A and B: Two new nitrogen-containing Withanolides from the leaves of Datura stramonium L.

Thirteen new withanolides, stramoniumlides A-M (1-13) alongside fourteen previously identified analogs from the extracts of Datura stramonium L. leaves. Notably, compounds 1 and 2 were nitrogen-containing withanolides that linked adenine and proline in steroids for the first time. Structural determination was accomplished through detailed spectroscopic analysis, including NMR and MS techniques, combined with comparative studies against published spectroscopic data for configuration assignment. Bioassay results showed that active ingredients 1, 2, 10, 22 and 26 inhibited NO production by LPS-stimulated BV2 cells to a greater extent than positive active ingredients (IC50 = 8.04 μM). Among them, compound 2 significantly suppressed the production of TNF-α and IL-6. In molecular docking analysis, compound 2 exhibited strong affinity for the TLR4 protein, with a binding energy of -11.2 kcal/mol, which was further supported by a KD value of 15.8 × 10-6 M in the isothermal titration calorimetry (ITC) assay. Concurrently, compound 2 was found to significantly inhibit the expression of TLR4, as demonstrated by immunofluorescence assay. In addition, Western blotting was employed to assess the suppression of the TLR4/MyD88/NF-κB pathway, thereby elucidating the molecular mechanism underlying the anti-inflammatory effect.