RESUMEN
A total of 17 structurally diverse clerodane diterpenoids, including ten undescribed clerodane diterpenoids (tinopanoids K-T, 1-10) and seven known compounds (11-17), were isolated from the vines and leaves of Tinospora crispa. Compound 3 has not only bear the dominant substituents of γ-hydroxy-α, ß-unsaturated-γ-lactone with anti-inflammatory activity, but also a ternary epoxy structure at C-3/C-4. The planar structures and relative configurations of the clerodane diterpenoids were elucidated by spectroscopic data interpretation. The absolute configurations of compounds 1, 4, 8 and 13 were determined by single-crystal X-ray crystallographic, while that of compound 3 was determined using computed ECD data and single crystal X-ray diffraction of related p-bromobenzoate ester (3a). Subsequently, all compounds were evaluated for their inhibitory effect on nitric oxide (NO) production of LPS-activated BV-2 cells, and compounds 3 and 8 exhibited better NO inhibitory potency, with IC50 values of 5.6 and 13.8 µM than the positive control minocycline (Mino, IC50 = 22.9 µM). The corresponding results of western blot analysis and qRT-PCR revealed that compound 3 can significantly inhibit the inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) protein expressions, mRNA levels of pro-inflammatory cytokins of tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6) and interleukin 1ß (IL-1ß). The underlying mechanism by which compound 3 exerted anti-neuroinflammatory effects was investigated by western blot and immunofluorescence assay, which suggested compound 3 inhibited LPS induced neuroinflammation via the suppression of toll-like receptor 4 (TLR4) dependent Signal Transducer and Activator of Transcription 3 (Stat3) and mitogen-activated protein kinase (MAPK) signaling pathways, and the activation of Heme Oxygenase-1 (HO-1) mediated signals.
