Tinopanoids K-T, clerodane diterpenoids with anti-inflammatory activity from Tinospora crispa

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 gamma-hydroxy-alpha, beta-unsaturated-gamma-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 mu M than the positive control minocycline (Mino, IC50 = 22.9 & mu;M). 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-alpha (TNF-alpha), interleukin 6 (IL-6) and interleukin 1 beta (IL-1 beta). 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.

Automated summary

A total of 17 structurally diverse clerodane diterpenoids were isolated from Tinospora crispa, including ten newly described compounds. Compound 3 exhibited anti-inflammatory activity with a ternary epoxy structure at C-3/C-4. The structures and configurations of these compounds were determined using spectroscopic data interpretation and single-crystal X-ray crystallography. Compound 3 showed inhibitory effects on neuroinflammation by modulating TLR4, Stat3, MAPK, and HO-1 signaling pathways.