The dichloromethane extract of Callicarpa longissima rich in diterpenoid phenols exerts anti-inflammatory effect via inhibiting the TLR4/NF-κB signaling pathway

Ethnopharmacological relevance: Callicarpa longissima is a typical Yao ethnomedicine that has been used to treat arthritis in China. Our previous study found that the dichloromethane extract (DCME) of C. longissima showed anti-inflammatory activity in vitro. However, the anti-inflammatory mechanism and detailed chemical composition of DCME remain unclear, which lead to the original interest of this study. Aim of the study: The study aimed to evaluate the anti-inflammatory properties of the DCME from C. longissima and further explore the accurate chemical components responsible for this active extract. Materials and methods: The anti-inflammatory activity of DCME in vivo was tested with carrageenan-induced mice paw edema model. Its anti-inflammatory mechanism was explored with LPS-stimulated RAW264.7 macrophages model. The compounds in DCME were isolated by repeated column chromatography and their structures were identified on the basis of nuclear magnetic resonance spectroscopy. The anti-inflammatory activities of the isolates in vitro were also tested by suppressing releases of inflammatory mediators (NO, IL-6 and TNF-alpha) in RAW264.7 macrophages model. In addition, the molecular docking analysis, which evaluated the potential interaction between the compounds and Toll-like receptor 4 (TLR4) and nuclear factor xB (NF-xB), was performed. Results: DCME effectively alleviated the mice paw edema induced by carrageenan. In LPS-stimulated RAW264.7 cells, DCME significantly decreased the production of interleukin (IL)-6 and tumor necrosis factor alpha (TNF-alpha) via inhibiting their mRNA transcription, down-regulated the expression of TLR4 and myeloid differentiation factor 88, inhibited the phosphorylation of alpha inhibitor of NF-xB (IxB alpha), NF-xB p65, and degradation of IxB alpha. Twelve diterpenoid phenols were identified from DCME, and they not only showed different inhibitory effects on the production of NO, IL-6 and TNF-alpha in LPS-stimulated RAW264.7 cells, but also could bind to TLR4 and NF-xB as analyzed by molecular docking. Conclusions: Taken together, DCME from C. longissima could inhibit inflammatory response both in vitro and in vivo, which is mainly attributed to the synergistic effect of abundant diterpenoid phenols through inhibiting the TLR4/NF-xB signaling pathway, and might be a promising agent for the treatment of inflammatory diseases.