The flavonoid-enriched extract from the root of Smilax china L. inhibits inflammatory responses via the TLR-4-mediated signaling pathway

Ethnopharmacological relevance: Smilax china L. has been used clinically to treat various inflammatory disorders with a long history. Aim of the study: To investigate the mechanisms underlying anti-inflammatory action of the extract from the herb. Materials and methods: The extract was identified and quantified using the Ultra Performance Liquid Chromatography-Photo Diode Array-Mass Spectrometer method. The anti-inflammatory activities were examined in xylene-induced mouse ear edema and cotton ball-induced rat granuloma. The inflammatory mediators, pro-inflammatory cytokines and TLR-4-mediated signals in LPS-stimulated RAW264.7 macrophages were determined using ELISA, real-time PCR, Western blot and/or immunofluorescence, respectively. Results: The extract was found to enrich flavonoids (44.3%, mainly astilbin, engeletin, isoastilbin, cinchonain Ia, quercetin-3-O-a-L-rhamnopyranoside and chlorogenic acid). The flavonoid-enriched extract (FEE) inhibited xylene-induced mouse ear edema and cotton ball-induced rat granuloma, and suppressed LPS-induced overrelease and/or overexpression of tumor necrosis factor-alpha, cyclooxygenase-2, inducible nitric oxide synthase, interleukin-1 beta and interleukin-6 in RAW264.7 macrophages. Mechanistically, FEE suppressed protein overexpression of TLR-4 and its downstream signals, MyD88 protein, phosphorylated inhibitory kappa B-alpha, NF-kappa B-P65 and MAPK p38, as well as phosphorylation of phosphoinositide 3-kinase (PI3K) p85 alpha at Tyr(607) and Akt at Ser(473) in LPS-stimulated macrophages. The mode of the anti-inflammatory action of FEE was similar to that of TAK-242 (a selective TLR-4 inhibitor). Conclusions: The present results demonstrate that FEE inhibit inflammatory responses via the TLR-4-mediated signaling pathway. Our findings go a new insight into the mechanisms underlying anti-inflammatory action of the herb, and provide a better understanding of its use for inflammatory diseases.