Identification of anti-inflammatory fractions of Geranium wilfordii using tumor necrosis factor-alpha as a drug target on Herbochip® - an array-based high throughput screening platform

Background: Geranium wilfordii is one of the major species used as Herba Geranii (lao-guan-cao) in China, it is commonly used solely or in polyherbal formulations for treatment of joint pain resulted from rheumatoid arthritis (RA) and gout. This herb is used to validate a target-based drug screening platform called Herbochip(R) and evaluate anti-inflammatory effects of Geranium wilfordii ethanolic extract (GWE) using tumor necrosis factor-alpha (TNF-alpha) as a drug target together with subsequent in vitro and in vivo assays. Methods: A microarray-based drug screening platform was constructed by arraying HPLC fractions of herbal extracts onto a surface-activated polystyrene slide (Herbochip(R)). Using TNF-alpha as a molecular probe, fractions of 82 selected herbal extracts, including GWE, were then screened to identify plant extracts containing TNF-alpha-binding agents. Cytotoxicity of GWE and modulatory effects of GWE on TNF-alpha expression were evaluated by cell-based assays using TNF-alpha sensitive murine fibrosarcoma L929 cells as an in vitro model. Results: The in vivo anti-inflammatory effects of GWE were further assessed by animal models including carrageenan-induced hind paw edema in rats and xylene-induced ear edema in mice, in comparison with aspirin. The hybridization data obtained by Herbochip(R) analysis showed unambiguous signals which confirmed TNF-alpha binding activity in 46 herbal extracts including GWE. In L929 cells GWE showed significant inhibitory effect on TNF-alpha expression with negligible cytotoxicity. GWE also significantly inhibited formation of carrageenan-induced hind paw edema and xylene-induced ear edema in animal models, indicating that it indeed possessed anti-inflammatory activity. Conclusion: We have thus validated effectiveness of the Herbochip(R) drug screening platform using TNF-alpha as a molecular target. Subsequent experiments on GWE lead us to conclude that the anti-RA activity of GWE can be attributed to inhibitory effect of GWE on the key inflammatory factor, TNF-alpha. Our results contribute towards validation of the traditional use of GWE in the treatment of RA and other inflammatory joint disorders.