Inhibition of cytokine secretion by scrophuloside A3 and gmelinoside L isolated from Verbascum blattaria L. by high-performance countercurrent chromatography

The purpose of this study was to analyze the HPLC-DAD-ESI-Q-TOF-MS/MS profile of the methanolic extract obtained from the aerial parts of Verbascum blattaria L. and develop efficient high-performance countercurrent chromatography (HPCCC) methods for isolation of its major iridoid glycosides. Additionally, the potential anti-inflammatory activities of the extract and two of the isolated compounds were evaluated using a human neutrophil model. LC-MS analysis led to the identification of 35 constituents, of which 18 iridoids, eight saponins, three phenylethanoids, four flavonoids and two phenolic acids. Scropolioside F (A, 2 mg), scrophuloside A(3) (B, 16 mg) and gmelinoside L (C, 13 mg) were obtained from 300 mg of the crude methanolic extract by HPCCC with the help of two different biphasic solvent systems: n-hexane/ethyl acetate/n-butanol/water (1/2/1/2; v/v/v/v) and n-hexane/ethyl acetate/methanol/water (1/19/1/19; v/v/v/v). The crude methanolic extract (100 mu g/mL) down-regulated lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNF-alpha) release in a comparable extent to dexamethasone (25 mu M). Gmelinoside L (50 mu M) was more active than scrophuloside A(3), producing inhibition percentages of 43.52% and 41.28% on interleukin 8 (IL-8) and TNF-alpha level, respectively. The HPCCC methods developed in the current work may serve to large scale isolation of iridoid glycosides from Verbascum species or other iridoid abundant-genera.