Screening and identification of Caulis Sinomenii bioactive ingredients with dual-target NF-κB inhibition and β2- AR agonizing activities.

Caulis Sinomenii (CS) is a valuable traditional medicine in China. Its extract can act as an anti-inflammatory agent and a vascular smooth muscle relaxant. However, the underlying mechanisms remain unknown. In this study, we developed a simple dual-target method based on ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry combined with a dual-target bioactive screening assay for anti-inflammatory and antispasmodic activities to characterize the chemical structure of various bioactive compounds of CS rapidly. Seven potential NF-κB inhibitors were identified, including laudanosoline-1-O-xylopyranose, 6-O-methyl-laudanosoline-1-O-glucopyranoside, menisperine, sinomenine, laurifoline, magnoflorine and norsinoacutin. Furthermore, IL-6 and IL-8 assays confirmed the anti-inflammatory effects of these potential NF-κB inhibitors, in which laudanosoline-1-O-d-xylopyranose and menisperine were revealed as novel NF-κB inhibitors. Among the seven identified alkaloids, three potential β2 -adrenergic receptor agonists, including sinomenine, magnoflorine and laurifoline, were characterized using a luciferase reporter system to measure for the activity of β2 -adrenergic receptor agonists. Finally, sinomenine, magnoflorine and laurifoline were identified not only as potential NF-κB inhibitors but also as potential β2 -adrenegic receptor agonists, which is the first time this has been reported. Molecular dynamic simulation and docking results suggest that the three dual-bioactive constituents could not only inhibit Pseudomonas aeruginosa PAK strain-induced inflammatory responses via a negative regulation of the Braf protein that participates in MAPK signaling pathway but also activate the β2 -adrenegic receptor. These results suggest that CS extract has dual signaling activities with potential clinical application as a novel drug for asthma.