Identification of key anti-inflammatory components of green prickly ash fruit based on zebrafish model and activity tracking strategy

As a famous medicinal and edible spice, Green prickly ash fruit (GPAF) is widely used as anti-inflammatory agent in Asian countries. However, the key active components have not yet been identified. An animal model was established using a transgenic zebrafish line, Tg(llyz: DsRED2), with neutrophils labeled by red fluorescent protein to evaluate the anti-inflammatory activity. The main chemical components in GPAF were analyzed and identified by ultra-high performance liquid chromatography-quadrupole electrostatic field orbitrap-mass spectrometry technology (UHPLC-QE-Orbitrap-MS). The drug-likeness, absorption, distribution, metabolism, and elimination properties of these identified compounds were further screened using the SwissADME tool and dynamic molecular docking. Finally, the key components of GPAF were evaluated inflammation inhibition using the zebrafish model. Using the established zebrafish model, we found that GPAF extract exhibited significant anti-inflammatory effects. 13 components were identified by UHPLC-QE-Orbitrap-MS in GPAF extract. 7 components screened by ADME screening exhibit good parameters of passive gastrointestinal absorption (HIA) and brain entry (BBB). The results of dynamic molecular docking showed that Toddaline, Hydroxy-α-sanshool, and Hydroxy-β-sanshool could dock stably with the three core targets of AKT1, VEGFA and MAPK3. Finally, We confirmed the anti-inflammation activities of Toddaline, Hydroxy-α-sanshool, and Hydroxy-β-sanshool in the zebrafish model. A new activity-directed strategy was applied to identify three new anti-inflammation components of GPAF as Toddaline, Hydroxy-α-sanshool, and Hydroxy-β-sanshool. The results may supply the strong scientific evidence for quality control and health product development of GPAF.