Guided isolation of new iridoid glucosides from Anarrhinum pubescens by high-performance thin-layer chromatography-acetylcholinesterase assay

Plants are an important source of natural iridoids. This study demonstrates for the first time the acetylcholinesterase (AChE) inhibitory activity of iridoids belonging to the class of antirrhinosides. As iridoids distinguish the chemical composition of most species of the Plantaginaceae family, the active AChE inhibitors were investigated in the hydro-alcoholic extract of Anarrhinum pubescens Fresen. High-performance thin-layer chromatography (HPTLC) in combination with the AChE inhibition assay is a time and material saving methodology, and thus was employed to directly point to the individual enzyme inhibitors occurring in the plant. The effect-directed screening successfully discovered three active metabolites. These were characterized as antirrhinoside-derived iridoids. Two of these are here reported as newly isolated natural compounds. Identification of the two new metabolites was based on analysis of their collected spectroscopic data (HRMS, 1D and 2D NMR). Their structures were elucidated to be 6-O-, 6'-O-di-trans-cinnamoyl-antirrhinoside (1) and 5-O-, 6-O-difoliamenthoyl-antirrhinoside (3), while the previously known compound 6-O-foliamenthoyl-(6'-O-cinnamoyl)-antirrhinoside (2) was assigned by extensive analysis of its HRMS and HRMS/MS data. The activity of the isolated compounds was referred to the known AChE inhibitor rivastigmine, i.e. their activity were calculated and expressed as values equivalently to rivastigmine. This neuroprotective potential of iridoids mediated through AChE inhibition promote them to compete as natural curatives for neurodegenerative disorders like Alzheimer's disease.