High-performance thin-layer chromatography combined with effect-directed assays revealed bioactivity profiles of Salvia aegyptiaca, S. verbenaca, and S. officinalis.

The effect-directed profiling of aqueous ethanol extracts of Tunisian Salvia aegyptiaca and Salvia verbenaca aerial parts and Tunisian and Hungarian Salvia officinalis leaves was achieved by high-performance thin-layer chromatography (HPTLC) coupled with radical scavenging (using DPPH• free radical) and enzyme inhibitory (acetylcholinesterase, AChE) bioactivity assays. The mixture of toluene - ethyl acetate - methanol - formic acid, 11:2:6:1 (V/V) was used as a mobile phase for the separation of bioactive zones. The characterization of the compounds present in the active zones was performed by derivatization with aluminum chloride or natural product-polyethylene glycol (NP-PEG) reagent, and HPTLC-heated electrospray ionization-high resolution tandem mass spectrometry (HPTLC-HESI-HRMS/MS). Compounds in the nine antioxidant zones could be characterized by derivatization as phenolic compounds and tentatively identified by MS as caffeic acid derivatives, or flavonoid glycosides or glucuronides. Rosmarinic acid and luteolin 7-O-glucuronide were detected in S. aegyptiaca and S. officinalis extracts, while apigenin 7-O-glucuronide and luteolin 7-O-glucoside were identified in S. aegyptiaca and S. verbenaca. The co-presence of an unknown caffeic acid derivative, hispidulin 7-O-glucuronide and luteolin 7-O-glucoside was proved within a single zone of S. officinalis extract. Additionally, an unknown rosmarinic acid derivative and an unknown luteolin derivative were detected in S. officinalis and S. verbenaca, respectively. These compounds exhibited weak AChE inhibitory activity, with luteolin 7-O-glucuronide demonstrating the strongest effect.