Three types of Salvia leaves were profiled using high-performance thin-layer chromatography (HPTLC). The researchers, based out of the HUN-REN Center for Agricultural Research (Budapest, Hungary) and the University of Gabes (Gabes, Tunisia), published their research into the Journal of Chromatography B (1).
Salvia is an herb in the mint family, typically known for its hallucinogenic effects (2). More than 900 species of Salvia have been found throughout the world, with the salvia extract market being estimated to be worth $500 million in 2023 (3). This growth has been driven by increased demand for natural ingredients in different industries, such as personal care and food and beverage. Three Salvia strains, S. aegyptiaca L., S. verbenaca L., and S. officinalis L., used for medicinal purposes. These strains have been used for treating diseases and conditions including inflammation, seizures, diabetes, and diarrhea, among others. Salvia herbs are used because of its biologically active compounds, particularly phenolic acids, flavonoids, and triterpenes, among others. While these herbal medicines have been recognized for their healing properties and having abundance of a wide range of compounds, there is a lack of information on the biological, biochemical, and chemical profiles of their polar or non-polar extracts.
High-performance thin-layer chromatography (HPTLC) is one of the most widely used analytical techniques for the qualitative and quantitative analysis of environmental samples, food, and natural products. This technique comes with advantages such as simple sample preparation, short analysis time, and automation during the execution of the analytical steps. Few studies have been conducted on salvia extracts using HPTLC–(bio)assays.
In this study, researchers coupled HPTLC with radical scavenging (using DPPH• free radical) and enzyme inhibitory (acetylcholinesterase, AChE) bioactivity assays to analyze Salvia strains. 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 analyzed using this combination of techniques. 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 compounds present in the active zones were characterized using 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 were characterized using derivatization as phenolic compounds and tentatively identified by MS as caffeic acid derivatives, 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. Within a single zone of S. officinali extract, the co-presence of an unknown caffeic acid derivative, hispidulin 7-O-glucuronide and luteolin 7-O-glucoside was shown. An unknown rosmarinic acid derivative and an unknown luteolin derivative were also detected in S. officinalis and S. verbenaca, respectively.
The HPTLC hyphenations provided a fast and simple means of obtaining chemical and biological fingerprints of aqueous ethanol extracts of the three species, the researchers wrote. The profiles were species-specific, allowing for easier distinction. Compounds in bioactive zones obtained through the HPTLC-related assays were identified using HPTLC–high resolution tandem mass spectrometry (HRMS/MS) as phenolic compounds, namely flavonoids and phenolic acids.
(1) Reguigui, A.; Baglyas, M.; Cselőtey, A.; et al. High-Performance Thin-Layer Chromatography Combined with Effect-Directed Assays Revealed Bioactivity Profiles of Salvia aegyptiaca, S. verbenaca, and S. officinalis. J. Chromatogr. B 2025, 1261, 124653. DOI:
(2) Holland, K.; Goodwin, M. What Is Salvia Divinorum? Healthline 2024.
(3) Salvia Extract Market By Product Type, By End-Use Industry, Global Market Size, Share, Growth, Trends, Statistics Analysis Report, By Region, and Segment Forecasts 2024 – 2033. DataHorizzon 2024.
