Efficient extraction and purification of flexibilane and tigliane diterpenoids from Stillingia lineata using sequential SFE-CO2 and SFC-CO2

Abstract

Quite a number of methods of extraction and separation are currently used to isolate and purify secondary metabolites from complex mixtures, but "green" technologies are still far from being used routinely in public research laboratories. In recent years, supercritical fluid extraction (SFE) and chromatography (SFC) have gained increasing interest in production of high-value plant extracts for pharmaceutical, cosmetic and food industries. SFC enables fast and reliable separation of biomolecules from complex mixtures [1,2]. Supercritical carbon dioxide (SC-CO2) is an eco-friendly, chemically inert, nontoxic and noninflammable fluid. As a green material, SC-CO2 has low viscosity and high diffusivity that make it suitable as a solvent for SFE and as a mobile phase for SFC. Previous chemical investigation of Stillingia lineata ((Lam.) Müll. Arg. Euphorbiaceae) leaves led to the isolation of the rare C20-flexibilane tonantzitlolones (TZLs) (PKC activators and potent anti-tumor agents) [3] and tigliane derivatives (strong inhibitors of Chikungunya viral replication) [4 – 6]. This plant was selected to validate our green approach aiming to specifically extract, then isolate diterpenoids of interest. According to a pre-determined experimental plan, sequential SFE-CO2 was carried out by varying pressure, temperature, flow rate and step duration. For each experiment, an ethanol step gradient was performed, affording six fractions (Figure below). Identification of compounds was achieved by LC-UV-HRMS [2], then compared with standards and classified into different chemical classes (TZLs, diterpenes, chlorophylls and glycosylated flavonoids). Their relative proportions were subsequently determined by measurement of UV peak areas, allowing selecting optimal SFE conditions, and diterpene-enriched fraction for further SFC purifications. This approach not only has the benefit of using eco-friendly methodologies, but it also allows a concurrent prefractionation during the extraction step.