Unlocking the Unique Potential of Thymus pannonicus: Exploring the Efficacy of Supercritical CO(2) Extraction, with and Without Pre-Treatments.

Since ancient times, many plant species within the Thymus genus have been used due to their numerous health benefits, such as antimicrobial, anti-inflammatory, antiseptic, or diuretic activity. While many of the species within this genus were well known and described, Thymus pannonicus All. or Pannonian thyme remains relatively unexplored despite its unique chemical composition and activity. T. pannonicus is a small shrub, spread over central and eastern Europe, with a diverse, location-dependent chemical composition. Within this study, the citral chemotype of T. pannonicus was used, which up to our knowledge was found and described only in Northern Serbia. Therefore, in order to explore the composition of T. pannonicus low-polar extracts and to present a preliminary insight into their bioactivity, supercritical carbon dioxide (ScCO(2)) extraction was applied (with and without pre-treatments), followed by GC-MS analysis. Different ScCO(2) extraction pressures were tested (100, 150, 200, 250, and 300 bar) in order to determine the most suitable conditions for the extraction of T. pannonicus's low-polar fraction. Additionally, in order to maximize the yield of the extraction, two types of ScCO(2) extraction pre-treatments were investigated (enzymatic and microwave). The highest extraction yield obtained from untreated plant material was 3.01% (w/w), and it was obtained at conditions of 150 bar and 40 °C, while the 4% (v/w) enzymatic pre-treatment, with the same conditions, provided a yield of 3.89%. For all of the obtained extracts, the GC-MS analysis showed that oxygenated monoterpenes and sesquiterpenes were the two most dominant groups with principal bioactive compounds such as (E)-citral (18.95-38.17%), (Z)-citral (6.68-14.66%), β-bisabolene (8.2-14.4%), and nerol (6.08-9.67%). The extracts that exhibited the highest concentration of principal bioactive compounds ((Z)-citral, and (E)-citral) were further analyzed for anticancer potential, using short- and long-term cell viability observations on liver cancer cells.