Unlocking the Unique Potential of Thymus pannonicus: Exploring the Efficacy of Supercritical CO2 Extraction, with and Without Pre-Treatments

Since ancient times, many plant species within the <i>Thymus</i> 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, <i>Thymus pannonicus</i> All. or Pannonian thyme remains relatively unexplored despite its unique chemical composition and activity. <i>T. pannonicus</i> is a small shrub, spread over central and eastern Europe, with a diverse, location-dependent chemical composition. Within this study, the citral chemotype of <i>T. pannonicus</i> was used, which up to our knowledge was found and described only in Northern Serbia. Therefore, in order to explore the composition of <i>T. pannonicus</i> low-polar extracts and to present a preliminary insight into their bioactivity, supercritical carbon dioxide (ScCO₂) extraction was applied (with and without pre-treatments), followed by GC-MS analysis. Different ScCO₂ extraction pressures were tested (100, 150, 200, 250, and 300 bar) in order to determine the most suitable conditions for the extraction of <i>T. pannonicus</i>'s low-polar fraction. Additionally, in order to maximize the yield of the extraction, two types of ScCO₂ extraction pre-treatments were investigated (enzymatic and microwave). The highest extraction yield obtained from untreated plant material was 3.01% (<i>w</i>/<i>w</i>), and it was obtained at conditions of 150 bar and 40 °C, while the 4% (<i>v</i>/<i>w</i>) 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.