Enzyme-assisted optimized supercritical fluid extraction to improve Medicago sativa polyphenolics isolation

The aim of this study was to develop an effective Medicago sativa extraction method using enzyme-assisted supercritical fluid extraction (EA-SFE). The response surface methodology (RSM) based on Box-Behnken design was chosen to optimize the parameters of extraction process, i.e., extraction temperature, pressure and percentage of co-solvent for total phenolics content and antioxidants. Commercial enzyme preparation kemzyme was applied to M. sativa leaves. The extraction of antioxidant phenolics in optimal conditions from the enzymatically hydrolyzed M. sativa leaves was carried out by supercritical fluid extraction (SFE) with 96% ethanol as a co-solvent. Optimal extraction parameters for TPC and the antioxidant activity were: the temperature of 68 °C, the pressure of 205 bar and 15.5% of the co-solvent addition. For the sake of comparison, conventional extraction was performed. The obtained results showed that the use of the commercial enzyme formulation allowed the content of phenolic compounds in the leaves extract increase as compared to the control. HPLC-MS/MS characterization revealed that extraction of enzyme digested material was much more efficient (142.69 ± 5.11 μg/g) than extraction of non-digested material (94.17 ± 4.80 μg/g) or the use of conventional method (57.42 ± 2.25 μg/g). Our work offers an effective enzyme-assisted supercritical fluid extraction protocol for the enhanced release of biological active compounds from plant cells.