Supercritical CO2 extraction of plant bioactives using hydroethanolic mixtures as cosolvent/suspension medium. 1. Case study on the extraction of red clover (Trifolium pratense L.) isoflavones

This study investigated the extraction of bioactive compounds from suspensions of finely disrupted red clover (Trifolium pratense L.) using SuperCritical (sc) CO₂ modified with hydroethanolic mixtures and compared the results with those obtained from a packed bed extraction. Total solids, phenolics, flavonoids, and isoflavones (formononetin and biochanin A) were quantified, together with the antioxidant activity of the extracts. A 30-min static extraction of hydroethanolic suspensions of red clover pressurized with CO₂ at 313 K or 323 K and from 20 MPa to 35 MPa, was followed by a 300-min dynamic extraction stage in which a solvent mixture with the same composition as the CO₂-rich gaseous phase in the extraction vessel was continuously fed. The extraction of suspensions was pronouncedly more selective than a packed bed extraction using the same dynamic extraction solvent mixture for recovering phenolics (from (28−70) mg·g⁻¹ in suspension versus from (14−18) mg·g⁻¹ GAE/substrate in packed bed), total flavonoids (from (30−58) mg·g⁻¹ versus from (11−20) mg·g⁻¹ QE/substrate), and isoflavones (from (41−92) mg·g⁻¹ versus from (8.5–17) mg·g⁻¹ isoflavones/substrate). Despite these differences, the antioxidant activity (ORAC method) of the extracts was similar for both methods (from (32−33) mg·g⁻¹ in suspension versus from (29−40)x10⁻⁶ mol·g⁻¹ TE /dry substrate in packed bed). The study identified water content in the liquid phase as the primary factor influencing extraction rate and yield. In conclusion, the extraction of bioactive compounds from red clover suspensions using scCO₂ modified with hydroethanolic mixtures demonstrated higher selectivity and yield compared to packed bed extraction.