Supercritical CO2-Based Extraction and Detection of Phenolic Compounds and Saponins from the Leaves of Three Medicago varia Mart. Varieties by Tandem Mass Spectrometry

A comparative metabolomic study of three varieties of alfalfa (Medicago varia Mart.) was performed via extraction with supercritical carbon dioxide modified with ethanol (EtOH) and the detection of bioactive compounds via tandem mass spectrometry. Several experimental conditions were investigated in the pressure range of 50–250 bar, with ethanol used as a co-solvent in an amount of 1% of the total volume in the liquid phase at a temperature in the range of 31–70 °C. The most effective extraction conditions were as follows: a pressure of 250 Bar and a temperature of 60 °C for M. varia. M. varia contains various phenolic compounds and sulfated polyphenols with valuable biological activity. Tandem mass spectrometry (HPLC-ESI–ion trap) was applied to detect the target analytes. A total of 103 bioactive compounds (59 polyphenols and 44 compounds belonging to other chemical groups) were tentatively identified in extracts from aerial parts of alfalfa. For the first time, twenty-one chemical constituents from the polyphenol group (flavones: Formononetin, Chrysoeriol, Cirsimaritin, Cirsiliol, Cirsilineol, tricin-O-hexoside, Apigenin C-glucose C-deoxyhexoside, Apigenin 7-O-diglucuronide, 2′-Hydroxygenistein 4′,7-O-diglucoside, etc.) and six from other chemical groups (saponins: Soyasaponin II, Soyasaponin gamma g, Soyasaponin I, Soyasaponin Bd, Soyaysaponin beta g, etc.) were identified in the aerial parts of M. varia.