Used by the temperature and ethanol concentration in the extraction buffer. Accordingly, we had been able to define an optimal protocol based on the extraction of red chicory powder at four C for 30 min making use of 50 ethanol containing 2 tartaric acid as the solvent, matching the efficiency of your gold-standard protocol determined by methanol acidified with 2 HCl below precisely the same conditions (no considerable difference observed within a t-test, p 0.05). We characterized the extracts by evaluating their stability over time when stored as pure extracts, JPH203 manufacturer three-fold concentrates, or lyophilized powders at two diverse tem-Molecules 2021, 26,14 ofperatures (4 and 23 C). We discovered that the lyophilization of aqueous extracts (extraction buffer = 2 tartaric acid in water with no ethanol) followed by storage at four C preserved the anthocyanin contents for 6 months, whereas the storage of pure extracts or three-fold concentrates revealed a robust unfavorable impact on anthocyanin stability triggered by the greater storage temperature and by the presence of ethanol inside the extraction buffer. By lowering the water activity of the matrix via the sublimation of water molecules at low temperatures, lyophilization reduces the reactivity of anthocyanins, which includes their conversion to colorless hemiketal and chalcone types that take place naturally in aqueous environments [16]. This freeze-drying technique has already been applied effectively by other people to preserve the anthocyanin content material of other plant matrices for six months, including extracts of sweet cherry [17] and elderberry [18]. Thus, though probably the most efficient extraction procedure necessary a solvent containing 50 ethanol, the presence of ethanol Fmoc-Gly-Gly-OH Cancer limits the postextraction stability of anthocyanins over time when stored as pure extracts, concentrates, or lyophilized powder. The degradation kinetics of anthocyanins in the presence of growing concentrations of ethanol have already been associated with the disruption of -interactions among the aromatic rings [19]. In an aqueous answer, these interactions stack the planar structures of anthocyanins (a phenomenon referred to as self-association), shielding their cores from nucleophilic attacks that may bring about hydrolysis or oxidation. Ethanol is thought to interfere with this stacking phenomenon to indirectly bring about irreversible degradation with the chromophores, triggering the color loss we observed in the pure extracts and concentrates containing 50 ethanol. When employing water containing 2 tartaric acid, the temperaturedependent degradation of anthocyanins was ameliorated, particularly when stored as a lyophilized powder (a number of t-tests, p 0.05). We, thus, selected storage at 23 C in our optimized sustainable protocol. The total anthocyanin content of red chicory leaf extracts prepared utilizing our optimized sustainable protocol (70.1 1.eight mg/100 g LFW) was larger than previously reported. For example, Lavelli [11] accomplished maximum yields of 65.3 mg/100 g LFW by extraction with 50 methanol containing four formic acid at room temperature, whereas Migliorini et al. [9] achieved maximum yields of 73.53 0.13 mg/100 g LFW by extraction with water acidified with acetic acid (pH two.5 at 62.4 C). Red chicory leaves have previously been shown to accumulate numerous anthocyanins, especially cyanidin-3-O-galactoside, cyanidin-3-O-glucoside, cyanidin-3-O-(6-malonyl)glucoside, cyanidin-3-O-rutinoside, cyanidin-3,5-di-O-(6-O-malonyl)-glucoside, cyanidin3-O-(-O-acetyl)-glucoside, and cyanidin-3-O-gluc.