Optimization of extraction condition to improve blackthorn (Prunus spinosa L.) polyphenols, anthocyanins and antioxidant activity by natural deep eutectic solvent (NADES) using the simplex lattice mixture design method

In the present study, a statistical tool called the simplex lattice mixture design method was used to create a new formulation of Natural Deep Eutectic Solvent (NADES) that is derived from a combination of three compounds (lactic acid, maltose, and water) to extract phenolic compounds, including the anthocyanin content, as well as their antioxidant activity from blackthorn fruits (Prunus spinosa L.). The optimal condition for the highest compounds and antioxidant activity was obtained by simultaneous optimization of the ternary mixture containing lactic acid: maltose: water (66:16:16 v/v/v), which was the most suitable for simultaneous maximization of maximum total phenolic content (TPC) (134 ± 1.6 mg GAE/g) and antioxidant activity (AA) showing a 141 ± 1.4 mmol/TE g, which were measured using the DPPH assay. The ternary mixture containing lactic acid: maltose: water (33:33:33 v/v/v), which was the most suitable for simultaneous maximization of the total anthocyanin content (TAC) (120.3 ± 1.3 mg C3G/g)/TE g. The ANOVA analysis of the mixture design showed that all three responses (TPC, TAC, and AA) were statistically significant, with determination coefficients of 94.41 %, 94.96 %, and 98.38 %, respectively, and fit the quadratic regression models. A chromatographic analysis of the NADES extract of the blackthorn fruit revealed that phenolics (vanillic acid, chlorogenic acid, caffeic acid, and gallic acid) and anthocyanin (Cyanidin-3-O-glucoside, Cyanidin-3-O-rutinoside, Peonidin-3-O-glucoside and Delphidin-3-O-β-O-glucoside) are the most prevalent bioactive compounds. These results indicate that the simplex lattice mixture design effectively optimized the phenolic and anthocyanin content, thereby preserving their antioxidant activity. This optimization is valuable, as it can potentially enhance the nutritional and functional value of blackthorn fruit extracts that are rich in phenolic compounds and have antioxidant properties. These discoveries could be used in the creation of functional food, dietary supplement, and natural antioxidant for several fields.