*4.3. Experimental Designs*

## 4.3.1. Factorial Design

Initially, the supercritical fluid pressure and cosolvent effects in the global extraction yield were investigated; for that, a 22 factorial design was used. The pressure was evaluated at 120, 180, and 240 bar and ethanol, water, and a mixture of ethanol and water (1:1 *v*/*v*) were used as cosolvents, aiming to promote the extraction of polar compounds. The choice of these variables was supported by reports on the optimization of supercritical extraction [71,76,77]. In this step, the carbon dioxide temperature was 40 ◦C for all extractions. The experiments were performed in triplicate at the factorial design's central point. The response surface was then associated with the regression equation. The evaluation of the effects of the variables and their interactions was achieved through an analysis of variance (ANOVA) [78].

## 4.3.2. Box–Behnken Design

The second step of this study was comprised of a Box–Behnken design, which was used for the extraction process optimization to obtain the compound, *p*-anisic acid. This experimental design consisted of a fractional factorial design method in three levels [55]. Three variables were evaluated, resulting in a total of 15 experiments. The pressure was evaluated at 200, 250, and 300 bar; the temperature was evaluated at 40, 50, and 60 ◦C; and an average particle size of 42, 60, and 150 mesh (0.423, 0.303, and 0.125 mm, respectively) were used. All 15 experiments used 4.7% (*w*/*w*) ethanol as the cosolvent since *p*-anisic acid

is highly soluble in this solvent. These process variables were chosen based on previous studies that used RSM to analyze the SFE of plant material [32,79–81].

A polynomial equation was fitted into the experimental data. The optimal extraction condition was achieved in terms of the highest selectivity of the interest compound (*p*-anisic acid). The matrix design was developed in Minitab® software.

## *4.4. Chemical Analysis*

To investigate the chemical composition of the extracts, each was separated by column chromatography. Both the extracts and the fractions were analyzed by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC).
