*2.1. Step I—Design of the Experiments and the Chemical and Biological Evaluations* 2.1.1. Factorial Design

First of all, a factorial design 22 was performed evaluating the pressure and modifier effects in the global yield of the extraction process. The experiments resulted in 7 extracts with a global yield between 1.25 and 2.49% *w*/*w* (extract/plant). These results are presented in Table 1 together with the experimental design matrix, the levels of each factor, and their combinations determined by the factorial design.


**Table 1.** Factorial 22 design matrix and observed responses.

<sup>a</sup> gram of crude extract from 100 g of dried flowers; <sup>b</sup> S/F is the solvent-to-feed ratio.

The lowest yield value was obtained at the lowest pressure (120 bar), using water as the cosolvent, while the largest yield was obtained at the extraction process with the highest pressure (240 bar), using ethanol as the cosolvent. The factorial design data were processed using the Minitab® statistical software and the analysis of variance (ANOVA) proved that both factors, pressure, and the cosolvent were statistically significant in the global yield of the supercritical fluid extraction. The effects were evaluated using a linear regression and the contour plot can be viewed in Figure 1.

**Figure 1.** Contour plot for global extraction yield as a function of CO2 pressure and modifier.

The model fitted to the experimental data was presented as a coefficient of determination, R2, which was equal to 0.9981. The adjusted coefficient of determination had a value of 0.9944, which means that only 0.56% of the variations were not explained by the model used for the contour plot, which is presented in Equation (1):

$$\text{global yield} \left( \% \frac{w}{w} \right) = 1.81281 + 0.32906 \text{ P} + 0.29094 \text{ M} + 0.06094 \text{ P M} \tag{1}$$

where P is the pressure and M is the modifier (values for coded variables).

#### 2.1.2. Extract Purification and Chemical Analyses

The 7 extracts were analyzed by HPLC and in all of them, a compound with a retention time of about 10.6 min was detected (Supplementary Material—Figures S1–S7). In the first step of this study, the compound was only analyzed qualitatively. After a comparative analysis with different standards, the most common compound was identified as *p*-anisic acid. Once the compound was identified in all extracts, the following steps involved its purification by silica gel column chromatography, thin-layer chromatography (TLC), and HPLC for each fraction.

Most of the identified compounds in the raw extracts, including the *p*-anisic acid, were only identified in the ethyl acetate fraction. Therefore, this fraction was submitted once again to column chromatography with a gradient of solvents in increasing order of polarity, and 10 fractions were collected. According to the results of the HPLC analysis, *p*-anisic acid was detected in subfractions 4 and 5, obtained with a ratio of hexane-to-ethyl acetate of 40:60 and 20:80 *v*/*v* as solvents, respectively (Supplementary Material—Figures S8–S9). In subfraction 2, a compound that presented as orange in color in the TLC analysis (Supplementary Material—Figure S10) after staining with sulfuric vanillin was not identified in the HPLC analysis under the studied conditions (Supplementary Material). Nevertheless, this subfraction was chosen for the antimicrobial activity tests, along with subfraction 4, the ethyl acetate fraction, and all 7 crude extracts. We did not use subfraction 5 for the antimicrobial assays due to the lower yield compared to subfraction 4, since both have a similar chromatographic profile.
