4.4.2. High-Performance Liquid Chromatography (HPLC)

The extracts were analyzed in Agilent 1200 Series liquid chromatography equipped with a U.V. detector. The separation was carried out in a C18 column (4.6 mm × 250 mm × 5 μm). The mobile phase used a binary system consisting of water (A) and acetonitrile (B), both with 2% acetic acid, in gradient mode of 80–20% of B in 30 min, with a 1.0 mL·min−<sup>1</sup> flow. The injected sample volume was 5 μL and the detector was set at 258 nm. The content of *p*-anisic acid in the extracts was evaluated with a calibration curve (R2 = 0.9986) containing 0.6, 0.45, 0.3, 0.15, and 0.06 mg·mL−<sup>1</sup> of standard *<sup>p</sup>*-anisic acid (99% purity, Sigma Aldrich). The extracts were diluted in acetonitrile at 10 mg·mL−<sup>1</sup> for their analysis by HPLC.

#### *4.5. Antimicrobial Activity*

The indirect bioautography method [83] was used to indicate the antimicrobial activity of the supercritical fluid extracts from *A. mearnsii* flowers. The analysis was performed to evaluate the activity against the microorganisms *Staphylococcus aureus* (ATCC 25923) and *Escherichia coli* (ATCC 25922). In this analysis, the extracts were applied to a thin layer chromatography (TLC) plate and were submitted to a run with dichloromethane as the mobile phase. After the solvent evaporation, the TLC plates were plunged into the culture medium inoculated with the microorganisms and were incubated for 24 h at 37 ◦C. The inoculum was prepared with a suspension of the microorganisms of 1.0 × 104 CFU·mL−<sup>1</sup> and was incorporated in the Mueller–Hinton agar. After the growth phase, a solution of

INT (p-iodonitrotetrazolium violet) was added for better visualization of the inhibition halos [84]. Amoxicillin 0.1 mg·mL−<sup>1</sup> was used as qualitative positive control while the culture medium inoculated with the microorganism was used as a negative control, without the presence of the extracts.

The antimicrobial activity was determined by the minimum inhibitory concentration (MIC) using a dilution method on microplates [85]. The microorganism inoculum was prepared with the colonies in a saline solution and the Mueller–Hinton broth, resulting in a final concentration of 1.0 × 104 CFU·mL<sup>−</sup>1. In each microplate well, 100 <sup>μ</sup>L of the Mueller– Hinton broth containing inoculums, followed by 100 μL of the extract solubilized in Tween 20, as well as water (at final concentrations of 0.75, 1.5, 3.0, 6.0, 12.0, and 24 mg·mL<sup>−</sup>1), were introduced. Other concentrations were used for the fractions obtained after the column chromatographic separation, defined according to each fraction weight. For the ethyl acetate, the final concentrations were 1.8, 3.6, 7.3, 14.7, 29.5, and 59.2 mg·mL−1; for the fraction obtained with hexane:ethyl acetate (80:20), named "subfraction 2", the final concentrations were 2.2, 4.4, 8.9, 17.9, 35.9, and 71.3 mg·mL−1; and the fraction obtained with hexane:ethyl acetate (60:40), named "subfraction 4", was tested at concentrations of 0.73, 1.4, 2.9, 5.9, 11.8, and 23.6 mg·mL<sup>−</sup>1.
