Extraction and Isolation of Compound 1

The fresh leaves without stems (1.6 kg) were subjected to a turbo extraction for 5 min in an industrial blender, using EtOH: H₂O (1:1, v/v) at a plant: solvent proportion of 1:1 (w/v). Subsequently, the hydroethanolic extract (HE) was filtered and the volume was concentrated by rotaevaporation (model v-700, Buchi^®). With the HE, a liquid-liquid extraction was made with three solvents of different degrees of polarity. Through this process, the dichloromethane (CH₂Cl₂), ethyl acetate (AcOEt), and n-butanol (n-BuOH) fractions were obtained.

For the isolation of compound 1, the n-BuOH fraction (2 g) was selected and submitted into a column chromatography (silica gel 60) with gradient elution (30%–0% CH₂Cl₂: 70%–100% AcOEt and 95%–0% AcOEt: 5%–100% MeOH) resulting in nine fractions (G1–G9). The fractions G3 (520.7 mg) and G4 (372.5 mg) were purified separately through a process of size-exclusion column chromatography using Sephadex LH-20 as stationary phase and MeOH as solvent. The process of purification ended with five subfractions (F1–F5) for both primary fractions, G3 and G4. Between them, the subfractions F4 (from G3) and F5 (from G4) showed the same chemical profile by TLC, characterized by the presence of just one yellow band. These subfractions were reunited and submitted to a final purification process through preparative high-performance liquid chromatography (HPLC-UV) (Shimadzu^®) using an isocratic elution (80% Milli-Q water and 20% Acetonitrile), a semipreparative column C18 Luna Phenomenex^® (250 mm × 10 mm, 5 µm), flow of 4 ml/min and analysis time of 25 min. All fractions obtained during the entire purification process, were monitored by thin layer chromatography (TLC) (Merk silica gel 60) and the bands were visualized by UV (254 and 365 nm). After the purification, the isolation of compound 1 (34.8 mg) was confirmed by UPLC-DAD system used was a Shimadzu Model LC-20AD, with DAD detector SPD-M20A and software LabSolutions. Then, a Phenomenex Kinetex RP-18 column (150 mm × 4.6 mm, 2.6 μm particle size) equipped with a Phenomenex security guard column (4.0 mm × 2.0 mm i.d.) was used. The eluents were: (A) trifluoroacetic acid (TFA) 0.3% and (B) acetonitrile. The following gradient (v/v) was applied: 7%–15% B, 0–3 min; 15%–20% B, 3–12 min; 20%–22% B, 12–30 min; 30 min total analysis time. Flow elution was 0.7 ml/min, and 12 μl of each sample was injected. The UV-DAD detector was programmed to wavelength 200–500 nm and the chromatograms were plotted at 254 and 340 nm. The samples were resuspended in methanol: water, 1:1 (v/v) and the final concentration was 2 mg/ml for the extracts. HPLC-grade acetonitrile and trifluoracetic acid (TFA) were provided from J. T. Baker (Brazil). Water was purified with a Milli-Q system (Millipore). Samples and solvents were filtrated through a membrane (pore-size 0.45 μm) and degassed. The analyses were performed in triplicate. The UPLC-DAD analysis confirmed a purity percentage of approximately 93% ( Figure S1 , Supplementary Material ). The detailed conditions of all column chromatography are described in Table S1 , in Supplementary Material .