*3.7. NMR Spectroscopy*

NMR spectra were obtained using a Bruker Avance III Ascend 600 MHz (14.1 T) spectrometer (Billerica, MA, USA) equipped with a 5 mm inverse probe. Each sample had its 1H 90◦ pulse calibrated and transmitter frequency o ffset calculated = 1881.69 Hz (SP1) and 2822.59 Hz (SP2), and the value was used to all following experiments, which were conducted at 70 ◦C with samples (20 mg/mL) previous solubilized with deuterium oxide (D2O) without spinning. 1D-NMR spectra were obtained using a presat sequence pulse (zgpr) to suppress solvent peak. Presat experiments were performed with a spectral width (SWH) of 4795.396 Hz, size of fid (TD) = 64k, and number of scans (NS) = 8. 2D-NMR homonuclear (1H–1H) COSY (Correlation Spectroscopy) analysis were performed using Bruker's cosygpprqf pulse sequence with SWH = 4795.396 Hz, TD = 2048 (F2) × 256 (F1), number of dummy scans (DS) = 16, NS = 16, and relaxation delay = 1.5 s. 2D-NMR heteronuclear (1H–13C) HSQCed (Edited Heteronuclear Single Quantum Coherence) analysis were performed using Bruker's hsqcedetgpsisp2.2 pulse sequence with SWH = 4795.396 Hz (F2) × 16667.754 Hz (F1), TD = 2048 (F2) × 256 (F1), DS = 16, NS = 32, and relaxation delay = 1.0 s. HMBC (Heteronuclear Multiple Bond Correlation) analysis were performed using Bruker's hmbcgpndqf pulse sequence with SWH = 4789.272 Hz (F2) × 20124.867 Hz (F1), TD = 2048 (F2) × 256 (F1), DS = 16, NS = 46, and relaxation delay = 1.0 s. The chemical shift of 1H and 13C were expressed in δ (ppm) relative to TMSP (trimethylilsilylpropionate) as an internal standard (δ = 0 ppm). NMR spectra analysis was conducted using Bruker's TopSpin v 4.0.6 software (Bruker, Billerica, MA, USA). The proposed structures were designed using ChemSketch v.12.0 software (ACD/Labs, Toronto, ON, Canada).

## *3.8. Cell Culture*

The RAW 264.7 macrophage cell line (ATCC number TIB-71) was cultured in DMEM supplemented with FBS (10% v/v) and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin). The cells were incubated at 37 ◦C in a humidified atmosphere with 5% CO2. For maintenance of the cells, the culture medium was changed every three days, and the cells were further subcultured at the 80% confluency using a cell scraper.

#### *3.9. MTT Reduction Test*

The ability of RAW 264.7 macrophages to reduce MTT was evaluated according to the previously described method of Mosmann [65] to analyze the e ffect of SPs on cell viability. Initially, the cells were seeded in 96-well plates at a density of 1 × 10<sup>4</sup> /well. After treatment with the SPs at the di fferent concentrations tested (12.5–100 μg/mL) for 24 h, the culture medium was replaced with 100 μL of MTT (1 mg/mL dissolved in DMEM). Then, the cells were incubated for 4 h at 37 ◦C. Subsequently, the culture supernatant was discarded, and the crystals of formazan were solubilized in ethanol, 100 μL/well. Absorbance was measured with an Epoch microplate spectrophotometer (Biotek Instruments Inc., Winooski, VT, USA) at 570 nm. Cell viability was calculated in relation to the negative control using the formula: % viability = (Atest/AControl) × 100, in which Atest corresponds to the absorbance of the experimental group and Acontrol corresponds to the absorbance of the negative control.
