Luna 5 m

The analyses of ElectroSpray Ionization High-Resolution Mass Spectrometry (ESI-HRMS) were performed in a MicrOTOFQ-II Bruker Daltonics mass spectrometer (Q-Tof analyzer, Bremen, Germany) coupled to a Shimadzu HPLC (Kyoto, Japan) system consisting of two pumps LC-20AD, automatic injector SIL-20A, column oven CTO-20A and controller CBM-10A. A Phenomenex Luna 5 µm (150 × 3 mm, 100 Å particle size) column was used. The column oven was kept at 30 °C, and chromatography was performed with a flow of 500 µL/min using MeOH:H₂O as the mobile phase in a gradient of 0 min 20% of MeOH held until 5 min, from 5 to 30 min 20 to 100% MeOH. The mass spectrometer was operating in electrospray positive mode, with nebulization and drying gas at 4 Bar and 8 L/min, respectively. The capillary voltage was set to 4500 V, and the drying temperature was set to 200 °C. The collision cell and quadrupole energy were set to 12 eV and 6 eV, respectively. Samples were prepared by dissolving 50 µL of the supernatant collected directly from the extracts in 950 µL of MeOH, and filtered (Millex PTFE, 0.45 µm); 10 µL of this sample was injected into the equipment. The HPLC-MS raw data were analyzed by XCMS online software 21, and multivariate analyses were performed using Unscrambler software version 10 (CAMO Analytics, Oslo, Norway).

The fraction that had the highest antioxidant activity, F_A, was filtered through 0.45-µm filters (Whatman, GE Healthcare). RP-HPLC analysis was done using a Spectra System^TM HPLC (Thermo Fisher Scientific Inc.), with a reversed phase C₁₈ column (250 mm×4.6 mm, Luna 5 µM; Phenomenex, Torrance, CA, USA). Peptides were eluted with a discontinuous gradient comprising mobile phase A (0.1%, V/V, trifluoroacetic acid (TFA) in distilled deionized water) and mobile phase B (70%, V/V, ACN in 0.05%, V/V, TFA) at a flow rate of 0.7 mL/min. TFA and ACN were purchased from Sigma-Aldrich, Merck. The gradient started at A:B=100:0 (V/V), changing linearly to A:B=90:10 (V/V) 14 mL, then to A:B=70:30 (V/V) 8.75 mL and finally to A:B=60:40 (V/V) 6 mL. Peptides were detected by measuring the absorbance at 280 nm (A_{280 nm}). Chromatographic analyses were done by ChromQuest software, v. 5.0 (16 ). Four subfractions, F₁, F₂, F₃ and F₄, were obtained, lyophilized and their antioxidant activities were determined by DPPH and ABTS radical scavenging assays.