*2.5. Antioxidant Properties*

Antioxidant capacity by DPPH and ABTS methods, total phenols, flavonoids and spermidine content, and polyphenolic compounds profile of the lulo fruit and homogenized and nonhomogenized juices were determined.

For determination of total phenols and flavonoids content and antioxidant capacity by DPPH and ABTS method, antioxidants were extracted from fruit or from juice by diluting the samples in an 80:20 (*v/v*) methanol-water solution at a 1:10 ratio (*w/v*) and centrifuged at 10,000 rpm and 20 ◦C for 5 min (Selecta, "Medifriger BL-S"). Subsequent analyses were carried out on the supernatant (extract) by triplicate.

Total phenol content was determined following the Folin–Ciocalteu method [16,17]. For this procedure, 0.125 mL of extract, 0.125 mL of Folin–Ciocalteu reagen<sup>t</sup> (Sigma-Aldrich, Saint Louis, MO, USA), and 0.5 mL of double-distilled water were mixed and allowed to react for 6 min. After that, 1.25 mL of 7% (*w/v*) sodium carbonate solution and 1 mL of double distilled water were added. Absorbance was measured in a spectrophotometer (Thermo Scientific, Helios Zeta U/Vis, Loughborough, UK) at 765 nm after 90 min. A standard gallic acid curve ranging from 0 to 500 mg/L was obtained. Results were expressed in milligrams of gallic acid equivalent (GAE) per gram of sample.

Flavonoid content was determined following the method described by Luximon-Ramma et al. [18]. In this case, 1.5 mL of extract and 1.5 mL of a 2% (*w/v*) aluminum chloride solution in methanol were mixed and left in the dark for 10 min. Absorbance was measured on a spectrophotometer (Thermo Scientific, Helios Zeta U/Vis, Loughborough, UK) at 368 nm. The resulting data were compared to a standard quercetin curve ranging from 0 to 350 mg/L. The results were expressed in milligrams of quercetin equivalent (EQ) per gram of sample.

Antioxidant capacity by DPPH (2.2-diphenyl-1-picrylhydrazyl) radical was determined following the method described by Kuskoski et al. [19] and Stratil et al. [20], with some modifications. A blend made up of 0.1 mL of the extract, 0.9 mL of methanol, and 2 mL of a 100 μM methanol–DPPH (39.4 μg/mL) solution was prepared. After 60 min of reaction time, absorbance was measured at 517 nm in a spectrophotometer (Thermo Scientific, Helios Zeta UV/Vis, Loughborough, UK). The results were expressed as milligrams of Trolox equivalent (TE) per gram of sample, using the Trolox calibration curve within a 0 to 500 mg/L concentration range.

Antioxidant capacity by ABTS (2.2--azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) radical was evaluated following the method described by Re et al. [21]. A solution containing 7 mM of ABTS radical and 2.45 mM of potassium persulfate was prepared and left in the dark at room temperature for 16 h. ABTS+ was mixed with phosphate buffer to reach an absorbance of 0.70 ± 0.02 at 734 nm. Then, 0.1 mL of extract was added to 2.9 mL of ABTS+ solution. Absorbance was measured at 734 nm in a spectrophotometer (Thermo Scientific, Helios Zeta UV/Vis, Loughborough, UK) after 0, 3, and 7 min of reaction time. The results were expressed as mg of Trolox equivalent (TE) per gram of sample.

Polyphenolic compounds profile and spermidine content were determined by liquid chromatography—mass spectrometry (LC-MS) analysis. First, phenolic compounds were extracted according to the procedure described by Rodrigues et al. [22] and Svobodova [23], with some modifications. In brief, 5 g of sample were mixed with 20 mL of methanol/water (80:20 *v/v*) solution by stirring (Ultra-Turrax, Staufen, Germany) at 150 rpm and room temperature for 1 h. This mix was centrifuged (Beckman Coulter AvantiTM J-25, Hamburg, Germany) at 3864× *g* and 20 ◦C for 5 min and the supernatant taken. The extraction procedure was repeated five times. Finally, the supernatant was filtered using a Whatman No. 1 paper filter and, subsequently, a 0.45 μm nylon filter and then directly injected into the HPLC equipment.

The equipment used for separation and identification of phenolic compounds was an (Agilent 1290 HPLC Technologies series infinity System LC, Santa Clara, CA, USA) system with a MS detector and a C18 (1.7 μm, 2.1 × 50 mm, Waters) UHPLC (Ultra High-Performance Liquid Chromatography) column. A flow rate of 0.4 mL/min and an injection volume of 5 μL at 30 ◦C were applied. The solvents employed were 0.1% formic acid in water (A) and 0.1% formic acid in methanol (B). The applied gradient elution was 10% B (0 min), 10% B (5 min), 100% B (12 min); 100% B (18 min), 10% B (18.5 min), 10% B (25 min). An automated calibration was performed using an external calibrant delivery system (CDS) which infuses calibration solution prior to sample introduction. The selected system was an (AB SCIEX Triple TOF™ 5600 MS, Santa Clara, CA, USA) which was used for data acquisition in both positive and negative modes over a mass range of 80–1000 m/z, under the following conditions: both negative and positive ion modes; ion source gas 1 (GC1): 55 psi; ion source gas 2 (GC2): 55 psi; gas curtain 1:25 psi at 400 ◦C; negative ion spray voltage (ISVF): −4500; collision energy (CE): −50; positive ion spray voltage (ISVF): 5500; collision energy (CE): 30; accumulation time for both positive and negative modes set at 100 ms. The MS used the IDA (Information-Dependent-Acquisition) acquisition method, the survey scan type (TOF-MS, Time of Fligh-Mass- Spectrometry) and the dependent scan type (product ion) with collision energy set at 50 V/30 V. Likewise, spermidine was quantified using the LC-MS/MS (Liquid Chromatography—Mass Spectrometry) method by means of direct extrapolation on the standard curve. The results were expressed in mg/L. Determinations were done by triplicate.
