Repuestos

5.3.4.1. Spectrophotometric analyses: total phenolic compounds (TP), total flavonoids (TF), total monomeric anthocyanins (TMA) and polymeric color (PC)

For total phenolic compounds and total flavonoids analyses of the CO2/EtOH/H2O elderberry pomace extracts, solutions were prepared by dissolving the dried extracts in EtOH (for TP) or methanol (MeOH) (for TF), and then adding ddH2O until a 50:50 (v/v) final proportion.

Total phenolic compounds in these extracts were quantified according to the Folin-Ciocalteu’s method, and following the procedure proposed by Singleton and Rossi (1965) with some modifications, based on Cheung et al. (2003). Up to 0.5 mL aliquots of each extract sample were introduced into test tubes, and aqueous EtOH (50:50) was added as

necessary to bring the total volume to 0.5 mL. The Folin-Ciocalteu’s reagent (0.5 mL) was added and the mixture vortexed. After 3 min, 0.5 mL of saturated Na2CO3 (~17%) was added to the mixture and it was made up to 5.0 mL by adding distilled water. The reaction was kept in the dark for 90 min, after which absorbance was read at 725 nm. Results were designated by TP and expressed as gallic acid equivalents (GAE).

Total flavonoids in the PSE extracts were measured by an assay developed by Zhishen et al. (1999). Up to 1.0 mL aliquots of each extract sample were introduced into test tubes containing 4 mL ddH2O, and aqueous MeOH (50:50) was added as necessary to bring the total volume to 5.0 mL; 0.3 mL of aqueous NaNO2 (5:95) was added to the flask, followed by 0.3 mL of aqueous AlCl3 (10:90) and 2 mL of 1 M NaOH. The reaction solution was immediately diluted with the addition of 2.4 ml of ddH2O and thoroughly mixed, and its absorbance was measured at 510 nm. Results were designated by TF and expressed as epicatechin equivalents (ECE).

The CO2/EtOH/H2O elderberry pomace extracts were also analyzed by the pH differential method (Giusti and Wrolstad, 2001) to quantify total monomeric anthocyanins (TMA) and polymeric color (PC). For TMA quantification, extracts were diluted in potassium chloride buffer pH 1.0 and in sodium acetate buffer pH 4.5 using the appropriated dilution factor.

Absorbance measurements were taken at 520 and 700 nm, after 15 min. TMA contents were determined considering the molar extinction coefficient and molecular weight of cyanidin-3-glucoside chloride (26900 L/(cm.mol) and 449.2 g/mol, respectively), and expressed as CyG equivalents (CyGE). For PC evaluation, extracts were diluted in distilled water using the appropriated dilution factor. Potassium metabisulfite solution and water were added to two separate samples and absorbances were measured at 420, 520 and 700 nm, after 15 min.

An UV/VIS spectrophotometer (Jasco V-530, Japan) was employed for all measurements which were run in triplicate, and results were expressed in percentage (w/w, d.b.).

5.3.4.2. Analysis by high-performance liquid chromatography, coupled to photodiode array and electrospray ionization mass spectrometry detectors (HPLC-PDA-ESI/tandem MS)

Anthocyanins and rutin identification for the CO2/EtOH/H2O (0:80:20) extract was carried out on a SURVEYOR LC equipped with a Surveyor MS Pump (MSPUMP) and a PDA detector Surveyor PDA Plus (Thermo Finnigan) and interfaced with a QITMS mass spectrometer (LCQ Advantage Ion Max MS/MS, Thermo Finnigan) equipped with an API-ES ionization chamber, which were controlled by the LCQ Xcalibur software.

Separation was performed on an Spherisorb ODS2 column, 150×2.1 mm, 3 μm particle size and a Spherisorb ODS2 guard cartridge, 10×4.6 mm, 5 μm particle size (Waters, MA, USA) at 293 K, using 2% aqueous formic acid (A) and methanol (B) as mobile phase. A discontinuous gradient of 5-15% B in A (0-10 min), 15-25% B (10-15 min), 25-50% B (15-40 min), 50-80% B (40-50 min) and 80% B (50-60 min) isocratically was used at a flow rate of 200 μL/min. The first detection was made with a PDA in a wavelength range 200-600 nm, followed by a second detection in the mass spectrometer.

Mass analyses were obtained in the negative ion mode. The mass spectrometer was programmed to perform two consecutive scans: full mass (m/z 50-2000) and MS² of the most abundant ion in the full mass. Source voltage was 5 kV and the capillary voltage and temperature were -10 V and 563 K. Nitrogen was used as sheath and auxiliary gas at flow rate of 13 and 2 (Finnigan arbitrary units), respectively. The normalized energy of collision was 35% using helium as collision gas.

5.3.4.3. HPLC-PDA analysis

PSE extracts were analyzed in a Gilson apparatus equipped with a diode-array detector and data treatment was carried out with software Unipoint^® 2.10 Gilson. An ODS-2 column (250×4.6 mm i.d., 5 μm, Spherisorb S5, Waters, MA, USA) at 298 K and a C18 guard cartridge (30×4 mm i.d., 4 μm, Hichrom, Berkshire, UK) were used. A mobile phase, constituted by aqueous formic acid (5%, v/v) (A) and MeOH (B), was used with a discontinuous gradient of 5-15% B (0-10 min), 15-25% B (10-15 min), 25-50% B (15-40 min) and 50-80% B (40-50 min), followed by an isocratic elution during 10 min, at a flow rate of 1 mL/min. Samples were adjusted to pH ~2 with HCl and microfiltered (0.20 μm) before HPLC injection. Anthocyanins were detected from their chromatographic and ultraviolet (UV) spectral properties and the major anthocyanins, CyG, CyS and CySG, were identified by comparing with the HPLC-PDA-ESI/tandem MS results. Quantification of CyG, CyS, CySG and total anthocyanins (TA) was carried out from a calibration curve prepared using the CyG standard, at 520 nm, and expressed as cyanidin-3-glucoside equivalents (CyGE), in percentage (w/w, d.b.). Quantification of rutin (R) was carried out by using a calibration curve prepared from the rutin standard, at 360 nm. The assay standard deviation was obtained by the central experimental point extract injected three times.

5.3.4.4. Antioxidant activity: DPPH assay

The method described by Blois (1958) was employed in which aliquots (100 µL) of extracts were added to 500 µL of a methanolic solution (500 µM) of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical and 1.4 mL of methanol. After mixing for 30 s, the reaction mixture was incubated in the dark at room temperature for 30 min and the absorbance measured at 517 nm on a spectrophotometer (U-2000 model, Hitachi, Tokyo, Japan). The extracts reducing activities were estimated from the decrease in absorbance, and the results expressed as IC50 values, defined as the amount of extract that decreased by 50% the initial absorbance of the DPPH radical solution, at 517 nm. All assays were performed in triplicate.

5.4. Results and discussion