L ascorbic acid

Differences in the antioxidant activity between the freshly frozen and differently dried sprout and needle extracts were measured using a single electron transfer-based FRAP (ferric ion reducing antioxidant power) method, which measures the ability of an antioxidant to reduce ferric (FeIII) to ferrous (FeII) ions [46 (link)]. The reaction mixture contained the sample, 20 mmol/L FeCl₃·6H₂O (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) and 10 mmol/L 2,4,6-Tris (2-pyridyl)-s-triazine (TPTZ) (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) in 300 mmol/L acetate buffer pH 3.6. The formation of ferrous-tripyridyltriazine complex in the reaction mixture is measured by absorbance at 593 nm in 96-microplate format with three technical replicates of each sample on the plate and series of dilutions to fit the sample to the standard curve. FeSO₄·7H2O (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) was used as a standard compound and L(+)-ascorbic acid (150 µM and 800 µM) (VWR Chemicals) as a control, and the results are expressed as µmol/L Fe(II) equivalents.

Tetraethylorthosilicate (TEOS), cetyltrimethylammonium bromide (CTAB), sodium hydroxide (NaOH), chloroauric acid (HAuCl₄), silver nitrate (AgNO₃), hydrochloric acid (HCl) and sodium citrate dibasic trihydrate, gold chloride trihydrate (HAuCl₄·3H₂O), sodium citrate tribasic dehydrate, silver nitrate (AgNO₃), l-ascorbic acid (AA), hydrogen peroxide solution (H₂O₂, 30 wt%), (3-mercaptopropyl), trimethoxysilane (MPTMS, 95%), phosphate buffered saline (PBS, pH 7.4), hydrochloric acid (HCl, 37%), sulphuric acid (H₂SO₄, 96%), acetone and 2-propanol were obtained from VWR International, UK. Deionized (DI) water was purified using the Millipore Milli-Q gradient system (>18.2 MΩ).

Pentaerythritol-tetrakis(3-mercaptopropionate) (“tetrathiol”), triallyl-1,3,5-triazine-2,4,6-(1H,3H,5H)-trione (“triallyl”), 2-(boc amino) ethanethiol, and methanol (99.8%) were obtained from Sigma-Aldrich (Brøndby, Denmark). Lucirin TPO-L was obtained from BASF (Ludvigshafen, Germany). Acetonitrile (ACN), trifluoroacetic acid (TFA), DL-dithiothreitol (D9779, DTT), iodoacetamide (I670-9, IAA), immunoglobulin G (56,834-25MG, IgG, from human serum), trypsin (T1426, from bovine pancreas), and salts were purchased from Sigma-Aldrich (Stockholm, Sweden). L( +)-Ascorbic acid (99.0–100.5%) was from VWR chemicals (Stockholm, Sweden). Water (MQ H₂O) was purified in a Millipore Synergy® 185 (Bedford, MA, USA) to a resistivity of 18.2 MΩ·cm at 25 °C. DEAE Affi-Gel® Blue Gel (DEAE) used for IgG extraction was obtained from Bio-RAD, Hercules, USA. Pierce® C18 Tips, 100 µL bed size, were from Thermo Fisher Scientific (Rockford, USA). Kinesis Tubing PEEK (TM) natural 1/32 inch × 0.015 inch connecting syringe and microchip was purchased from Fisher Scientific (Stockholm, Sweden). MALDI matrices 2,5-dihydroxybenzoic acid (DHB) and α-cyano-4-hydroxycinnamic acid (HCCA) and MALDI plate were obtained from Bruker Daltonics (Bremen, Germany).

Wet pomace (WP) was collected from an industrial olive extraction unit (olive mill) at Ribatejo, Portugal, operating on a two-phase centrifugation process. The WP was kept refrigerated at −15 °C in polyethylene bags until use. An industrial sample of dried and extracted (n-hexane) wet pomace (P-Ind) was collected from a pomace oil recovery unit at Alentejo, Portugal.
Quinine hemisulphate monohydrate (> 98%, Fluka, Sigma-Aldrich Corp., St. Louis, MO, USA), ethylenediamine (EDA, >99.5%, Fluka, Sigma-Aldrich Corp., St. Louis, MO, USA), 2,2-diphenyl-1-picrylhydrazyl (DPPH, 95%, Alfa Aesar, Kandel, Germany), L-ascorbic acid (AA, 99%, VWR, Radnor, PA, USA), gallic acid (97.5%, Sigma, Sigma-Aldrich Corp., St. Louis, MO, USA), D-glucose monohydrate (for biochemistry and microbiology, Merck, Darmstadt, Germany), N,N-dimethylformamide (DMF, >99.8%, Sigma-Aldrich Corp., St. Louis, MO, USA), and D₂O (>99.9%, Sigma-Aldrich Corp., St. Louis, MO, USA) were used as received. All other reagents and solvents were of analytical grade and were purified and/or dried by standard methods. Ultrapure water (Milli-Q, Millipore; Merck KGaA, Darmstadt, Germany) was used in all the experiments (synthesis and analysis).

Differences in the antioxidant activity between inner and outer bark extracts obtained from the storage experiments were measured using a SET-based FRAP (Ferric ion reducing antioxidant power) method, which measures the ability of an antioxidant to reduce ferric (FeIII) to ferrous (FeII) ions [43 (link)]. The reaction mixture contained the sample, 20 mM FeCl₃·6H₂O (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) and 10 mM 2,4,6-Tris(2-pyridyl)-s-triazine (TPTZ) (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) in 300 mM acetate buffer pH 3.6. The formation of ferrous-tripyridyltriazine complex in the reaction mixture is measured by absorbance at 593 nm in 96-microplate format with three technical replicates of each sample on the plate and series of dilutions to fit the sample to the standard curve. FeSO₄·7H₂O (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) was used as a standard compound and l(+)-ascorbic acid (150 μM and 800 μM) (VWR Chemicals) as a control and the results are expressed as μmol/L Fe(II) equivalents.

The analytical standards caffeic acid, (+) catechin, ellagic acid, ferulic acid, gallic acid, trans para-coumaric acid, 3,4-dihydroxybenzoic acid (protocatechuic acid), urolithin A, urolithin B [3-hydroxyl-6H-benzo(c)chromen-6-one], 4-hydroxyl-3-methoxycinnamaldehyde (coniferyl aldehyde), trans-3,5-dimethoxy-4-hydroxycinnamaldehyde (sinapic aldehyde), scopoletin, syringic aldehyde, syringic acid, vanillic acid, vanillin, and the internal standard 1,2,3-¹³C₃ ferulic acid were purchased from Sigma Aldrich (Taufkirchen, Germany). Urolithin C and urolithin D were obtained from Biozol (Eching, Germany). Methanol (BAKER ANALYZED LC-MS Reagent), water (HiPerSolv CHROMANORM for HPLC MS grade), acetonitrile (Ultra Gradient HPLC Grade), and L(+) ascorbic acid were purchased from VWR (Darmstadt, Germany). Formic acid, ammonium formate, ß glucuronidase from Helix pomatia (type HP 2, aqueous solution), ethyl acetate, disodium ethylenediaminetetraacetic acid (EDTA), magnesium sulfate (anhydrous), sodium acetate, and Dulbecco's phosphate buffered saline were all obtained from Sigma Aldrich.

Ferric reducing antioxidant power (FRAP) assay is based on single-electron transfer and measures the ability of an antioxidant to reduce ferric (FeIII) to ferrous (FeII) ion [35 (link)]. Samples, with three technical replicates in a 96-well format, were used in the assay as described by Vaario et al. [36 (link)]. The samples were mixed with 20 mM FeCl₃·6H₂O and 10 mM 2,4,6-tris (2-pyridyl)-s-triazine (TPTZ) (both from Sigma-Aldrich Chemie GmbH, Steinheim, Germany) in 300 mM acetate buffer pH 3.6. The absorbance was measured at 594 nm with a fluorescence microplate reader (Varioskan Flash, Thermo Scientific) after the formation of the ferrous-tripyridyltriazine complex in the reaction mixture. FeSO₄·7H₂O (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) was used as a standard compound and L(+)-ascorbic acid (150 µM and 800 µM) (VWR Chemicals) as a control and the results are expressed as µmol Fe(II) equivalents per 100 g.