Viniferin

The solvents used for polyphenol extraction and liquid chromatographic mass spectrometry (LC-MS/MS) analysis, methanol, acetonitrile and formic acid, were purchased from Merck (Darmstadt, Germany). 2,2-diphenyl-1picrylhydrazyl (DPPH), used to determine the antioxidant capacity, was purchased from Thermo Fisher (Kandel, Germany). The polyphenols used as standards, aminobenzoic acid, acetylsalicylic acid, caffeic acid, chlorogenic acid, ellagic acid, gallic acid, p-coumaric acid, protocatechuic acid, salicylic acid, trans-ferulic acid, vanillic acid, apigenin, epicatechin, aesculetin, catechin hydrate, isorhamnetin, kaempferol, luteolin, polydatin, quercetin, resveratrol, rutin, syringaldehyde and viniferin, were purchased from Sigma-Aldrich (Madrid, Spain). The Mili-Q water used in all the solutions was purified with the Merck Millipore Milli-Q™ Reference Ultrapure Water Purification System model Z00QSVC01 (Darmstadt, Germany).

The phenolic standards including malvidin-3-O-glucoside, procyanidin dimers B1, gallic acid, (+)catechin, (−)epicatechin, procyanidin dimers B2, (−)-epigallocatechin, caffeic acid, myricetin, quercetin, quercetin 3-glucoside, vanillic acid, trans-p-coumaric acid, 3,4-dihydroxybenzoic acid, polydatin, kaemperol, cis-resveratrol, trans-resveratrol, trans-4-hydroxy-3-methoxycinnamic acid and viniferin were purchased from Sigma-Aldrich (St. Louis, MO, USA). The water was purified using a Milli-Q system (Millipore, Billerica, MA, USA). Ortho-phosphoric acid was obtained from Tianjin kemio chemical reagent Co., Ltd. (Tianjin, China). Acetonitrile was obtained from Fisher Scientific (Fair Lawn, NJ, USA). Acetaldehyde, DNPH-Acetaldehyde hydrazine standard, DNPH (30% water, m/m) and bovine serum albumin were obtained from Sigma-Aldrich (St. Louis, MO, USA), while the DNPH (30% water, m/m) was purified by recrystallization from Acetonitrile. All above solvents employed were high-performance liquid chromatography (HPLC grade). DPPH was purchased from Sigma-Aldrich (St. Louis, MO, USA).

Stilbenes and ABA were quantified starting from 200 mg of frozen leaves. Reversed-phase high-performance liquid chromatography analysis was performed on a 1,260 Agilent Technologies system. Chromatographic separation was performed with a Phenomenex (Torrance, CA) Luna C18 column (150 × 2.1 mm, 3 μm particle size), with a C18 SecurityGuard column (4.0 × 3.0 mm ID), operated at room temperature. Elution was carried out using aqueous formic acid (0.1% v/v; mobile phase A) and acetonitrile (mobile phase B). HPLC analysis was performed using a gradient from 20 to 60% of mobile phase B in 15 min, then from 60 to 100% of B in 4 min; after washing for 5 min with solvent B, the column was re-equilibrated. The flow rate was 0.2 mL/min and injection volume was 10 μL. A triple quadrupole mass spectrometer (Varian 310-MS TQ Mass Spectrometer) equipped with an ESI interface operated in negative ion mode was used. The detection of analytes was carried out in multiple reaction monitoring (MRM) mode by monitoring two transitions for each compound. The concentration of analytes was quantified using an external calibration method. Original standards of resveratrol (purity ≥99%), polydatin (purity ≥95%), and viniferin (purity ≥95%), purchased from Sigma-Aldrich, were used to prepare the calibration curves. Similarly, the ABA content was quantified as described by Siciliano et al. (2015 (link)).