Trans piceid

Methanol and acetonitrile gradient grade for liquid chromatography were supplied by Merck (Darmstadt, Germany). Water used for all solutions was obtained from a Milli-Q water purification system (Millipore) (Woburn, MA, USA). Acetic solution 50% for HPLC and standards of trans-resveratrol, trans-piceatannol trans-pterostilbene and trans-piceid were purchased from Sigma-Aldrich (St. Louis, MO, USA). All standards presented a purity ≥95%. The chemical structures of the molecules are presented in Table 5. The stock solutions were prepared in methanol and standard solutions by diluting with water.

The fully ground powder of grape leaves was dried at −105 °C for 24 h; the samples were weighed and then extracted in methanol (Tedia, Fairfield, USA) away from light at 4 °C overnight. The insoluble solid was discarded by low-temperature centrifugation at 4000 × g for 15 min. The clear methanol extracts were filtered through a 0.22 µm membrane film and stored in sample bottles. HPLC determination was conducted using an Agilent 1260 Infinity HPLC system (Agilent, USA). Stilbene was separated from the filtered samples (10 µL) using a binary gradient of solvent A (acetonitrile) and solvent B (ultrapure water). The wavelength for fluorimetric determination was 306 nm. The gradient conditions were based on a previous study^{79 (link)}. The retention times were confirmed using standard samples of trans-resveratrol and trans-piceid (Sigma-Aldrich, USA). The stilbenoid concentration was determined based on the peak area.

Extracellular content of trans-resveratrol and trans-piceid was determined as described by Pietrowska-Borek et al. [3 (link),6 (link)]. For this, 20 µL of diluted and filtered (Anopore 0.2 µm) samples were analyzed by HPLC in the UV–VIS range using a LiChrospher 100 RP-18 column (250 × 4 mm, 5 µm; Merck, Darmstadt, Germany). Gradient elutions were performed with 0.05% TFA (solvent A) and 0.05% TFA in methanol:acetonitrile (60:40 v/v; solvent B): 0 min, 10% B; 5 min, 15% B; 40 min, 35% B; 45 min, 65% B; 50 min, 65% B; and 55 min, 10% B, setting the flow rate at 1 mL min⁻¹. To determine the intracellular content of trans-resveratrol and trans-piceid, 200 mg of freeze-dried cells were extracted overnight with 4 mL of methanol at 4 °C with continuous shaking, and then 20 µL of each sample was analyzed on a LiChrospher 100 RP-18 column as described above. trans-Resveratrol and trans-piceid were identified (at 308 nm) and quantified by comparison with standards of commercial trans-resveratrol (Sigma-Aldrich, St. Louis, MO, USA) and trans-piceid (ChromaDex, Los Angeles, CA, USA) using respective calibration curves.

Reagents of analytical grade used for HPLC analyses included the standards trans-piceid, ɛ-viniferin, and trans-resveratrol (Sigma-Aldrich, St. Louis, MI, USA). Other used reagents were methyljasmonate (MeJA), cis-jasmonate (cJA), dihydrojasmonic acid (2HJA), cellulase from Trichoderma viride (TvC), sodium orthovanadate (NaVO) (Sigma-Aldrich, St. Louis, MI, USA), naphthaleneacetic acid (NAA), 6-benzylaminopurine (BAP), sucrose, agar, yeast extract, bacto-peptone, magnesium sulphate heptahydrate, potassium dihydrogen phosphate, sodium dodecylsulphate (SDS) (Sigma-Aldrich, St. Louis, MI, USA; Duchefa BIOCHEMIE B.V, Netherlands; Imuna, Slovakia, Merck, Germany). Water was purified by the Direct-Q^® 3 UV Water Purification System (Merck Millipore, Darmstadt, Germany). Reference standard solutions of polyphenols were prepared in mixture methanol:water (1:1, v/v) and stored at 4 °C in dark.

Stilbenoids standards trans-resveratrol, trans-piceid, trans-pterostilbene, ε-viniferin used for the identification of stilbenoids in grape berry extracts along with analytical grade methanol, acetonitrile, and ethyl acetate were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Acetonitrile (ACN) and methanol (MeOH) were HPLC supergradient purity. Catechin, epiCatechin, vanillic acid, protocatechuic acid, 4-hydroxybenzoic acid, gallic acid, syringic acid, p-coumaric acid, trans-resveratrol, trans-piceid, caffeic acid, ferulic acid, piceatannol, rutin, myricetin, quercetin, kaemferol, isorhamnetin, and perchloric acid were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Malvidine -3,5-diglukoside was purchased from Indofine Chemical Company. Inc. (Hillsborough, NJ, USA).
Other used chemicals were at least analytical grade and were obtained from local suppliers (Lachema, Penta, Prague, Czech Republic).
A stock standard solution was prepared by accurately weighting about 10 mg of each phenol in 25 mL volumetric flask. The standard was dissolved in 10 mL of acetonitrile, bringing up to volume with distilled water [27 (link)].

The flavan-3-ols and stilbenes were analyzed in an HPLC Agilent 1200 series system equipped with DAD (Agilent, Waldbronn, Germany), and coupled to an AB Sciex 3200 TRAP (Applied Biosystems, Foster City, CA, USA) with triple quadrupole, turbo spray ionization (electrospray assisted by a thermonebulization, Agilent, Waldbronn, Germany) and a mass spectroscopy system (ESI-MS/MS, Agilent, Waldbronn, Germany). They were previously isolated from wine by SPE on C18 cartridges (Waters Sep-Pak Plus, 820 mg of adsorbent, Saint-Quentin En Yvelines, France) and then separated in an Ascentis-C18 column (4.6 × 150 mm; 2.7 μm particle; Supelco, Germany), thermostated at 16 °C and with a flow rate of 0.3 mL/min [45 (link)].
Pure standards were used for identification and quantification (calibration curves): (+)-catechin, (−)-epicatechin, trans-resveratrol and trans-piceid from Sigma Aldrich (Tres Cantos, Madrid, Spain); (+)-gallocatechin and (−)-epigallocatechin from Phytolab (Vestenbergsgreuth, Germany); procyanidins B1 and B2 from Extrasynthese (Genay, France). cis-Resveratrol and cis-piceid were obtained by UV irradiation (366 nm during 5 min in quartz vials) of a 25% MeOH solution of their trans isomers.