Avicularin

The following substances and solvents were used in the study: procyanidins B1, B2, C1, A1, A2, A4, (+)-catechin, (–)-epicatechin, quercetin, quercitrin (quercetin-3-O-rhamnoside), isoquercitrin (quercetin-3-O-glucoside), avicularin (quercetin-3-O-arabinofuranoside), guaiaverin (quercetin-3-O-arabinopyranoside), rutin (quercetin-3-O-rutinose), reynoutrin (quercetin-3-O-xyloside), kaempferol, nicotiflorin (kaempferol-3-O-rutinoside), afzelin (kaempferol-3-O-rhamnoside), astragalin (kaempferol-3-O-glucoside), chlorogenic acid (3-O-caffeoylquinic acid), neochlorogenic acid (5-O-caffeoylquinic acid), cryptochlorogenic acid (4-O-caffeoylquinic acid), p-coumaric acid, arbutin, hydroquinone, α-amyrin, β-amyrin, β-sitosterol, lupeol, erythrodiol, maslinic acid, oleanolic acid, acetonitrile, acetone, and methanol, which were purchased from Sigma–Aldrich (Steinheim, Germany); hyperoside (quercetin-3-O-galactoside), procyanidin B3, uvaol, friedelin, 6″-O-acetylisoquercitrin (quercetin-3-O-(6″-acetylglucoside)), betulin, and betulinic and corosolic acids from Extrasynthese (Genay, France); ursolic acid from Carl Roth (Karlsruhe, Germany); and trifluoroacetic acid from Merck (Darmstadt, Germany). All used chemicals were of HPLC grade. Ultrapure water used in this study was prepared with Milli–Q^® 180 (Millipore, Bedford, MA, USA) water purification system.

All the solvents, reagents, and standards used were of analytical grade. The following substances were used in the study: Ethanol 96% (v/v) (AB Strumbras, Kaunas, Lithuania), procyanidin C1, procyanidin B2, quercetin, hyperoside, avicularin, quercitrin, kaempherol 3-O-glucoside, luteolin 7-O-glucoside, phloridzin, formic acid, acetonitrile, (+)-catechin, (-)-epicatechin, rutin, isoquercitrin, chlorogenic acid, p-coumaric acid, caffeic acid, hydrochloric acid, Hoechst33342, propidium iodide, glucose, temozolomide, DMSO and KCl (Sigma-Aldrich, Steinheim, Germany), Dulbecco’s Modified Eagle Medium (DMEM) with Glutamax, foetal bovine serum, penicillin-streptomycin, Versene solution, antibiotic-antimycotic solution (Anti-Anti) were of Gibco brand and purchased from Thermo Fisher Scientific, Waltham, MA, USA. During the study, we used purified de-ionized water prepared with the Milli–Q^® (Millipore, Bedford, MA, USA) water purification system.

Crabapple leaf samples (approximately 0.8–1.0 g fresh weight) were subjected to extraction with 10 mL extraction solution (methanol: water: formic acid: trifluoroacetic acid= 70: 27: 2: 1)58 (link) at 4 °C in the dark for 72 h, shaking every 6 h. The supernatant was isolated by filtration through filter paper and a further filtration through a 0.22 μm Millipore^TM filter (Billerica, MA, USA). For the HPLC analysis, trifluoroacetic acid: formic acid: water (0.1: 2: 97.9) was used as mobile phase A and trifluoroacetic acid: formic acid: acetonitrile: water (0.1: 2: 48: 49.9) was used as mobile phase B. The gradients used were as follows: 0 min, 30% B; 10 min, 40% B; 50 min, 55% B; 70 min, 60% B; 30 min, 80% B. Detection was performed at 520 nm for anthocyanin and 350 nm for flavonol58 (link), respectively. All samples were analyzed in three biological triplicates (extracted from three different batches of leaves).
In this study, we employed HPLC-ESI (±)-MS2 analysis to identify the kinds of compounds by standards and comparing their spectroscopic data to literature59 60 (link). Cyanidin-3-O-glucoside, quercetin-3-O-glucoside, avicularin, phloridzin, quercetin (Sigma-Aldrich, Germany), Procyanidin B2 (Sigma-Aldrich, UK) was used as standards.