Procyanidin a2

(–)-Epicatechin, (+)-catechin, gallic acid, procyanidin A1, procyanidin A2, procyanidin B1, procyanidin B2, procyanidin C1, 2.2-diphenyl-1-picrylhydrazyl (DPPH), Folin-Ciocalteu reagent (2 mol/l), p-(Dimethylamino)cinnamaldehyde (DMAC), CD₃OD and acetonitrile of chromatographic grade were purchased from Sigma-Aldrich (St. Louis, MO, USA). 2.2′-Azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) was purchased from Aladdin Inc (Shanghai, China). Double-distilled water (ddH₂O) was used in all experiments and samples for HPLC-DAD and LC-ESI-Q-TOF-MS were filtered through a 0.22 μm membrane before injection. All the other reagents were of analytical grade and were bought from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

Four reagent grade (GR) mineral salts such as KH₂PO₄ (≥99.5%), KCl (≥99.5%), K₂SO₄ (≥99.0%), and MgSO₄ (≥99.0%) were used as foliar nutrients in the field experiment.
All of the solvents and reagents used were GR or higher grade quality. The standard materials of gallic acid (≥99%), procyanidin B2, epicatechin (≥98%), 4-hydroxylbenzaldehyde (≥99%), rutin (≥98%), ferulic acid (≥99.5%), kaempferol-3-glucoside (97.0%), and quercetin-3-glucoside (≥98%) were purchased from Aladdin (Shanghai, China), and procyanidin A2 (≥98.0%) from Sigma–Aldrich (Saint Louis, United States). The reference materials of cyanidin-3-glucoside (≥98%) and cyanidin-3-rutinoside (≥90%) were obtained from Macklin-Lab (Shanghai, China).

Methanol, acetonitrile, and acetic acid were from Merck, Germany, with 98% purity, HPLC grade. Gallic acid, procyanidin A2, proanthocyanidins, vanillin, (+)-catechin (C), cyanidin-3-glucoside, procyanidin B2, (−)-epicatechin (EC), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), Folin-Ciocalteu reagent, 1,1-diphenyl-2-picryldydrazyl (DPPH), tripyridyl-triazine (TPTZ) were supplied from Sigma-Aldrich (St. Louis, MO, USA). Other chemicals and solvents used were analytical grade from local suppliers (Sinopharm, Guangzhou, China).

Analytical phenolic standards (caffeic acid, cinnamic acid, chlorogenic acid, daidzein, (−)-epicatechin, ferulic acid, gallic acid, genistein, hesperetin, p-coumaric acid, p-hydroxybenzoic acid, kaempferol, phloretin, procyanidin A2, procyanidin B2, protocatechuic acid, quercetin, quercetin-3-O-glucoside, quercetin 3-O-galactoside, resveratrol, rutin, syringic acid and vanillic acid) were obtained from Sigma-Aldrich (St. Louis, MO, USA). The (+)-catechin hydrate, cyanidin chloride and delphinidin chloride were purchased from Cayman Chemicals (Ann Arbor, MI, USA). Acacetin, apigenin, luteolin, LCMS-grade methanol, acetone (ACS grade), naringenin and naringenin chalcone were purchased from Fisher Scientific (Pittsburgh, PA, USA). Ultra-pure water (with a resistivity of 18 MΩ.cm and a TOC level < 5 ppb) for all the experiments was prepared from a Milli-Q purification system (Millipore, MA, USA).

The analytical grade methanol for extraction was purchased from Avantor Performance Materials (Gliwice, Poland). HPLC grade solvents (acetonitrile, formic acid, orthophosphoric acid) used for UHPLC and HPLC analyses were obtained from Avantor Performance Materials and Sigma-Aldrich (Seelze, Germany/St. Louis, MO, USA). HPLC grade standards of esculin, fraxin, (‒)-epicatechin, procyanidin A2 and ascorbic acid were purchased from Sigma-Aldrich and Phytolab (Vestenbergsgreuth, Germany). All reagents for scavenging assays, the phosphate buffered saline (PBS) and the OPD substrate for peroxidase were purchased from Sigma-Aldrich. Peroxynitrite (ONOO⁻) was synthesized according to Pryor et al. (1995). A Pierce BCA Protein Assay Kit was obtained from Thermo Scientific (Waltham, MA, USA). All immune reagents for 3-nitrotyrosine (3-NT) detection were purchased from Abcam (Cambridge, UK). Redistilled water was used in all analyses. For chemical tests requiring constant temperature, the samples were incubated in a BD 23 incubator (Binder, Tuttlingen, Germany). Scavenging assays and activity tests in blood plasma models were performed in 96-well microplates using a SPECTROstar Nano microplate reader (BMG LabTech, Ortenberg, Germany).

Sigma Aldrich Chemical Co. (St. Louis, MO, USA) provided Folin–Ciocalteu reagents, sodium carbonate, fructose, glucose, DPPH (2,2-Diphenyl-1-picrylhydrazyl), AAPH (2,2′-azobis (2-methyl-propanimidamide) dihydrochloride), fluorescein, and Trolox. In addition, J.T. Baker Chemical Co. (Temixco, Mexico) supplied solvents, including methanol (≥99%) and ethanol (≥99%), while Sigma Aldrich Chemical Co. (St. Louis, MO, USA) supplied formic acid (≥98%), isopropanol (≥99%), acetonitrile (≥99%), and acetone (≥98%). Specific polyphenols, such as quercitin (≥95%), were purchased from the HWI group (Rülzheim, Germany); rutin (≥97%), kaempferol (≥97%), catechin (≥97%), and epicatechin (≥98%) were purchased from Toronto Research Chemicals Inc. (Toronto, ON, Canada); procyanidin A2 (≥90%) and procyanidin B2 (≥90%) were also purchased from Sigma Aldrich Chemical Co. (St. Louis, MO, USA); and caffeic acid (≥98%), vanillic acid (≥98%), and resveratrol (≥99%), were also purchased from TCI America (Portland, OR, USA).

Procyanidin A2 and 3-(4-hydroxyphenyl)propionic acid were purchased from Sigma-Aldrich (Shanghai, China). RPMI-1640 medium, DMEM medium and fetal bovine serum were purchased from Gibco Life Technologies (California, USA). Human ox-LDL was purchased from Yiyuan Biotechnologies (Guangzhou, China). The kits for total cholesterol (TC) and free cholesterol (FC) measurement were purchased from Applygen Technologies Inc., (Beijing, China). A ROS kit was purchased from Beyotime Institute of Biotechnology (Shanghai, China). The kit for determination of MDA equivalent level was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The ELISA kits of IL-6 and IL-1β were obtained from R&D System (Minneapolis, MN, USA). SYBR-Green qPCR Master Mix and RevertAid First Strand cDNA Synthesis Kit were obtained by TransGen Biotech (Beijing, China). The primary antibodies against NF-κB-p65 and β-actin were obtained from Cell Signal Technology Inc. (Beverly, MA). All other reagents were of analytical grade and obtained locally.