Neochlorogenic acid

Folin–Ciocalteu’s reagent, methanol, ethyl acetate, formic acid, acetic acid, gallic acid, Na₂CO₃, and AlCl₃ were purchased from POCH S.A. (Gliwice, Poland). Standards of apigenin, luteolin, rutin, rosmarinic acid, caffeic acid, and chlorogenic acid for HPTLC, 2,2-diphenyl-1-picrylhydrazyl (DPPH), Trolox, carrageenan, and Tween-80 were purchased from Sigma-Aldrich (Poland). HPTLC silica gel 60 F₂₅₄ plates, trifluoroacetic acid, acetonitrile, and standards for HPLC (chlorogenic acid, neochlorogenic acid, rosmarinic acid, caffeic acid, luteolin-7-O-glucoside, apigenin-7-O-glucoside, acacetin-7-O-glucoside, luteolin, and apigenin) were purchased from Merck (Germany). The tablets of “Diclophenac” (produced by chemical and pharmaceutical factory “Chervona zirka”, Kharkiv, Ukraine) and “Metamizole sodium (Analgin)” (produced by pharmaceutical factory “Darnitsa”, Kyiv, Ukraine) were purchased from the pharmacies in Ukraine. All the reagents and solvents were of analytical grade.

Chokeberry fruits were bought at the local market. Alginic acid sodium salt (very low viscosity) was obtained from Alfa Aesar (Kandel, Germany), while low-methoxyl pectin (Genu pectin type LM-5-CS) was from CP Kelco (Lille Skensved, Denmark). Calcium chloride, ethanol, sodium acetate, potassium chloride and ammonium acetate were from Gram-mol (Zagreb, Croatia). Sodium carbonate was purchased from T.T.T. (Sveta Nedelja, Croatia). Potassium persulfate and Folin-Ciocalteu reagent were procured from Kemika (Zagreb, Croatia). Cupric chloride, neocuproine and 2,4,6-tri(2-pyridyl)-s-triazine (TPTZ) were bought from Acros Organic (Geel, Belgium). methanol (HPLC grade) was from J.T. Baker (Deventer, The Netherlands) and orthophosphoric acid (HPLC grade, >85%) was from Fisher Scientific (Loughborough, UK). Hydrochloric acid (37%) and methanol were bought from Carlo Erba Reagents (Sabadell, Spain) and acetic acid (>99.5%) was from Alkaloid (Skopje, North Macedonia). Trolox, 2,2-diphenyl-1-picrylhydrazil, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, 4-dimethyl-amino-cinnamaldehyd and most of the standards used for HPLC analysis (chlorogenic acid, quercetin and rutin) were from Sigma-Aldrich (St. Louis, MO, USA), while the standards of cyanidin-3-galactoside, neochlorogenic acid and hyperoside were products of Extrasynthese (Genay, France).

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.

A total of 14 A. quinata leaves samples were collected from different areas in Gongju and Muju, South Korea. All samples were authenticated by Professor Jong Seong Kang (College of Pharmacy, Chungnam National University, Daejeon, Korea), as shown in Figure S1 and Table S1 (Supplementary Materials). The samples were deposited in the pharmaceutical analysis laboratory, College of Pharmacy, Chungnam National University, with a code number of AQLS. The harvested samples were dried in an LTO-Do-150S oven (Labtech-one, Daejeon, Korea) at 45 °C for 3 days. After drying, the samples were pulverized into a fine powder, then stored at 4 °C until use. Mouse anti-ICAM-1 (#sc-8439), mouse VCAM-1 (#sc-13160), mouse anti-NF-kB p65 (sc-514451), and anti-phospho-eNOS antibody were purchased from Santa Cruz Technology, Inc. (Santa Cruz, CA, USA). The reference standards of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, rutin isochlorogenic acid C, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and a buffer solution of formic acid were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). HPLC-grade methyl alcohol and acetonitrile were purchased from Burdick & Jackson (Muskegon, MI, USA). Water was purified using a Milli-Q system (Milford, MA, USA).

Chemical reagents (acetonitrile, L-ascorbic acid, methanol, acetic acid, and formic acid) were purchased from Sigma-Merck, Poznań, Poland. For the qualitative and quantitative content of polyphenols the following standards were used: gallic acid, caffeic acid, isovanilic acid, chlorogenic acid, neochlorogenic acid, p-coumaric acid, 4-hydroxybenzoic acid, 3,4-dihydroxybenzoic acid, orientin (luteolin-8-glucoside), quercetin, quercetin-3-O-glucoside, procyanidin B2, luteolin, kaempferol (Sigma-Aldrich, Poznań, Poland), cyanidin-3-O-glucoside, cyanidin-3-O-galactoside, cyanidin-3-O-arabinoside (Polyphenols Laboratories AS, Sandnes, Norway), cyanidin-3-O-xyloside (Toronto Research Chemicals, North York, ON, Canada), quercetin-3-O-galactoside, quercetin-3-O-vicianoside, quercetin-3-O-rutinoside, (+)-catechin and (−)-epicatechin (Extrasynthese, Genay, France). All other chemicals were of analytical grade.

Neochlorogenic acid, gallic acid, protocatechuic acid, chlorogenic acid, caffeic acid, p-hydroxybenzoic acid, 1,3-dicaffeoylquinic acid, p-coumaric acid, rutin, quercetin 3-β-galactoside, ferulic acid, taxifolin, 3,4-dicaffeoylquinic acid, trans-m-coumaric acid, quercetin 3-α-L-rhamnoside, 3,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid, rosmarinic acid, myrcetin, luteolin, quercetin, trans-cinnamic acid, apigenin, kaempferol, fenofibrate, dimethyl sulfoxide (DMSO), ethylenediaminetetraacetic acid (EDTA), hematoxylin, eosin, and trizma base were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM), streptomycin, and penicillin were purchased from Invitrogen (Carlsbad, CA, USA). Other reagents were commercially available and of special grade.