Astragalin

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.

The compound 2-aminoethyl diphenylborinate (NTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH) and flavonoid standards (myricetin, naringenin, chrysin, and galangin) were purchased from Fluka (Steinheim, Germany); acetonitrile and formic acid (both of them MS grade), methanol (HPLC grade), sodium carbonate, hydrochloric acid, toluene, ethyl acetate, and Folin–Ciocalteu reagent from Merck (KGaA, Darmstadt, Germany); polyethyleneglycol 4000 (PEG), standards of phenolic compounds (rutin, hyperoside, astragalin, chlorogenic acid, caffeic acid, gallic acid, and ferulic acid) and Trolox standard from Sigma-Aldrich (Steinheim, Germany). All chemicals which purity is not previously emphasized were of analytical purity grade.
methanolic standard mixture of phenolic compounds was prepared with following final standard concentrations: galangin (50 ng/µL), chlorogenic acid, myricetin, chrysin and caffeic acid (100 ng/µL), gallic acid, ferulic acid and naringenin (200 ng/µL).
The cartridges for solid-phase extraction (SPE) were Strata C18–E (500 mg/3 mL) obtained from Phenomenex (Torrance, CA, USA). Ultrapure water was obtained from a TKA MicroPure (Thermo Fisher Scientific, Bremen, Germany) water purification system, 0.055 µS/cm. Syringe filters (13 mm, polyfluorotetraethylene (PTFE) membrane 0.45 µm) were purchased from Supelco (Bellefonte, PA, USA).

HPLC-grade methanol, acetonitrile, and chemicals were purchased from Merck, Mumbai, India. Reference standards of chlorogenic acid, rutin, isoquercetrin, nictoflorin, astragalin, quercetin were purchased from Sigma-Aldrich (St Louis, MO, USA). The chemical structures of the six constituents were listed in Figure 2.

Choline chloride, L-proline, succinic acid, methanol, sodium hydroxide, sodium carbonate, sodium-nitrite, 2,2-Diphenyl-1-picrylhydrazyl (DPPH), trolox, gallic acid, protocatechuic acid, syringic acid, chlorogenic acid, p-hydroxybenzoic acid, aesculetin, caffeic acid, isoorientin, rutin, vitexin, isoquercetin, p-coumaric acid, quercitrin, astragalin, rosmarinic acid, luteolin, quercetin, naringenin, kaempferol, and rutin were purchased from Sigma Aldrich Chemical Co. (St. Louis, MO, USA). Urea, ascorbic acid, and ethyl acetate were purchased from Betahem (Belgrade, Serbia). Lactic acid and tartaric acid were purchased from ICN Biomedicals Inc. (Aurora, OH, USA). Maleic acid and aluminum chloride were purchased from Acros Organics (Geel, Belgium). Folin–Ciocalteu reagent was purchased from Carlo Erba reagents (Milan, Italy). Glycerol was purchased from Zorka Pharma (Šabac, Serbia). All solvents and chemicals were analytical purity grade.