Quercetin 3 o rutinoside

ABTS (2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (Sigma-Aldrich, Poland); acetonitrile (Sigma-Aldrich, Poland); deionized water (Sigma-Aldrich, Poland); ethyl acetate (Merck, Poland); methanol (Merck, Poland); ortho-phosphoric acid (Chempur, Poland); phenolic compound standards (purity 99.5%—99.9%), including caffeic acid, chlorogenic acid, epigallocatechin, gallic acid, kaempferol, kaempferol-3-O-glucoside, quercetin, quercetin-3-O-glucoside, quercetin-3-O-rutinoside, and salicylic acid (Sigma-Aldrich, Poland); caffeine (Merck, Poland); and phosphate-buffered saline (Merck, Poland) were used in this study.

Formic acid, bile salts, soluble starch, (+)-arabinogalactan, tryptone, yeast extract, xylan from birchwood, L-cysteine hydrochloride monohydrate, guar gum, inulin, Tween 80, buffered peptone water, Dulbecco′s phosphate buffer saline (PBS), casein sodium salt from bovine milk, pectin from citrus fruits, mucin from porcine stomach-type III, CaCl₂, KCl, NaCl, NaHCO₃, anhydrous K₂HPO₄, KH₂PO₄, MgSO₄ monohydrate, FeSO₄ heptahydrate, resazurin redox indicator, quercetin, kaempferol, quercetin-3-O-rutinoside (aka rutin), phenylacetic acid, 4′-hydroxyphenylacetic acid, 3′-hydroxyphenylacetic acid, 3′,4′-dihydroxyphenylacetic acid, 3-phenylpropanoic acid, 3-(4′-hydroxyphenyl)propanoic acid, 3-(3′-hydroxyphenyl)propanoic acid, 3-(3′,4′-dihydroxyphenyl)propanoic acid (aka dihydrocaffeic acid), 4-hydroxybenzoic acid, 3-hydroxybenzoic acid, 3,4-dihydroxybenzoic acid (aka protocatechuic acid), and benzene-1,3,5-triol (aka phloroglucinol) were obtained from Sigma-Aldrich (St Louis, MO, USA). Isorhamnetin was from PhytoLab GmbH (Vestenbergsgreuth, Germany). All solvents and water were UHPLC-grade and were purchased from VWR International (Milan, Italy).

All of the solvents and chemicals that were used in this study were purchased from Sigma-Aldrich (Darmstadt, Germany).
The anthocyanin standards cyanidin-3-O-glucoside chloride, pelargonidin-3-O-glucoside chloride, cyanidin-3-O-galactoside (purity 90%), cyanidin-3-O-arabinoside (purity 97%), cyanidin-3-O-glucoside (purity 95%), and cyanidin (purity 95%) were purchased from Polyphenols AS (Sandnes, Norway). Chlorogenic acid, caffeic acid, quercetin-3-O-rutinoside, quercetin-3-O-glucoside, ellagic acid, rutin, and myricetin were also purchased from Sigma-Aldrich (Darmstadt, Germany).

ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (Sigma-Aldrich, Poland), acetonitrile HPLC purity (Sigma-Aldrich, Poznan, Poland), deionized water (Sigma-Aldrich, Poland), methanol HPLC purity (Merck, Warszawa, Poland), ortho-phosphoric acid 99.9% (Chempur, Piekary Śląskie, Poland), phenolics standards (purity 99.5%–99.9%) of gallic, chlorogenic, p-coumaric benzoic, ellagic, oenothein B, quercetin-3-O-rutinoside, myricetin, luteolin, quercetin, quercetin-3-O-glucoside and kaempferol (Sigma-Aldrich, Poland) and phosphate-buffered saline (Merck, Warszawa, Poland) were used.

Phenolic acids including, gallic acid, caffeic acid, ferulic acid, and chlorogenic acid were purchased from Sigma (St. Louis, MO, USA) and flavonoids including, kaempferol, kempherol-3-O-rutinoside, kempherol-3-O-glucoside, quercetin, quercetin-3-O-rutinoside, quercetin-3-O-glucoside, (+)-catechin, (−)-epicatechin and cyanidin-3-O-glucoside, were purchased from Sigma and from Extrasynthese Co. ™ (Geney, France).
The standard solutions (10 ppm) were prepared in methanol. All the solvents, as methanol and acetonitrile (Honeywell Riedel-de Haen), were LC-MS grade. Purified water was obtained from Milli-Q Plus™ System from Millipore (Milford, MA, USA). Formic acid was purchased from Aldrich (St. Louis, MO, USA).

ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (Sigma-Aldrich, Poland); acetonitrile at HPLC purity (Sigma-Aldrich, Poznan, Poland); deionized water (Sigma-Aldrich, Poland); methanol at HPLC purity (Merck, Poznan, Poland); ortho-phosphoric acid 99.9% (Chempur, Piekary Śląskie, Poland); phenolics standards (purity 99.5–99.9%) of gallic, chlorogenic, p-coumaric benzoic, ellagic, oenothein B, quercetin-3-O-rutinoside, myricetin, luteolin, quercetin, quercetin-3-O-glucoside, and kaempferol (Sigma-Aldrich, Poland); and phosphate-buffered saline (Merck, Poznan, Poland) were used.

Petals were harvested from the fully open flowers and were homogenized in extraction solution (0.1% HCl in methanol) followed by incubation at 4°C in the dark for 24 h. The supernatants was transferred to a clean tube and filtered through a 0.2 μm Tefion filter before analysis. The extract was analyzed using SHIMADZU HPLC with TSK ODS-80Ts QA (4.6 mm × 250 mm, 5 μm, Tosoh) column. The volume of injection was 10 μl. Milli-Q water containing 10% (v:v) formic acid was used as solution A and CH₃CN containing 0.1% (v:v) formic acid was used as solution B. The linear elution gradient method was as follows: 0–20 min, 10% B; 20–25 min, 30% B; 25–35 min, 10% B. at 520 and 360 nm for anthocyanins and flavonols, respectively. The quantification of the anthocyanins was achieved as cyanidin Chromatograms were acquired 3-O-glucoside (Sigma, USA) equivalents. The flavonol level was determined with quercetin-3-O-rutinoside (Sigma, USA) equivalents (Bogs et al., 2005 (link)). For the analysis of the anthocyanin content of tobacco flowers, the samples were extracted with acidic MeOH in the dark for 48 h. The level of anthocyanins was determined according to Zhang et al. (2009 (link)): Q_Anthocyanins = (A₅₃₀-0.25 × A₆₅₇) × M⁻¹. All analyses were performed with three biological replicates.