Rutin

The reagents used in the analysis satisfied all quality requirements and were of analytical grade. The following reagents were used in the study: ethanol 96% (v/v) (manufactured by Stumbras AB, Kaunas, Lithuania), the Folin–Ciocalteu reagent (Sigma-Aldrich Chemie, Steinheim, Germany), sodium carbonate (Na₂CO₃) (Carl Roth GmbH, Karlsruhe, Germany), gallic acid monohydrate (Sigma-Aldrich Chemie, Steinheim, Germany), 2,20-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), ferric chloride hexahydrate (FeCl₃·6H₂O), sodium acetate trihydrate (CH₃COONa·3H₂O) (Sigma-Aldrich Chemie, Steinheim, Germany), glacial acetic acid 99.8% (Lachner, Neratovice, Czech Republic), 2,4,6-tripyridyl-s-triazine (TPTZ) (Carl Roth GmbH, Karlsruhe, Germany), concentrated hydrochloric acid (Fluka-Chemie, Buchs, Switzerland), Trolox ((±)-6-hydroxy-2,5,7,8-tetramethylchromano-2-carboxylic acid) (Sigma-Aldrich, St. Louis, MO, USA), aluminum chloride hexahydrate (Fluka, Germany), hexamethylenetetramine (Sigma-Aldrich, Gillingham, UK), rutin (Carl Roth GmbH, Karlsruhe, Germany), sodium molybdate, sodium nitrite, sodium hydroxide (Chempur, Tarnowskie Gory, Poland); quercitrin, (+)-catechin, and (−)-epicatechin (Sigma-Aldrich, Steinheim, Germany). Purified water was produced using the Milli-Q^® water purification system (Millipore, Bedford, MA, USA).

Deionized water was produced using Millipore Simplicty UV station (Merck Millipore, Burlington, MA, USA). Acetonitrile, formic acid, ethanol was purchased from VWR (Radnor, PA, USA). Chlorogenic acid, hyperoside, rutin, gallic acid were purchased from Carl Roth (Karlsruhe, Germany). L-arginine, Tween-80 and aluminum chloride were purchased from Sigma-Aldrich (Sant Louis, MI, USA). Querectin was bought from Borschagovsky CPP (Kyiv, Ukraine) and fructose—from LLC Company ”Ukrhimsyre” (Kharkiv, Ukraine). Blood glucose, high-density lipoprotein cholesterol (Ch-HDL) and low-density lipoprotein cholesterol (Ch-LDL) (Felitis-Diagnostics, Ukraine) insulin (DRG, Germany) and triacylglycerols (TAG, Lachema, Czech Republic) were determined in blood serum using standard sets of reagents. Chemical standards used for HPLC analysis were previously isolated and identified in the Department of Pharmacognosy and Molecular Basis of Phytotherapy, Medical University of Warsaw.

In total, 40 mg of each plant material was directly extracted with 1 mL methanol-d₄ (99.8%, Deutero GmbH, Kastellaun, Germany), containing 0.935 mM HMDS (hexamethyl disiloxane) as the internal standard. After 30 s of vigorous mixing with vortex genie 2 (Scientific industries, Bohemia, NY, USA), 15 min of ultrasonic bath extraction was applied. The samples were centrifuged for 10 min at 14,000 rpm (5415 C Eppendorf, Eppendorf AG, Hamburg, Germany) to separate the plant powder. Then, 0.73 mL of the supernatants was transferred to Deu-Quant-5-7 NMR tubes (Deutero).
For method development, a mixture of chlorogenic acid (Sigma Aldrich, Steinheim, Germany, 1.73 mg/mL) and rutin (Roth, Karlsruhe, Germany, 3.20 mg/mL) was solved in methanol-d₄.

References used for the HPLC-MS analysis were purchased from Sigma Aldrich (St. Louis, MO, USA): Chlorogenic acid, p-coumaric acid, caffeic acid, rutin, apigenin, quercetin, isoquercitrin, quercitrin, hyperoside, kaempferol, myricetol, and fisetin, Roth (Karlsruhe, Germany): Ferulic acid, sinapic acid, gentisic acid, gallic acid, patuletin, luteolin or from Dalton (Toronto, ON, Canada): cichoric acid, caftaric acid. HPLC grade solvents, analytical grade acids used for mobile phases and Folin-Ciocâlteu reagent were purchased from Merck (Darmstadt, Germany), together with sodium carbonate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate and aluminium chloride used for antioxidant assays. ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) ≥98% purity, potassium peroxodisulfate (≥99% purity), DPPH, and Trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid; ≥97% purity) also used in antioxidant tests were purchased from Sigma Aldrich (Schnelldorf, Germany). Gallic acid monohydrate (99.5%) was purchased from Serva, (Heidelberg, Germany).

The extraction solvent was of p.a.-grade purity: acetone (JT Baker, Deventer). The solvents used for HPLC analysis were of HPLC-grade purity: water (JT Baker, Deventer), acetonitrile (JT Baker, Deventer), and trifluoroacetic acid (Roth, Karlsruhe). Rutin (Roth, Karlsruhe), quercetin (Kemika, Zagreb) and quercitrin (Roth, Karlsruhe) were used for the calibration curves.

The reagents used in the assays met all quality requirements and were of analytical grade. The following reagents were used in the study: ethanol 96% (v/v) (manufactured by SC Vilniaus degtinė, Vilnius, Lithuania), Folin–Ciocalteu reagent, gallic acid monohydrate, ferric chloride hexahydrate (FeCl₃ × 6H₂O), sodium acetate trihydrate (CH₃COONa × 3H₂O), DPPH (2,2-diphenyl-1-picrylhydrazyl), Trolox ((±) -6-hydroxy-2,5,7,8-tetramethylchromano-2-carboxylic acid), hexamethylenetetramine, chlorogenic acid, caffeic acid, phloridzin, quercetin, quercitrin, kaempferol-3-O-glycoside (+)-catechin, (−)-epicatechin, and (−)-epicatechin gallate (Sigma-Aldrich, Steinheim, Germany), sodium carbonate (Na₂CO₃), rutin (Carl Roth GmbH, Karlsruhe, Germany), glacial acetic acid 99.8% (Lachner, Neratovice, Czech Republic), 2,4,6-tripyridyl-s-triazine (TPTZ) (Alfa Aesar, Karlsruhe, Germany), concentrated hydrochloric acid, aluminum chloride hexahydrate (Fluka-Chemie, Buchs, Switzerland), sodium molybdate, sodium nitrite, and sodium hydroxide (Chempur, Tarnowskie Gory, Poland). Purified water was prepared, using the Milli-Q^® water-purification system (Millipore Co., Bedford, MA, USA).