Neochlorogenic

Trifluoracetic acid, acetonitrile, methanol, and ethanol were provided from Penta (Prague, Czech Republic). α-Amylase and neutral detergent solution package (containing sodium lauryl sulphate, EDTA disodium, sodium borate, sodium phosphate dibasic together with triethylene glycol) were purchased from Ankom Technology (Macedon, NY, USA). Folin-Ciocalteu reagent, AlCl₃·6H₂O were purchased from Penta (Prague, Czech Republic). The substance 2,2’-azinobis(3-ethylbenzo-thiazoline-6-sulfonic acid) diammonium salt (ABTS), radical 2,2-diphenyl-1-picrylhydrazyl (DPPH), and standard 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox) were provided from Sigma-Aldrich (St. Louis, MI, USA). Phenolic and sugar standards were as follows: Epigallocatechin, catechin, epicatechin, rutin, quercetin, kaempferol, neochlorogenic, chlorogenic, gallic, protocatechuic, p‑hydroxybenzoic, vanillic, caffeic, syringic, p-coumaric, ferulic, sinapic, ellagic, o-coumaric, cinnamic acids and protocatechuic acid ethyl ester; D(+)-maltose, D(+)-glucose, D(−)-fructose, D(+)-rhamnose, D(+)-xylose, D(+)-saccharose, all were purchased from Sigma-Aldrich (St. Louis, MI, USA). All standards and solvents used in this study were of HPLC-grade (purity ≥ 98.5–99.0%). Total dietary fiber and resistant starch assay kits were provided from Megazyme International Ireland Ltd. (Wicklow, Ireland).

Lyophilised and powdered samples (about 250 mg) were extracted with 2 mL of methanol using sonication (10 min at 25 ± 2 °C) and centrifuged for 10 min at 15,000 rpm. The supernatant was filtered through 0.22 μm filter (Millex^®GP, Millipore, Merck, Darmstadt, Germany). RP-HPLC analyses was carried out according to Simlat et al.^{34 (link)} using a Merck–Hitachi liquid chromatograph (LaChrom Elite, Hitachi, Tokyo, Japan) equipped with a DAD detector L-2455 and Purospher^®RP-18e (250 × 4 mm/5 mm) column (Merck, Darmstadt, Germany). Quantification was carried out at λ = 254 nm for phenolic acids and λ = 370 nm for flavonoids. Identification was performed through a comparison of the retention times of the peaks with authentic reference compounds and co-chromatography with standards. Quantification consisted of measurement of the peak area with reference to the standard curve derived from five concentrations (0.03125–0.5 mg ml⁻¹). Commercially available standards of the phenolic acids: chlorogenic, neochlorogenic (Sigma–Aldrich, St Louis, MO, USA), isochlorogenic A (ChromaDex, Irvine, CA, USA), and rosmarinic (Sigma–Aldrich, St Louis, MO, USA), as well as the flavonoids isoquercetin, and quercitrin (Sigma–Aldrich, St Louis, MO, USA), were used to generate the calibration curves. The analysis was repeated three times.

All HPLC solvents were from J.T. Baker (Phillipsburg, NY, USA) and were HPLC grade. The ethanol (99.9%) used for extraction procedure and other used chemicals were an analytical grade. Standard of nicotine (purity: 98.40%), and chlorogenic acid (purity: 97.2%) were obtained from Dr. Ehrenstorfer GmbH (Augsburg, Germany) while standards of cryptochlorogenic (purity: ≥98%) and neochlorogenic (purity: 98%) were obtained from Sigma-Aldrich (Steinheim, Germany). The standard of rutin (purity: 97%) was purchased from Acros Organics (Geel, Belgium) and nicotiflorin (purity: ≥95.0%) was obtained from PhytoLab GmbH & Co. KG (Vestenbergsgreuth, Germany). The ultrapure water for the experiments was obtained from a Milli-Q water purification system Simplicity 185 by Millipore (Bedford, MA, USA).