Tartary buckwheat (Fagopyrum tataricum, Gaertn.) samples were obtained from Breeding Station (harvested 2015, Palikije, Poland). The material originated from tartary buckwheat domestic cultivar from Slovenia (Rangus, Šentjernej) and it consisted all of morphological parts of tartary buckwheat, which include leaves, flowers, stalk and roots. The material was dried and all results were calculated as g/100 g of dry matter (d.m.). These parts were ground by using laboratory grinder (Foss, Sweden).
Thermostable α-amylase (Novozymes, Bagsvaerd, Denmark) was used for digestion of starch. The reagents used to determine the content of neutral detergent fiber (NDF) were: sodium dodecyl sulfate (C12H25NaO4S, Sigma-Aldrich, Saint Louis, USA), neutral disodium versenate dehydrate (C10H14N2Na2O8*10H2O), disodium tetraborate decahydrate (Na2B4O7*10 H2O), disodium hydrogen phosphate (Na2HPO4) and ethylene glycol (Poch, Gliwice, Poland). Reagents used to determine the content of ADF were 1 N sulfuric acid (H2SO4, 1 N, Poch, Gliwice, Poland) and N-cetyl-N,N,N-trimethylammoniumbromid (C19H42BrN, Merck, Darmstadt, Germany). Reagents used to determine the content of ADL were: sulfuric acid (72%), and acetone (Poch, Gliwice, Poland). Determination of polyphenolic contents was performed using the reagents and standards of acetonitrile, methanol, 2,6-dihydroxybenzoic acid, 3,4-dihydroxybenzoic acid, 3,5-dihydroxybenzoic acid, 4-hydrobenzoic acid, caffeic acid, catechin, chlorogenic acid, fagopyrin, ferulic acid, myricetin, gallic acid, isovanilic acid, isovitexin, kaempferol, luteolin, p-coumaric acid, procyanidin B2, quercetin, quercetin 3-D galactoside, rutin, syringic acid and vitexin, purchased from Sigma Aldrich (Steinheim, Germany).
The Content of Neutral Detergent Fibre
The content of NDF, consisting of cellulose, hemicellulose and ADL, was determined using the detergent method according to Van Soest and Wine, and Dziedzic et al. [6, 7]. Thermostable α-amylase was used to digest starch. The content of NDF, ADF and ADL was analysed using chemical reagents. Hemicellulose (H) content was calculated from the difference between NDF and ADF, while cellulose (C) content was calculated as the difference between ADF and ADL. Analyses were conducted using a Fibertec System M 1020 apparatus by Tecator (Foss, Sweden).
Extraction and Analysis of Polyphenolic Substances
The extraction was performed by mixing 0.2 g of each sample with 10 mL of solvent (60 °C, 1 h). Methanol, ethanol, acetone and their aqueous solutions are usually used to extract bioactive substances from plants [8,9,10,11,12]. In our study we used two solvents for extraction of polyphenolic substances: methanol (Germany, Sigma Aldrich) and water. Methanol was chosen based on previous studies [8, 13], while water was selected because it is the natural solvent in the human body . The samples (morphological parts of tartary buckwheat) were incubated in methanol or water for 1 h at 60 °C. Subsequently, the samples were centrifuged (4000 x g) and filtered (0.45 μm, Millipore). Reversed-phase (C18 column) ultra-high-performance liquid chromatography electrospray ionisation mass spectrometry (RP–UHPLC–ESI-MS) analysis was performed using a Dionex UltiMate 3000 UHPLC (Thermo Fisher Scientific, Sunnyvale, CA, USA) coupled to a Bruker maxis impact ultrahigh resolution orthogonal quadrupole-time-of-flight accelerator (qTOF) equipped with an ESI source and operated in negative-ion mode (Bruker Daltonik, Bremen, Germany). The RP chromatographic separation was achieved with a Kinetex™ 1.7 lm C18 100 A, LC column 100 _ 2.1 mm (phenomenex, Torrance, CA, USA). The ESI-MS settings were as follows: capillary voltage 4500 V, nebulizing gas 1.8 bar, and dry gas 9 l/min at 200°C. The scan range was from mass-to-charge ratio (m/z) 80–1200. The mobile phase was composed of water containing 0.1% formic acid (A) and acetonitrile (B). The flow rate was 0.2 ml/min with a gradient elution of 5–95% B over 20 min. The sample injection volume was 3 μL. The column temperature was set to 40 °C. The ESI-MS system was calibrated using sodium formate cluster ions introduced by loop-injection at the beginning of the LC-MS run. The LC-MS data were processed using Data Analysis 4.1 software (Bruker Daltonik, Bremen, Germany). Molecular ions [M-H] - were extracted from full scan chromatograms and peak areas were integrated. The compounds present in each sample were identified by comparing their retention times with those of standards, and based on molecular mass and structural information from the MS detector.
Experiments were conducted with three replications. Each value was the mean of three independent trials. One-way analysis of variance (ANOVA) was performed. Hierarchical cluster analysis was carried out using Ward amalgamation rule with the Euclidean distance (d) measure. Tree plots were scaled to a standardized scale (dlink/dmax*100). Non-hierarchical cluster analysis (k-means clustering) was performed to form a grouping of wheat fiber samples. V-fold cross-validation algorithm was used to determine the best number of clusters. Principal component analysis (PCA) technique was used to reduce the dimensionality of data and to present the samples in a new coordinate system. Statistica software, Version 10, StatSoft Inc. (OK, USA) was used to carry out statistical analysis.