Plant Material

Investigations were carried out on twenty five wheat (T. aestivum L.) varieties, including twelve of winter and thirteen of spring wheat varieties (Tables 2 and 3). They were grown at the experimental field of the Institute of Soil Science and Plant Cultivation — State Research Institute in Pulawy, located in Osiny (Lublin province, Poland, 51° 52′ 02″ N, 22° 05′ 25″' E) under organic system, while four cultivars of winter wheat were cultivated in conventional system, too. The plant materials were collected from each variety at flag leaf sheath opening — BBCH 47 (Biologische Bundesanstalt, Bundessortenamt and Chemical Industry) scale. Directly after harvesting, the plant material was frozen in the laboratory freezer (−18 °C) and then lyophilized using Freeze Dryer-Gamma 2-16 LSC (Martin Christ Gefriertrocknungsanlagen GmbH, Germany). Plant materials were powdered using Ultra Centrifugal Mill ZM 200 (Retsch, Germany), defatted in a Soxhlet extractor with chloroform, and then used for extraction.

Chemical Reagents

Methanol and chloroform of the analytical purity grade, acetonitrile hypergrade, and formic acid (>98%, MS-grade) for liquid chromatography (LC)–MS were purchased from J.T. Baker (Deventher, Netherlands). Ultra-pure water was prepared in-house with a Milli-Q water purification system (Millipore Co.). DPPH^· was obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). The thin-layer chromatography–2,2-diphenyl-1-picrylhydrazyl (TLC–DPPH^•) test was performed on the surface of aluminum-backed silica gel 60 F₂₅₄ plates (Merck, Darmstadt, Germany). The standards of chlorogenic acid (5-O-caffeoylquinic acid, ≥95%) and rutin (≥ 94%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Other chemical reagents were purchased from POCH S.A. (Gliwice, Poland).

Extraction and Purification of Wheat Samples

Deffated and milled wheat aerial parts (100 mg each) were extracted with 80% methanol (25 mL) at 1500 psi solvent pressure, 100 °C cell temp., flush 150%, and three static cycles, using an automated extractor, ASE 200 (Dionex, Sunnyvale, CA). The extracts (25 mL) were collected in vials, and then, the solvent was evaporated under reduced pressure using vacuum evaporator, at 40 °C. The dried extracts were suspended in Milli-Q water (10 mL) and purified by solid-phase extraction (SPE) using C18 Sep-Pak cartridges (1 cm³, 360 mg, Waters Corp., Milford, MA) to remove non-phenolic compounds. The cartridges were first washed with 4 mL of 0.5% methanol to remove carbohydrates and then washed with 4 mL of 80% methanol to elute phenolics. The phenolic fractions were evaporated to dryness using vacuum evaporator, redissolved in 1 mL of 60% methanol and stored in a freezer at −20 °C before analysis. Directly before chromatographic analyses, each fraction was sonicated for 5 min and then centrifuged at 16,000 × g for 10 min using Sigma 3-16KL centrifuge (Sigma Laborzentrifugen GmbH, Osterode am Harz, Germany). For each variety, three independent samples were prepared.

Determination of Phenolics by UPLC–PDA–ESI–MS Analysis

Quantitative analyses of free phenolic acids (caffeic acid derivatives) and flavonoids were carried out using a Waters ACQUITY UPLC system (Waters Corp., Milford, MA, USA), equipped with a PDA and a triple-quadrupole mass detector (Acquity TQD, Waters Corp.). Wheat phenolic fractions at a concentration of 100 mg of dried weight of plant material per mL (mg DW/mL) were separated on an Acquity HSS C18 (1.8 μm, 100.0 × 2.1 mm, Waters) column, which was maintained at 40 °C. The injection volume was of 2.0 μL. The elution (0.4 mL/min) was carried out with a gradient of solvent B (acetonitrile with 0.1% formic acid) in solvent A (water with 0.1% formic acid): 7% B in 4 min, 7 to 17% B from 4 to 22 min, 17 to 55% B from 22 to 29.95 min, 55 to 95% B from 29.95 to 30 min, 95% B from 30 to 32 min, 95 to 7% B from 32 to 32.05 min, and 7% B from 32.05 to 35 min. The PDA was operated in the range of 220−450 nm, with the resolution of 3.6 nm. Mass analyses were performed applying ESI(−) ionization mode, using the following conditions: capillary voltage, 2.8 kV; cone voltage, 35 V; source temp., 140 °C; desolvation temp., 350 °C; cone gas flow (nitrogen), 100 L/h; and desolvation gas flow, 800 L/h. UPLC analyses were performed in triplicate. The identification of chromatographic peaks was made on the basis of their UV spectra, ESI–MS/MS fragmentation patterns and literature, and comparison with authentic standards (either commercially available or previously isolated compounds) [13]. The determination of individual phenolics was carried out using external standard method and was based on peak area calculated from selected UV chromatograms. Caffeic acids derivatives were quantified using 5-caffeoylquinic acid (UV detection at 320 nm), and flavonoids were quantified using rutin (UV detection at 255 nm), as a group standards. Each calibration curve was prepared from triplicate analyses of standard working solutions at seven different concentrations over the range of 1.693–270.7 μg/mL for 5-caffeoylquinic acid and 1.601–256.1 μg/mL for rutin. All data were acquired and processed using Waters MassLynx 4.1 software.

TLC–DPPH^• Test and Image Processing

The standard of rutin (5 μL) and each wheat, and the SPE purified phenolic fractions (30 μL) were applied to silica TLC plates by means of a micropipette. As a mobile phase, we used acetonitrile–water–chloroform–formic acid 60:15:10:5 (v/v/v/v). The plates were developed at 90 mm and dried before derivatization. TLC plates were immersed for 5 s in 0.2% (w/v) DPPH^• radical methanolic solution. After removing DPPH^• excess, the plates were kept in the dark for 30 min and then scanned seven times, every 10 min. The test was performed in triplicate. The results of TLC–DPPH^• test were saved in jpg documents and then processed by free ImageJ program expended by the National Institutes of Health in the USA. First, the images were turned into 8-bit ones and next denoised by median filter (radius, 5 pixels). Then, the baseline drift was removed. The images processed in the aforementioned way were then inverted. For quantitative comparisons, the areas under the common peaks were measured using the “Wand Tool” after drawing the baseline with “Straight Line Selection Tool.”

Statistical Analysis

The linearity of each calibration curve was determined by plotting peak area against concentration. A 1/X weighted linear regression analysis of data was used to calculate the slope, y-intercept, and coefficient of determination (r²) for both 5-caffeoylquinic acid and rutin. The limit of quantification (LOQ) of the assays was accepted as the lowest concentration of the calibration series with a precision of ±20% [15]. The experimental results were expressed as mean ± SD. The differences among cultivars were evaluated on the basis of analysis of variance and Tukey's confidence intervals for individual phenolics. Statistical significance was declared at P ≤ 0.05.

Tentative Identification and Quantification of Phenolic Compounds

By means of UPLC–PDA–ESI–MS/MS system (Waters Corp., USA), a number of phenolic compounds were tentatively identified and in the all researched varieties the phenolic acids and the flavonoids were quantified as 5-caffeoylquinic acid and as rutin, respectively. Quantitative analyses were carried out using a seven-point calibration curves, which showed excellent fitting to a first-order polynomial equations. The linearity of response, which was very high (r² > 0.999) for both 5-caffeoylquinic acid and rutin in tested ranges, and other calibration parameters are shown in Table 2. The identification of 19 compounds in resulted chromatograms was made according to our previous article [13], i.e., on the basis of their UV spectra, ESI–MS/MS fragmentation patterns and literature, and comparison with authentic standards. Thus, seven free phenolic acids and twelve flavone glycosides were identified in the wheat varieties (Table 1, Figure 1). Content of individual and total phenolics was significantly different between varieties (Tables 2 and 3). The average concentrations of total phenolic acids and flavonoids were definitely higher in spring wheat cultivars than in the winter varieties. Content of total phenolic acids changed in the narrow from (332.01 ± 1.69) to (1377.96 ± 4.14) μg/g dry weight (DW) for spring wheat varieties and from (241.96 ± 3.89) to (570.80 ± 5.18) μg/g DW for winter wheat varieties. Analysis of the data disclosed that total phenolic acids were higher in Trappe, Kandela, and Bombona variety. The sum of 4-O-caffeoylquinic acid (3) and 3-O-feruoylquinic acid (4) was present in higher amounts (Table 3). Besides this, p-coumaric acid (5) represented 9.9–29.1% of total phenolic acids content in spring wheat and 19.3–53.6% in winter wheat varieties. The sum of 4-O-caffeoylquinic acid (3) and 3-O-feruoylquinic acid (4) constituted 28.9–45.6% in spring wheat and 9.6–38.9% in winter wheat varieties. Concentrations of other determined phenolic acids were considerably lower.

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UPLC–UV chromatograms of wheat (Triticum aestivum) cv. Kandela, (A) at 320 nm for phenolic acids, (B) at 255 nm for flavonoids. Peak numbers correspond to the numbering of compounds in the Table 1

Citation: Acta Chromatographica Acta Chromatographica 31, 2; 10.1556/1326.2017.00416

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Content of total flavonoids was higher than phenolic acids and ranging from (4698.6 ± 33.5) to (13753.7 ± 72.1) μg/g for spring and from (2612.8 ± 22.2) to (6451.8 ± 10.2) μg/g DW for winter wheat varieties (Table 4). There were large differences in the flavonoid content among the tested wheat cultivars. Higher total flavonoids concentration (13753.7 ± 72.1 μg/g, DW) was recorded in Kandela variety followed by Ostka Smolicka (12484.1 ± 80.7 μg/g, DW) and Łagwa (9201.3 ± 29.1 μg/g, DW), respectively. Isoorientin (11) and luteolin C-hexoside O-deoxyhexoside (14) were found to be the main flavonoids in researched varieties. Isoorientin (11) represented 16.4–33.5% of total flavonoids content in spring wheat and 5.0–27.4% in winter wheat cultivars. Luteolin and its glycosides might be used also as cancer chemopreventive agents [16]. Moheb et al. [6] demonstrated that isoorientin was the major constituent (42% of total flavonoids) of wheat leaves (in leaf development stage), too. These authors suggest that the relatively high abundance of isoorientin in wheat leaves attests to its use as a potential source of active natural health-promoting compounds. Besides this, mainly in North America, wheat leaves are used as a source of juice (wheatgrass juice) and elsewhere as a beneficial healthy supplement (http://www.wheatgrass.ca/). Flavonoids, which have been accumulated in the plants in lower amounts, were also characterized by a smaller variability within the group of the analyzed cultivars. Among the examined wheat cultivars, the smallest total content of the tested compounds was recorded for Arkadia, which was the most infected by fungal pathogens occurring on the leaves. According to Winkel-Shirley [17], flavonoids and other phenolic derivatives play important roles in plant's interaction with its environment, as defense against pathogens and pests, as signaling with microorganisms.

Our research on the influence of cultivars and production system on phenolic concentrations showed large differences in the content of phenolics in the tested cultivars of winter wheat (Table 5). The analysis showed a significant variability of their amount among individual cultivars. Despite the lack of meaningful differences in the compared production systems, there was a tendency to accumulate more phenolic acids in the organic system and more flavonoids in the conventional system. Flavonoids are also known to accumulate when plants are exposed to synthetic compounds such as diphenyl ether herbicides [18]. The level of only one phenolic acid (4-O-feruoylquinic acid, 8) was higher in varieties cultivated in conventional system. Besides this, 3-O-caffeoylquinic acid (1) and 5-O-feruoylquinic acid (7) were below limit of quantification. Zuchowski et al. [10] confirmed the thesis that organically produced wheat grains contain higher levels of phenolic acids. They reported also that comparing the ferulic and p-coumaric acid, as well as total phenolic acids content, varieties grown in the organic system, had usually their higher level. In other previous studies, it was demonstrated that the concentration of defence-related natural products in conventionally produced crop are often thought to be lower than in crops grown by organic methods [19, 20].

DPPH^• Scavenging Activity

In order to determine antioxidant activity of wheat samples, TLC–DPPH^• test was performed. According to our previous studies, which focused on Triticum species, the analyzed wheat varieties contain phenolic acids, flavone glycosides, or acylated flavone glycosides [13]. The activity of the acylated glycosides also differed depending on the amount of sugar moieties attached to aglycone, as well as on the sugar type [21]. In our study, the activity of the separated compounds has been expressed for the first time for each variety as the ratio of area under common peak to the area under rutin's peak. Then, the sum of active compounds for each variety was determined. Quantitative data obtained for 25 wheat varieties were presented in Table 3. The results of TLC–DPPH^• test (Figure 2) indicated that spring wheat varieties are generally a richer source of compounds with direct antiradical properties than winter varieties, the average activity being 3.76 and 2.85, respectively. Average antioxidant activity for the spring wheat cultivars was in a range from 1.98 to 4.89, in relation to rutin's activity (1.00). Comparing the activity of individual wheat varieties, the relationships between the content and the type occurring in the phenolics and the level of antioxidant activity were observed. A significant (P ≤ 0.05) correlation between antiradical capacity and total phenolic acids content was identified. Interestingly, Trappe, Kandela and Bombona, which exhibited the highest total phenolic acids, mainly 5-O-caffeoylquinic acid (2), p-coumaric acid (5), and the sum of 4-O-caffeoylquinic acid (3) and 3-O-feruoylquinic acid (4), registered the highest DPPH radical scavenging activity. According to a review by Rice-Evans et al. [22], hydroxycinnamic acids were esterified with quinic acid, with free hydroxyl groups exhibiting strong antioxidant properties. Moreover, these varieties were found to be rich in luteolin glycosides, isoorientin (11) and luteolin C-hexoside O-deoxyhexoside (14). Furthermore, the presence of acylated flavone glycosides no. 15 and 17 increases activity of variety. According to Ciesla et al. [23], flavonoid glycosides acylated with hydroxycinnamic acids are stronger free radical scavengers in comparison with the corresponding non-acylated polyphenols. Comparing the antioxidant activity of four winter wheat cultivars (Arkadia, Bamberka, Jantarka, Sailor) grown in both farming systems, we have shown that the analyzed varieties had higher average activity in conventional crops: 2.86, 4.08, 4.99 and 3.50, respectively.

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The results obtained after processing TLC video scans with ImageJ program, including: (A) TLC plate image for 13 spring wheat varieties and rutin, after processing; (B) line profile plot obtained for one (cv. Trappe) of the variety; (C) and line profile plot obtained for rutin (standard)

Citation: Acta Chromatographica Acta Chromatographica 31, 2; 10.1556/1326.2017.00416

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