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Original Article

Antioxidant activity, anthocyanin profile, and mineral compositions of colored wheats

Vladamir P. Shamanina, Zeynep Hazal Tekin-Cakmakb,c, Salih Karasuc, Inna V. Pototskayaa, Elena I. Gordeevad, Artem O. Vernera, Alexey I. Morgounove, Mustafa Yamanf, Osman Sagdicc, Hamit Koksela,b*

aOmsk State Agrarian University, Omsk 644008, Russia;

bDepartment of Nutrition and Dietetics, Health Sciences Faculty, Istinye University, İstanbul 34010, Türkiye;

cDepartment of Food Engineering, Faculty of Chemical and Metallurgical Engineering, Davutpasa Campus, Yildiz Technical University, Istanbul 34349, Türkiye;

dCereal Functional Genetics Group, Institute of Cytology SB RAS, Novosibirsk 630090, Russia;

eScience Department, S. Seifullin Kazakh Agrotechnical University, Astana 010011, Kazakhstan;

fDepartment of Nutrition and Dietetics, Health Sciences Faculty, Istanbul Sabahattin Zaim University, Istanbul 34303, Türkiye

Abstract

The antioxidant activities (ABTS and CUPRAC) of 40 different colored wheat genotypes were investigated, and some of the diverse wheat genotypes were selected to investigate their anthocyanin profiles and mineral compositions. The ABTS values of free and bound fractions were in the range of 15.61-157.36 mg TE/100 g and 33.26-189.48 mg TE/100 g, respectively. For the free and bound fractions of the colored wheat samples, the CUPRAC values were determined between 25.73 and 229.20 mg TE/100 g, and between 82.00 and 348.93 mg TE/100 g, respectively. The anthocyanin profiles of the colored wheats varied depending on the genotypes. Cyanidin-3-O-glucoside chloride was abundant in the samples w3, w8, w17, w18 and w20 while malvidin-3-O-glucoside chloride was the major anthocyanin for the samples w13, w23, w14, w30 and w34. Cyanidin-3-O-glucoside chloride was significantly higher in w18 than others (p<0.05). Cyanidin-3,5-di-O-glucoside was only found in the free extracts of the two samples (w17 and w20). The best accessions with high antioxidant activity and anthocyanin profile were identified for their potential utilization in wheat breeding programs. According to the results, the w17 sample (line BW 49880–Dark colored P, F4) was chosen as the best sample due to its highest antioxidant activity (p<0.05) and the best anthocyanin profile. The macronutrients Ca, K, and Mg highly varied among the genotypes studied. The trace elements, Zn and Fe were significantly higher in purple-grained lines than that of the red-grained variety. The study’s outcomes are likely to support breeding programs to generate new wheat cultivars with greater nutritional benefits.

Key words: ABTS, anthocyanin profile, colored wheats, CUPRAC, mineral compositions

*Corresponding Author: Hamit Koksel, Department of Nutrition and Dietetics, Health Sciences Faculty, Istinye University, İstanbul, Turkey. Email: hamit.koksel@istinye.edu.tr

Received: 6 November 2023; Accepted: 13 February 2024; Published: 27 February 2024

DOI: 10.15586/qas.v16i1.1414

© 2024 Codon Publications
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0). License (http://creativecommons.org/licenses/by-nc-sa/4.0/)

Introduction

Wheat and other cereal grains are good sources of phenolic chemicals, which have been linked to various nutritional benefits (Călinoiu and Vodnar, 2018). Phenolic acids and flavonoids, concentrated in the grain’s outer layers and removed as bran after milling, are the major phenolic components present in wheat (Suchowilska et al., 2020). Flour and semolina, generally refined milling products, are used to make bread and pasta, respectively, while bran is mainly fed to animals. In order to receive the nutritional advantages of cereals, they must be consumed as whole grain products (Papageorgiou and Skendi, 2018).

Colored wheats (blue, black, and purple colored) have relatively large concentrations of phytochemical substances, primarily found in the grain’s outer layers. According to several studies, anthocyanins may be critical in avoiding a variety of degenerative diseases since they have an anticancer impact through various anti-inflammatory and antioxidant activities. It is reported that eating foods high in anthocyanins may lower the risk of developing disorders affecting the immunological, digestive, neurological, endocrine, sensory, urinary, and circulatory systems, as well as cancer (Li and Beta, 2011; Liu et al., 2021).

Two complimentary Pp genes (Colored pericarp), Pp3/TaMyc1 and Pp-1/Myb, which are located on chromosome 2AL and the 7th homologous group (7AS, 7BS, and 7DS, respectively), are responsible for the color of these grains. The dominant allele of the Pp3/TaMycA1 gene was transferred to bread wheat from tetraploid wheat Triticum turgidum L. subsp. abyssinicum Vavilov (Martinek et al., 2014; Morgounov et al., 2020). The existence of anthocyanins in the aleurone layer of the grain gives wheat its blue color. The Ba gene (Blue aleurone), which regulates the production of anthocyanins in the aleurone layer in common wheat, was introduced into that crop from Triticum boeoticum and several plants of the genus Agropyron L. (Zeven, 1991; Dhua et al., 2021). Since anthocyanins in blue-grained and purple-grained lines are located in different layers of the grain shell, in the aleurone layer and the pericarp, respectively, these lines were crossed with each other for anthocyanin pyramiding in the same genome (Gordeeva et al., 2022).

Anthocyanins are natural water-soluble flavonoids. An analysis of anthocyanins using HPLC-MS revealed that the purple grains had a greater content of cyanidin and pelargonidin, while the blue grains had a greater content of delphinidin (Zhang et al., 2022; Sytar et al., 2018). Anthocyanin pigments are significant in agricultural plant response to adverse environmental factors. Anthocyanins are present in cells in glycosylated forms. They support sustaining cell turgor under drought conditions (Adzhieva, 2015). According to Chalker-Scott (1999), anthocyanins are frequently transiently present at particular developmental phases. They can be brought on by various environmental conditions, which are low temperatures, water stress, UVB, visible radiation, and water stress.

Although wheat is among the most important grains in people’s diets, information on the genotypic variation in phytochemical profile, especially anthocyanin contents of different wheat genotypes, is limited. These constituents strongly affect the total antioxidant activities of wheat genotypes, which influence the health benefits of wheat-based food products. In the present study, the total antioxidant activity (ABTS and CUPRAC) of 40 different colored genotypes were tested, and 9 diverse wheat varieties and experimental lines were selected among them to investigate their anthocyanin profiles and mineral compositions. Therefore, this study aimed to investigate the best accessions with high antioxidant capacity, good anthocyanin profile, and mineral composition among 40 different colored wheat genotypes for potential use in breeding programs, which is also the study’s novelty. A successful breeding program is expected to combine these features with good agronomic properties, such as high yield and resistance to biotic and abiotic stress factors in newly developed colored wheat cultivars.

Materials and Methods

Materials

Plant samples

The descriptions of the plant materials are presented in Table S1, which was also published by Gordeeva et al. (2020) and Shamanin et al. (2022). These lines resulted in a two-stage crossing of donor-colored lines with recurring promising variations of Siberian Selection Element 22, Aina, Tobol’skaya, and breeding line BW49880 (CIMMYT). The anthocyanin pericarp lines were chosen in the F2 and BC1F2 generations based on phenological markers of colored grain coloring, dark-red coleoptiles, and molecular SSR markers surrounding the Pp-1D and Pp3 genes. The selection of new lines with anthocyanin blue pigments of grains in the whole spikes was carried out in the F2 and BC1F2 generations after crossing and subsequent backcrossing with parental varieties. The resulting purple and blue-grained lines were crossed with each other to obtain black-grained (dark-colored) lines.

Chemical material

ABTS solution, CuCl2, neocuproine, NH4Ac, acetone, diethyl ether, ethyl acetate, and hexane were bought as analytical grade (Sigma-Aldrich, Bornem, Belgium). NaOH, HCl, absolute ethyl alcohol, methanol (analytical grade), formic acid, acetonitrile, and acetic acid (glacial) were bought from Merck Co. (Darmstadt, Germany).

Extraction

Extraction of free and bound fractions was performed, as reported by Shamanin et al. (2022). The extracts were placed in amber-colored vials and stored at -18°C until analysis.

The antioxidant activities

ABTS scavenging activity

The ABTS scavenging activity of colored wheats was determined as described by Rice-Evans and Miller (1997). The absorbance was determined at 734 nm using a UV spectrophotometer (Shimadzu 150 UV-1800, Kyoto, Japan), and the data were reported in mg of TE per 100 g of wheat.

CUPric-reducing antioxidant capacity

For colored wheat extracts, the CUPric-reducing antioxidant capacity (CUPRAC) determination was carried out with the method described by Apak et al. (2004). The absorbance measurements were taken at 450 nm with a UV spectrophotometer (Shimadzu 150 UV-1800, Kyoto, Japan). The data were reported in mg of TE per 100 g of wheat.

Measurement of individual anthocyanin compounds

Individual anthocyanins were identified by HPLC systems (LC-20AD pump, SPDM20A DAD detector, SIL-20A HT autosampler, CTO-10ASVP column oven, DGU-20A5R degasser, and CMB-20A communications bus module (Shimadzu Corp., Kyoto, Japan). The mobile phase consisted of solutions A (7.5% formic acid in acetonitrile) and B 7.5% formic acid in deionized water). The mobile phase gradient program used to separate anthocyanins is given in Table 1.

Table 1. HPLC gradient program for anthocyanins.

Time (min) Mobile phase A (%) Mobile phase B (%)
0.01 3 97
1.00 3 97
12.00 15 85
24.00 25 85
28.00 30 70
45.00 3 97

The HPLC conditions defined by Simões et al. (2009) were selected. An Intersil® ODS column (C18, 250mm×4.6mm length and 5 μm particle size, GL Sciences, Tokyo, Japan) was used for the separation. The column oven temperature was 20°C, the flow rate was 1 mL/min, and the chromatograms were recorded at 520 nm. The free and bound extracts were filtered through a 0.20-μm filter and injected (20 µL) into HPLC. Six standards were utilized for anthocyanin separation and quantification: delphinidin-3-O-galactoside, cyanidin-3-O-glucoside, cyanidin-3-O-rutinoside, petunidin-3-O-glucoside, pelargonidin-3-O-glucoside, peonidin-3-O-glucoside. The anthocyanin concentrations are reported in µg /100 g wheat.

Mineral composition analysis

The analysis of macro- and microelements (Ca, K, Mg, Fe, Zn, Cu, Mn) of wheat samples was carried out according to Karpiuk et al. (2016) and Mandizvo and Odindo (2019). The analysis was repeated two times for each sample. Samples were prepared using a Milestone Ethos UP microwave system. The content of chemical elements was analyzed by atomic absorption spectrometry (AACCI, 2000) with flame atomization on a ContrAA 800 D instrument (Analytik Jena, Germany). The content of elements is given in mg per kg of dry matter.

Statistical analysis

All analyses were carried out in duplicates. The mean values and standard deviations were presented in the tables. All results were analyzed using ANOVA, and Tukey’s post hoc test determined the significance of mean differences and t-test in SPSS version 9.0 (SPSS Inc. Chicago, IL). Pearson correlation coefficients and their significance were calculated for some tested variables (antioxidant activity and mineral content).

Results and Discussion

The antioxidant activities (ABTS and CUPRAC)

Antioxidant activities of extracts were determined by ABTS and CUPRAC assays. The ABTS assay has been widely preferred in assessing the antioxidant capacity of foods because of the stability of the ABTS radical (Schaich et al., 2015). In the CUPRAC assay, the antioxidant reacts by donating an electron, transforming Cu2+ to Cu+.

The ABTS values of the free extracts and bound extracts of the colored wheat samples are given in Table 2. The ABTS values of the free extracts and bound extracts of colored wheat samples were determined between 15.61 and 157.36 mg TE/100 g, 33.26 and 189.48 mg TE/100 g, respectively. The ABTS value of the free fraction of w17 was significantly higher than the other colored wheat samples (p < 0.05). The total ABTS values were between 61.86 and 320.97 mg TE/100 g. The ABTS of bound extracts in red, blue, and purple wheat samples reported by Geyik et al. (2023) were 100.10, 207.49 mg, and 239.96 mg TE/100 g, respectively, while the antioxidant activities determined using ABTS assay in their free fractions were 52.70, 87.77, and 76.87 mg TE/100 g, respectively.

Table 2. ABTS scavenging activity values of colored wheat genotypes.

# of sample Free
(mg TE/100 g)
Bound
(mg TE/100 g)
Total*
(mg TE/100 g)
w1 41.19±0.5K 143.55±0.4F 184.74±0.9DE
w2 34.56±0.7Q 154.41±0.7E 188.97±1.4D
w3 45.29±0.5H 38.94±0.1UVWX 84.23±0.6QR
w4 40.29±0.6M 43.72±0.4QRSTU 84.01±1.0QRS
w5 26.69±0.3V 49.96±0.6OP 76.65±0.9STUVW
w6 43.94±0.4I 38.94±0.7UVWX 82.88±1.1QRST
w7 28.88±0.7U 34.56±0.1XY 63.44±0.8XY
w8 32.42±0.7R 44.67±0.6QRS 77.09±1.3RSTUV
w9 23.71±0.4X 38.32±0.6VWX 62.03±1.0Y
w10 40.85±0.2LM 35.46±0.7WXY 76.31±0.9TUVW
w11 124.53±0.3B 42.31±0.1RSTUV 166.84±0.4F
w12 32.70±0.4R 47.26±0.1OPQ 79.96±0.5RSTU
w13 29.44±0.7T 40.63±0.9STUV 70.07±1.6VWX
w14 37.42±0.7P 35.34±0.8WXY 72.76±1.5UVW
w15 40.96±0.1L 139.79±0.3F 180.75±0.4E
w16 42.26±0.3J 189.48±0.8C 231.74±1.1C
w17 157.36±0.7A 163.61±0.1D 320.97±0.8A
w18 67.94±0.1D 159.01±0.2DE 226.95±0.3C
w19 38.04±0.0O 45.97±0.1PQR 84.01±0.1QRS
w20 71.65±0.1C 219.16±2.2A 290.81±2.3B
w21 34.33±0.6Q 196.71±1.8B 231.04±2.4C
w22 33.99±0.6Q 66.28±1.3L 100.27±1.9MNO
w23 22.53±0.6Y 73.78±0.2JK 96.31±0.8NOP
w24 38.94±0.1N 75.00±0.3J 113.94±0.4JK
w25 44.17±0.1I 49.84±0.8OP 94.01±0.9OP
w26 28.60±0.6U 33.26±0.5Y 61.86±1.1Y
w27 48.73±0.3G 56.03±0.1MN 104.76±0.4LM
w28 25.79±0.1W 47.60±0.4OPQ 73.39±0.5UVW
w29 30.23±0.5S 59.08±1.8M 89.31±2.3PQ
w30 16.46±0.8Z 95.53±0.5G 111.99±1.3JKL
w31 52.04±0.8E 139.12±1.3F 191.16±2.1D
w32 42.83±0.6J 88.90±1.1H 131.73±2.7G
w33 30.11±0.1S 39.50±0.6TUVW 69.61±0.7VWX
w34 45.85±0.4H 71.31±0.1JKL 117.16±0.5IJ
w35 50.13±0.4F 51.92±0.2NO 102.05±0.6MN
w36 25.33±0.1W 44.05±1.4QRST 69.38±1.5WXY
w37 34.39±0.5Q 82.15±0.4I 116.54±0.9IJ
w38 38.66±0.0N 69.11±0.7KL 107.77±0.7KLM
w39 37.37±0.6P 85.30±0.7HI 122.67±1.3HI
w40 41.08±0.9KL 83.95±1.3I 125.03±2.2GH

Means with different letters within the same column differ significantly (p < 0.05). *Total: sum of the ABTS values of free and bound fractions.

A comparative study was carried out across laboratories by Sharma et al., (2023) on colored wheat lines to validate their phytochemicals and antioxidant activity. The results indicated that the colored wheat lines had higher antioxidant activity (ABTS values) than white wheat lines across the laboratories. Within the colored wheat lines studied black wheat lines had the best nutraceutical profile across the laboratories, with an observed order of black > blue > purple > white. In line with the results of Sharma et al. (2023), in the present study, the w20 sample had black-colored grains and had the highest ABTS values in the bound extracts.

The CUPRAC values of the free and bound extracts of colored wheat samples are presented in Table 3. For the free fraction of colored wheat samples, the CUPRAC values were determined between 25.73 and 229.20 mg TE/100 g, while the values for the bound fractions were between 82.00 and 348.93 mg TE/100 g. The CUPRAC values of the free fraction of w18 and w20 were significantly higher than the other colored wheat samples (p <0.05). The total CUPRAC values of colored wheat extracts were calculated between 107.73 and 494.13 mg TE/100 g. Zengin et al. (2017) determined the CUPRAC values of 7 different wheat cultivars in the 116.03 to 242.47 mg TE/100 g wheat grain range. The total CUPRAC values of w16, w18, and w20 were not significantly different and were significantly higher than the other colored wheat samples (p < 0.05).

Table 3. CUPRAC values of colored wheat genotypes.

# of sample Free
(mg TE/100 g)
Bound
(mg TE/100 g)
Total*
(mg TE/100 g)
w1 103.87±0.0EFGHI 124.40±0.7W 228.27±0.7EFGHIJKL
w2 110.80±0.2DEFG 164.13±0.4P 274.93±0.6CDEFGHI
w3 60.67±0.1LMNO 83.87±0.2AJ 144.54±0.3BCDE
w4 116.67±0.0CDEF 216.93±0.8H 333.60±0.8CDEFGH
w5 72.40±0.2JKLMN 102.80±0.5AC 175.20±0.7FGHIJKL
w6 128.67±0.3BCDE 176.40±0.3N 305.07±0.6AB
w7 67.33±0.6KLMNO 235.60±0.3F 302.93±0.9BCD
w8 150.27±0.1B 251.60±0.7E 401.87±0.8FGHIJKL
w9 130.80±0.3BCDE 159.00±0.0Q 289.80±0.3FGHIJKL
w10 95.60±0.0FGHIJK 199.33±0.1L 294.93±0.1EFGHIJKL
w11 134.80±0.2BCD 152.13±0.0R 286.93±0.2DEFGHIJKL
w12 78.00±0.0HIJKLM 203.87±0.2K 281.87±0.2CDEFGHIJK
w13 66.80±0.0KLMNO 128.40±0.1U 135.20±0.1GHIJKL
w14 44.67±0.0NOPQ 122.27±0.0Y 126.93±0.0L
w15 56.13±0.1MNOP 179.60±0.0M 235.73±0.1CDEFGHIJKL
w16 145.20±0.2BC 348.93±0.2A 494.13±0.4A
w17 77.08±0.0HIJKLM 275.33±0.2B 352.41±0.2BC
w18 229.20±0.4A 254.53±0.3C 483.73±0.7A
w19 106.27±0.3DEFGH 209.47±0.1I 315.73±0.4BCDEF
w20 228.40±0.3A 253.73±0.3D 482.13±0.3A
w21 90.53±0.1FGHIJKL 147.60±0.2S 238.13±0.3CDEFGHIJK
w22 100.93±0.0EFGHIJ 102.00±0.0AD 202.93±0.0FGHIJKL
w23 108.80±0.0DEFG 114.00±0.1Z 222.80±0.1DEFGHIJKL
w24 84.93±0.10GHIJKLM 90.27±0.0AH 175.20±0.1GHIJKL
w25 91.60±0.0FGHIJK 205.73±0.2J 297.33±0.2CDEFG
w26 27.33±0.0PQ 88.67±0.1AI 116.00±0.1KL
w27 92.13±0.0FGHIJK 102.80±0.1AC 194.93±0.1FGHIJKL
w28 56.13±0.1MNOP 100.67±0.2AE 156.80±0.3IJKL
w29 119.33±0.0CDEF 152.40±0.1R 271.73±0.1CDEFGHI
w30 86.00±0.00GHIJKLM 127.33±0.1V 213.33±0.1EFGHIJKL
w31 39.07±0.0OPQ 84.40±0.0AJ 123.47±0.0JKL
w32 95.60±0.1FGHIJK 226.53±0.2G 322.13±0.3BCDEF
w33 103.33±0.0EFGHI 104.67±0.1AB 208.00±0.1EFGHIJKL
w34 74.80±0.0IJKLM 171.87±0.2O 246.67±0.2CDEFGHIJ
w35 111.60±0.10HIJKLM 134.80±0.0T 246.40±0.1CDEFGHIJ
w36 110.00±0.1DEFG 123.60±0.0X 233.60±0.1CDEFGHIJKL
w37 56.13±0.0MNOP 105.73±0.1AA 161.87±0.1HIJKL
w38 68.93±0.1KLMNO 98.53±0.1AF 167.47±0.2HIJKL
w39 86.53±0.0GHIJKL 96.40±0.0AG 182.93±0.0GHIJKL
w40 25.73±0.0Q 82.00±0.1AK 107.73±0.1L

Means with different letters within the same column differ significantly (p < 0.05).

*Total: sum of the ABTS values of free and bound fractions.

As indicated above, ABTS and CUPRAC analyses were carried out on the free and bound fractions of 40 colored wheat samples (Tables 2 and 3). The free, bound, and total phenolic contents, as well as the free and bound phenolic profiles of these colored wheat samples, were reported earlier by Shamanin et al. (2022). Considering the data in the cited publication and the data in Tables 2 and 3, nine samples with different phenolic composition and antioxidant capacities were selected to represent these samples and used for further detailed anthocyanin analysis. These samples were also selected to cover different colored (red, purple, dark purple, blue, light brown, and black) grains for breeding programs.

Anthocyanin Profile

The distribution of anthocyanins in the free extracts of different colored wheat samples is shown in Table 4. No anthocyanins were detected in the bound extracts of the colored wheat samples used in this study. Seven different anthocyanin standards were utilized to characterize the wheat samples’ anthocyanin profile. As can be seen from Table 4, the wheat samples exhibited different anthocyanin distributions.

Table 4. Anthocyanin profile in the free extracts of colored wheat lines (µg/100 g).

Kuromanin Cyanidin Keracyanin Delphinidin Peonidin Callistephin Malvidin
Cyanidin-3-O-glucoside chloride Cyanidin-3,5-di-O-glucoside Cyanidin-3-O-rutinoside chloride Delphinidin 3-O-β-D-glucoside chloride Peonidin 3-O-glucoside chloride Pelargonidin 3-O-glucoside chloride Malvidin-3-O- glucoside chloride
w3 5.60±0.03g n.d. n.d. n.d. n.d. n.d. n.d.
w13 7.71±0.01f n.d. 6.13±0.03d n.d. 4.42±0.00g 6.21±0.00e 31.88±0.02e
w17 254.2±0.3b 162.2±0.3b 161.2±0.2a 132.4±0.2a 132.0±0.3a 111.8±0.3a 6.68±0.01g
w18 106.1±0.5c n.d. 7.27±0.01c 57.61±0.03b 31.23±0.09d 14.52±0.01d 79.66±0.03d
w20 326.2±0.2a 251.1±0.2a 90.91±0.05b 56.28±0.01c 46.75±0.13b 1.11±0.00g 236.0±0.4a
w23 77.09±0.08d n.d. n.d. 45.25±0.04d 39.42±0.20c 58.61±0.12b 151.9±0.1b
w24 14.37±0.00e n.d. n.d. n.d. 29.89±0.11e 17.04±0.01c 128.8±0.2c
w30 8.03±0.01f n.d. n.d. n.d. n.d. n.d. 17.61±0.01f
w34 4.48±0.02g n.d. n.d. n.d. 10.35±0.01f 2.84±0.01f 17.82±0.01f

Means with different letters within the same column differ significantly (p < 0.05).

n.d.: Not detected.

Colored wheats have been linked to a variety of health advantages, the majority of which are related to their antioxidant characteristics (Saini et al., 2021). The greatest total anthocyanin content (TAC) value was reported in deep purple, blue, and purple wheats, respectively. Amber and red wheat had the lowest TACs (Syed et al., 2013). In blue wheat samples, the most abundant anthocyanin pigments were reported as delphinidin-3-galactoside, delphinidin-3-glucoside, delphinidin-3-rutinoside, and malvidin-3-glucoside (Abdel-Aal et al., 2016; Ficco et al., 2014; Sharma et al., 2020) whereas cyanidin-3-glucoside, cyanidin-3-(6-malonyl glucoside), cyanidin-3-rutinoside, peonidin-3-glucoside, and peonidin-3-(6-malonyl glucoside) were reported as the primary anthocyanins in purple wheat samples (Abdel-Aal et al., 2018; Abdel-Aal et al., 2016; Hosseinian et al., 2008). In the present study, cyanidin-3,5-di-O-glucoside, cyanidin-3-O-glucoside chloride, cyanidin-3-O-rutinoside chloride, delphinidin 3-O-β-D-glucoside chloride, malvidin-3-O-glucoside chloride, pelargonidin 3-O-glucoside chloride, and peonidin 3-O-glucoside chloride were present in the colored wheat samples (Table 4).

In a previous study on purple wheat bran, cyanidin was determined as the most abundant aglycone, followed by peonidin. In contrast, the other common aglycones (delphinidin, petunidin, pelargonidin, and malvidin) were also found, although in much lower amounts than cyanidin and peonidin. Cyanidin-3-O-glucoside chloride was significantly higher in w18 than in the others (p<0.05). Geyik et al. (2023) also found that the significant anthocyanin of blue and purple wheat extracts in the free fraction was cyanidin-3-O-glucoside chloride. Abdel-Aal et al. (2018) claimed that a significant proportion of acylated anthocyanins in purple wheats was claimed to improve their stability during handling and processing.

In the present study, the correlations were also calculated between antioxidant activities (ABTS and CUPRAC) and individual anthocyanin contents of the colored wheat samples. The correlations between CUPRAC values and anthocyanins were not significant. However, there were some significant correlations between the samples’ ABTS values and anthocyanin contents. The correlations between ABTS values and all anthocyanins were r= 0.65-0.91, except malvidin-3-O-glucoside chloride. All of these correlations are significant (p <0.05). Similarly, Sharma et al. (2018) also reported that the ABTS assay positively correlated with TAC for anthocyanin in bio-fortified colored wheat samples.

While cyanidin-3-O-glucoside chloride was the most abundant anthocyanin for four colored wheat samples (w17, w18, w20, and w23), malvidin-3-O-glucoside chloride was determined as the most abundant anthocyanin in the four samples (w18, w20, w23, and w24). The w17 and w20 samples generally had high levels of anthocyanin contents except malvidin-3-O-glucoside chloride for w17 and pelargonidin 3-O-glucoside chloride for w20. Cyanidin-3,5-di-O-glucoside was detected in significant amounts only in dark purple w17 (162.16 µg/100 g) and black-colored w20 (251.12 µg/100 g) samples. Of the 7 anthocyanins, cyanidin-3-O-glucoside chloride was detected only in sample w3. Peonidin 3-O-glucoside chloride was detected in all colored wheat samples except w3 and w30. The results of this study and the literature information proved that peonidin 3-O-glucoside chloride is an essential anthocyanin for purple and black wheats (Eliášová et al., 2020). Delphinidin 3-O-β-D-glucoside chloride, an essential anthocyanin for blue and purple wheats, was detected in significant amounts in 4 wheat samples. This result was similar to the literature findings (Abdel-Aal et al., 2006; Boligon et al., 2014). Pelargonidin 3-O-glucoside chloride generally had a similar trend to peonidin 3-O-glucoside chloride. Pelargonidin 3-O-glucoside chloride was detected in all colored wheats except for the w3 and w30 samples. Malvidin-3-O-glucoside chloride was detected in all samples except the red-colored w3 sample.

This study indicated that the distribution and amount of individual anthocyanins in colored wheat samples are significantly affected by the genotype. Similar results have also been reported in the literature (Abdel-Aal et al., 2018). In colored wheat bran, cyanidin was discovered to be the major aglycone, and peonidin was the second-highest aglycone. However, the other frequent aglycones (delphinidin, petunidin, pelargonidin, and malvidin) were also found in colored wheats, although at much lower amounts than cyanidin and peonidin (Abdel-Aal et al., 2018).

Mineral composition

The colored-grain genotypes’ Macro- and microelement contents are presented in Table 5. The macronutrients Ca, K, and Mg strongly varied depending on genotypic differences between the studied samples. Potassium had the highest concentration (3247 and 4210 mg/kg) among all macro-elements in the colored wheat lines. The w3 sample had the highest K content, while the w13 sample had the lowest K content. The concentrations of other macro-elements were as follows: Mg was in the range of 996-1533 mg/kg), Ca was in the range of 497-790 mg/kg. The w18 sample had the highest Ca content (790 mg/kg). The wheat samples tested in the present study were relatively rich in Mg and Ca. Among the micro-elements, Cu concentration was the lowest (3.0-5.0 mg/kg). Fe, Mn, and Zn concentrations were in the range of 51.0-65.5 mg/kg, 29.0-46.0 mg/kg, and 31.5-56.5 mg/kg, respectively.

Table 5. The content of macro- and microelements in grains of colored wheat lines

# of sample The concentration of macro- and microelements (mg/kg of dry matter)
K Ca Mg Fe Cu Mn Zn
w3 4210±95.0a 597±12.5ab 1533±87.0a 51.0±2.0a 5.0±0.3a 46.0±3.0a 31.5±4.5a
w13 3247±49.5b 619±45.5ab 996±54.0c 53.5±5.5a 3.5±0.5a 29.0±1.0a 37.0±6.0a
w17 3529±46.0b 575±51.5ab 1228±49.5abc 61.0±1.0a 3.5±0.5a 37.5±2.5a 47.0±2.0a
w18 3252±81.0b 790±62.0a 1133±39.5bc 53.5±0.5a 3.5±0.5a 33.0±8.0a 44.5±0.5a
w20 3485±53.0b 590±51.0ab 1242±35.5abc 65.5±7.5a 3.0±0.0a 43.0±9.0a 48.0±2.0a
w23 3530±49.5b 645±48.0ab 1332±50.5ab 61.5±2.5a 4.0±1.0a 33.0±2.0a 56.5±6.5a
w24 3433±51.5b 687±42.0ab 1268±60.5abc 54.0±5.0a 3.5±0.5a 39.5±2.5a 38.0±8.0a
w30 3459±59.5b 500±21.0b 1179±47.5bc 54.5±5.5a 3.0±0.0a 33.5±0.5a 42.0±0.0a
w34 3284±58.0b 497±36.5b 1236±54.5abc 52.0±3.0a 4.0±0.0a 34.0±1.0a 37.5±7.5a

Means with different letters within the same column differ significantly (p < 0.05).

The wheat genotypes obtained by crossing donors of anthocyanin biosynthesis genes with the variety Element 22 and CIMMYT line BW49880 (w17, w18, w20, w23) had the most intense grain color (dark purple and black). These genotypes are characterized by an increased content of Zn (44.5–56.5 mg/kg) and Fe (53.5–65.5 mg/kg). Genotypic differences were revealed in the accumulation of Fe and Zn in the grain of purple wheat genotypes; some had relatively higher Fe and Zn contents. This is a desirable feature targeted by various breeding programs worldwide that are interested in the mineral biofortification of wheat (Cakmak et al., 2010; Velu et al., 2019). The contents of trace elements Zn and Fe in individual genotypes of purple-grained samples were significantly higher than that of the red-grained variety Element 22, which can be explained by the formation of additional chelate bonds of anthocyanins with metals zinc and iron (metal chelation), which is deposited in the outer layers of grain and prevents anthocyanin degradation during grain ripening (Loskutov & Khlestkina, 2021). Therefore, the selected purple wheat samples (w13, w17, w18, w20, w23, w24, w30, w34) can be used as promising genotypes for wheat breeding to improve the micronutrient profile in terms of Zn and Fe of wheat-based end-products. The natural variation in Zn in the wheat grain rarely exceeds 30–35 μg/g. In the current study, the colored wheat sample, w23, had a Zn concentration exceeding 50 μg/g, which will benefit human health (Abugalieva et al., 2021). Fe and Zn had some correlations with all anthocyanins in the range of r= 0.48-0.86 for Fe and r= 0.32-0.59 for Zn. The highest correlation of Fe was with cyanidine-3-O-glucoside chloride (r=0.86), and the highest correlation of Zn was with delphinidin-3-O-β-D-glucoside chloride (r=0.59). The correlations between Zn and individual anthocyanins were not statistically significant. However, the correlations of Fe with cyanidin-3-O-glucoside chloride (r= 0.86), cyanidin-3,5-di-O-glucoside (r= 0.81), cyanidin-3-O-rutinoside chloride (r= 0.67), and delphinidin 3-O-β-D-glucoside chloride (r= 0.66) were significant (p< 0.05). There seem to be some positive correlations between Fe and anthocyanin contents of the colored wheat. However, further studies with different genotypes grown in diverse locations are needed to confirm this conclusion.

Wheat grains are generally poor in essential minerals such as Mn, Fe, Cu, and Zn, resulting in malnutrition in terms of minerals. This is an important problem that affects a significant part of the human population, whose nutrition is based on wheat as the primary food product. Hence, healthy wheat crops with high concentrations of essential minerals should be prioritized for better human nutrition (El-Soda and Aljabri, 2022).

Conclusions

Although wheat is one of the most important grains in the human diet, information on the genotypic variation of wheat in phenolic content and especially in anthocyanin contents is limited. Thus, further studies are needed to investigate the variations in phenolic and anthocyanin contents and the mineral composition of wheat genotypes from different origins. These genotypes can be utilized in breeding programs to develop new wheat varieties suitable for functional food products. The results indicated that relatively high genotypic variations among colored wheats concerning the total antioxidant activity, anthocyanins profiles, and mineral composition provide a sound basis for developing wheat varieties with health benefits.

In the present study, the best accessions with high antioxidant capacity and anthocyanin profile were identified for their further potential utilization in breeding programs. According to the study results, the w17 (line BW 49880–Dark purple P, F4) was chosen as the best sample because it had the highest antioxidant activity, anthocyanin, and mineral content. There were also other wheat samples (e.g., w20) with good antioxidant activity and anthocyanin profile among the tested samples. The ABTS value of the free fraction of w17 was significantly higher than the other colored wheat samples (p <0.05). The w17 and w20 samples generally had high levels of anthocyanin contents except malvidin-3-O-glucoside chloride for w17 and pelargonidin 3-O-glucoside chloride for w 20. The wheat genotypes obtained by crossing donors of anthocyanin biosynthesis genes with the variety Element 22 and CIMMYT line BW49880 (w17, w18, w20, w23) had the most intense grain color (dark purple and black). These genotypes are also characterized by an increased content of Zn (44.5–56.5 mg/kg) and Fe (53.5–65.5 mg/kg). The results of the present study are expected to support the breeding programs’ efforts to develop new wheat varieties with improved nutritional properties. The material used in the present study is also tested for agronomic properties such as yield and resistance to biotic and abiotic stress factors in the wheat breeding program of Omsk State Agrarian University. It is expected to combine these features in newly developed colored wheat cultivars.

Author Contributions

V.P.S.: Conceptualization, project administration, supervision, resources, funding acquisition, Z.H.T.-C.: Investigation, formal analysis, data curation, writing—original draft, S.K.: Writing the original draft, review & editing, I.V.P.: Conceptualization, review & editing, E.I.G.: Mineral composition analysis, A.O.V.: Resources, A.I.M.: Review & editing, M.Y.: Investigation, formal analysis, O.S.: Conceptualization, review & editing and H.K.: Conceptualization, project administration, writing the original draft, writing, review & editing. All authors have read and agreed to the submitted version of the manuscript.

Funding

This research was funded by the Ministry of Science and Higher Education of the Russian Federation (Agreement No. 075-15-2021-534; May 28, 2021).

Conflicts of Interest

The authors declare no conflict of interest.

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Supplementary

Table S1. The description of the plant materials.

# Cultivar/Line, Grain Color and Generation Origin
w1 cv Pam’yati Azieva-Red, St Saratovskaya29/Lutescens 99/80-1.7
w2 cv Duet-Red, St Individual selection from the hybrid population Erythrospermum 59 // Tselinnaya 20/ANK 102
w3 cv Element 22-Red, St (Granıt X Saratovskaya29) X [Erythrospermum59 X (Tselinnaya 20 X Tertsiya)]
w4 cv Tobol’skaya-Red, St Red Lutescens 123-S/Omskaya20
w5 cv Saratovskaya 29-Red, St Albidum 24/Lutescens 55/11
w6 cv Aina-Red, St Tselinnaya Yubilienaya/2*Pastor /3/ Babax/Lr43 // Babax
w7 cv Seri 82-Red, St Kavkaz/(Sib) Buho // Kalyansona/Bluebırd
w8 line10 Element_22-Blue4Th(1) Element_22//S29_ (4Th/4D)/Element_22
w9 line 11 Element_22 Blue4Th(1) Element_22//S29_ (4Th/4D)/Element_22
w10 cv Balda T.dicoccum Individual selection from the hybrid population Belka (T. dicoccum )/Svetlana (T. durum ) // Belka
w11 line Emmer-ColoredTri15744 T.durum Tri15744 /T.dicoccum cv Gremme//*2 T.dicoccum K-25516
w12 cv Saratovskaya 29-ColoredP(5) i:S29_P
w13 line Aina–ColoredPF, BC1F8 Aina *2/i:S29 PF
w14 line Element 22-ColoredPF(8), BC1F8 Element 22 *2/i:S29_PF
w15 line Saratovskaya 29-ColoredPF(2) i:S29_PF
w16 line Tobol’skaya–ColoredPF, BC1F8 Tobol’skaya *2/i:S29_PF
w17 line BW 49880–Dark coloredP, F4 BW 49880 *2/S29_4Th) // BW 49880 *2/i:S29_P
w18 line Element 22-Black, F4 Element 22 *2/S29_4Th) // Element 22 *2/i:S29_PF
w19 line BW 49880–ColoredPF, BC1F8 BW 49880 *2/i:S29_PF
w20 line BW 49880–BlackP+4Th, F4 BW 49880 *2/S29_4Th) // BW 49880 *2/i:S29_P
w21 line BW 49880-Dark ColoredP+4Th, F4 BW 49880 *2/S29_4Th) // BW 49880 *2/i:S29_P
w22 line Element 22-ColoredPF, BC1F8 Element 22 *2/i:S29_ PF
w23 line BW 49880-Dark ColoredP+4Th, F4 BW 49880 *2/S29_4Th) // BW 49880 *2/i:S29_P
w24 line BW 49880-Light brownP+4Th, F4 BW 49880 *2/S29_4Th) // BW 49880 *2/i:S29_P
w25 line BW 49880-ColoredPF(10-7), BC1F4 BW 49880 *2/i:S29 PF
w26 line BW 49880-ColoredPF(10-4-1), BC1F5 BW 49880 *2/i:S29 PF
w27 line Element 22-ColoredPF, BC1F3 Element 22 *2/i:S29_PF
w28 line Element 22-ColoredPF, BC1F3 Element 22 *2/i:S29_PF
w29 line Element 22 -ColoredPF(2-7), BC1F4 Element 22 *2/i:S29_PF
w30 line Element 22-ColoredPF(2-8), BC1F4- Element 22 *2/i:S29_PF
w31 line Element 22 -ColoredPF(2-10), BC1F4 Element 22 *2/i:S29_PF
w32 line Element 22-ColoredPF(2-2-1), BC1F5 Element 22 *2/i:S29_PF
w33 line Element 22-ColoredPF(2-2-7), BC1F5 Element 22 *2/i:S29_PF
w34 line Element 22-ColoredPF(2-3-8), BC1F5 Element 22 *2/i:S29_PF
w35 line Element 22-ColoredPF(2-3-12), BC1F5 Element 22 *2/i:S29_PF
w36 line Element 22-ColoredPF, (2-3-17) BC1F5 Element 22 *2/i:S29_PF
w37 line Element 22-Colored (2-4-6), BC1F5 Element 22 *2/i:S29_PF
w38 line Element 22-ColoredPF, (2-5-1) BC1F5 Element 22 *2/i:S29_PF
w39 line Element 22-ColoredPF, (2-5-4), BC1F5 Element 22 *2/i:S29_PF
w40 line Element 22-ColoredPF, (2-5-14), BC1F5 Element 22 *2/i:S29_PF