Progress in Nutrition 2017; Vol. 19, N. 4: 450-459 DOI: 10.23751/pn.v19i4.5847 © Mattioli 1885

Original article

Phytochemical content and antioxidant activity of einkorn (Triticum monococcum ssp. monococcum), (Triticum aes- tivum L.), and (Triticum durum Desf.) Yunus Şahin1, Arzu Yıldırım2, Buhara Yücesan3, Nusret Zencirci1, Şerafettin Erbayram4, Ekrem Gürel1 1Department of Biology, Faculty of Art and Sciences, Abant İzzet Baysal University, Bolu, - E-mail: nzencirci@yahoo. com; 2Department of Field Crops, Faculty of Natural and Agricultural Sciences, Abant İzzet Baysal University, Bolu, Turkey; 3Department of Science and Technology, Faculty of Natural and Agricultural Sciences, Abant İzzet Baysal University, Bolu, Turkey; 4Bolu Quality and Feed Industry Corporation, Bolu, Turkey; 5Current Address: Department of Biochemistry, School of Medicine, Altınbaş University, Bakırköy, İstanbul, Turkey.

Summary. Introduction. Wheat (Triticum ssp.), a major , and its marginally grown hulled ancestor einkorn (Triticum monococcum ssp. monococcum), have bioactive compounds reducing and preventing chronic diseases such as diabetes, cancer, and cardio vascular diseases, besides highly desired nutritional properties. Methods. We evaluated the total phenolics and flavonoids and quantified their phenolic acids,α -tocopherol by high perfor- mance liquid chromatography, and their 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity. Results. Ferulic acid (148.67-764.04 µg/g), p-coumaric acid (5.06-54.09 µg/g), and total phelonic content (2.06-8.11 µmol GAE/g) of einkorn were significantly higher than bread and durum wheat (p<0.05). Conclusion. Results suggested that einkorn is a rich gene resource for the improvement of modern healthier wheat cultivars.

Key words: bioactive compounds, bread wheat, durum wheat, einkorn, phenolic acids, Triticum monococcum ssp. monococcum

Introduction gene resources for wheat improvement (4), for in- stance, 7A, having a resistance, Wheat (Triticum ssp.), cultivated for centuries in has been introduced into modern wheat cultivars (5). the Middle-East, Central Asia, Europe, and North- Unfortunately, its cultivation is marginalized into Kas- Africa, is one leading staple crop. With the evolu- tamonu, Bolu, Bilecik, and Sinop provinces of Turkey, tion from wild ancestors, cultivated einkorn (Triticum today (6). There are, too, some limited einkorn (Triti- monococcum spp. monococcum), (Triticum dico- cum monococcum spp. monococcum) production regions ccum Schrank.), durum (Triticum durum Desf.), and in the , , , and (7-9). bread (Triticum aestivum L.) wheat contributed highly Wheat has healthy compounds in human diet, to the diet –and, of course, to the health– of human such as fiber, phytochemicals, antioxidants etc. (10). (1). Einkorn (Triticum monococcum spp. monococcum), Fiber eases digestion and, therefore, decreases the the first cultivated wheat resulted in the modern du- intestinal cancer risks (11). Phytochemicals inhibits rum and bread (2). Einkorn was first domes- radical oxidation with DNA or other cell components ticated around the Karacadag Mountains, Diyarbakir, and protects the cells from the deleterious com- Turkey (3). It, with its capability of adapting to ad- pounds. Bioactive phenolic acids, tocols (vitamin E), verse environmental conditions and possessing rich and carotenoid compounds in wheat showing antioxi- Polyphenols and antioxidant activities of wheat species 451 dant activity (12, 13), prevent or reduce the risk for Materials and methods chronic diseases: type 2 diabetes, ischemic heart dis- ease, colon and breast cancers (14-17). Furthermore, sample collection some bioactive compounds, phenolic acids, antioxi- Five bread (Triticum aestivum L. Cvs.: Akbaşak, dants, and vitamin E exist in all wheat, though more in Bayraktar, Gerek 79, Seval, Demir-2000), five durum wild wheat speciess (18,19). (Triticum durum Desf, Cvs.: C-1252, Altin 40-98, Wheat has been traditionally preferred by con- ANK-98, Kiziltan-91, Altıntaş) cultivars, and two sumers for grain products so far. As more attention einkorn (Triticum monococcum spp. monococcum) pop- has been given to wheat cultivars with strong , ulations from Seben-Bolu and ihsangazi-Kastamonu protein content, composition, and resistance to (Table 1) were studied. Bread and durum wheat cul- biotic and abiotic stresses in bread wheat (20, 21), and tivars were provided by Central Research Institute yellow-colored product in durum wheat (22), for Agricultural Research (CRIFC), Ankara and by health compounds such as fibers, phytochemicals, Anatolian Research Institute (ARI), Eskişehir. Two and bioactive compounds have been underestimated. different einkorn populations originating from Se- Here, therefore, the aim of this study was to investi- ben-Bolu and ihsangazi-Kastamonu were provided gate 1) differences among total phenols, flavonoids, by Bolu Quality and Feed Industry Corporation, antioxidant activities of 12 different einkorn, durum, Turkey. and bread wheat populations/cultivars, and 2) quantify their ferulic, p-coumaric, and α-tocopherol composi- Materials and reagents tions by reverse-phase high performance liquid chro- Folin-Ciocalteu, Merck (Darmstad, Germany), matography (HPLC). was used to evaluate total phenolic content of each extract and gallic acid, Merck (Darmstad, Germany), for construction of the calibration curve in total phe-

Table 1. Types, species, genomes, names, and locations of wheat entries.

Common Name Species and Subspecies Genome Population or Cultivar Location

Einkorn T. monococcum ssp. monococcum AA ID - 1 İhsangazi / Kastamonu

Einkorn T. monococcum ssp. monococcum AA ID - 2 Seben / Bolu

Bread wheat T. aestivum L. AABBDD Akbasak 073 - 44 CRIFC1, Ankara

Bread wheat T. aestivum L. AABBDD Bayraktar CRIFC1, Ankara

Bread wheat T. aestivum L. AABBDD Gerek - 79 ARI2, Eskişehir

Bread wheat T. aestivum L. AABBDD Seval CRIFC1, Ankara

Bread wheat T. aestivum L. AABBDD Demir - 2000 CRIFC1, Ankara

Durum wheat T. durum Desf. AABB C - 1252 CRIFC, Ankara

Durum wheat T. durum Desf. AABB Altintas ARI, Eskişehir

Durum wheat T. durum Desf. AABB Altin 40 - 98 CRIFC1, Ankara

Durum wheat T. durum Desf. AABB ANK - 98 CRIFC1, Ankara

Durum wheat T. durum Desf. AABB Kiziltan - 91 CRIFC1, Ankara

1CRIFC: Central Research Institute for Agricultural Research; 2ARI: Anatolian Research Institute 452 Y. Şahin, A. Yıldırım, B. Yücesan, et al. nol evaluation. 2-Dipheny-1-picryhydrazyl radical flasks and hexane was evaporated. Dry residues were (DPPH) from Sigma-Aldrich (Steinheim, Germany) dissolved in 0.5 mL of methanol and transferred into was used to perform antioxidant activity. Catechol, the glass vials (1.50 mL) through nylon filter (0.22 used for constructing calibration curve in total flavo- µm) for HPLC analysis (24). noid evaluation and ferulic acid, p-coumaric acid, and α-tocopherol were purchased from Sigma-Aldrich Determination of total phenolic content (Steinheim, Germany). TPC of extracts was determined using Folin- Ciocalteau procedure (25). Briefly, 20 μL of extracts (1 Extractions and sample preparation for testing mg/mL) was mixed with 1.58 mL distilled water. Then, All wheat were extracted for free phenolic mixture was oxidized with the addition of 100 μL of content (FPC), bound phenolic content (BPC), total Folin-Ciocalteau reagent. The mixture was neutralized flavonoid content (TFC), phenolic acids (ferulic acid with the addition of 300 μL of saturated Na2CO3 (w/v) and p-coumaric), and α-tocopherol quantification by solution after 2 minutes of reaction. The mixtures were HPLC. Phenolic acids generally exist in a free, esteri- allowed to stand at ambient temperature for 120 min- fied or glycosylated form in . The bound phenolic utes until the characteristic blue color developed. The acids of wheat were extracted with alkaline hydrolysis absorbance of resulting blue-colored mixtures was after liberated free phenolic acids with methanol. measured at 765 nm. Gallic acid was used as a calibra- a. Extraction of wheat grains for FPC, TFC, and tion standard and results were represented as micro- DPPH assay: moles (µmol) gallic acid equivalent (GAE) per gram All wheat grains were de-hulled and grounded into of . Data were summarized as mean ± SD a fine powder. 30 g of each grounded sample were of three replications. extracted by soxhlet for 24 hours at ambient temper- ature with 300 mL of 100% methanol. Methanolic Determination of total flavonoid content extracts were evaporated and stored at -20˚C. TFC of extracts was determined according to b. Extraction for BPC: Chang et al. (26). Briefly, 500 µL of extracts (1 mg/

The residues from the soxhlet extraction were used mL) was reacted with 5% NaNO2 (w/v), after 5 min-

for the bound phenolic extraction. Bound phenolics utes, 10% AlCl3 (w/v) was added for 1 minute, fol- were then released by alkaline hydrolysis using 4 M lowed by 4% NaOH (w/v). The mixtures were allowed NaOH before extraction. After adjusting the pH of to stand at ambient temperature for 30 minutes un- alkaline solutions to 2.0 by 6 N HCl, the mixtures til their characteristic pink color was developed. The were extracted with ethyl acetate and diethyl ether absorbance of solutions was measured at 510 nm. (1:1, v/v) until a white color was observed. Subse- Catechol was used as a calibration standard and re- quently, solvent was evaporated at 30˚C to dryness sults were represented as micromoles (µmol) catechol under nitrogen gas and stored at -20˚C (23). equivalent (CTE) per gram of whole wheat. Data were c. Extraction for α-tocopherol: reported as mean ± SD of three replications. Approximately 0.4 g of each sample was weighed for Briefly, 10 mg of extracts was dissolved in 10 ml α-tocopherol extraction, 200 µL of catechol (0.2 g/ of 100% methanol and solutions were diluted to 200, mL) and 5 mL of 0.5 mol/L methanolic KOH was 100, 50, 25, or 12.5 µg/mL. Subsequently, 500 µL of added to solutions and stirred. The solutions were each diluted solutions were reacted with 1.50 mL of subsequently placed into a water bath at 80˚Cfor 15 1 M DPPH solution in 100% methanol (w/v) for 30 minutes and shaken on the vortex every 5 minutes minutes. The absorbance of solutions was measured at thrice with 1 min resting interval. Then, 1 mL dis- 517 nm (27). The control was the ascorbic acid. Data tilled water and 5 mL hexane were added into the were reported as percentage of DPPH inhibition as in solutions before they were prepared. The mixtures the following, where

were stirred for 1 minute, then the 3 mL of hex- DPPH %inhibition=[(Absorbancecontrol-Absorb-

ane aliquot of each samples was transferred into the ancesample/Absorbancecontrol)]×100 Polyphenols and antioxidant activities of wheat species 453

Phenolic acids (Ferulic acid, p-coumaric), and followed by Altin 40-98 (0.63 ± 0.05 µmol GAE/g), α-tocopherol analyses by HPLC Ferulic and p-coumaric ID-1 (0.63 ± 0.03 µmol GAE/g), ID-2 (0.62 ± 0.02 acid analyses were performed based on a pre-described µmol GAE/g), Akbasak (0.55 ± 0.02 µmol GAE/g), method (23) with some modifications. The samples C-1252 (0.51 ± 0.01 µmol GAE/g), Ank-98 (0.47 and standards at 10 µL were injected using ACC-3000 ± 0.04 µmol GAE/g), Bayraktar (0.46 ± 0.03 µmol Autosampler attached to Ultimate 3000 HPLC sys- GAE/g), Demir-2000 (0.44 ± 0.03 µmol GAE/g), tem equipped with 3000 pump and diode array detec- Seval (0.42 ± 0.06 µmol GAE/g), Altintas (0.42 ± tor, Dionex (Olten, Switzerland). The phenolic acids 0.03 µmol GAE/g), and Gerek-79 (0.29 ± 0.02 µmol were separated on a reverse phase Ace 5 C18 Column GAE/g), respectively. Overall, no significant dif- (150 mm × 4.6 mm). Eluent A was ultra-distillated ferences existed among einkorn, bread, and durum water while eluent B was acetonitrile, both containing wheats, though within each species for cultivars and/ 0.1% formic acid (v/v). The solvent gradient was at a or populations (p<0.05). flow rate of 800 µL/min, with 0 min 100% A; 10 min BPC of wheat populations and cultivars varied 85% A; 15 min 50% A; 20 min 100% A. α-tocopherol from 1.60 ± 0.30 µmol GAE/g to 7.49 ± 0.08 µmol analysis was performed according to a previously de- GAE/g with an average of 3.56 ± 0.09 µmol GAE/g. scribed method (23). It was separated on reverse phase The highest amount of BPC was in ID-1 (7.49 ± 0.08 Ace 5 C18 Column (150 mm × 4.6 mm). Eluent A was µmol GAE/g), and followed by ID-2 (6.37 ± 0.06 96% methanol and 4%ultradistillated water. Column µmol GAE/g), ANK-98 (3.34 ± 0.13 µmol GAE/g), temperature was set at 36˚C and the solvent gradient, Akbasak (3.33 ± 0.01 µmol GAE/g), Gerek-79 (3.16 at a flow rate of 800 µl/min, was as the following: 0-20 ± 0.05 µmol GAE/g), Demir-2000 (3.14 ± 0.07 µmol min 100% A. GAE/g), Seval (2.93 ± 0.05 µmol GAE/g), Altintas (2.91 ± 0.05 µmol GAE/g), C-1252 (2.85 ± 0.01 µmol Statistical analysis GAE/g),and Kiziltan-91 (2.83 ± 0.05µmol GAE/g), A completely randomized block design with three Altin 40-98 (2.73 ± 0.13 µmol GAE/g), Bayraktar replications was run for each character of all wheat (1.60 ± 0.30 µmol GAE/g), respectively. Bound phe- samples studied. Mean differences were separated by nolic, on the average, was almost 7-fold higher than Duncan’s Multiple Range test using SPSS version free phenolic of wheat populations and cultivars and, 18.0 (SPSS Inc. Chicago, IL). All data, then, were re- ranged between 2.06 ± 0.27 µmol GAE/g and 8.11 ported as mean ± standard deviations, based on three ± 0.06 µmol GAE/g, while TPC (ranged 2.06-8.11 replications. µmol GAE/g) was 4.07 ± 0.08 µmol GAE/g. While einkorn populations were significantly higher than bread and durum wheat cultivars (p<0.05), there was Results and discussion no significant differences among bread and durum

Total phenolic contents Phenol compounds are mainly concentrated in the cell wall of cereal grain as bound form and cannot be easily extracted with ethanol, methanol, or acetone but be released by alkali hydrolysis (23). Phenolic contents of wheat cultivars and popu- lations were determined as free and bound (Figure 1). BPC was significantly higher than FPC (p<0.05). FPC in wheat cultivars and populations ranged be- tween 0.29 ± 0.02 and 0.75 ± 0.03 µmol GAE/g. The Figure 1. Free, bound, and total phenolic content of wheat cul- tivars and populations (mean ± SD). The vertical bars represent average was 0.52 ± 0.03 µmol GAE/g. Kızıltan-91 had the standard deviation of each data point. Bars with no common the highest FPC (0.75 ± 0.03 µmol GAE/g), and was letters are significantly different (p < 0.05). 454 Y. Şahin, A. Yıldırım, B. Yücesan, et al. wheat cultivars (p<0.05). However, differences within and the lowest one was Gerek-79 (0.04 ± 0.02 µmol bread and durum wheat cultivars were evident. CTE/g). While there were no significant differences Einkorn, durum, and bread wheat investigated in among einkorn, bread wheat and durum wheat; culti- this study showed similarities with TPC values of ten vars and/or populations within each species were dif- diploid einkorns, another diploid T. urartu Tum., two ferent from each other (P<0.05). tetraploids, and two hexaploid wheat species and acces- That the studies up to date used different stand- sions (extracted with ethanol and ranged 570-1012 mg ards in total phenolics and flavonoids made compari- GAE/kg dry matter) reported by Yilmaz et al. (28) and sons among studies difficult. Dinelli et al. (13) report- there was significant differences between wild wheat ed TFC (bound+free) of 22 different old and modern and modern wheat species and accessions (p<0.05). common wheat cultivars ranged between 872-1715 Also, there was significant differences between wild µmol cathecin equivalent/100g in grains were signifi- and modern wheat cultivars and lines reported by Cic- cantly different (p<0.05). That result showed similar- coritti et al. (29) showed similarity (p<0.001). Ranged ity in our results for the variation within species and from 2.55 to 8.58 µmol GAE/g of emmer, einkorn cultivars. The range of TFC of 12 different emmers (collected from the same region -western Blacksea), were 1.06-2.29 µmol cathecin equivalent/g, of 6 differ- and bread wheat landraces reported by Serpen et al. ent einkorn landraces were 0.80-1.59 µmol cathecin (30), however, TPC values of our einkorns were about equivalent/g and of 2 bread wheat cultivars were 1.32- 2.2-fold higher than those values of einkorn popula- 1.35 µmol cathecin equivalent/g (30). These results tions. In a study by Adom et al. (31), TPC values of were higher than the cultivars and populations investi- some durum and bread wheat varieties ranged from gated in our study and there was significant difference 7.09-8.59 µmol GAE/g were similar to our results as between emmer and einkorn landraces (p<0.05). Dif- well. On the other hand, there was another study indi- ferences in these two studies might be attributed to the cating that our einkorn TPC was higher than those of differences in the standards, populations, and cultivars. eight soft red cultivars (32). The differ- ences for the TPC of cultivars and populations may be DPPH radical scavenging activity due to environment X genotype interaction. DPPH assay are widely evaluated the free radical scavenging effectiveness of various antioxidant sub- Total flavonoid content stances. The reduction capability on the DPPH radical Flavonoids act on several cellular activities such as is determined by the decrease in its absorbance at 517 cell signal transduction, apoptosis, and reactive oxygen. nm induced by antioxidants. The decrease in absorb- Consumption of high flavonoid food reduces chronic ance of DPPH radical caused by antioxidants is due to diseases including cancer and alzhemier (33, 34). the reaction between antioxidant molecules and radi- TFC of wheat cultivars and populations were cals. expressed as micromoles CTE per gram of whole wheat (Figure 2). TFC of wheat populations and cul- tivars were between 0.04 ± 0.02 µmol CTE/g -0.39 ± 0.01µmol CTE/g. The average TFC was 0.23± 0.02 µmol CTE/g of whole wheat. The highest amount of TFC was in Altintas (0.39 ± 0.01µmol CTE/g), fol- lowed by C-1252 (0.35 ± 0.03 µmol CTE/g), ID-2 (0.33 ± 0.04 µmol CTE/g), ID-1 (0.28 ± 0.02 µmol CTE/g), Bayraktar (0.28 ± 0.01 µmol CTE/g), ANK- 98 (0.28 ± 0.02 µmol CTE/g), Akbasak (0.26 ± 0.02 µmol CTE/g), Altin (0.18 ± 0.02 µmol CTE/g), Seval Figure 2. Total flavonoid content of wheat cultivars and popu- lations (mean ± SD). The vertical bars represent the standard (0.15 ± 0.01 µmol CTE/g), Demir-2000 (0.12 ± 0.03 deviation of each data point. Bars with no common letters are µmol CTE/g), Kiziltan-91 (0.09 ± 0.01 µmol CTE/g), significantly different (p < 0.05). Polyphenols and antioxidant activities of wheat species 455

The percentage of DPPH radical inhibition of Altin 40-98 (21.10%) had highest one, but Altintas wheat populations and cultivars (Table 2) indicated (2.22%) lowest one. At 200 µg/ml concentration, Al- that, at minimum concentration (12.5 µg/ml), wheat tin 40-98 (22.34%) and Kiziltan-91 (21.43%) showed populations and cultivars significantly tended to scav- the highest scavenging activity. However, Bayrak- enge of DPPH radicals (p<0.05). Until 200 µg/ml con- tar (9.68%) and Altintas (10.69) had the lowest one centration, there was a significant increase in scaveng- among wheat species and cultivars. There was no sig- ing activity of DPPH radicals (p<0.05). At minimum nificant difference between einkorn, bread, and durum concentration (12.5 µg/ml), ID-2 (2.79%), Gerek-79 wheat cultivars and populations (p<0.05). (2.41%), and Kiziltan-91 (2.39%) showed highest The data obtained in DPPH test, by various pro- scavenge activity of DPPH radicals than those of oth- cedures and solvents in different studies make com- ers, respectively. On the other hand, Altintas (0.06%) parisons difficult. In this study, data were expressed as and Seval (0.15%) showed the lowest scavenging ac- the percentage inhibition of DPPH radicals, because tivity. At 25 µg/ml, Demir-2000 (5.53%) and Seval scavenge 50% of radical (IC50) values of wheat culti- (5.37%) had the highest scavenge activity, respectively, vars and populations were higher than the literature however, Altintas (0.28%) had the lowest scaveng- (data not shown). However, in comparison of DPPH ing activity in the wheat cultivars and populations. radical scavenging activity of T. aestivum, T. turgidum At concentration of 50 µg/ml, Kiziltan-91 (6.62%), ssp. durum, T. turgidum ssp. dicoccum, and T. monococ- Demir-2000 (5.72%) and ID-2 (5.54%) showed high- cum reported by Ciccoritti et al. (29) ranged between iest scavenge activity, respectively, but Altintas (0.42%) 87±3 and 93±8, data represented as ED50 (mg of wheat had the lowest one. At concentration of 100 µg/ml, milled grain required to obtain 50% DPPH scaveng-

Table 2. The percentage inhibition of radical scavenging activity of cultivars and populations at different concentration.

Entries % of DPPH inhibition1

12.5 µg/mL 25 µg/mL 50 µg/mL 100 µg/mL 200 µg/mL

Ascorbic acid 96.29 96.86 96.57 96.57 96.57

ID - 1 0.35cd 2.51c 4.35ab 9.44cde 13.89bc

ID - 2 2.79a 3.87b 5.54a 9.47cde 16.43b

Akbasak 073 - 44 0.61bc 0.76f 1.60bc 4.30fg 11.62cde

Bayraktar 0.66bc 1.54e 1.51bc 4.97efg 9.68e

Gerek - 79 2.41a 2.35c 4.28ab 7.94def 15.52b

Seval 0.15d 5.37a 4.04ab 9.56cde 12.47cd

Demir - 2000 0.91b 5.53a 5.72a 11.85bcd 16.11b

C - 1252 0.75bc 2.55c 2.55bc 8.41def 16.62b

Altintas 0.06d 0.28g 0.42c 2.22g 10.69de

Altin 40 - 98 0.38cd 1.97d 2.26bc 21.10a 22.34a

ANK - 98 0.43cd 0.87f 3.98ab 13.64bc 16.52b

Kızıltan - 91 2.39a 3.73b 6.62a 16.31b 21.43a

1Mean values with the same letter within the column are not significantly different from each other at P < 0.05. 456 Y. Şahin, A. Yıldırım, B. Yücesan, et al. ing on dry basis), showed that there was no significant 2.40 µg/g) had the highest ferulic acid content, fol- difference between wheat species (p<0.05). In another lowed by ID-2 (762.89 ± 14.17 µg/g), which were sig- study reported by Yilmaz et al. (28) of T. monococcum, nificantly higher than those of other wheat cultivars T. aestivum L., and T. durum Desf. extracted with 3 (p<0.05). Moreover, Demir-2000 (554.82 ± 8.55 µg/g) different solvents revealed the same result as our study. had the highest amount of ferulic acid among bread On the other hand, there was significantly difference wheat cultivars and ANK-98 (480.11 ± 31.30 µg/g) between cultivars and landraces of emmer, einkorn, possessed the highest amount of ferulic acid among and bread wheat in the assessment of antioxidant durum wheat cultivars. There were significant dif- acitivities by 2,2’-azino-bis(3-ethylbenzothiazoline- ferences among einkorn and other bread and durum 6-sulphonic acid)(ABTS•+) assay reported by Serpen wheat species and their cultivars (p<0.05). However, et al. (30) (p<0.05). there were no differences between bread and durum wheat species and cultivars (p<0.05). Einkorn had 1.8- Ferulic acid and p-coumaric content fold more ferulic acid than bread and durum wheat. Wheat phytochemicals are mainly composed of Highest p-coumaric content among wheat cul- phenolic acids. They highly contribute to the antioxi- tivars and species was in ID-1 (54.09 ± 1.76 µg/g) dant properties. Ferulic and p-coumaric acid are the and ID-2 (47.64 ± 2.31 µg/g), which were signifi- most abundant chemicals in the wheat kernels, respec- cantly higher than those of other wheat species and tively. In this study, phenolic acids, ferulic acid, and cultivars (p<0.05). Furthermore, Demir-2000 (33.45 ± p-coumaric acid were determined within wheat. 1.23 µg/g) had the highest amount of p-coumaric acid Ferulic acid contents of wheat cultivars and popu- among bread wheat cultivars and Altin 40-98 (23.83 ± lations (Table 3) were expressed as total (free+ bound) 0.73 µg/g) had the highest ferulic acid among durum ferulic acid content. The average ferulic acid content wheat cultivars. On the average, concentration of p- was 485.63± 9.76µg/g of whole wheat. ID-1 (764.04± coumaric was 25.18 ±1.06 µg/g. Furthermore, einkorn

Table 3. Average concentration (µg/g) of bioactive compounds in einkorn, bread, and durum wheat cultivars and populations.

Entries ferulic acid1 p-coumaric1 α-tocopherol1

ID-1 764.04 ± 2.40a 54.09 ± 1.76a 1.41 ± 0.07c

ID-2 762.89 ± 14.17a 47.64 ± 2.31b 3.16 ± 0.70bc

Akbasak 073 - 44 478.76 ± 23.90d 15.28 ± 0.35g 1.59 ± 0.70c

Bayraktar 148.67 ± 13.99g 5.06 ± 0.01i 3.81 ± 1.40b

Gerek - 79 476.50 ± 6.52c 29.38 ± 0.06d 4.01 ± 1.19b

Seval 460.08 ± 1.11de 26.45 ± 1.82de 3.27 ± 0.12bc

Demir - 2000 554.82 ± 8.55b 33.45 ± 1.23c 9.51 ± 0.49a

C - 1252 439.73 ± 17.09e 22.32 ± 1.22f 7.68 ± 0.70a

Altintas 467.20 ± 16.26de 15.30 ± 0.11g 3.09 ± 0.70bc

Altin 40 - 98 397.80 ± 12.18f 23.83 ± 0.73ef 3.51 ± 0.70bc

ANK - 98 480.11 ± 31.30d 11.32 ± 1.59h 4.38 ± 1.40b

Kiziltan - 91 396.96 ± 29.95f 18.08 ± 1.55g 1.66 ± 0.70c 1Data represented as mean ± SD, mean values with the same letter within the column are not significantly different from each other at P < 0.05. Polyphenols and antioxidant activities of wheat species 457 had 2.5-fold higher p-coumaric than bread and durum vars, which was similar to ours (p<0.05). Demir-2000 wheat species and cultivars. While significant differ- and C-1252 showed similar results to hard wheat cul- ences were observed between einkorn populations and tivars, Einkorn, emmer, and soft wheat cultivars (19), cultivars of bread / durum wheat (p < 0.05), there was ranged for α-tocopherol between 5.50-11.90 µg/g of no significant difference between durum and bread α-tocopherol. But, other cultivars and populations had wheat cultivars (p < 0.05). lower values than those. These differences may be ex- Similar results were observed in ten diploid plained by genotype and environmental differences. einkorns, another diploid T. urartu Tum., two tetra- ploids, and two hexaploid wheat species and accessions, which ranged between 471.1-724.9 mg/kg dry matter Conclusion in ferulic acid and 102.1-10.8 mg/kg dry matter in p- coumaric (28). Similar results occurred for ferulic acid Preference of consumers in preventing and con- and p-coumaric (35) and for Maryland soft red win- trolling chronic diseases by cereal based diets may ter wheats. They ranged between 455.92-621.47 µg/g successfully be carried out by cultivating new wheat for ferulic (32) and 25.05-54.21 µg/g for p-coumaric, cultivars rich in bioactive compounds. In this study, lower than those of our einkorn populations. Further- einkorn populations had significantly higher ferulic, more, ferulic acid values in this study were similar to p-coumaric and TPCs than modern bread and du- those of emmer and einkorn species reported by Ser- rum wheat cultivars (p<0.05). Results here suggested pen et al. (30). While they ranged, there, between the possibility of production of popu- 232.66- 775.30 µg/g. p-coumaric they were higher lations, and hopefully possibility of cultivars rich in than those of 25.05-54.21 µg/g our study. However, particular health beneficial component(s) may provide concentrations of ferulic acid of einkorn populations benefit to the consumers. In addition, higher TPC of reported in this study were about 2.5-fold higher than einkorn may offer novel wheat genetic resources for those einkorn landraces. the improvement of new wheat cultivars and the de- velopment of wheat-based functional foods.

α - tocopherol content Acknowledgements Vitamin E (tocopherols and tocotrienols) com- pounds play an important role in the antioxidation of The authors are grateful to the Abant Izzet Baysal Uni- cell membrane and lipoproteins. They also have many versity Research Foundation (Project No: 2014.03.01.715) and Bolu Quality and Feed Industry Corporation, Bolu, Turkey. beneficial functions to human health, such as prevent- ing cancer, cardiovascular diseases, a protective effect lowering LDL cholesterol by inhibiting cholesterol bi- References osynthesis, and expressing an antioxidant activity (36). The highestα -tocopherol in this study was with- 1. Pirgozliev V, Rose SP, Pellny T, Amerah AM, Wickramasin- in Demir-2000 (9.51±-0.49 µg/g of whole wheat), ghe M, Ulker M, Rakszegi M, Bedo Z, Shewry PR and whereas the lowest was within ID-1 (5.06 ± 0.01 µg/g Lovegrove A. Energy utilization and growth performance of chickens fed novel wheat inbred lines selected for differ- of whole wheat). The average of all was 3.92±0.41 ent pentosan levels with and without xylanase supplementa- µg/g. No difference existed among einkorn, durum, tion. Poult Sci. 2015; 94: 1–8. and bread wheat species, but among cultivars and pop- 2. Salamini F, Ozkan H, Brandolini A, Schäfer-Pregl R and ulations (p<0.05). Martin W. Genetics and geography of wild cereal domes- t cat on n the . Nat Rev Genet. 2002; 3: 429–441. In a study, Hejtmankova et al. (24), with two i i i 3. Heun M, Schäfer-Pregl R, Klawan D, Castagna R, Ac- einkorn, two emmer, and three spring wheat cultivars cerbi M, Borghi B and Salamini F. Site of Einkorn Wheat (ranged between 5.69-8.57 mg/kg dry matter) report- Identified by DNA Fingerprinting. Science. ed significant differences between species and culti- 1997; 278: 1312–1314. 458 Y. Şahin, A. Yıldırım, B. Yücesan, et al.

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