Unraveling the Bioactive Profile, Antioxidant and DNA Damage Protection Potential of Rye (Secale cereale) Flour

Six different solvents were used as extraction medium (water, methanol, ethanol, acidified methanol, benzene and acetone) to check their phenolics extraction efficacy from flour of two rye cultivars. Rye extracts with different solvents were further analyzed for the estimation of phytochemicals and antioxidant properties. Different tests (TPC, TAC, DPPH, FRAP, ABTS, RPA and CTC) were performed to check the antioxidant properties and tannin contents in extracts. A bioactive profile of a rye cultivar indicated the presence of total phenolic compounds (0.08–2.62 mg GAE/g), total antioxidant capacity (0.9–6.8 mg AAE/g) and condensed tannin content (4.24–9.28 mg CE/100 g). HPLC was done to check phenolics in rye extract with the best solvent (water), which indicated the presence of Catechol (91.1–120.4 mg/100 g), resorcinol (52–70.3 mg/100 g), vanillin (1.3–5.5 mg/100 g), ferulic acid (1.4–1.5 mg/100 g), quercetin (4.6–4.67 mg/100 g) and benzoic acid (5.3 mg/100 g) in rye extracts. The presence of DNA damage protection potential in rye extracts indicates its medicinal importance. Rye flour could be utilized in the preparation of antioxidant-rich health-benefiting food products.


Introduction
Frequent use of unhealthy food products (junk foods), less physical activity, a busy working schedule and deficiency of health-benefiting nutrients in a daily diet may heighten susceptibility to chronic diseases [1,2]. The addition of whole grains and grain-based healthy food products are favorably supported in diet charts, as they provide significant amounts of protein, fibers, carbohydrates, minerals and bioactive compounds. Rye (Secale cereale) is considered an important traditional cereal crop, and is cultivated worldwide. Rye stands in second place as a cereal grain after wheat, whose flour is utilized for the preparation of bakery products, especially bread and biscuits [3]. Rye (Secale cereale) belongs to the Poaceae family and its genus is Secale. Rye crops are believed to have originated in southwestern Asia and are widely cultivated in Europe, North America and Asia [4]. Rye crops are well known for their adaptability toward harsher environmental conditions. The crops can germinate well at 1.11-3.33 • C. Rye grains are oval/wedge shaped and light to dark brownish in color. Rye grains are genetically related to wheat and barley grains [5]. However, the major difference lies in the size of grains, their nutritional composition and the organoleptic properties they possess. For the proper growth of rye grains, the preferred soil types are light loam/sandy, and well drained/fairly dry soil with a pH ranging from 4.5-8.2. Rye can grow in sandy soils with less nutrients than other soil types. Rye grains are capable of tolerating saline conditions, a low pH and a high concentration of aluminum. Being a long-day plant, rye crops require 40-60 days to shift in their reproductive stage. The vegetation period for rye grains may vary from 120-150 days.
Rye is considered a stress-tolerant and disease-resistant cereal crop [6]. Rye grains have been scrutinized as a good source of fiber, and contain proteins, minerals and bioactive phenolic compounds that have been well documented for their health-benefiting effects [7][8][9][10][11]. Hundreds of bioactive phytochemicals from the extracts of natural resources have been identified and being studied for their health-benefiting antioxidant properties [10,[12][13][14]. Liu et al. [15] demonstrated that health-benefiting properties of food products are mainly due to the syringic and additive effects of bioactive compounds. Cereal grains and their milling fractions may possess mixtures of bioactive compounds. The bioactive profile of rye grains and milling fractions indicates the presence of ferulic acid, sinapic acid, p-Coumaric acid, syringic acid and vanillic acid, followed by p-hydroxybenzoic acid [16][17][18][19]. The unique characteristic feature of these specific phytochemicals is their solvent specificity, as some of them are soluble in an aqueous phase whereas other are either soluble in organic solvents or a combination of aqueous and organic phases [20][21][22][23][24][25][26][27][28][29]. Scantiness of scientific information regarding the effect of different solvent types on rye phenolics incited us to design the present work. The objective of the present research work is to analyze the effect of different solvents on rye phenolics, screening for specific bioactive compounds and their quantifications using HPLC analysis.

Materials and Methods
Rye cultivars (black and white) were collected (IARI, 28.08 • N; 77.12 • E; 228.61 m) from PUSA, New Delhi. Grains were washed well with tap water, oven dried (Rescholar, India) at 45 • C for 48 h and stored in cross-zip airtight pouches/plastic containers for further experimental work.

Sample Preparation
Rye grains from different cultivars were milled to fine flour (Bajaj, India) and passed through a sieve (60BSS) to obtain a uniform size of flour particles (250 micron). Defattation of different rye flour samples (black and white) was carried out with Hexane (1:4 w/v; 10 min; thrice). Defatted samples were dried in a hot air oven (40 • C for 15-24 h, Rescholar, India) and stored (deep freezer, −20 • C, Vestfrost, India). Thereafter, defatted samples were processed using absolute benzene, ethanol, acetone, methanol, acidified methanol (HCl-Methanol; 1:99 v/v) and water in ratio of 1:10 w/v at 50 • C for 40 min in a water bath (Rescholar, India). The flour-solvent mixture was filtered (Whatman No. 1 filter paper 100125R, HiMedia) and extracts were stored in sample-storing vials at 4 • C in a refrigerator (Samsung, India). Rye flour extracts prepared in different solvents were named water rye extract (WRE), ethanol rye extract (ERE), methanol rye extract (MRE), acetone rye extract (ARE), acidified methanol rye extract (AMRE) and benzene rye extract (BRE), respectively. The entire process is represented in Figure 1.

Total Phenolic Content (TPC) and HPLC Analysis
TPC in rye extracts (WRE, ERE, MRE, ARE, AMRE and BRE) were analyzed using the FCR (Folin-Ciocalteu reagent) method [31]. FCR (500 µL) was added in an aliquot (100 µL) of each rye extract (WRE, ERE, MRE, ARE, AMRE and BRE) followed by an aqueous sodium carbonate solution addition (1500 µL, 20%). The resulting reaction mixture (extracts and reagents) was kept undisturbed (20 min) in the dark at ambient temperature. After 20 min, distilled water was added to prepare the final volume up to a mark (10 mL). Absorbance of blue colored extract-reagent mixture was recorded (765 nm; Shimadzu, India). Gallic acid (mg/mL stock, HiMedia) was used (standard). Results were expressed as mg GAE (gallic acid equivalent) g −1 dry weight basis (dwb).
The TAC of rye extracts (WRE, ERE, MRE, ARE, AMRE and BRE) was determined [34]. The standard used during the TAC assay was ascorbic acid. A reagent was prepared to evaluate the antioxidant properties in rye extracts using ammonium molybdate (4 mM), conc. H 2 SO 4 (0.6 M) and sodium hydrogen orthophosphate (28 mM). Rye extracts (100 µL) were allowed to react with the TAC reagent (3 mL). The resulting TAC reagent-extract mixture was warmed (95 • C/90 min). Ascorbic acid (mg/mL) was used as the standard. The TAC value of rye extracts was calculated from the equation generated, and results were expressed as mg AAE/g. Absorbance of the rye extract-reagent mixture and standard solution was recorded (695 nm).

Reducing Power Activity (RPA)
The RPA of rye extracts (WRE, ERE, MRE, ARE, AMRE and BRE) was determined [35]. Rye extracts (100 µL) were allowed to react with an aqueous potassium ferricyanide (K 3 [Fe(CN) 6 ]) solution (1%, 100 µL) followed by incubation in a water bath (50 • C/30 min). The reaction mixture was cooled at room temperature followed by addition of trichloroacetic acid (1% 100 µL) and aqueous ferric chloride solutions (0.1%, 100 µL). The colored mixture was incubated (15 min), which was diluted with distilled water after incubation to prepare the final volume (10 mL). Absorbance of green colored complex was noted (700 nm). The standard used during the RPA assay was Quercetin. The RPA was calculated from the equation generated and results were expressed as mg QE/g. Absorbance of the extract-reagent mixture and standard solution was recorded (700 nm).

Condensed Tannin Content (CTC)
The CTC of rye extracts (WRE, ERE, MRE, ARE, AMRE and BRE) was assessed using the Vanillin (C 8 H 8 O 3 )-HCl method [37]. Rye extracts (100 µL) were allowed to react with Vanillin-HCl (1:0.5 v/v). The reaction mixture was kept undisturbed at room temperature (15 min). Absorbance of the extract-reagent mixture against a blank was recorded (500 nm). For the preparation of the standard curve, catechin was used as the standard. CTC in rye extracts was expressed as mg CE (catechin equivalent)/100 g.
DNA retention (%) = Intensity of supercoiled DNA with the oxidative radical and extract Intensity of supercoiled DNA (control) × 100

Statistical Analysis
Experiments were performed in triplicate to generate results. Afterwards, mean value and standard deviation were calculated. Tukey's test was used to test significant differences among experiments. Differences among means were considered statistically significant at a 5% level. The score plot, loading plot and correlation for determining the relationship was generated using Minitab software 18.

Bioactive Compounds
Rye extracts (BR and WR) were subjected to prelim phytochemical assessment, with the results reported in Table 1. The WRE showed the presence of coumarins, tannins, sugar, saponins and protein, whereas the ERE and ARE showed only saponins. Further, BRE showed the presence of flavonoids, protein and saponins, followed by the MRE, which showed only two compounds (i.e., coumarins and saponins). The AMRE indicated the presence of coumarins, flavonoids, sugars, tannins and saponins.
Phenolic profiles of substrates like fruits/vegetables and cereal grains are gaining more attention among researchers, industries and consumers because of their significance to human health. In the present study, six different extraction mediums (ethanol, acetone, methanol, acidified methanol, water and benzene) were used to extract out phenolic compounds from rye cultivars (BR and WR). Among the selected rye cultivars, black rye (BR) possessed a higher amount of phenolic compounds in the different extraction mediums than the white rye (WR). Extraction mediums used during the experimental work of the present study played an important role when extracting phenolics. Water proved to be the better solvent for extracting total phenolics (TPC) from rye flour as compared to acidified methanol, methanol, ethanol, acetone and benzene. The highest TPC was observed in water extracts (BR (2.62 mg GAE/g); WR (2.14 mg GAE/g)), followed by acidified methanol extracts (BR (1.68 mg GAE/g); WR (1.55 mg GAE/g)), methanol (BR (1.42 mg GAE/g); WR (1.00 mg GAE/g)) and ethanol (BR (0.33 mg GAE/g); WR (0.27 mg GAE/g)). As compared to water, neither acidified methanol, methanol, ethanol, acetone nor benzene were efficient enough to extract out a significant amount of phenolics from rye flour (Figure 2a). TPC in water rye extracts (WRE) of BR (2.62 mg GAE/g) and WR (2.14 mg GAE/g) was in consistent with the results of Mishra et al. [39], who reported a maximum TPC of 2.19 mg/g in water extracts of rye. The TPC (1-1.42 mg GAE/g) in the MREs (BR, WR) agreed with the finding of Zielinski et al. [40], who reported 1.35-1.47 mg/g TPC in 80% methanolic extracts. Total phenolic content in methanolic (80%) extracts of rye varied from 0.98-3.36 mg GAE/g in reports by Kulichova et al. [12] and Ragaee et al. [41]. Michalska et al. [17] reported the effect of phosphate buffered saline (PBS) and methanol (80%) on recovery of phenolics from rye grains. The amount of phenolics was 2.31 mg/g for rye extracts prepared in PBS and 1.43 mg/g for methanolic extract. The CTC in BR and WR extracts was observed to be 4.24-9.28 mg CE/100 g (Figure 2b). The highest CTC was noted in acetone extracts of BR (9.28 mg CE/100 g) and WR (8.74 mg CE/100 g), whereas the lowest amount was noted in benzene extracts of BR (4.80 mg CE/100 g) and WR (4.24 mg CE/100 g).

Antioxidant Properties
Different extracts of rye (WRE, ERE, MRE, ARE, AMRE and BRE) were assessed using TAC, DPPH, RPA, ABTS and FRAP assays. These antioxidant assays are either specific color-forming or decolorizing tests that have been widely adopted for screening antioxidant properties in natural extracts via free radical scavenging [2,20,51]. Antioxidant properties of rye extracts are reported in Table 4. DPPH activity is expressed as percent inhibition, indicated by the ability of an extract to decolorize and convert a purple-colored DPPH reagent to mustard yellow (stabilized form) [58][59][60]. Among extracts (WRE, ERE, MRE, ARE, AMRE and BRE) of selected rye cultivars (BR and WR), WRE showed maximum activity against DPPH at 84% for the BR and 80.3% for the WR. Percent inhibition activity against DPPH radical was observed to be 64.8-74.3% for the AMRE, followed by 38.7-43.5% for the MRE, 21.3-31.4% for the ERE, 14.8-26% for the ARE and 10.7-14.1% for the BRE. Further, another antioxidant assay ABTS was performed to check the percent inhibition activity in rye extracts. Percent (%) inhibition against ABTS was also observed to be higher in water extracts of both rye cultivars: 89.7% for black and 87.6% for white. Rye extracts (AMRE, MRE, ERE, ARE and BRE) other than water also possessed antioxidant potential, as indicated by activity in the black and white rye extracts. Extracts prepared from black rye showed activity/percent inhibition as: AMRE (75.3%), MRE (66.8%), ERE (61.4%), ARE (49.2%) and BRE (22.8%). Extracts prepared from white rye showed activity/percent inhibition as: AMRE (69.2%), MRE (61.2%), ERE (54%), ARE (42.7%) and BRE (18.1%). Overall, water proved the most efficient solvent, followed by acidified methanol and methanol. Mishra et al. [39] studied cold water extracts prepared from rye cultivars, reporting 47.98% inhibition in extracts during a DPPH assay and 97.54% inhibition during an ABTS assay. The difference in percent inhibition may have been due to climatic conditions and geographical distributions. The TAC value indicates the potential of extracts to reduce from a Mo (VI) form to a Mo (V) form. The TAC of BR and WR was observed to be 0.9-6.8 mg AAE/g. The TAC for black rye extracts was observed as: WRE (6.8 mg AAE/g), ERE (1.8 mg AAE/g), MRE (3.9 mg AAE/g), ARE (1.7 mg AAE/g), AMRE (4.9 mg AAE/g) and BRE (0.9 mg AAE/g). Similarly, the TAC in white rye extracts was observed as: WRE (6.1 mg AAE/g), ERE (1.6 mg AAE/g), MRE (3.9 mg AAE/g), ARE (1.6 mg AAE/g), AMRE (4.5 mg AAE/g) and BRE (0.9 mg AAE/g). The FRAP of BR and WR extracts (WRE, ERE, MRE, ARE, AMRE and BRE) ranged from 1.1-8.2 mg FeSO 4 ·7H 2 O equivalent/g. The lowest value was observed in white rye flour extracted with benzene (1.1 mg FeSO 4 ·7H 2 O equivalent/g), whereas a higher FRAP was observed in black rye flour extracted with water (8.2 mg FeSO 4 ·7H 2 O equivalent/g). The FRAP value of an extract represents its capability to reduce Fe 3+ to Fe 2+ . Kulichova et al. [12] studied FRAP values of rye extracts that ranged from 2.27-5.36 mg Trolox equivalent/g. The RPA values of different rye extracts ranged from 0.7 to 8.7 mg QE/g. Kulichova et al. [12] reported a reducing power value of rye extracts in the range of 0.87-20.1 mg Trolox equivalent/g.

Relationship between Bioactive Compounds and Antioxidant Properties
Statistical software Minitab was used to draw a score plot. Six different dots were recorded that indicated the efficacy of different solvents used to extract rye phenolics in the present study ( Figure 4). To ease understanding, two different circles were drawn in which the properties or solvents that have positive relationships to each other are shown inside each circle. Pearson analysis was done to evaluate the correlation among different properties of rye extracts. Significant correlation was observed between the TPC and antioxidant assays (FRAP, r = 0.992 p < 0.01; TAC, r = 0.990; RPA, r = 0.969 p < 0.01; DPPH, r = 0.968 p < 0.01; ABTS, r = 0.882 p < 0.01) ( Table 5).

DNA Damage Protection Potential (DDPP)
Fenton's reagent is extensively used as a DNA degrading agent [45,61] during DDPPA. Rye extracts prepared using water were subjected to DNA protection against the damaging activity of Fenton's reagent on pBR322 (model DNA). Non-availability of bands during gel electrophoresis confirmed the damaging activity, whereas their appearance showed the presence of DDPP. The DDPP of rye extracts during electrophoresis is represented in Figure 5, shown by the appearance of bands in Lane 4 and Lane 5. Figure 6 represents the DDPP (%) of aqueous rye extracts. Published literature has also confirmed the presence of DDPP in extracts prepared from various botanical resources [45,62]. However, no report on DDPP in rye extracts has previously been published.

Conclusions
Comparison of different solvents (water, ethanol, methanol, acetone, acidified methanol and benzene) indicates water to be an efficient solvent for the liberation of phenolic compounds from selected rye cultivars. Maximum antioxidant properties were also observed in water extracts. The presence of specific phytochemicals in rye flour makes it a healthbenefiting substrate that can be used in the preparation of various products for human use. For processing flour, the major solvent used at the domestic as well as the commercial scale is water. Efficacy of water for phenolic extraction from rye flour could prove important from an industrial point of view, as it is easily available and cost effective.