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MS identification and UHPLC-Online ABTS antioxidant activity guided mapping of barley polyphenols
Accepted Manuscript Q-TOF LC/MS identification and UHPLC-Online ABTS antioxidant activity guided mapping of barley polyphenols Shiwangni Rao, Abishek B. Santhakumar, Kenneth A. Chinkwo, Christopher L. Blanchard PII: DOI: Reference:
S0308-8146(18)30975-0 https://doi.org/10.1016/j.foodchem.2018.06.011 FOCH 22972
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
14 March 2018 2 June 2018 4 June 2018
Please cite this article as: Rao, S., Santhakumar, A.B., Chinkwo, K.A., Blanchard, C.L., Q-TOF LC/MS identification and UHPLC-Online ABTS antioxidant activity guided mapping of barley polyphenols, Food Chemistry (2018), doi: https://doi.org/10.1016/j.foodchem.2018.06.011
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Q-TOF LC/MS identification and UHPLC-Online ABTS antioxidant activity guided mapping of barley polyphenols Shiwangni RAOab, Abishek B. SANTHAKUMARab*, Kenneth A. CHINKWOab, and Christopher L. BLANCHARDab a
School of Biomedical Sciences, Charles Sturt University, Wagga Wagga, 2650, New South Wales, Australia Australian Research Council (ARC) Industrial Transformation Training Centre (ITTC) for Functional Grains, Graham Centre for Agricultural Innovation, Charles Sturt University, Wagga Wagga, 2650, New South Wales, Australia b
* indicates corresponding author. Correspondence: Dr. Abishek B. Santhakumar BMedSc, MMedSc (MLS), GCTE, PhD Lecturer in Haematology School of Biomedical Sciences Discipline Leader and Researcher ARC ITTC for Functional Grains Charles Sturt University, Locked Bag 588, Bldg. 288, Room 238 Wagga Wagga, NSW 2678, Australia Tel: +61 2 6933 2678 Email: [email protected] Co-Author email address: Shiwangni Rao ([email protected]) Kenneth A. Chinkwo ([email protected]) Christopher L. Blanchard ([email protected])
Abbreviations: 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid), ABTS; 2,4,6- Catechin equivalents, CE; 2,2-diphenyl1-picrylhydrazyl, DPPH; Ferric reducing ability of plasma assay, FRAP; Gallic acid equivalents, GAE; Reactive oxygen species, ROS; Total anthocyanin content, TAC; Trolox equivalents, TE; Total phenolic content, TPC; Total proanthocyanidin content, TPAC; Tris(2-pyridyl)-s-triazine, TPTZ; Quad Time of flight liquid chromatography mass spectra, Q-TOF LC/MS; Ultra-high performance liquid chromatography, UHPLC
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Abstract The polyphenol composition and antioxidant activity of seven Australian-grown barley varieties were characterized in this study. UHPLC with an online ABTS system was used to identify individual polyphenols while simultaneously measuring their antioxidant activity. The Q-TOF LC/MS system was utilized to identify the phenolic compounds that demonstrated substantial antioxidant activity. The variety, Hindmarsh, showed the highest total phenolic content and antioxidant activity. There was no significant difference observed amongst the other varieties in their total phenolic content, however, they did have significant variation in proanthocyanidin content and antioxidant activity (p < 0.05). Prodelphinidin B3 was the most abundant polyphenol with the highest antioxidant activity amongst all the barley varieties tested. Other polyphenols identified with antioxidant activity included procyanidin, glycosides of catechin and flavan-3-ols. Polyphenol characterization of Australian grown barley varieties demonstrated that they have significant antioxidant activity, hence, promoting the value of whole grain barley as a potential functional food ingredient.
Keywords: Barley, UHPLC-online ABTS, antioxidant, cereals, Q-TOF LC/MS
Chemical Compounds Caffeic acid (PubChem CID:689043); Catechin (PubChem CID:9064); Ellagic acid (PubChem CID:5281855); Ferulic acid (PubChem CID:445858); Gallic acid (PubChem CID:370); Isoscoparin-2"-O-glucoside (PubChem CID:72193659); P-coumaric acid (PubChem CID:637542); Procyanadin B2 (PubChem CID:122738); Prodelpinidin B (PubChem CID:5089687); Prodelpinidin B3 (PubChem CID:13831063).
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1. Introduction Barley (Hordeum vulgare L.) is primarily cultivated for its starch-rich seeds, however, its utilisation as malt has taken precedence over its value as a food grain. Global barley production in the cropping year 2016/2017 averaged to 146.20 million metric tons. Australia was the second largest contributor followed by Russia, and exports 65% of its barley production (Statista, 2018). Like most cereal grains, polyphenols are present in the bran layer of whole grain barley, in free and glycosylated bound forms. Polyphenols that are found in pigmented and nonpigmented barley varieties range from simple phenolic acids to complex dimers of proanthocyanidins (Kim, et al., 2007). Studies have shown polyphenols to have antioxidant activity which has been attributed to their ability to scavenge reactive oxygen species (ROS) (Bahadoran, et al., 2013). In particular, the hydroxyl groups present in the polyphenol structures readily donate hydrogen ions in order to stabilise free radicals (Pereira, et al., 2009). In biological systems, polyphenols can act as antioxidants by inhibiting enzymes associated with free radical production. The mechanism by which polyphenols inhibit these enzymes is by either forming hydrogen bonds with the hydroxyl groups or with the benzenoid rings (Parr & Bolwell, 2000). There has been an increased interest in barley consumption due to its reported nutritional properties. A number of studies have reported barley to have antioxidant (Bonoli, et al., 2004; Butsat & Siriamornpun, 2010; Dvorakova, et al., 2008a), anti-inflammatory (Zhu, et al., 2015) and chemopreventive (Choi, et al., 2014; Jeong & Lee, 2013) properties; suggesting whole grain barley may have a role as a potential functional food. Polyphenolic composition and antioxidant properties of barley have been studied in varieties grown around the world (Dvorakova, et al., 2008b; Kim, et al., 2007; Zhao, et al., 2008). However, there
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has only been limited studies on the identification of phenolic compounds found in Australian-grown barley and quantification of their individual antioxidant activities. In the present study, polyphenol profiles of grain from seven commercially grown Australian barley varieties were investigated using UHPLC and Q-TOF LC/MS. The antioxidant capacity of individual phenolic compounds were quantified using an ABTS online system coupled to the UHPLC system.
2. Materials and Methods 2.1 Materials Acetone, acetonitrile, ethanol, methanol, sodium hydroxide, sodium peroxide, sodium carbonate, anhydrous sodium acetate, potassium persulfate, hydrochloric acid, sulphuric acid and acetic acid were sourced from Chem Supply Pty Ltd (Port Adelaide, South Australia, Australia). Folin-Ciocalteu reagent, Trolox (6-Hydroxy-2,5,7,8-tetramethylchroman-2carboxylic
acid),
2,2-diphenyl-1-picrylhydrazyl,
DPPH;
ABTS
(2,2′-Azino-bis
(3-
ethylbenzothiazoline-6-sulfonic acid), TPTZ (2,4,6-Tris(2-pyridyl)-s-triazine), vanillin, gallic acid, ferulic, caffeic, catechin, epicatechin, protocatechuic acid, o-coumaric, p-coumaric, sinapic, syringic, vanillic, iso-vanillic, quercetin, naringenin, rutin and trans-cinnamic were sourced from Sigma-Aldrich (St Louis, Missouri, USA). Barley samples from field trials in Wongarbon and Condobolin, New South Wales, 2015, were provided by National Variety Trials and New South Wales Department of Primary Industries (NSW, Australia). These field yield trials were commissioned by the Grains Research and Development Center (GRDC) to evaluate barley varietal cultivation suitability assessment based on yield. All the varieties were cultivated under the same conditions and harvested at the same time. The varieties analysed from Wongarbon included Commander, Compass (Location 1 – L1), Hindmarsh, Latrobe, Westminster, Schooner and Gairdner, while Compass (Location 2 – L2) was sourced from Condobolin.
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2.1 Polyphenol Extraction Whole kernel barley samples were finely ground in a Perten Laboratory Mill 3000 (Hägersten, Sweden) with a 0.5 mm sieve. The method for extracting phenolic compounds was adapted from previously described studies (Rao, et al., 2018; Zhou, et al., 2014) with minor modifications. A 1 g sample of flour was defatted with 20 mL hexane overnight and then air-dried. Defatted samples were then extracted three times with 20ml of an acetone, water and acetic acid solution (70:29.5:0.5 v/v/v) at a ratio of 20:1 (v/w) on a magnetic stirring plate for 1 hour at room temperature (25C). Samples were then centrifuged at 11, 963 g for 10 minutes. The supernatants were pooled and evaporated using a rotary vacuum (Rotavapor R-210 BUCHI Labortechnik, Flawil, Switzerland) then freeze-dried (ChristAlpha 2-4 LD Plus freeze dryer, Biotech International, Germany) to powder. The samples were reconstituted in 50% methanol for chemical analysis and stored at -20C until required. The extraction and chemical analysis were performed in triplicate for all samples.
2.2 Total Phenolic Content (TPC) The total phenolic content was determined using procedures described by Qiu, et al. (2010) with minor modifications. Briefly 125 l of the Folin-Ciocalteu reagent was added to 125 L of sample and 500 L of deionized water. Following 6 minutes of incubation, the mixture was neutralised by adding 1.5 mL of 7% sodium carbonate solution and topped with 1 mL of deionized water. After a 90-minute incubation in the dark, the absorbance was measured at 725 nm on a microplate reader (BMG Labtech Fluostar Omega, Offenburg, Germany). The total phenolic content of the barley samples were expressed as mg 100 g-1of gallic acid equivalents (GAE).
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2.3 Total proanthocyanidins Content (TPAC) The vanillin assay adapted from Sun, et al. (1998) and Min, et al. (2011) was used to quantify total proanthocyanidin content. Briefly, 0.2 mL of barley sample was combined with 0.5 mL of 1% (w/v) vanillin in methanol and 0.5 mL of 25% sulphuric acid in methanol. The mixture was gently vortexed and incubated in a 37C water bath for 15 minutes. The absorbance of the sample was then measured at 500 nm using a microplate reader. Total proanthocyanidin content was quantified as mg 100 g-1 (+)-catechin equivalents (CE) of the sample and were calculated using a (+)-catechin standard curve.
2.4 2, 2-diphenyl-1-picrylhydrazyl (DPPH) Antioxidant Assay Free radical scavenging activity was quantified using a DPPH assay adapted from Sompong, et al. (2011). Barley extracts resuspended in 50% methanol (100 µl) were vortexed with 3500 µl of a DPPH (4.37mg in 100 ml of methanol) radical solution. The mixture was incubated at room temperature in the dark for 30 min. The absorbance of the mixture was read at 715 nm at room temperature using a microplate reader. DPPH free radical scavenging ability was quantified expressed as µmol 100g-1 Trolox equivalents (TE).
2.5 Ferric Reducing Ability of Plasma Assay (FRAP) The FRAP assay was modified using methods by Sompong, et al. (2011). The FRAP reagent was prepared by mixing 100 mL of acetate buffer (300 mM, pH 3.6), 10 mL TPTZ solution (10 mM TPTZ in 40 mM HCl), 10 mL FeCl3.6H2 O (20 mM) in a ratio of 10:1:1 with 12 mL of deionized water. The FRAP reagent (1.8 mL) was mixed with 60 L of the sample and 180 L deionized water and gently vortexed. After incubation at 37C for 40 minutes, the absorbance was measured at 593 nm using a microplate reader. The ferric reducing ability was expressed as µmol 100g-1 TE Trolox equivalents (TE).
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2.6 UHPLC with online ABTS and Q-TOF LC/MS Polyphenol profiling and quantification was undertaken as described by Rao, et al. (2018) and conducted on an Agilent UHPLC system using a C18 Poroshell120 column (3.0 mm x 100 mm, 2.7 m) (Agilent Technologies, CA, USA). The UHPLC system was assembled with a UV-vis photodiode array detector (PDA) and autosampler. The system was combined with an external binary pump, coil column and UV-vis detector, injecting ABTS solution simultaneously at a flow rate of 0.38 mL min-1. The antioxidant activity of individual polyphenols were determined using an ABTS system after passing through the UHPLC system. Mobile phase A comprised of deionized water and 0.1% acetic acid. Mobile phase B comprised of acetonitrile with 0.1% acetic acid. Samples of 5.7 L was injected into the system at a gradient elution of 0-1 min, 0-10%; B; 1-5 min, 10-26% B; 5-7.5 min, 26-35% B; and 7.5-13 min, 35-100% B. Standards were used to quantify identified compounds. For unknown compounds (peaks P1 to P9), gallic acid equivalents was used as a measure for quantification. Trolox was used to quantify the ABTS radical scavenging activity and was expressed as mg 100 g-1 Trolox equivalents (TE). Absorbances were measured at 280 nm for polyphenols and 414 nm for ABTS activity. Mass spectra of the unknown peaks P1 to P9 were determined using the above-mentioned UHPLC System connected to Agilent 6530 Accurate-Mass Q-TOF LC/MS (Agilent Technologies, CA, USA). Peak identification was performed in negative mode, nitrogen gas nebulisation was set at 45psi with a flow of 5L/min at 300ºC while the sheath gas was set at 11L/min at 250ºC. The capillary and nozzle voltage was set at 3.5kV and 500V respectively. A complete mass scan ranging from m/z 50 to1300 was used. Compounds of the unknown peaks were analysed and tentatively identified from the time of flight ESI mass spectra using Agilent Mass Hunter Qualitative Analysis software.
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2.7 Statistical Analysis Statistical analysis was performed using one-way analysis of variance (ANOVA), followed by Tukey’s posthoc multiple comparisons test using GraphPad Prism 7 software (GraphPad Software Inc, California, USA). The results are reported as mean ± standard deviation. Statistical significance was determined at a level of p < 0.05.
3. Results and Discussion 3.1 Total phenolic content (TPC) Analysis of the TPC in the current study showed that Hindmarsh had the highest TPC at 130.80 ± 6.89 mg 100g-1 GAE, followed by Compass L1 (121.10 ± 8.90 mg 100g-1 GAE) and Latrobe (115.8 ± 6.18 mg 100g-1 GAE) (p < 0.05) (Table 1). Meanwhile, Gairdner exhibited the lowest TPC followed by compass L2 from Condobolin (80.2 ± 16.11 and 83.81 ± 14.78 mg 100g-1 GAE respectively). Studies conducted on barley from Canada and China have reported similar TPC values of 114 and 196 mg 100g-1 respectively (Hao & Beta, 2012; Madhujith & Shahidi, 2009). In contrast, a study by Dvorakova, et al. (2008b) reported lower TPC values for barley varieties from the Czech Republic ranging from 28.47 to 49.91 mg 100g-1. While the study conducted by Maillard, et al. (1996) on barley varieties from France report TPC content as high as 1071 mg 100g-1 GAE. This variation in TPC can result from factors such as cultivation environment, variety, extraction procedure and extraction solvent (Bonoli, et al., 2004; Kim, et al., 2007). A significant variation in the TPC values due to a variation in cultivation environment was obeserved in varieties, compass L1 and L2 in this study (Table 1). Kim, et al. (2007) investigated variation in phenolic content due to variety and found that TPC ranged from 19.1 mg 100g-1 GAE to 40.38 mg 100g-1 GAE in 127 hulled and hull-less pigmented barley. It was also observed that unhulled barley had higher phenolic content (26.86 mg 100g-1 GAE) than hulled barley (20.70 mg 100g-1 GAE). In addition to varietal differences, different solvents
8
used in the extraction procedures has been demonstrated to have a varying effect on the TPC. Bonoli, et al. (2004) demonstrated that in barley the TPC ranged from 29 to 68 mg 100g-1 with 75% acetone showing the highest TPC followed by 75% ethanol and 75% methanol. Furthermore, the current study mimics barley for malt where the husk is intact and comprises approximately 20% of the sample weight (Fox, 2009), hence a contributing factor to the slightly lower total phenolic content in comparison to the values reported by Madhujith and Shahidi (2009).
Table 1. Polyphenol content and antioxidant activity of Australian barley varieties. TPC (mg 100 g-1 GAE)
TPAC (mg 100 g-1 CE)
DPPH (µmol 100 g-1 TE)
FRAP (µmol 100 g-1 TE)
121.10 ± 8.90a 83.81 ± 14.78b 130.80 ± 6.89a 115.80 ± 6.18a 87.52 ± 14.00b
2.93 ± 0.48c 4.07 ± 0.88ac 5.31 ± 0.23a 2.25 ± 0.29cd 2.54 ± 1.40cd
632.10 ± 62.67ab 343.50 ± 51.65d 655.10 ± 78.87b 538.30 ± 35.23ac 635.70 ± 83.15b
502.30 ± 67.62ab 500.30 ± 94.11a 536.20 ± 30.45a 430.60 ± 14.94bc 515.10 ± 83.23a
Schooner 95.92 ± 7.58b 3.26 ± 1.86cb 498.60 ± 52.53c Gairdner 80.20 ± 16.11b 5.12 ± 1.50ab 432.50 ± 57.33cd Different letters in columns indicate significant differences at p < 0.05.
384.20 ± 45.09cd 329.40 ± 32.96d
Compass L1 Compass L2 Hindmarsh LaTrobe Westminster
3.2 Total Proanthocyanidins Content (TPAC) Proanthocyanidins have been found in barley and in particular complexs of prodelphinidin and procyanidin have been detected in proanthocyanidin-rich varieties (Goupy, et al., 1999; Quinde-Axtell & Baik, 2006). The current study observed that Hindmarsh (5.31 ± 0.23 mg 100g-1 CE) and Gairdner (5.12 ± 1.50 mg 100g-1 CE) had the highest levels of TPAC compared to other varieties (p < 0.001) (Table 1). Similarly, Kim, et al. (2007) reported TPAC in pigmented barley from Korea that range from 1.58 mg 100g-1 CE to 13.18 mg 100g1
CE. Contrastingly, Yoshida, et al. (2010) extracted free and bound phenolic acids using a
range of extraction solvents including hexane/dichloromethane (1:1 v/v), acetone/water/acetic acid (70:29.5:0.5 v/v/v) and 80% ethanol that resulted in higher proanthocyanidin values
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ranging from 12.2 mg 100g-1 CE to 80 mg 100g-1 CE in non-pigmented hulled and hull-less barley grown in Japan. The observed variation in TPAC could still be attributed to the choice of solvent during the extraction process. In addition,
Commander
was observed to have
considerably
low amount
of
proanthocyanidins (0.641 ± 0.22 mg 100g-1 CE) in comparison to other varieties. Consequently presenting Commander grains as a suitable candidate for malting, as low proanthocyanidin has been associated with low cloudiness in beer (Øverland, et al., 1994).
3.3 DPPH Antioxidant Scavenging ability In this study three antioxidant assays, DPPH, FRAP and ABTS were performed. While DPPH and FRAP assays quantified the overall free radical scavenging activity, the online ABTS was designed to quantify the antioxidant activity of individual phenolic compounds present in the crude barley extracts. The results using the DPPH assay for the overall antioxidant activity demonstrated suggestive differences between the varieties tested at p < 0.05 (Table1). Hindmarsh exhibited the highest free radical scavenging activity at 655.10 ± 78.87 µmol 100g-1 TE, followed by Westminster at 635.70 ± 83.15 µmol 100g-1 TE and Compass L1 at 632.10 ± 62.67 µmol 100g-1 TE. Gairdner and Compass L2 recorded the lowest DPPH free radical scavenging ability at 432.50 ± 57.33 and 343.50 ± 51.65 µmol 100g-1 TE respectively. The free radical scavenging activity of Australian barley is comparable to barley from Canada (Madhujith & Shahidi, 2009), China (Zhao, et al., 2008) and Italy (Bonoli, et al., 2004) where similar values have been reported. Contrastingly, studies conducted in Japan (Yoshida, et al., 2010) have reported a higher DPPH value of 1501µmol 100g-1 TE (Yoshida, et al., 2010). While the study by Mareček, et al. (2017) has reported lower DPPH values of 130 µmol 100g-1 TE to 160 µmol 100g-1 TE in the Czech Republic spring barley. Therefore, as stated earlier cultivation
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environment, extraction solvent and procedure can have a significant impact on the phenolic compounds that are extracted and its relative antioxidant activity.
3.4 Ferric Reducing Ability of Plasma Assay (FRAP) The antioxidant activity of the barley varieties was measured by their ability to reduce Fe (III) (Table 1). The results demonstrated statistical differences between the varieties tested at p < 0.001. As observed with the TPC, Hindmarsh and Westminster exhibited the highest FRAP antioxidant activity at 536.20 ± 30.45 µmol 100g-1 TE and 515.10 ± 83.23 µmol 100g-1 TE respectively. Similar to the DPPH assay, Schooner and Gairdner exhibited the lowest antioxidant activity at FRAP values of 384.20 ± 45.09 µmol and 329.40 ± 32.96 µmol 100g-1 TE respectively. The two compass varieties exhibited significant difference in DPPH but did not vary significantly in FRAP antioxidant activity, this is believed to be a result of variation in regulation of specific phenolic compounds influenced by the cultivation location. Overall these results correlate with the TPC findings where Hindmarsh had the highest TPC and Gairdner had the lowest. While TPC, TPAC, DPPH and FRAP values of varieties such as Commander, Westminster and Latrobe varied between assays, Hindmarsh consistently exhibited higher levels across all the assays.
3.5 UHPLC with online ABTS A number of polyphenols were detected by the UHPLC-online ABTS system, seven of which were confirmed by standards and the others with substantial antioxidant activity were identified using Q-TOF LC/MS. The identified phenolic compounds included gallic acid, catechin, caffeic acid, ferulic acid, trans-cinnamic acid, o-coumaric acid and ellagic acid (Table 2). The above listed phenolic compounds have also been detected in barley from other countries such as America (Quinde-Axtell & Baik, 2006), Czech Republic (Dvorakova, et al., 2008b), Japan (Yoshida, et al., 2010) and Canada (Hao & Beta, 2012). 11
The unknown peaks (P1 to P9) were tentatively identified as p-coumaric acid isomer, isoscoparin-2"-O-glucoside, catechin dihexoside, catechin-5-O-glucoside, isoorientin-7-Ogentiobioside, prodelphinidin B3, prodelphinidin B, procyanidin B2 and apigenin 6-Carabinoside 8-C-glucoside respectively (Table 2). These identified polyphenolic compounds have been reported in previous studies (Ferreres, et al., 2008; Gangopadhyay, et al., 2016; Quinde-Axtell & Baik, 2006). The study by Ferreres, et al. (2008) identified glycosylated flavones
in barley leaves of which isoorientin-7-O-gentiobioside, isoscoparin-2"-O-
glucoside and apigenin 6-C-arabinoside 8-C-glucoside were found in the whole kernel barley samples in the current study. In addition, the current study also found derivatives of catechin, prodelphinidin and Procyanidin that have been reported in barley grains by Gangopadhyay, et al. (2016) and Quinde-Axtell and Baik (2006).
Table 2. Peak identification using 6530 Accurate-Mass Q-TOF LC/MS Peak
Tentative Identification
Formula
m/z
P1 P2 P3 P4 P5 P6 P7 P8 P9
P-coumaric acid isomer Isoscoparin-2"-O-glucoside Catechin dihexoside Catechin-5-O-glucoside Isoorientin-7-O-gentiobioside Prodelpinidin B3 Prodelpinidin B Procyanadin B2 Apigenin 6-C-arabinoside 8-C-glucoside
C9H8O3 C28H32O16 C27H33O16 C21H24O11 C11H20O7 C30H26O13 C30H26O13 C30H26O12 C20H30O7
164.0717 625.2015 613.1797 451.1259 771.3704 593.1312 593.1312 577.1355 563.1419
Retentio n time 4.27 4.47 4.76 5.28 5.54 5.63 5.63 6.32 6.74
Average Mass 164 624 614 452 772 593 593 577 563
In the current study, catechin-5-O-glucoside and prodelphinidin B3 (Figure 2) were the most abundant polyphenols in the seven Australian barley varieties followed by caffeic acid. Other studies by Hao and Beta (2012); Quinde-Axtell and Baik (2006); Yoshida, et al. (2010) have reported ferulic acid to be the most abundant phenol present in barley which, as mentioned previously, can be attributed to factors such as variety, environment and extraction procedure. Furthermore, in the current study, significant variation was observed with
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catechin-5-O-glucoside, prodelphinidin B3 and prodelphinidin B levels across the seven varieties investigated (Table 3). The varieties Schooner, Westminster, Compass L2 and Commander recorded the highest content of catechin-5-O-glucoside followed by Latrobe, Compass, Hindmarsh and Gairdner. Hindmarsh, Commander, Compass L2 and Westminster recorded the highest content of prodelphinidin B3 with Gairdner and Compass L1 recording similarly low levels of prodelphinidin B3. In addition, it was observed that ellagic acid was not detected from the variety Compass L2 from Condobolin but was present in Compass L1 sample from Wongarbon.
Figure 2. Mass Spectra of Prodelphinidin B3
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Table 3. Quantification of Australian barley polyphenols (mg 100g-1) using UHPLC detected at 280nm. Peaks
Commander
GA
0.36 ± 0.02a
P1
Compass L1
Compass L2
Hindmarsh
LaTrobe
Schooner
Westminster
Gairdner
0.35 ± 0.00a
0.39 ± 0.02a
0.39 ± 0.02a
0.35 ± 0.00a
0.35 ± 0.01a
0.36 ± 0.03a
0.35 ± 0.01a
0.96 ± 0.21b
0.89 ± 0.18b
0.73 ±0.13b
1.05 ± 0.16b
1.13 ± 0.15b
0.97 ± 0.17b
1.05 ± 0.10b
0.84 ± 0.03b
P2
1.95 ± 0.05c
1.77 ± 0.19c
2.24 ±0.42v
2.28 ± 0.13c
1.78 ± 0.92c
1.95 ± 0.19c
1.98 ± 0.29c
1.34 ± 0.31c
P3
0.65 ± 0.07d
0.43 ± 0.06d
0.62 ±0.07d
0.46 ± 0.04d
0.53 ± 0.02d
0.48 ± 0.09d
0.54 ± 0.07d
0.48 ± 0.02d
P4
6.80 ± 1.49eh
4.43 ± 1.15ef
7.62 ±1.39eh
4.18 ± 0.48f
5.08 ± 0.79efh
12.81 ± 5.76g
7.56 ± 5.24h
3.96 ± 0.89f
P5
3.03 ± 0.74i
3.41 ± 0.34i
3.72 ±1.64i
2.82 ± 0.99i
2.85 ± 0.84i
02.29 ± 055i
3.68 ± 0.69i
2.78 ± 0.33i
P6
9.71 ± 3.53j
6.98 ± 2.43kl
9.04 ±1.30jk
10.76 ± 3.43j
8.76 ± 2.98jk
15.08 ± 0.59l
9.76 ± 3.58j
6.61 ± 1.44kl
P7
0.72 ± 0.28m
0.45 ± 0.02m
1.54 ±0.18mn
3.60± 1.78n
2.78 ± 0.64mn
0.63± 0.22m
0.59 ± 0.21m
0.64 ± 0.11m
P8
1.29 ± 0.26p
1.00 ± 0.20p
0.70 ±0.12p
1.40 ± 0.19p
1.26 ± 0.09p
0.89 ± 0.31p
1.25 ± 0.69p
0.85 ± 0.18p
Cat
0.48 ± 0.08q
0.63 ± 0.06q
0.57 ±0.05q
1.51 ± 0.18q
1.06 ± 0.23q
0.92 ± 0.40q
0.83 ± 0.58q
0.56 ± 0.07q
P9
1.26 ± 1.08r
0.67 ± 0.10r
1.89 ±0.48r
0.76 ± 0.31r
0.88 ± 0.16r
0.96 ± 0.07r
0.69 ± 0.29r
0.66 ± 0.07r
CAF
3.30 ± 0.39s
3.16 ± 0.37s
1.42 ±0.23s
3.95 ± 1.57s
2.95 ± 0.70s
2.89 ± 0.40s
3.11 ± 1.05s
3.34 ± 0.66s
p-C
0.91 ± 0.13t
0.87 ± 0.05t
0.52 ±0.10t
0.85 ± 0.06t
0.77 ± 0.17t
0.91 ± 0.07t
0.98 ± 0.09t
0.89 ± 0.08t
FA
0.48 ± 0.03u
0.50 ± 0.01u
0.88 ±0.06u
0.46 ± 0.03u
0.65 ± 0.21u
0.51 ± 0.01u
0.51 ± 0.01u
0.55 ± 0.08u
t-Cin
0.36 ± 0.00v
0.36 ± 0.00v
0.37 ±0.01v
0.36 ± 0.00v
0.40 ± 0.04v
0.36 ± 0.01v
0.36 ± 0.00v
0.36 ± 0.00v
o-C
0.42 ± 0.09w
0.36 ± 0.00w
0.40 ±0.01w
0.48 ± 0.02w
0.47 ± 0.03w
0.39 ± 0.01w
0.42 ± 0.01w
0.40 ± 0.01w
EA
0.45 ± 0.01x
0.46 ± 0.00x
n
0.45 ± 0.01x
0.45 ± 0.02x
0.46 ± 0.00x
0.46 ± 0.00x
0.46 ± 0.01x
CAT, catechin; FA, ferulic acid; GA, gallic acid; CAF, caffeic acid; p-C, p-coumaric; o-C, o-coumaric; EA, ellagic; n, detected in negligible amounts. Different letters in rows indicate significant differences at p < 0.05.
14
It was observed that the amount of the phenolic compound present did not necessarily correlate to its antioxidant activity (Figure 1 and Table 4). For instance, phenolic compounds such as catechin-5-O-glucoside was recorded as the second most abundant phenolic compound but was identified as the least active in scavenging for free radicals. Whereas catechin dihexoside, isoorientin-7-O-gentiobioside, procyanidin B2 and ellagic had significantly higher antioxidant activity in comparison to their phenolic content ranking. In addition, significant differences were seen in the antioxidant activity of catechin dihexoside, isoorientin-7-O-gentiobioside, prodelphinidin B and B3 across the varieties studied. This observation aligns with the study conducted by Goupy, et al. (1999), that also found prodelphinidin B3 to be the most active free radical scavenging polyphenol followed by procyanidin B3 and C among nine barley varieties from France.
Figure 1. Shiwangni. UHPLC Chromatogram of barley variety Hindmarsh. The chromatogram illustrates polyphenol detection at 280 nm (top), and the relative online ABTS chromatogram at 414 nm below.
15
Table 4. Quantification of antioxidant activity using UHPLC-Online ABTS profiling (mg 100g-1 TE) detected at 414nm. Peaks Commander
Compass L1
Compass L2
Hindmarsh
LaTrobe
Schooner
Westminster
Gairdner
P1
1.66 ± 0.54a
1.43 ± 0.25a
1.21 ± 0.09a
1.69 ± 0.30a
1.94 ± 0.33a
1.43 ± 0.25a
1.86 ± 0.19a
1.18 ± 0.21a
P2
2.95 ± 0.60b
2.00 ± 0.37b
4.09 ± 0.70b
2.45 ± 0.12b
1.93 ± 0.90b
2.58 ± 0.33b
2.82 ± 0.78b
1.59 ± 0.11b
P3
4.20 ± 1.65cd
3.13 ± 0.94cd
2.93 ± 0.43c
8.39 ± 3.55d
5.19 ± 1.78cd
3.64 ± 1.35cd
2.58 ± 0.82c
2.28 ± 0.94c
P4
1.76 ± 0.14e
2.21 ± 0.32e
5.59 ± 0.83e
5.00 ± 0.79e
2.60 ± 1.33e
3.19 ± 0.52e
1.98 ± 0.51e
1.34 ± 0.02e
P5
9.51 ± 3.93fg
5.82 ± 2.83g
8.73 ± 1.65gh
14.09 ± 5.37f
12.47 ± 3.07fh
2.28 ± 0.98g
4.51 ± 3.53gi
4.86 ±2.43gi
P6
22.53 ± 1.64jm
22.87 ± 3.99j
18.68 ± 2.03l
31.23 ± 4.10k
26.27 ± 3.01jk
17.56 ± 1.56lm
19.39 ± 4.58lm
16.89 ±5.66lm
P7
29.79 ± 4.96no
7.09 ± 2.841np 13.16 ± 1.91op
10.01 ± 9.86no
12.84 ± 2.44o
15.03 ± 2.71n
4.39 ± 7.52n
5.04 ± 1.65n
P8
28.10 ± 0.44q
8.04 ± 0.24q
7.10 ± 0.68q
9.15 ± 0.63q
8.42 ± 1.63q
16.76 ± 1.10q
7.70 ± 2.24q
6.01 ± 0.80q
Cat
14.59 ± 1.78r
5.84 ± 0.54r
7.32 ± 0.54r
8.82 ± 0.13r
7.05 ± 2.01r
17.02 ± 1.28r
6.77 ± 1.11r
4.09 ± 0.81r
P9
03.56 ± 1.70s
2.33 ± 0.69s
6.51 ± 0.99s
4.55 ± 1.89s
4.11 ± 0.69s
1.88 ± 1.16s
2.65 ± 2.05s
2.11 ± 0.35s
EA
03.34 ± 0.29t
3.29 ± 0.24t
n
3.33 ± 0.18t
3.57 ± 0.29t
3.22 ± 0.28t
3.17 ± 0.10t
2.86 ± 0.21t
CAT, catechin; EA, ellagic acid; n, detected in negligible amounts. Different letters in rows indicate significant differences at p < 0.05.
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4. Conclusion The current study demonstrates that Australian barley varieties have significant variation in polyphenol content and antioxidant activity. Hindmarsh showed the highest total phenolic content which correlated to its high antioxidant activity. Other varieties such as Compass, Latrobe and Westminster also had comparable phenolic content and antioxidant activity. The UHPLC-online ABTS coupled with the Q-TOF LC/MS was used to identify compounds that readily scavenged free radicals. It was observed that in the barley variety Compass L1 and L2 cultivation environment resulted in variability of phenolic compounds and antioxidant activity. In addition, the current study also determined that the phenolic compound prodelphinidin and its isomer were the most predominant polyphenol and its relative antioxidant activity was largely responsible for the overall variation in antioxidant activity of the seven varieties of Australian barley studied.
Acknowledgements The authors would like to acknowledge Mr Neal Sutton from Australian Nationa Variety Trials and Mrs Jennifer Puma from the NSW Department of Primary Industries for providing the barley samples. The authors would also like to acknowledge Dr Lachlan Schwartz and Mr. Michael Loughlin for their invaluable assistance. Funding: This work was funded by the Australian Research Council Industrial Transformations Centre for Functional Grains [Identifier Number: IC140100027]. Shiwangni Rao is a recipient of the Australian Research Council Industrial Transformations Centre for Functional Grains scholarship through Charles Sturt University. The authors declare no conflict of interest.
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Maillard, M. N., Soum, M. H., Boivin, P., & Berset, C. (1996). Antioxidant Activity of Barley and Malt: Relationship with Phenolic Content. LWT - Food Science and Technology, 29(3), 238-244. Mareček, V., Mikyška, A., Hampel, D., Čejka, P., Neuwirthová, J., Malachová, A., & Cerkal, R. (2017). ABTS and DPPH methods as a tool for studying antioxidant capacity of spring barley and malt. Journal of Cereal Science, 73, 40-45. Min, B., McClung, A. M., & Chen, M.-H. (2011). Phytochemicals and Antioxidant Capacities in Rice Brans of Different Color. Journal of Food Science, 76(1), C117C126. Øverland, M., Heintzman, K. B., Newman, C. W., Newman, R. K., & Ullrich, S. E. (1994). Chemical Composition and Physical Characteristics of Proanthocyanidin-free and Normal Barley Isotypes. Journal of Cereal Science, 20(1), 85-91. Parr, A. J., & Bolwell, G. P. (2000). Phenols in the plant and in man. The potential for possible nutritional enhancement of the diet by modifying the phenols content or profile. Journal of the Science of Food and Agriculture, 80(7), 985-1012. Pereira, D. M., Valentão, P., Pereira, J. A., & Andrade, P. B. (2009). Phenolics: From chemistry to biology. Molecules, 14(6), 2202-2211. Qiu, Y., Liu, Q., & Beta, T. (2010). Antioxidant properties of commercial wild rice and analysis of soluble and insoluble phenolic acids. Food Chemistry, 121(1), 140-147. Quinde-Axtell, Z., & Baik, B. K. (2006). Phenolic compounds of barley grain and their implication in food product discoloration. Journal of Agricultural and Food Chemistry, 54(26), 9978-9984. Rao, S., Callcott, E. T., Santhakumar, A. B., Chinkwo, K. A., Vanniasinkam, T., Luo, J., & Blanchard, C. (2018). Profiling polyphenol composition and antioxidant activity in Australian-grown rice using UHPLC-Online ABTS system. Journal of Cereal Science. Sompong, R., Siebenhandl-Ehn, S., Linsberger-Martin, G., & Berghofer, E. (2011). Physicochemical and antioxidative properties of red and black rice varieties from Thailand, China and Sri Lanka. Food Chemistry, 124(1), 132-140. Statista. (2018). World barley production since 2008. In, vol. 2018). https://www.statista.com/statistics/271973/world-barley-production-since-2008/: Statista. Sun, B., Ricardo-da-Silva, J. M., & Spranger, I. (1998). Critical factors of vanillin assay for catechins and proanthocyanidins. Journal of Agricultural and Food Chemistry, 46(10), 4267-4274. Yoshida, A., Sonoda, K., Nogata, Y., Nagamine, T., Sato, M., Oki, T., Hashimoto, S., & Ohta, H. (2010). Determination of Free and Bound Phenolic Acids, and Evaluation of Antioxidant Activities and Total Polyphenolic Contents in Selected Pearled Barley. Food Science and Technology Research, 16(3), 215-224. Zhao, H. F., Fan, W., Dong, J. J., Lu, J., Chen, J., Shan, L. J., Lin, Y., & Kong, W. B. (2008). Evaluation of antioxidant activities and total phenolic contents of typical malting barley varieties. Food Chemistry, 107(1), 296-304. Zhou, Z., Chen, X., Zhang, M., & Blanchard, C. (2014). Phenolics, flavonoids, proanthocyanidin and antioxidant activity of brown rice with different pericarp colors following storage. Journal of Stored Products Reserach, 59, 120-125. Zhu, Y., Li, T., Fu, X., Abbasi, A. M., Zheng, B., & Liu, R. H. (2015). Phenolics content, antioxidant and antiproliferative activities of dehulled highland barley (Hordeum vulgare L.). Journal of Functional Foods, 19, 439-450.
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Research Highlights
Polyphenol content of eight Australian-grown barley varieties were characterised
Antioxidant activity of polyphenols was identified using UHPLC-online ABTS system
Phenolic composition and antioxidant activity exhibited significant variation
Variety Hindmarsh had higher polyphenolic content and antioxidant activity
Prodelphinidin B3 had the highest abundance and antioxidant activity
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S0308-8146(18)30975-0 https://doi.org/10.1016/j.foodchem.2018.06.011 FOCH 22972
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
14 March 2018 2 June 2018 4 June 2018
Please cite this article as: Rao, S., Santhakumar, A.B., Chinkwo, K.A., Blanchard, C.L., Q-TOF LC/MS identification and UHPLC-Online ABTS antioxidant activity guided mapping of barley polyphenols, Food Chemistry (2018), doi: https://doi.org/10.1016/j.foodchem.2018.06.011
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Q-TOF LC/MS identification and UHPLC-Online ABTS antioxidant activity guided mapping of barley polyphenols Shiwangni RAOab, Abishek B. SANTHAKUMARab*, Kenneth A. CHINKWOab, and Christopher L. BLANCHARDab a
School of Biomedical Sciences, Charles Sturt University, Wagga Wagga, 2650, New South Wales, Australia Australian Research Council (ARC) Industrial Transformation Training Centre (ITTC) for Functional Grains, Graham Centre for Agricultural Innovation, Charles Sturt University, Wagga Wagga, 2650, New South Wales, Australia b
* indicates corresponding author. Correspondence: Dr. Abishek B. Santhakumar BMedSc, MMedSc (MLS), GCTE, PhD Lecturer in Haematology School of Biomedical Sciences Discipline Leader and Researcher ARC ITTC for Functional Grains Charles Sturt University, Locked Bag 588, Bldg. 288, Room 238 Wagga Wagga, NSW 2678, Australia Tel: +61 2 6933 2678 Email: [email protected] Co-Author email address: Shiwangni Rao ([email protected]) Kenneth A. Chinkwo ([email protected]) Christopher L. Blanchard ([email protected])
Abbreviations: 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid), ABTS; 2,4,6- Catechin equivalents, CE; 2,2-diphenyl1-picrylhydrazyl, DPPH; Ferric reducing ability of plasma assay, FRAP; Gallic acid equivalents, GAE; Reactive oxygen species, ROS; Total anthocyanin content, TAC; Trolox equivalents, TE; Total phenolic content, TPC; Total proanthocyanidin content, TPAC; Tris(2-pyridyl)-s-triazine, TPTZ; Quad Time of flight liquid chromatography mass spectra, Q-TOF LC/MS; Ultra-high performance liquid chromatography, UHPLC
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Abstract The polyphenol composition and antioxidant activity of seven Australian-grown barley varieties were characterized in this study. UHPLC with an online ABTS system was used to identify individual polyphenols while simultaneously measuring their antioxidant activity. The Q-TOF LC/MS system was utilized to identify the phenolic compounds that demonstrated substantial antioxidant activity. The variety, Hindmarsh, showed the highest total phenolic content and antioxidant activity. There was no significant difference observed amongst the other varieties in their total phenolic content, however, they did have significant variation in proanthocyanidin content and antioxidant activity (p < 0.05). Prodelphinidin B3 was the most abundant polyphenol with the highest antioxidant activity amongst all the barley varieties tested. Other polyphenols identified with antioxidant activity included procyanidin, glycosides of catechin and flavan-3-ols. Polyphenol characterization of Australian grown barley varieties demonstrated that they have significant antioxidant activity, hence, promoting the value of whole grain barley as a potential functional food ingredient.
Keywords: Barley, UHPLC-online ABTS, antioxidant, cereals, Q-TOF LC/MS
Chemical Compounds Caffeic acid (PubChem CID:689043); Catechin (PubChem CID:9064); Ellagic acid (PubChem CID:5281855); Ferulic acid (PubChem CID:445858); Gallic acid (PubChem CID:370); Isoscoparin-2"-O-glucoside (PubChem CID:72193659); P-coumaric acid (PubChem CID:637542); Procyanadin B2 (PubChem CID:122738); Prodelpinidin B (PubChem CID:5089687); Prodelpinidin B3 (PubChem CID:13831063).
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1. Introduction Barley (Hordeum vulgare L.) is primarily cultivated for its starch-rich seeds, however, its utilisation as malt has taken precedence over its value as a food grain. Global barley production in the cropping year 2016/2017 averaged to 146.20 million metric tons. Australia was the second largest contributor followed by Russia, and exports 65% of its barley production (Statista, 2018). Like most cereal grains, polyphenols are present in the bran layer of whole grain barley, in free and glycosylated bound forms. Polyphenols that are found in pigmented and nonpigmented barley varieties range from simple phenolic acids to complex dimers of proanthocyanidins (Kim, et al., 2007). Studies have shown polyphenols to have antioxidant activity which has been attributed to their ability to scavenge reactive oxygen species (ROS) (Bahadoran, et al., 2013). In particular, the hydroxyl groups present in the polyphenol structures readily donate hydrogen ions in order to stabilise free radicals (Pereira, et al., 2009). In biological systems, polyphenols can act as antioxidants by inhibiting enzymes associated with free radical production. The mechanism by which polyphenols inhibit these enzymes is by either forming hydrogen bonds with the hydroxyl groups or with the benzenoid rings (Parr & Bolwell, 2000). There has been an increased interest in barley consumption due to its reported nutritional properties. A number of studies have reported barley to have antioxidant (Bonoli, et al., 2004; Butsat & Siriamornpun, 2010; Dvorakova, et al., 2008a), anti-inflammatory (Zhu, et al., 2015) and chemopreventive (Choi, et al., 2014; Jeong & Lee, 2013) properties; suggesting whole grain barley may have a role as a potential functional food. Polyphenolic composition and antioxidant properties of barley have been studied in varieties grown around the world (Dvorakova, et al., 2008b; Kim, et al., 2007; Zhao, et al., 2008). However, there
3
has only been limited studies on the identification of phenolic compounds found in Australian-grown barley and quantification of their individual antioxidant activities. In the present study, polyphenol profiles of grain from seven commercially grown Australian barley varieties were investigated using UHPLC and Q-TOF LC/MS. The antioxidant capacity of individual phenolic compounds were quantified using an ABTS online system coupled to the UHPLC system.
2. Materials and Methods 2.1 Materials Acetone, acetonitrile, ethanol, methanol, sodium hydroxide, sodium peroxide, sodium carbonate, anhydrous sodium acetate, potassium persulfate, hydrochloric acid, sulphuric acid and acetic acid were sourced from Chem Supply Pty Ltd (Port Adelaide, South Australia, Australia). Folin-Ciocalteu reagent, Trolox (6-Hydroxy-2,5,7,8-tetramethylchroman-2carboxylic
acid),
2,2-diphenyl-1-picrylhydrazyl,
DPPH;
ABTS
(2,2′-Azino-bis
(3-
ethylbenzothiazoline-6-sulfonic acid), TPTZ (2,4,6-Tris(2-pyridyl)-s-triazine), vanillin, gallic acid, ferulic, caffeic, catechin, epicatechin, protocatechuic acid, o-coumaric, p-coumaric, sinapic, syringic, vanillic, iso-vanillic, quercetin, naringenin, rutin and trans-cinnamic were sourced from Sigma-Aldrich (St Louis, Missouri, USA). Barley samples from field trials in Wongarbon and Condobolin, New South Wales, 2015, were provided by National Variety Trials and New South Wales Department of Primary Industries (NSW, Australia). These field yield trials were commissioned by the Grains Research and Development Center (GRDC) to evaluate barley varietal cultivation suitability assessment based on yield. All the varieties were cultivated under the same conditions and harvested at the same time. The varieties analysed from Wongarbon included Commander, Compass (Location 1 – L1), Hindmarsh, Latrobe, Westminster, Schooner and Gairdner, while Compass (Location 2 – L2) was sourced from Condobolin.
4
2.1 Polyphenol Extraction Whole kernel barley samples were finely ground in a Perten Laboratory Mill 3000 (Hägersten, Sweden) with a 0.5 mm sieve. The method for extracting phenolic compounds was adapted from previously described studies (Rao, et al., 2018; Zhou, et al., 2014) with minor modifications. A 1 g sample of flour was defatted with 20 mL hexane overnight and then air-dried. Defatted samples were then extracted three times with 20ml of an acetone, water and acetic acid solution (70:29.5:0.5 v/v/v) at a ratio of 20:1 (v/w) on a magnetic stirring plate for 1 hour at room temperature (25C). Samples were then centrifuged at 11, 963 g for 10 minutes. The supernatants were pooled and evaporated using a rotary vacuum (Rotavapor R-210 BUCHI Labortechnik, Flawil, Switzerland) then freeze-dried (ChristAlpha 2-4 LD Plus freeze dryer, Biotech International, Germany) to powder. The samples were reconstituted in 50% methanol for chemical analysis and stored at -20C until required. The extraction and chemical analysis were performed in triplicate for all samples.
2.2 Total Phenolic Content (TPC) The total phenolic content was determined using procedures described by Qiu, et al. (2010) with minor modifications. Briefly 125 l of the Folin-Ciocalteu reagent was added to 125 L of sample and 500 L of deionized water. Following 6 minutes of incubation, the mixture was neutralised by adding 1.5 mL of 7% sodium carbonate solution and topped with 1 mL of deionized water. After a 90-minute incubation in the dark, the absorbance was measured at 725 nm on a microplate reader (BMG Labtech Fluostar Omega, Offenburg, Germany). The total phenolic content of the barley samples were expressed as mg 100 g-1of gallic acid equivalents (GAE).
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2.3 Total proanthocyanidins Content (TPAC) The vanillin assay adapted from Sun, et al. (1998) and Min, et al. (2011) was used to quantify total proanthocyanidin content. Briefly, 0.2 mL of barley sample was combined with 0.5 mL of 1% (w/v) vanillin in methanol and 0.5 mL of 25% sulphuric acid in methanol. The mixture was gently vortexed and incubated in a 37C water bath for 15 minutes. The absorbance of the sample was then measured at 500 nm using a microplate reader. Total proanthocyanidin content was quantified as mg 100 g-1 (+)-catechin equivalents (CE) of the sample and were calculated using a (+)-catechin standard curve.
2.4 2, 2-diphenyl-1-picrylhydrazyl (DPPH) Antioxidant Assay Free radical scavenging activity was quantified using a DPPH assay adapted from Sompong, et al. (2011). Barley extracts resuspended in 50% methanol (100 µl) were vortexed with 3500 µl of a DPPH (4.37mg in 100 ml of methanol) radical solution. The mixture was incubated at room temperature in the dark for 30 min. The absorbance of the mixture was read at 715 nm at room temperature using a microplate reader. DPPH free radical scavenging ability was quantified expressed as µmol 100g-1 Trolox equivalents (TE).
2.5 Ferric Reducing Ability of Plasma Assay (FRAP) The FRAP assay was modified using methods by Sompong, et al. (2011). The FRAP reagent was prepared by mixing 100 mL of acetate buffer (300 mM, pH 3.6), 10 mL TPTZ solution (10 mM TPTZ in 40 mM HCl), 10 mL FeCl3.6H2 O (20 mM) in a ratio of 10:1:1 with 12 mL of deionized water. The FRAP reagent (1.8 mL) was mixed with 60 L of the sample and 180 L deionized water and gently vortexed. After incubation at 37C for 40 minutes, the absorbance was measured at 593 nm using a microplate reader. The ferric reducing ability was expressed as µmol 100g-1 TE Trolox equivalents (TE).
6
2.6 UHPLC with online ABTS and Q-TOF LC/MS Polyphenol profiling and quantification was undertaken as described by Rao, et al. (2018) and conducted on an Agilent UHPLC system using a C18 Poroshell120 column (3.0 mm x 100 mm, 2.7 m) (Agilent Technologies, CA, USA). The UHPLC system was assembled with a UV-vis photodiode array detector (PDA) and autosampler. The system was combined with an external binary pump, coil column and UV-vis detector, injecting ABTS solution simultaneously at a flow rate of 0.38 mL min-1. The antioxidant activity of individual polyphenols were determined using an ABTS system after passing through the UHPLC system. Mobile phase A comprised of deionized water and 0.1% acetic acid. Mobile phase B comprised of acetonitrile with 0.1% acetic acid. Samples of 5.7 L was injected into the system at a gradient elution of 0-1 min, 0-10%; B; 1-5 min, 10-26% B; 5-7.5 min, 26-35% B; and 7.5-13 min, 35-100% B. Standards were used to quantify identified compounds. For unknown compounds (peaks P1 to P9), gallic acid equivalents was used as a measure for quantification. Trolox was used to quantify the ABTS radical scavenging activity and was expressed as mg 100 g-1 Trolox equivalents (TE). Absorbances were measured at 280 nm for polyphenols and 414 nm for ABTS activity. Mass spectra of the unknown peaks P1 to P9 were determined using the above-mentioned UHPLC System connected to Agilent 6530 Accurate-Mass Q-TOF LC/MS (Agilent Technologies, CA, USA). Peak identification was performed in negative mode, nitrogen gas nebulisation was set at 45psi with a flow of 5L/min at 300ºC while the sheath gas was set at 11L/min at 250ºC. The capillary and nozzle voltage was set at 3.5kV and 500V respectively. A complete mass scan ranging from m/z 50 to1300 was used. Compounds of the unknown peaks were analysed and tentatively identified from the time of flight ESI mass spectra using Agilent Mass Hunter Qualitative Analysis software.
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2.7 Statistical Analysis Statistical analysis was performed using one-way analysis of variance (ANOVA), followed by Tukey’s posthoc multiple comparisons test using GraphPad Prism 7 software (GraphPad Software Inc, California, USA). The results are reported as mean ± standard deviation. Statistical significance was determined at a level of p < 0.05.
3. Results and Discussion 3.1 Total phenolic content (TPC) Analysis of the TPC in the current study showed that Hindmarsh had the highest TPC at 130.80 ± 6.89 mg 100g-1 GAE, followed by Compass L1 (121.10 ± 8.90 mg 100g-1 GAE) and Latrobe (115.8 ± 6.18 mg 100g-1 GAE) (p < 0.05) (Table 1). Meanwhile, Gairdner exhibited the lowest TPC followed by compass L2 from Condobolin (80.2 ± 16.11 and 83.81 ± 14.78 mg 100g-1 GAE respectively). Studies conducted on barley from Canada and China have reported similar TPC values of 114 and 196 mg 100g-1 respectively (Hao & Beta, 2012; Madhujith & Shahidi, 2009). In contrast, a study by Dvorakova, et al. (2008b) reported lower TPC values for barley varieties from the Czech Republic ranging from 28.47 to 49.91 mg 100g-1. While the study conducted by Maillard, et al. (1996) on barley varieties from France report TPC content as high as 1071 mg 100g-1 GAE. This variation in TPC can result from factors such as cultivation environment, variety, extraction procedure and extraction solvent (Bonoli, et al., 2004; Kim, et al., 2007). A significant variation in the TPC values due to a variation in cultivation environment was obeserved in varieties, compass L1 and L2 in this study (Table 1). Kim, et al. (2007) investigated variation in phenolic content due to variety and found that TPC ranged from 19.1 mg 100g-1 GAE to 40.38 mg 100g-1 GAE in 127 hulled and hull-less pigmented barley. It was also observed that unhulled barley had higher phenolic content (26.86 mg 100g-1 GAE) than hulled barley (20.70 mg 100g-1 GAE). In addition to varietal differences, different solvents
8
used in the extraction procedures has been demonstrated to have a varying effect on the TPC. Bonoli, et al. (2004) demonstrated that in barley the TPC ranged from 29 to 68 mg 100g-1 with 75% acetone showing the highest TPC followed by 75% ethanol and 75% methanol. Furthermore, the current study mimics barley for malt where the husk is intact and comprises approximately 20% of the sample weight (Fox, 2009), hence a contributing factor to the slightly lower total phenolic content in comparison to the values reported by Madhujith and Shahidi (2009).
Table 1. Polyphenol content and antioxidant activity of Australian barley varieties. TPC (mg 100 g-1 GAE)
TPAC (mg 100 g-1 CE)
DPPH (µmol 100 g-1 TE)
FRAP (µmol 100 g-1 TE)
121.10 ± 8.90a 83.81 ± 14.78b 130.80 ± 6.89a 115.80 ± 6.18a 87.52 ± 14.00b
2.93 ± 0.48c 4.07 ± 0.88ac 5.31 ± 0.23a 2.25 ± 0.29cd 2.54 ± 1.40cd
632.10 ± 62.67ab 343.50 ± 51.65d 655.10 ± 78.87b 538.30 ± 35.23ac 635.70 ± 83.15b
502.30 ± 67.62ab 500.30 ± 94.11a 536.20 ± 30.45a 430.60 ± 14.94bc 515.10 ± 83.23a
Schooner 95.92 ± 7.58b 3.26 ± 1.86cb 498.60 ± 52.53c Gairdner 80.20 ± 16.11b 5.12 ± 1.50ab 432.50 ± 57.33cd Different letters in columns indicate significant differences at p < 0.05.
384.20 ± 45.09cd 329.40 ± 32.96d
Compass L1 Compass L2 Hindmarsh LaTrobe Westminster
3.2 Total Proanthocyanidins Content (TPAC) Proanthocyanidins have been found in barley and in particular complexs of prodelphinidin and procyanidin have been detected in proanthocyanidin-rich varieties (Goupy, et al., 1999; Quinde-Axtell & Baik, 2006). The current study observed that Hindmarsh (5.31 ± 0.23 mg 100g-1 CE) and Gairdner (5.12 ± 1.50 mg 100g-1 CE) had the highest levels of TPAC compared to other varieties (p < 0.001) (Table 1). Similarly, Kim, et al. (2007) reported TPAC in pigmented barley from Korea that range from 1.58 mg 100g-1 CE to 13.18 mg 100g1
CE. Contrastingly, Yoshida, et al. (2010) extracted free and bound phenolic acids using a
range of extraction solvents including hexane/dichloromethane (1:1 v/v), acetone/water/acetic acid (70:29.5:0.5 v/v/v) and 80% ethanol that resulted in higher proanthocyanidin values
9
ranging from 12.2 mg 100g-1 CE to 80 mg 100g-1 CE in non-pigmented hulled and hull-less barley grown in Japan. The observed variation in TPAC could still be attributed to the choice of solvent during the extraction process. In addition,
Commander
was observed to have
considerably
low amount
of
proanthocyanidins (0.641 ± 0.22 mg 100g-1 CE) in comparison to other varieties. Consequently presenting Commander grains as a suitable candidate for malting, as low proanthocyanidin has been associated with low cloudiness in beer (Øverland, et al., 1994).
3.3 DPPH Antioxidant Scavenging ability In this study three antioxidant assays, DPPH, FRAP and ABTS were performed. While DPPH and FRAP assays quantified the overall free radical scavenging activity, the online ABTS was designed to quantify the antioxidant activity of individual phenolic compounds present in the crude barley extracts. The results using the DPPH assay for the overall antioxidant activity demonstrated suggestive differences between the varieties tested at p < 0.05 (Table1). Hindmarsh exhibited the highest free radical scavenging activity at 655.10 ± 78.87 µmol 100g-1 TE, followed by Westminster at 635.70 ± 83.15 µmol 100g-1 TE and Compass L1 at 632.10 ± 62.67 µmol 100g-1 TE. Gairdner and Compass L2 recorded the lowest DPPH free radical scavenging ability at 432.50 ± 57.33 and 343.50 ± 51.65 µmol 100g-1 TE respectively. The free radical scavenging activity of Australian barley is comparable to barley from Canada (Madhujith & Shahidi, 2009), China (Zhao, et al., 2008) and Italy (Bonoli, et al., 2004) where similar values have been reported. Contrastingly, studies conducted in Japan (Yoshida, et al., 2010) have reported a higher DPPH value of 1501µmol 100g-1 TE (Yoshida, et al., 2010). While the study by Mareček, et al. (2017) has reported lower DPPH values of 130 µmol 100g-1 TE to 160 µmol 100g-1 TE in the Czech Republic spring barley. Therefore, as stated earlier cultivation
10
environment, extraction solvent and procedure can have a significant impact on the phenolic compounds that are extracted and its relative antioxidant activity.
3.4 Ferric Reducing Ability of Plasma Assay (FRAP) The antioxidant activity of the barley varieties was measured by their ability to reduce Fe (III) (Table 1). The results demonstrated statistical differences between the varieties tested at p < 0.001. As observed with the TPC, Hindmarsh and Westminster exhibited the highest FRAP antioxidant activity at 536.20 ± 30.45 µmol 100g-1 TE and 515.10 ± 83.23 µmol 100g-1 TE respectively. Similar to the DPPH assay, Schooner and Gairdner exhibited the lowest antioxidant activity at FRAP values of 384.20 ± 45.09 µmol and 329.40 ± 32.96 µmol 100g-1 TE respectively. The two compass varieties exhibited significant difference in DPPH but did not vary significantly in FRAP antioxidant activity, this is believed to be a result of variation in regulation of specific phenolic compounds influenced by the cultivation location. Overall these results correlate with the TPC findings where Hindmarsh had the highest TPC and Gairdner had the lowest. While TPC, TPAC, DPPH and FRAP values of varieties such as Commander, Westminster and Latrobe varied between assays, Hindmarsh consistently exhibited higher levels across all the assays.
3.5 UHPLC with online ABTS A number of polyphenols were detected by the UHPLC-online ABTS system, seven of which were confirmed by standards and the others with substantial antioxidant activity were identified using Q-TOF LC/MS. The identified phenolic compounds included gallic acid, catechin, caffeic acid, ferulic acid, trans-cinnamic acid, o-coumaric acid and ellagic acid (Table 2). The above listed phenolic compounds have also been detected in barley from other countries such as America (Quinde-Axtell & Baik, 2006), Czech Republic (Dvorakova, et al., 2008b), Japan (Yoshida, et al., 2010) and Canada (Hao & Beta, 2012). 11
The unknown peaks (P1 to P9) were tentatively identified as p-coumaric acid isomer, isoscoparin-2"-O-glucoside, catechin dihexoside, catechin-5-O-glucoside, isoorientin-7-Ogentiobioside, prodelphinidin B3, prodelphinidin B, procyanidin B2 and apigenin 6-Carabinoside 8-C-glucoside respectively (Table 2). These identified polyphenolic compounds have been reported in previous studies (Ferreres, et al., 2008; Gangopadhyay, et al., 2016; Quinde-Axtell & Baik, 2006). The study by Ferreres, et al. (2008) identified glycosylated flavones
in barley leaves of which isoorientin-7-O-gentiobioside, isoscoparin-2"-O-
glucoside and apigenin 6-C-arabinoside 8-C-glucoside were found in the whole kernel barley samples in the current study. In addition, the current study also found derivatives of catechin, prodelphinidin and Procyanidin that have been reported in barley grains by Gangopadhyay, et al. (2016) and Quinde-Axtell and Baik (2006).
Table 2. Peak identification using 6530 Accurate-Mass Q-TOF LC/MS Peak
Tentative Identification
Formula
m/z
P1 P2 P3 P4 P5 P6 P7 P8 P9
P-coumaric acid isomer Isoscoparin-2"-O-glucoside Catechin dihexoside Catechin-5-O-glucoside Isoorientin-7-O-gentiobioside Prodelpinidin B3 Prodelpinidin B Procyanadin B2 Apigenin 6-C-arabinoside 8-C-glucoside
C9H8O3 C28H32O16 C27H33O16 C21H24O11 C11H20O7 C30H26O13 C30H26O13 C30H26O12 C20H30O7
164.0717 625.2015 613.1797 451.1259 771.3704 593.1312 593.1312 577.1355 563.1419
Retentio n time 4.27 4.47 4.76 5.28 5.54 5.63 5.63 6.32 6.74
Average Mass 164 624 614 452 772 593 593 577 563
In the current study, catechin-5-O-glucoside and prodelphinidin B3 (Figure 2) were the most abundant polyphenols in the seven Australian barley varieties followed by caffeic acid. Other studies by Hao and Beta (2012); Quinde-Axtell and Baik (2006); Yoshida, et al. (2010) have reported ferulic acid to be the most abundant phenol present in barley which, as mentioned previously, can be attributed to factors such as variety, environment and extraction procedure. Furthermore, in the current study, significant variation was observed with
12
catechin-5-O-glucoside, prodelphinidin B3 and prodelphinidin B levels across the seven varieties investigated (Table 3). The varieties Schooner, Westminster, Compass L2 and Commander recorded the highest content of catechin-5-O-glucoside followed by Latrobe, Compass, Hindmarsh and Gairdner. Hindmarsh, Commander, Compass L2 and Westminster recorded the highest content of prodelphinidin B3 with Gairdner and Compass L1 recording similarly low levels of prodelphinidin B3. In addition, it was observed that ellagic acid was not detected from the variety Compass L2 from Condobolin but was present in Compass L1 sample from Wongarbon.
Figure 2. Mass Spectra of Prodelphinidin B3
13
Table 3. Quantification of Australian barley polyphenols (mg 100g-1) using UHPLC detected at 280nm. Peaks
Commander
GA
0.36 ± 0.02a
P1
Compass L1
Compass L2
Hindmarsh
LaTrobe
Schooner
Westminster
Gairdner
0.35 ± 0.00a
0.39 ± 0.02a
0.39 ± 0.02a
0.35 ± 0.00a
0.35 ± 0.01a
0.36 ± 0.03a
0.35 ± 0.01a
0.96 ± 0.21b
0.89 ± 0.18b
0.73 ±0.13b
1.05 ± 0.16b
1.13 ± 0.15b
0.97 ± 0.17b
1.05 ± 0.10b
0.84 ± 0.03b
P2
1.95 ± 0.05c
1.77 ± 0.19c
2.24 ±0.42v
2.28 ± 0.13c
1.78 ± 0.92c
1.95 ± 0.19c
1.98 ± 0.29c
1.34 ± 0.31c
P3
0.65 ± 0.07d
0.43 ± 0.06d
0.62 ±0.07d
0.46 ± 0.04d
0.53 ± 0.02d
0.48 ± 0.09d
0.54 ± 0.07d
0.48 ± 0.02d
P4
6.80 ± 1.49eh
4.43 ± 1.15ef
7.62 ±1.39eh
4.18 ± 0.48f
5.08 ± 0.79efh
12.81 ± 5.76g
7.56 ± 5.24h
3.96 ± 0.89f
P5
3.03 ± 0.74i
3.41 ± 0.34i
3.72 ±1.64i
2.82 ± 0.99i
2.85 ± 0.84i
02.29 ± 055i
3.68 ± 0.69i
2.78 ± 0.33i
P6
9.71 ± 3.53j
6.98 ± 2.43kl
9.04 ±1.30jk
10.76 ± 3.43j
8.76 ± 2.98jk
15.08 ± 0.59l
9.76 ± 3.58j
6.61 ± 1.44kl
P7
0.72 ± 0.28m
0.45 ± 0.02m
1.54 ±0.18mn
3.60± 1.78n
2.78 ± 0.64mn
0.63± 0.22m
0.59 ± 0.21m
0.64 ± 0.11m
P8
1.29 ± 0.26p
1.00 ± 0.20p
0.70 ±0.12p
1.40 ± 0.19p
1.26 ± 0.09p
0.89 ± 0.31p
1.25 ± 0.69p
0.85 ± 0.18p
Cat
0.48 ± 0.08q
0.63 ± 0.06q
0.57 ±0.05q
1.51 ± 0.18q
1.06 ± 0.23q
0.92 ± 0.40q
0.83 ± 0.58q
0.56 ± 0.07q
P9
1.26 ± 1.08r
0.67 ± 0.10r
1.89 ±0.48r
0.76 ± 0.31r
0.88 ± 0.16r
0.96 ± 0.07r
0.69 ± 0.29r
0.66 ± 0.07r
CAF
3.30 ± 0.39s
3.16 ± 0.37s
1.42 ±0.23s
3.95 ± 1.57s
2.95 ± 0.70s
2.89 ± 0.40s
3.11 ± 1.05s
3.34 ± 0.66s
p-C
0.91 ± 0.13t
0.87 ± 0.05t
0.52 ±0.10t
0.85 ± 0.06t
0.77 ± 0.17t
0.91 ± 0.07t
0.98 ± 0.09t
0.89 ± 0.08t
FA
0.48 ± 0.03u
0.50 ± 0.01u
0.88 ±0.06u
0.46 ± 0.03u
0.65 ± 0.21u
0.51 ± 0.01u
0.51 ± 0.01u
0.55 ± 0.08u
t-Cin
0.36 ± 0.00v
0.36 ± 0.00v
0.37 ±0.01v
0.36 ± 0.00v
0.40 ± 0.04v
0.36 ± 0.01v
0.36 ± 0.00v
0.36 ± 0.00v
o-C
0.42 ± 0.09w
0.36 ± 0.00w
0.40 ±0.01w
0.48 ± 0.02w
0.47 ± 0.03w
0.39 ± 0.01w
0.42 ± 0.01w
0.40 ± 0.01w
EA
0.45 ± 0.01x
0.46 ± 0.00x
n
0.45 ± 0.01x
0.45 ± 0.02x
0.46 ± 0.00x
0.46 ± 0.00x
0.46 ± 0.01x
CAT, catechin; FA, ferulic acid; GA, gallic acid; CAF, caffeic acid; p-C, p-coumaric; o-C, o-coumaric; EA, ellagic; n, detected in negligible amounts. Different letters in rows indicate significant differences at p < 0.05.
14
It was observed that the amount of the phenolic compound present did not necessarily correlate to its antioxidant activity (Figure 1 and Table 4). For instance, phenolic compounds such as catechin-5-O-glucoside was recorded as the second most abundant phenolic compound but was identified as the least active in scavenging for free radicals. Whereas catechin dihexoside, isoorientin-7-O-gentiobioside, procyanidin B2 and ellagic had significantly higher antioxidant activity in comparison to their phenolic content ranking. In addition, significant differences were seen in the antioxidant activity of catechin dihexoside, isoorientin-7-O-gentiobioside, prodelphinidin B and B3 across the varieties studied. This observation aligns with the study conducted by Goupy, et al. (1999), that also found prodelphinidin B3 to be the most active free radical scavenging polyphenol followed by procyanidin B3 and C among nine barley varieties from France.
Figure 1. Shiwangni. UHPLC Chromatogram of barley variety Hindmarsh. The chromatogram illustrates polyphenol detection at 280 nm (top), and the relative online ABTS chromatogram at 414 nm below.
15
Table 4. Quantification of antioxidant activity using UHPLC-Online ABTS profiling (mg 100g-1 TE) detected at 414nm. Peaks Commander
Compass L1
Compass L2
Hindmarsh
LaTrobe
Schooner
Westminster
Gairdner
P1
1.66 ± 0.54a
1.43 ± 0.25a
1.21 ± 0.09a
1.69 ± 0.30a
1.94 ± 0.33a
1.43 ± 0.25a
1.86 ± 0.19a
1.18 ± 0.21a
P2
2.95 ± 0.60b
2.00 ± 0.37b
4.09 ± 0.70b
2.45 ± 0.12b
1.93 ± 0.90b
2.58 ± 0.33b
2.82 ± 0.78b
1.59 ± 0.11b
P3
4.20 ± 1.65cd
3.13 ± 0.94cd
2.93 ± 0.43c
8.39 ± 3.55d
5.19 ± 1.78cd
3.64 ± 1.35cd
2.58 ± 0.82c
2.28 ± 0.94c
P4
1.76 ± 0.14e
2.21 ± 0.32e
5.59 ± 0.83e
5.00 ± 0.79e
2.60 ± 1.33e
3.19 ± 0.52e
1.98 ± 0.51e
1.34 ± 0.02e
P5
9.51 ± 3.93fg
5.82 ± 2.83g
8.73 ± 1.65gh
14.09 ± 5.37f
12.47 ± 3.07fh
2.28 ± 0.98g
4.51 ± 3.53gi
4.86 ±2.43gi
P6
22.53 ± 1.64jm
22.87 ± 3.99j
18.68 ± 2.03l
31.23 ± 4.10k
26.27 ± 3.01jk
17.56 ± 1.56lm
19.39 ± 4.58lm
16.89 ±5.66lm
P7
29.79 ± 4.96no
7.09 ± 2.841np 13.16 ± 1.91op
10.01 ± 9.86no
12.84 ± 2.44o
15.03 ± 2.71n
4.39 ± 7.52n
5.04 ± 1.65n
P8
28.10 ± 0.44q
8.04 ± 0.24q
7.10 ± 0.68q
9.15 ± 0.63q
8.42 ± 1.63q
16.76 ± 1.10q
7.70 ± 2.24q
6.01 ± 0.80q
Cat
14.59 ± 1.78r
5.84 ± 0.54r
7.32 ± 0.54r
8.82 ± 0.13r
7.05 ± 2.01r
17.02 ± 1.28r
6.77 ± 1.11r
4.09 ± 0.81r
P9
03.56 ± 1.70s
2.33 ± 0.69s
6.51 ± 0.99s
4.55 ± 1.89s
4.11 ± 0.69s
1.88 ± 1.16s
2.65 ± 2.05s
2.11 ± 0.35s
EA
03.34 ± 0.29t
3.29 ± 0.24t
n
3.33 ± 0.18t
3.57 ± 0.29t
3.22 ± 0.28t
3.17 ± 0.10t
2.86 ± 0.21t
CAT, catechin; EA, ellagic acid; n, detected in negligible amounts. Different letters in rows indicate significant differences at p < 0.05.
16
4. Conclusion The current study demonstrates that Australian barley varieties have significant variation in polyphenol content and antioxidant activity. Hindmarsh showed the highest total phenolic content which correlated to its high antioxidant activity. Other varieties such as Compass, Latrobe and Westminster also had comparable phenolic content and antioxidant activity. The UHPLC-online ABTS coupled with the Q-TOF LC/MS was used to identify compounds that readily scavenged free radicals. It was observed that in the barley variety Compass L1 and L2 cultivation environment resulted in variability of phenolic compounds and antioxidant activity. In addition, the current study also determined that the phenolic compound prodelphinidin and its isomer were the most predominant polyphenol and its relative antioxidant activity was largely responsible for the overall variation in antioxidant activity of the seven varieties of Australian barley studied.
Acknowledgements The authors would like to acknowledge Mr Neal Sutton from Australian Nationa Variety Trials and Mrs Jennifer Puma from the NSW Department of Primary Industries for providing the barley samples. The authors would also like to acknowledge Dr Lachlan Schwartz and Mr. Michael Loughlin for their invaluable assistance. Funding: This work was funded by the Australian Research Council Industrial Transformations Centre for Functional Grains [Identifier Number: IC140100027]. Shiwangni Rao is a recipient of the Australian Research Council Industrial Transformations Centre for Functional Grains scholarship through Charles Sturt University. The authors declare no conflict of interest.
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Research Highlights
Polyphenol content of eight Australian-grown barley varieties were characterised
Antioxidant activity of polyphenols was identified using UHPLC-online ABTS system
Phenolic composition and antioxidant activity exhibited significant variation
Variety Hindmarsh had higher polyphenolic content and antioxidant activity
Prodelphinidin B3 had the highest abundance and antioxidant activity
20