Bound Phenolics of Quinoa Seeds Released by Acid, Alkaline, and

Article pubs.acs.org/JAFC

Bound Phenolics of Quinoa Seeds Released by Acid, Alkaline, and Enzymatic Treatments and Their Antioxidant and α‑Glucosidase and Pancreatic Lipase Inhibitory Effects Yao Tang,†,§ Bing Zhang,§,# Xihong Li,† Peter X. Chen,§,⊥ Hua Zhang,§ Ronghua Liu,§ and Rong Tsao*,§ †

Key Laboratory of Food Nutrition and Safety, Ministry of Education of China, Tianjin University of Science and Technology, Tianjin 300457, China § Guelph Research and Development Centre, Agriculture and Agri-Food Canada, 93 Stone Road West, Guelph, Ontario, Canada N1G 5C9 # State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, Jiangxi, China ⊥ Department of Food Science, University of Guelph, 50 Stone Road East, Guelph, Ontario, Canada N1G 2W1 ABSTRACT: Unextractable phenolics from plant foods and their role in health benefits have become increasingly important. Meal residues of three quinoa seeds free of fat and extractable phenolics were subjected to acid, alkaline, and enzymatic hydrolyses. The total and individual phenolic compounds released were analyzed, and 19 phenolics, predominantly phenolic acids and several flavonoids, were identified. The concentration of bound phenolics was highest in black quinoa followed by red and white, regardless of the hydrolysis method. Higher phenolic contents also showed stronger antioxidant activities and inhibition of α-glucosidase and pancreatic lipase activities. Carbohydrases, that is, pectinase, xylanase and feruloyl esterase, which effectively liberated bound phenolics are known to be secreted by colonic bacteria, suggesting potential antioxidant and antiinflammatory effects by these compounds in the large intestine during colonic fermentation. These results can also be applied to treat foods high in bound phenolics to enhance bioaccessibility. KEYWORDS: quinoa, bound phenolics, hydrolysis, enzyme, antioxidant activities, α-glucosidase, pancreatic lipase

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INTRODUCTION Macronutrients such as proteins and polysaccharides as well as micronutrients including polyphenols, vitamins, and fatty acids in quinoa (Chenopodium quinoa Willd.) seeds have been well studied in recent years.1−6 Compositions of the lipophilic phytochemicals including fatty acids, tocopherols, and carotenoids in both quinoa seeds and leaves and their contribution to antioxidant activities have been analyzed.2,6 Hydrophilic phytochemicals including different forms of extractable phenolics and betacyanins of selected quinoa cultivars and their potential as antioxidants have also been investigated recently in our laboratory.1 At least 23 phenolic compounds were found in either free or conjugated forms (liberated by alkaline and/or acid hydrolysis), the majority of which were phenolic acids consisting of mainly vanillic acid, ferulic acid, and their derivatives as well as the main flavonoids quercetin, kaempferol, and their glycosides. Pigments in dark-colored quinoa seeds were confirmed to be betacyanins rather than anthocyanins.1 The potential health benefits of phytonutrients of quinoa have only recently become the subject of some studies. These compounds have been assigned antiobesity and antidiabetic claims.7,8 Phenolic compounds have been the main group of hydrophilic bioactives studied, and most research has only reported on the composition and various bioactivities of extractable phenolics. However, in recent years, the role of bound phenolics, those remaining in the residue of plant samples following organic solvent extraction, in human health has been increasingly recognized as important.9,10 Phenolic compounds in bound form, once released, may have similar antioxidant © XXXX American Chemical Society

activities and protective effects against low-density lipoprotein (LDL) cholesterol oxidation and radical-induced DNA breakage.11,12 Insoluble phenolics are covalently bound to cell wall structural components such as cellulose, hemicellulose (e.g., arabinoxylans), lignin, pectin, and rod-shaped structural proteins and, thus, are unextractable by solvents in most characterization studies. Unextractable or bound phenolics are considered part of plants’ self-protection mechanism.9,13 In the study of bound phenolics, alkaline and acidic hydrolyses are the most common means used to release phenolic compounds from the glycosidic and ester bonds, respectively.9,12,14 Acidic hydrolysis at elevated temperatures results in the loss of some phenolics and the generation of furfural and its derivatives.9,12,15 Treatment with different concentrations (1−4 M) of sodium hydroxide for various lengths of time has proven to be effective in releasing these bound phenolics.9,12,14,16 Bound phenolics can also be released by more specific and milder methods such as by carbohydrase enzymes including pectinases, cellulases, feruloyl esterase, and glucanases.9,13,17 Although chemical hydrolyses are good means for studying bound phenolics, these methods involve high temperature and harsh acidic and alkaline conditions unattainable in the human digestive system. Enzymatic hydrolyses occur during digestion in the stomach as well as fermentation in the large Received: December 4, 2015 Revised: February 4, 2016 Accepted: February 8, 2016

A

DOI: 10.1021/acs.jafc.5b05761 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Barn store in Guelph, ON, Canada (July 2013). The seeds (500 g) were ground separately with a commercial coffee blender into a fine powder and stored in polyethylene tubes at −80 °C prior to analysis. Extractable lipophilic and hydrophilic phytochemicals were removed according to previously reported methods.1,6 The resulting defatted and extractable-phenolics-free residues were dried and confirmed to contain no soluble phenolics (by HPLC) and were used in studying bound phenolic composition by hydrolysis with acid, alkaline, and enzymes separately. All hydrolysis reactions and assays were carried out in triplicate. Bound Phenolics by Acid Hydrolysis (ABP). The residue (2.0 g) was transferred into a 50 mL polypropylene tube (VWR, Mississauga, ON, Canada), mixed with 25 mL of 2 M HCl, heated at 85 °C for 1 h, brought to pH 2 with 10 M NaOH, and then centrifuged at 6000g for 5 min. The supernatant was extracted with 15 mL of diethyl ether/ ethyl acetate (DE/EA, 1:1, v/v) six times. The combined extract was dried under N2 in the dark, reconstituted in 2 mL of 70% methanol, and filtered through a Phenex-NY 4 mm, 0.2 μm, syringe filter (SigmaAldrich Co., St. Louis, MO, USA) prior to further analyses. This fraction contained acid-hydrolyzable phenolics (ABP). Bound Phenolics by Alkaline Hydrolysis (BBP). The residue (2.0 g) was treated with 25 mL of 2 M NaOH, hydrolyzed for 4 h at room temperature under a stream of N2, acidified to pH 2 with 6 M HCl, and then centrifuged at 6000g for 5 min. The supernatant was extracted with DE/EA six times similar to the procedures described above. The combined extract was evaporated to dryness under N2 in the dark and subsequently dissolved in 2 mL of 70% methanol. This was the alkaline-hydrolyzable bound phenolic fraction (BBP). Bound Phenolics by Enzymatic Hydrolysis. The residue (2.0 g) was hydrolyzed separately by different enzymes in 25 mL of buffer solution after proper dilutions. Hydrolyses were carried out in bottles rotating at 10−12 rpm at different temperatures in an incubator. The most suitable conditions for each enzyme were adopted from those recommended by the manufacturers, namely, in 50 mM sodium acetate buffer (pH 5.0) at 37 °C for pectinase (50 U/mL), Viscozyme L (2 U/mL), and cellulase (35 U/mL); in 50 mM phosphate−citrate buffer (pH 6.5) at 60 °C for 12 h for xylanase 10B (2 U/mL); and in 50 mM phosphate buffer (pH 6.0) at 60 °C overnight (ca. 12 h) for feruloyl esterase (0.2 U/mL). Upon completion, the hydrolysates were placed in a 105 °C oven and heated for 5 min to inactivate the enzymes. The mixtures were cooled in a refrigerator at 4 °C and then brought to room temperature before being partitioned with DE/EA six times, similar to the procedures described above. The combined extract was evaporated to dryness under N2 in the dark and dissolved in 2 mL of 70% methanol to obtain enzyme-hydrolyzable bound phenolic fractions. Determination of Total Phenolic Content (TPC). The TPC was determined using the Folin−Ciocalteu assay as described in previously published methods and expressed as milligrams of gallic acid equivalents per gram of quinoa seed (mg GAE/g) (r2 = 0.997).1,25 HPLC and LC-MS Analyses. Individual phenolics were analyzed using an Agilent HPLC series 1100 (Agilent, Waldbronn, Germany) system consisting of a degasser, a binary gradient pump, a thermostated autosampler, and a diode array detector (DAD) and ChemStation software. Separation was carried out on a Kinetex XB-C18 column (100 mm × 4.6 mm, 2.6 μm) (Phenomenex Inc., Torrance, CA, USA). The binary mobile phase consisted of 5% formic acid in water (v/v) (solvent A) and 95% methanol mixed with 5% acetonitrile (v/v) (solvent B). The solvent gradient was as follows: 0−40 min, 0−80% B; 40−42 min, 80%−100% B; 42−44 min, 100% B; 44−45 min, 100%− 0% B. The injection volume was 7 μL, and the flow rate was kept at 0.7 mL/min for a total run time of 50 min. Peaks were monitored at 280 nm (Figure 1), 360 nm, and 520 nm for the different phenolic compounds. Quantification was performed at 280 nm with external standards using calibration curves generated between 0.25 and 50 mg/L (r2 ≥ 0.9999). Identification and structural confirmation of the polyphenols were done using a Dionex UHPLC UltiMate 3000 liquid chromatograph interfaced to an amaZon SL ion trap mass spectrometer (Bruker Daltonics, Billerica, MA, USA). An Agilent Poroshell 120 column

intestine from enzymes produced by colonic microorganisms such as bifidobacteria and lactic acid bacteria in humans.18,19 Bound phenolic compounds released by the colonic microbial enzymes may also play important roles in gut health.20 Studies on phenolic compounds from other plants such as oats and beans have shown inhibitory effects on α-glycosidase and pancreatic lipase activities.21−24 α-Glucosidase is a key enzyme for the digestion of complex carbohydrates, whereas lipase inhibitors can suppress triglyceride absorption leading to potential antiobesity effects.7,24 Inhibition of these two enzymes is therefore a valid strategy in managing blood glucose level and obesity; both are key factors for type 2 diabetes. Although phytochemicals of quinoa are currently being investigated for their potential roles in human health, in-depth studies on the chemistry and biochemistry of different forms of phenolic compounds have not been carried out. Effects of bound phenolics in this pseudocereal grain and the possible antioxidant, antiobesity, and antidiabetic activities remain uninvestigated. The objectives of this study were therefore to characterize bound phenolic compounds of three quinoas with black, red, and white seed coat colors, as released by acid, alkaline, and enzymatic hydrolyses, and to evaluate their contribution to antioxidant activity and, potentially, to antiobesity and antidiabetic activities as assessed against their inhibitory effect on α-glycosidase and pancreatic lipase activities.

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MATERIALS AND METHODS

Chemicals and Enzymes. All standard reference materials including p-hydroxybenzoic acid, 3,4-dihydroxybenzoic acid, catechin, p-coumaric acid, ferulic acid, caffeic acid, vanillic acid, kaempferol, quercetin, 5-hydroxymethyl-2-furan-2-carbaldehyde (HMF), 2,2diphenyl-1-picrylhydrazyl (DPPH), trolox, 1,3,5-tri(2-pyridyl)-2,4,6triazine (TPTZ), L-ascorbic acid, fluorescein, and 2,2′-azobis(2methylpropionamidine) dihydrochloride (AAPH), as well as enzyme inhibition assay chemicals including rat intestinal acetone powder, pancreatic lipase, 4-methylumbelliferyl-α-D-glucoside (4-MUG) and 4-methylumbelliferyl oleate (4-MUO), acarbose, and orlistat were from Sigma-Aldrich (Oakville, ON, Canada). Sodium acetate, ferric chloride hexahydrate, sodium phosphate monobasic, sodium phosphate dibasic, and HPLC grade solvents, including methanol, formic acid, and hydrochloric acid (HCl), were purchased from Caledon Laboratories (Georgetown, ON, Canada). Xylanase 10B (GH10-CBM22, EC 3.2.1.8) from a genetically modified Clostridium thermocellum strain was supplied by NZYTech Ltd. (Lisboa, Portugal). One unit was defined by the supplier as the amount of enzyme liberating 1 μmol of reducing sugar (measured as xylose equivalent) from xylan per minute in 50 mM phosphate−citrate buffer (pH 6.5) at 60 °C. Feruloyl esterase 1A (CE1, EC 3.2.1.73) was from the Clostridium strain and purchased from the same company as Xylanase 10B. One unit of this enzyme was defined as the amount of enzyme required to release 1 μmol of ferulic acid per minute from O-(5-O-[(E)-feruloyl]-α-L-arabinofuranosyl)-(1→3)-O-β-D-xylopyranosyl(1→4)-D-xylopyranose (FAXX) in 50 mM phosphate buffer, pH 6.0, at 60 °C. Pectinase (P2611, EC 3.2.1.15) activity was estimated by measuring the release of 1 μmol of reducing sugar (galacturonic acid) from polygalacturonic acid per minute in 50 mM citrate buffer at pH 5.0 at 37 °C. Viscozyme L (V2010, EC 3.2.1.6) activity was measured by the amount of enzyme required under standard conditions (37 °C, pH 5.0) degrading barley β-glucan to reducing carbohydrates with a reduction power corresponding to 1 mM glucose/min. Cellulase (C2730, EC 3.2.1.4) activity was analyzed by releasing the reducing carbohydrates from β-glucan as was done with Viscozyme. Pectinase, Viscozyme, and cellulase were bought from Sigma-Aldrich (Oakville, ON, Canada). Sample Collection and Preparation. Three commercial quinoa (C. quinoa) seeds (white, red, and black) originated from the Andes mountains of South America were purchased from a local Bulk B


Article


Figure 1. HPLC chromatograms of bound phenolic extracts of black quinoa as detected at 280 nm. Peaks without a number were not identified. XEBP, bound phenolics released with xylanase 10B; PEBP, bound phenolics released with pectinase; FEBP, bound phenolics released with feruloyl esterase 1A; BBP, bound phenolics released with alkaline hydrolysis; ABP, bound phenolics released with acid hydrolysis. (150 mm × 4.6 mm, 2.7 μm) was used for chromatographic separation. The mass spectrometer electrospray capillary voltage was maintained at 4.5 kV and the drying temperature at 220 °C with a flow rate of 10 L/min. Nebulizer pressure was 40 psi. Nitrogen was used as both a nebulizing and drying gas. Helium was used as a collision gas at a pressure of 60 psi. The mass-to-charge ratio (m/z) was scanned across the range of m/z 50−1200 in enhanced resolution negative-ion auto MS/MS mode. The Smart Parameter Setting (SPS) was used to automatically optimize the trap drive level for precursor ions. The instrument was externally calibrated with ESI TuneMix (Agilent). The initial mobile phase conditions for the LC were 98% solvent A consisting of 0.1% formic acid in water and 2% solvent B consisting of 0.1% formic acid in acetonitrile (v/v). The gradient went to 98% solvent B in 30 min. The flow rate was maintained at 0.4 mL/min. UV monitoring was at 280 nm for the phenolic compounds. Data acquisition and processing were performed using Esquire Control software. Antioxidant Activities. The radical scavenging activity assay was done using a modified DPPH method reported earlier.1 The DPPH radical scavenging activity was expressed as micromoles of trolox equivalents (TE) per gram of original quinoa seed (μmol TE/g) (r2 = 0.999). The ferric reducing antioxidant power (FRAP) assay followed the same method as in a previous paper.1 The FRAP antioxidant activity was expressed as micromoles of ascorbic acid equivalents (AAE) per gram of quinoa (μmol AAE/g) (r2 = 1.000). The oxygen radical absorption capacity (ORAC) assay was conducted according to reported protocols.1 The ORAC values were expressed as micromoles of trolox equivalents per gram of quinoa sample (μmol TE/g) (r2 = 0.998). Enzyme Inhibition Assay. α-Glucosidase inhibitory activity was determined using a method reported in our previous study.26 In brief, crude enzyme from rat intestinal acetone powder was suspended in 0.1 M cold phosphate buffer (pH 6.9) at 25 mg/mL and hydrated for 1 h at 4 °C. After centrifugation at 10000g, the supernatant was collected as a working enzyme solution. Twenty-five microliters of sample (final concentrations of 10, 25, and 50 mg dried bound phenolics extract/mL) prepared from lyophilized extracts,26 acarbose (positive control), or solvent blank (70% methanol) was mixed with 75 μL of the substrate 4-MUG (0.5 mM) in the wells of a 96-well plate and incubated at 37 °C for 15 min while shaking. Afterward, 25 μL of the working enzyme solution was added into each well and allowed to incubate for 1 h at 37 °C. Finally, 125 μL of borate buffer (300 mM) was added to terminate the reaction. The fluorescence was measured at an excitation wavelength of 360 nm and an emission wavelength of 460 nm with a fluorescence spectrophotometer (Bio-Tek Instruments Inc., Winooski, VT, USA). The α-glucosidase inhibitory activity was expressed as IC50, which was calculated from the percent inhibition of the serial dilutions as mentioned above. IC50 is defined as the concentration of extract required to inhibit 50% of the enzyme activity and expressed as milligrams extract per milliliter solvent (mg/mL).

Lipase inhibitory activity was determined using a modified method by Kawaguchi et al.27 Pancreatic lipase (type II, from porcine pancreas) and 4-MUO served as the reaction enzyme and fluorogenic substrate, respectively. Briefly, the mixture of 225 μL of substrate (0.24 mM) and 25 μL of sample solution of bound phenolic extract, orlistat standard (positive control), or solvent blank was incubated at 37 °C for 15 min, followed by the addition of 25 μL of enzyme solution (0.55 mg/mL) in Tris-HCl buffer (0.1 M, pH 8.0) in each well. After reaction at 37 °C for 1 h, the fluorescence was measured according to the same method as described above. The lipase inhibitory activity was expressed in IC50 as similarly defined for the α-glucosidase. Data Analysis. Results were expressed as the mean value ± standard deviation of three independent replicates. One-way analysis of variance (ANOVA) was used to compare the means. Differences were considered significant at p ≤ 0.05. All statistical analyses were performed using SPSS (version 18.0, Chicago, IL, USA).

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RESULTS AND DISCUSSION Bound Phenolic Content and Composition. The TPC of ABP was higher than that of BBP, whereas the total phenolic index (TPI) of ABP was lower than that of BBP (Table 2). This is most likely due to the fact that the Folin−Ciocalteu method can be interfered with by the high levels of HMF, furfural, and their derivatives coproduced during thermal acidic hydrolysis.15 In general, TPC was highest in the black quinoa followed by red and white varieties in all fractions of bound phenolics, regardless of the hydrolysis methods. The same can be said for TPI (Table 2). Extractable phenolic compounds including free and conjugated phenolics have been characterized and reported in our previous paper. Identification and quantification of the unextractable or bound phenolics of the residue after solvent extraction were carried out using the same HPLC/LC-MS method.1 Bound phenolics released by acid, alkaline, and enzymes from the residues of the three different quinoa seeds are presented in Table 1 along with their retention times, UV/ vis, and mass spectral data. Most of the identified compounds, including peaks 1−3, 5−7, 10−12, 15, 20, and 21, were also identified in the extractable fractions of the same quinoa seeds.1 Peaks 8, 9, and 13 were newly identified as vanillic acid derivative, syringic acid, and sinapic acid, respectively, although they have been previously found in certain quinoa cultivars as extractable phenolics.3,5 Peak 14 had a deprotonated molecular ion of m/z 163 [M − H]−, and on the basis of the UV spectrum and retention time, it was tentatively identified as o-coumaric acid. Two new phenolic acids 8,5′-diferulic acid (peak 17) and C


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Table 1. Bound Phenolic Compounds Identified by HPLC-DAD-MS in Quinoa Seed Residues after Removal of Extractable Contentsa peak

retention time

name

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

5.445 5.985 6.014 7.976 8.244 9.382 11.268 11.732 13.597 14.442 15.02 17.14 18.058 20.928 23.272 23.371 24.314 25.000 25.812 26.393 29.388

protocatechuic acid HMF 2,4-dihydroxybenzoic acid vanillin derivativec 4-hydroxybenzoic acid catechin caffeic acid vanillic acid derivativec syringic acid vanillin p-coumaric acid ferulic acid sinapic acid o-coumaric acid prunetin/biochanin A derivativesc HMF derivativec 8,5′-diferulic acidc rosmarinic acid chlorogenic acid derivativec quercetin kaempferol

MW

m/z

MS/MS

153 125 153 151 137 289 179 167 197 151 163 193 223 163

109 109 109

330 325 320 350

154 126 154 152 138 290 180 168 198 152 164 194 224 164 284

290 330 325 365 365

386 360 354 302 286

385 359 353 301 285

UV/vis (nm) 260, 282 260, 262, 260, 279 270 260, 230, 262, 320 290, 245, 280, 290, 280 330, 290, 295, 260, 265,

295 295 290 300

295 280 290

137, 113 245 179, 135 108 123

149 164 119 283 109 193 161 191 301 193

treatmentb ABP, ABP ABP ABP, ABP, BBP, BBP, ABP, ABP, ABP ABP, ABP, BBP BBP, BBP, ABP ABP, BBP, BBP, ABP, ABP,

BBP, EBP

BBP, EBP BBP, EBP EBP EBP EBP BBP, EBP BBP, EBP BBP, EBP EBP EBP BBP, EBP EBP EBP BBP, EBP BBP

a Refer to Materials and Methods for details. Values are the mean ± SD, n = 3. ND, not detected. bABP, bound phenolics released with acid; BBP, bound phenolics released with alkaline; EBP, bound phenolics released with enzymes (either xylanase 10B or pectinase or feruloyl esterase 1A). c Identified tentatively by LC-MS only.

xylanase (Table 2). o-Coumaric acid was a minute phenolic acid released only by pectinase and by alkaline hydrolysis in black quinoa. Syringic acid was found in all but pectinase-treated samples. Although the majority of the bound phenolics were phenolic acids, some flavonoids were also found after hydrolysis of quinoa residues. Quercetin and kaempferol were both found in ABP and BBP, but enzymes, except pectinase on quercetin, were not effective in freeing these compounds from the residue. Catechin, on the other hand, was found only in BBP and in darker quinoas by xylanase and feruloyl esterase. Peak 15 was produced in all fractions but ABP, and it was tentatively identified as an isoflavone derivative (Table 2). Enzymatic hydrolysis occurs naturally during microbial decay of plants or colonic fermentation of plant foods. Colonic microbial enzymes are mostly carbohydrases, which are highly specific and effective in releasing bound phenolic compounds that are cross-linked with cellulose and hemicellulose in plant cell wall matrix.19 The efficacy and specificity on bound phenolics such as ferulic acid released by some enzymes produced from the human distal intestinal microbes have been studied,19,29−31 Among the carboxylases investigated in the present study, only xylanase 10B, pectinase, and feruloyl esterase were effective in breaking the bonds and releasing bound phenolic compounds. Viscozyme L and cellulase did not produce detectable amounts of phenolic compounds (data not shown). According to TPC and TPI of the samples treated with xylanase 10B, pectinase, and feruloyl esterase, pectinase was most effective, producing more than twice as much TPI as the other two enzymes in all three quinoas, but not in releasing protocatechuic acid and syringic acid. Similar results have been reported for pectinase in releasing more bioactive compounds such as polyphenols in corn, fruit juices and pomace, and coffee pulp.32−34 Enzymes combined with alkaline and acid hydrolysis were also used in rice and barley.16,35 Black quinoa contained

rosmarinic acid (peak 18), were positively identified by congruent retention time and UV and MS spectral data. Peak 19 showed the same UV spectrum and molecular ion [M − H]− of m/z 353 as chlorogenic acid while having a different retention time. The definite identity of this minor component is yet to be determined. Chlorogenic acid has been previously identified in quinoa leaves.4 Not all quinoa seeds had the same bound phenolic composition, and not all hydrolysis methods released the same phenolic profile. There were also bound phenolics such as protocatechuic acid that were only different quantitatively among the three quinoa varieties, regardless of the hydrolysis methods (Table 2). Protocatechuic acid, 4-hydroxybenzoic acid, ferulic acid, p-coumaric acid, and 8,5′-diferulic acid were produced by all hydrolysis methods in all three quinoa varieties except a few samples of lighter colored quinoas by enzymatic hydrolyses. Among these compounds ferulic acid was found to be more readily released by alkaline hydrolysis (194.35 μg/g, average of the three cultivars tested, the same hereinafter) and by pectinase (175.51 μg/g) at nearly the same yields compared to those by acid hydrolysis (86.99 μg/g) (Table 2), possibly because pectinase and alkaline hydrolysis act similarly through breaking the glycosidic bonds linking phenolic acids to galacturonic acid residues or to polysaccharides of the cell wall. However, different from the enzymatic action, alkalinic condition may cause loss of ferulic acid and other phenolics during hydrolysis.9,28 Xylanase and feruloyl esterase were not as effective as pectinase in ferulic acid release. Sinapic acid was detected only in BBP, whereas 2,4-dihydroxybenzoic and vanillin were found only in ABP (Table 2). Rosmarinic acid was identified for the first time in quinoa, but only as a bound phenolic by alkaline and enzymatic hydrolyses with xylanase, pectinase, and feruloyl esterase. Peak 19, a chlorogenic acid derivative, was released from all quinoa residues except by D


E

WQ

15.81 ± 0.84 13.88 ± 0.76

5.23 ± 0.26 4.61 ± 0.14

16.72 ± 1.36 13.16 ± 0.68

6.08 ± 0.31 4.77 ± 0.25

± ± ± ± ± 2.24 0.69 0.19 0.23 4.93

± 3.56

± 0.89 ± 0.52

6.35 ± 0.25 5.26 ± 0.22

17.36 ± 0.76 15.52 ± 0.91

30.72 ± 1.44

16.28 13.59 ND 72.52 ND 16.28 231.01 7.97 6.12 112.48

71.16 ± 3.12 3.88 ± 0.12

ND

WQ

5.76 ± 0.31 30.44 ± 1.43

ND 36.68 ± 1.68

47.41 ± 1.81

ND ND ND 1.44 ± 0.09 ND 4.56 ± 0.24 81.68 ± 4.36 ND ND ND

ND 8.88 ± 0.19

72.44 ± 2.12

ND

BQ 20.84 ± 0.96

± ± ± ± ± 1.44 0.68 0.73 0.36 1.25

28.52 ± 1.13 18.64 ± 0.97

ND 35.56 ± 1.45

51.92 ± 3.96

ND ND 28.92 21.22 20.76 5.12 58.44 ND ND ND

42.52 ± 3.36 3.61 ± 0.16

± ± ± ± ± 1.73 0.28 0.69 0.24 7.53

WQ

47.24 ± 2.64 21.52 ± 1.48

ND 51.32 ± 3.28 ND ND

ND ND

BQ

ND

ND ND

1.16 ± 0.05 ND

8.76 ± 0.32

± 0.85

± 0.13 ± 0.07

± 0.53

± 0.29

WQ

± 0.03 ± 0.37

± 0.08 ± 5.88

± 0.16 ± 0.25

9.88 ± 0.28

ND 0.36 4.84 ND ND 5.96 123.84 ND 0.72 8.48

ND 3.36 ± 0.16

ND

ND

ND ND

7.72 ± 0.35 ND

1.01 ± 0.03 20.28 ± 1.08 ND 1.32 ± 0.07

7.84 ± 0.36

6.52 ND ND ± 0.03 12.28 ND 2.28 ± 0.05 2.39 ND ND ± 0.64 21.52

± 0.11

43.72 ± 3.24 42.76 ± 2.86 0.52 ± 0.04 1.24 ± 0.04

ND

49.39 ± 2.08 43.64 ± 1.79

RQ

XEBP

ND 1.68 ND ND ND ND 1.28 ± 0.04 1.72 ND ND ND ND 1.61 ± 0.03 0.62 ND ND ND ND 11.24 ± 0.32 20.08

ND ND

ND

ND

60.22 ± 3.81 8.04 ± 0.47

ND ND 52.96 13.16 10.04 6.28 120.85 ND ND ND

56.36 ± 2.08 2.88 ± 0.12

63.64 ± 2.68 111.52 ± 4.56

24.60 ± 1.56

RQ

ABP

± 0.27 ± 0.12

± 0.24 ± 10.21

± 0.17 ± 0.03

12.93 ± 0.52 ND

12.04 ± 0.47 1.48 ± 0.16

8.57 ± 0.24

ND 5.44 1.28 ND ND 6.28 186.48 ND 1.36 7.32

10.92 ± 0.61 3.04 ± 0.15

ND

16.92 ± 0.84

RQ

PEBP BQ

± 0.32 ± 0.23

± 0.26 ± 9.45

± 0.19 ± 0.18

10.56 ± 0.45 ND

6.84 ± 0.29 2.21 ± 0.08

7.85 ± 0.24

ND 7.81 7.39 ND ND 8.05 216.21 ND 7.72 7.87

11.32 ± 0.48 2.44 ± 0.13

ND

17.32 ± 0.92

WQ

RQ

BQ

ND

ND

47.08 ± 1.68 42.07 ± 2.52

ND

± 0.16

± 0.13 ± 0.48

± 0.14

± 0.27

6.90 ± 0.16

7.46 ND ND 10.36 ND 0.04 2.22 0.56 15.25 ND ND 0.43 6.91

0.12

ND ND

ND ND

ND ND

8.17 ± 0.07 12.04 ± 0.52 13.79 ± 0.75 5.01 ± 0.04 5.48 ± 0.05 6.45 ± 0.62

ND

ND 2.99 ± 1.56 ± 0.17 ND ND ND ND ND ND ND ND 0.73 ± 10.78 ± 0.49 12.23 ± ND ND ND ND 8.05 ± 0.36 5.35 ±

ND ND ND 2.63 ± 0.84 44.24 ± 2.37 46.54 ± 1.43

ND

ND

FEBP

411.09 ± 2.62 468.08 ± 2.58 658.27 ± 11.45 289.92 ± 4.86 403.48 ± 2.05 575.15 ± 8.28 25.96 ± 0.81 127.69 ± 4.48 141.52 ± 4.62 190.32 ± 4.33 274.08 ± 9.27 313.81 ± 10.12 36.42 ± 1.74 130.09 ± 2.57 156.71 ± 1.87 686.94 ± 20.09 950.29 ± 21.45 1248.09 ± 9.29 739.41 ± 20.36 1175.85 ± 6.67 1445.51 ± 7.85 70.03 ± 3.49 337.88 ± 5.62 391.17 ± 9.29 463.81 ± 13.81 552.81 ± 10.52 578.64 ± 2.85 87.96 ± 0.88 376.96 ± 2.90 402.79 ± 9.03

26.32 ± 2.24

± 5.97

± 0.93 ± 9.16 ± 0.59

± 0.12

25.72 ± 2.04

± 6.44

± 0.12 ± 6.44 ± 0.88

± 0.04

± 0.76 ± 3.76

8.16 20.64 ND 2.29 ND 10.57 183.08 9.72 ND 92.68

± 0.84 ± 0.12

11.04 2.28 ND 0.68 ND 8.20 168.96 30.60 ND 116.16

64.08 ± 4.28 5.57 ± 0.36

ND

BQ 29.64 ± 1.04

RQ

32.32 ± 1.56

ND 6.64 ± 0.32

ND

ND

BBP

a

Values are the mean ± SD, n = 3; ND, not detected. ABP, bound phenolics released with acid; BBP, bound phenolics released with alkaline; XEBP, bound phenolics released with xylanase 10B; PEBP, bound phenolics released with pectinase; FEBP, bound phenolics released with feruloyl esterase 1A; WQ, white quinoa; RQ, red quinoa; BQ, black quinoa; TPI, total phenolic index (sum of individual phenolic concentrations excluding HMF and related compounds, μg/g quinoa); TPC, total phenolic content (μg/g quinoa).

TPI TPC

protocatechuic acid 2,4-dihydroxy benzoic acid vanillin der 4-hydroxybenzoic acid catechin caffeic acid vanillic acid der syringic acid vanillin p-coumaric acid ferulic acid sinapic acid o-coumaric acid prunetin/ biochanin A 8,5′-diferulic acid rosmarinic acid chlorogenic acid der quercetin kaempferol

phenolic compd

Table 2. Concentrations (μg/g) of Individual Bound Phenolics of Quinoa Seeds Hydrolyzed with Alkaline, Acid, and Enzymesa

Journal of Agricultural and Food Chemistry Article


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Journal of Agricultural and Food Chemistry the highest bound phenolic content of all three varieties regardless of the enzymes used (Table 2). Feruloyl esterase has been used for cleaving ester bonds between hydroxycinnamic acids (such as ferulic acid, caffeic acid, cinnamic acid, and chlorogenic acid) esterified to arabinoxylans and certain pectin present in plant cell walls.13 Interestingly, this enzyme was also found to produce some benzoic acids such as protocatechuic acid and 4-hydroxybenzoic acid from quinoa seed residues (Table 2). The enzyme mixture consisting of pectinase, amylase, and cellulase was used to hydrolyze bound phenolics and compared with thermal and alkaline hydrolysis in purple corn.34 This, however, does not address the effectiveness of individual enzymes and specificity in breaking bonds between phenolics and other components, like what was found in the present study. Antioxidant Activities of Bound Phenolics in Quinoa Seeds. Extractable phenolic compounds were higher in quinoa seeds with darker seed coat color, and that also contributed significantly to the higher antioxidant activity of dark-colored quinoa.1 The present study, however, focuses on the unextractable phenolics, that is, bound phenolics. The antioxidant activities of the bound phenolic compounds released by acid, alkaline, and enzymatic hydrolyses from white, red, and black quinoa seed residues were assessed using DPPH, FRAP, and ORAC methods (Figure 2). It was interesting to note that except for the pectinase fraction in FRAP assay, black quinoa showed the highest antioxidant activity in all assays regardless of hydrolysis method. White quinoa had the lowest antioxidant activity in all assays. Bound phenolics released by the three most effective enzymes showed in general weaker antioxidant activity as compared to those by acid or alkaline hydrolysis (Figure 2). The acid hydrolysis fraction (ABP) displayed a slightly higher antioxidant activity than the BBP fraction despite the fact that the latter had higher TPI as analyzed by HPLC (Table 2). The higher antioxidant activity of ABP might be from HMF and its derivatives as these compounds are formed during acid hydrolysis and are known to contribute to some TPC, FRAP, and ORAC activities.15 The antioxidant activities of the bound phenolic fractions were generally much lower than the extractable fractions for all three quinoa seeds, although the ORAC values were similar.1 Compared to the antioxidant activity of bound phenolics in other grains, they were only slightly lower than those of hard wheat flour but higher than those of buckwheat flour.14,36 The antioxidant activity of the bound phenolics warrants further study on the local antioxidant and anti-inflammatory effects in epithelial layers of the colon, where they can be potentially liberated. Inhibition of α-Glucosidase and Lipase Activities. α-Glucosidase and pancreatic lipase are important enzymes in the digestive tract and are involved in sugar and lipid digestion, respectively.24,26,37 Inhibition of these two enzymes by food bioactives such as phenolic compounds is suggestive of potential benefits in blood sugar and weight management, thus, ultimately in type 2 diabetes.24,37 Figure 3 shows the IC50 values of the BBP fraction of three the quinoa seeds against α-glucosidase and lipase activities. BBP was chosen because of its relatively higher TPC and TPI values as compared to that from the enzymatic hydrolysis and for its absence of interfering nonphenolic compounds such as HMF present in the ABP. Inhibition of α-glucosidase and lipase by food phenolics is generally weak compared to the drugs, for example, acarbose and orlistat.24 IC50 values of the bound phenolics in the three quinoas ranged from 37.58 to 55.58 mg/mL for α-glucosidase and were

Figure 2. Antioxidant activities of the bound phenolics of three quinoa cultivars liberated by alkaline, acid, and enzymatic treatments: (A, top) DPPH assay (values are expressed as μmol Trolox equivalents/g quinoa (μmol TE/g)); (B, middle) FRAP assay (values are expressed as μmol ascorbic acid equivalents/g quinoa (μmol AAE/g)); (C, bottom) ORAC assay (values are expressed as μmol Trolox equivalents/g quinoa (μmol TE/g)). Values are means ± SD, n = 3.

Figure 3. Inhibitory effect of bound phenolics released by alkaline hydrolysis (BBP) on α-glucosidase and lipase activities. IC50 is the concentration of the extracts (sugar-removed) that inhibits 50% of α-glucosidase or lipase activity. The IC50 values of acarbose and orlistat were originally reported in ref 26. Values are means ± SD, n = 3.

highly negatively correlated to TPC (r2 = −0.9819) and TPI (r2 = −0.8972), suggesting phenolics are the main inhibitors of α-glucosidase in the extracts. The inhibitory activity found in this study was slightly higher than those reported for free F


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health beneficial compounds in quinoa. Quinoa seeds not only contain many extractable phytochemicals with strong antioxidant activities, but the present study also showed that there were various bound phenolic acids and even flavonoids in quinoa. These compounds can be liberated by alkaline, acid, and enzymatic hydrolyses. The present study identified at least 19 bound phenolic compounds in the residue of quinoa, predominantly phenolic acids, but also flavonoids. Ferulic acid and p-coumaric acid, protocatechuic acid, 4-hydroxybenzoic acid, and 8,5′-diferulic acid were the major phenolic acids found in most fractions, and quercetin and kaempferol were the main flavonoids. The concentration of bound phenolics, estimated either by spectrophotometric assay or by HPLC, was highest in black quinoa followed by red and white, regardless of the hydrolysis method used. The findings of the present study, particularly the fact that bound phenolics can be released by certain carbohydrases such as pectinase, xylanase, and feruloyl esterase and that these enzymes can be potentially secreted by colonic bacteria, add important information to the discussion on the potential role of bioactives in the gut, especially in the colon. The contribution of the bound phenolics to the high antioxidant activity points to further studies on their role in gut health, especially in regulating biomarkers related to immune response. The inhibitory effects of bound phenolics of quinoa on α-glucosidase and pancreatic lipase activities also warrant studies on enhancing potential nutritional and health benefits by using carbohydrases or probiotic bacteria producing such. The results of this study also can be applied to treat food high in bound phenolics for enhanced bioaccessibility.

phenolics in raw Nura and Rossa beans and significantly higher than those reported for other grains, such as corn, barley, and black soybean.23,24 Similar results were found for lipase activity (Figure 3). The IC50 value against lipase also correlated to TPI of BBP of the studied quinoas (r2 = −0.9281), indicating phenolic compounds are most likely the contributing factor to the lipase inhibitory activity. Previous studies also reported that phenolic acids including ferulic acid, vanillic acid, and p-coumaric acid exerted strong inhibitory activity on pancreatic lipase.22 An attempt was also made to measure the inhibitory effect of individual phenolics against these two enzymes. However, the activities were relatively low for phenolic acids, with IC50 values being >200 μg/mL for both enzymes; inhibition by the two flavonols quercetin and kaempferol was observed but still significantly weaker than the synthetic drugs (acarbose and orlistat) (Figure 3). The IC50 values of acarbose and orlistat were 9.34 μg/mL and 1.14 ng/mL for α-glucosidase and pancreatic lipase, respectively, as reported in our recent study.26 This finding was consistent with previous studies by Cai et al., who reported the IC50 values to be 219.3 ± 11.8, 210.5 ± 6.9, and 251.2 ± 9.3 μg/mL for ferulic acid, p-coumaric acid, and vanillic acid for pancreatic lipase, respectively.22 Interestingly the same authors also found a synergistic effect among these phenolic acids, suggesting stronger inhibition on these enzymes is possible for mixed phenolics such as those produced by chemical hydrolysis and enzyme treatment in the present study. Further studies need to be carried out to confirm this. Inhibition of these two enzymes by the carbohydrasereleased phenolics in quinoa would be minimal considering the low concentrations (Table 2), and it is highly unlikely that these phenolics could reach the upper gut for α-glucosidase and lipase inhibition even if they were released in sufficient amount in the microbiome of the colon. However, findings of the present study do suggest that treatment by acid or enzyme hydrolysis or fermentation of foods high in bound and unavailable phenolics such as quinoa can increase the release of phenolics prior to consumption. TPC of the 70% methanol extractable fraction was ca. 14−23 mg/g, of which ca. 20% was free phenolics and ca. 80% was conjugated phenolics that could be detected only upon acid and alkaline hydrolysis.1 Even though the unextractable bound phenolics found in the present study were relatively low (TPC was 0.7−1.2 mg/g by acid or alkaline hydrolysis, Table 2), when considered together with the conjugated phenolics found in our earlier study, an average total of 85% phenolics in quinoa seeds is unavailable as free phenolics.1 Other foods may also contain high bound phenolics. More than 90% of the total phenolics in tortilla maize was found to be in bound form,9,10 but treatment by an enzyme mixture consisting of pectinase, amylase, and cellulase showed that it was effective in releasing bound phenolics in purple corn.34 Treating high-fiber plants with carbohydrase enzymes or bacteria producing such has been a common practice only in animal nutrition.38 To our knowledge, this is the first report that systematically examined bound phenolics of quinoa and compared the efficacy of different methods in releasing those otherwise unextractable compounds. As the role of bound phenolics in human health, particularly gut health, becomes more highly regarded because of the various antioxidant, anti-inflammatory, and other effects reported in recent studies, and as the health benefits of quinoa seeds continue to be explored, results from the present study add new information to the full understanding of

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AUTHOR INFORMATION

Corresponding Author

*(R.T.) Phone: (226) 217-8108. Fax: (226) 217-8180. E-mail: [email protected]. Funding

This project is supported by the A-Base Research (No. 1004) of Agriculture & Agri-Food Canada (AAFC). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Dyanne Brewer and Dr. Armen Charchoglyan, Advanced Analysis Center, University of Guelph, for providing mass spectra reports.

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REFERENCES

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Bound Phenolics of Quinoa Seeds Released by Acid, Alkaline, and

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