Characterization of phenolic compounds extracted from wheat protein fractions using high-performance liquid chromatography/liquid chromatography mass spectrometry in relation to anti-allergenic, anti-oxidant, anti-hypertension, and anti-diabetic properties

ABSTRACT

The purpose of this study was to identify novel natural bioactive phenolic compounds with anti-oxidant, anti-allergic, anti-hypertensive, and anti-diabetic properties in wheat protein fractions. Free and bound phenolic compounds were isolated from the albumin, glutelin-1, glutelin-2, prolamin, and globulin wheat protein fractions. The biological properties of the extracted phenolics were analyzed in vitro using 1,1-diphenyl-2-picryl-hydrazyl assays, enzyme-linked immunosorbent assays, angiotensin-1 converting enzyme assays, and α-amylase assays. The free and bound phenolic compounds were identified using liquid chromatography electrospray ionization tandem mass spectrometry methods. The aromatic rings in globulin were the highest in both before and after the removal of phenolic compounds (1.13 and 1.05 mg/g). The highest values of angiotensin-1 converting enzyme inhibitory and α-amylase inhibition (%) were obtained in glutelin-1 (73.17 and 96.41%, respectively) before removal phenolic compounds. The biological activity was affected by the presence or absence of phenolic compounds. Allergenicity was minimized in the presence of phenolic compounds. Correlation coefficients between wheat protein fractions and biological properties are described.

KEYWORDS:

• Phenolics
• Angiotensin-1 converting enzyme
• Allergenicity
• Liquid chromatography electrospray ionization tandem mass spectrometry
• 1,1-diphenyl-2-picryl-hydrazyl
• Protein fractions
• Antioxidant activity
• Wheat

Introduction

Phenolic compounds are abundant in plant-based foods and play essential roles as antioxidants for the prevention of heart disease.^[1–3] Phenolics are secondary metabolites that contain aromatic rings with one or more hydroxyl groups.^[4] The main subclass of phenolics is phenolic acids, which are found as amides or glycosides in bound form or esters in free form. Phenolic acids differ in the number and position of hydroxyl groups on the aromatic ring.^[5] Phenolic acid structures include hydroxycinnamic acid derivatives such as caffeic, ferulic, sinapic acids, and p-coumaric acids and hydroxybenzoic acid derivatives such as vanillic, gallic, protocatechuic, and syringic acids.^[6,7]

In wheat, phenolic compounds interact with proteins in complexes that alter the structural properties of the proteins.^[4] Wheat proteins are classified by Osborne according to the solubility of the protein fractions and include albumins, globulins, gliadins, and glutenins.^[8] Phenolic compounds form insoluble complexes with proteins that reduce IgE binding of allergens due to irreversible precipitation.^[9] The wheat gluten proteins, glutenins, and gliadins, are more complex than gluten proteins found in cereals such as rye and barley. The presence of gluten causes human immune diseases such as wheat allergies and celiac disease.^[10] Wheat intake can also protect against chronic diseases including cardiovascular disease.^[11] Wheat is also rich in polyphenols, which function as antioxidants to promote health.^[12] The major role of angiotensin-1 converting enzyme (ACE) in the renin-angiotensin (RA) system is to control blood pressure; various food sources contain ACE inhibitory peptides.^[13] Moreover, the enzyme α-amylase, which is found in the functions in the regulation of postprandial hyperglycemia,^[14,15] such as salivary and pancreatic secretions,^[16] by hydrolyzing oligosaccharides to maltose. α-amylase inhibitors elicit anti-diabetic effects by decreasing the level of glucose in the blood after a carbohydrate-rich meal.^[17] Therefore, consumption of wheat can protect against type 2 diabetes.^[18] The objectives of this study were as follows: (1) to identify and characterize extracted phenolic compounds from wheat protein fractions; and (2) to identify novel natural protein fractions in wheat flour with anti-allergenic, anti-oxidant, anti-hypertensive, and anti-diabetic potential.

Materials and methods

Plant materials

White wheat flour (moisture content 8.8%, protein 10.4%, fat 1.6%, ash 0.6%, and carbohydrates 78.6%) from Triticum aestivum was purchased from a local market in Irbid, Jordan. There were 10 plastic packages (1.5 kg) of white wheat flour purchased and stored at 4°C. Composite samples were prepared by mixing 100 g from each of the 10 packages for analysis.

Approximate chemical composition

Moisture, protein, fiber, fat, and ash were determined according to the official methods described in the Association of Official Analytical Chemists (AOAC).^[19] Total carbohydrates were calculated by difference.

Preparation of protein fractions from white wheat flour

Sequential extraction of protein fractions from white wheat flour samples was performed as described by Kwon et al.^[20] with minor modifications. Ten grams of white wheat flour were weighed in an aluminum dish on an analytical balance (Mettler PJ3000, Europe) and transferred to a beaker, then 100 mL of distilled water was added. The mixture was stirred using a magnetic stirrer for 1 h and centrifuged at 10,000 × g for 15 min (X32HK, Hermle Labortechnik GmbH, Germany). The supernatant and residue were separated by decanting and frozen at –18°C. The supernatant was lyophilized using a freeze drier (LFD-5508, Daihan Labtech Co. LTD, Korea). The residue was dissolved in 100 mL of NaOH (0.1 N, adjusted to pH 11 using a pH meter; Cyberscan, Eutech instrument pH 1500), stirred for 1 h and centrifuged at 10,000 × g for 15 min. The second supernatant was decanted into a beaker, frozen at –18°C, and lyophilized. The second residue was dissolved in 100 mL of 50% acetic acid, stirred for 1 h, and centrifuged at 10,000 × g for 15 min. The third supernatant was decanted into a beaker, frozen at –18°C and lyophilized. The third residue was dissolved in 100 mL of 80% ethanol, stirred for 1 h, and centrifuged at 10,000 × g for 15 min. The fourth supernatant was decanted into a beaker, frozen at –18°C, and lyophilized. The fourth residue was dissolved in 100 mL of 10% NaCl, stirred for 1 h, and centrifuged at 10,000 × g for 15 min. The fifth supernatant was decanted into a beaker, frozen at –18°C, and lyophilized. The fifth residue was discarded.

Extraction of free phenolic compounds from wheat protein fractions

The wheat protein fractions were extracted as described by Alu’datt et al.^[21] One gram of each wheat protein fraction was extracted with 25 mL of methanol for 1 h at 25°C in a water bath and centrifuged at 10,000 × g for 10 min (X32HK, Hermle Labortechnik GmbH, Germany). The supernatant was stored at –18°C until further analysis. The resultant phenolic compounds were designated free phenolic compounds at room temperature (FP-25°C). The residue remaining from methanol extraction at room temperature was extracted with 25 mL of methanol for 1 h at 50°C in a water bath and centrifuged at 10,000 × g for 10 min. The resultant supernatant was stored at –18°C until further analysis. The resultant phenolic compounds were designated free phenolic compounds using heat treatment (FP-50°C). The resultant residue was subjected to further extraction.

Extraction of bound phenolic compounds from wheat protein fractions

Bound phenolic compounds were extracted according to the method described by Alu’datt et al.,^[21] with modifications. The residue that remained after methanol and heat extraction (see above) was hydrolyzed in dilute alkaline solution (25 mL, pH 12.0, 0.1 N NaOH) for 12 h at 25°C in a water bath with shaking to extract bound phenolic compounds, and the extract was centrifuged at 10,000 × g for 10 min. The supernatant was frozen at –18°C and freeze-dried in a lyophilizer (LFD-5508, Daihan Labtech Co. LTD, Korea). The bound phenolic compounds in the lyophilized supernatant were extracted with 25 mL of methanol for 1 h at 25°C in a water bath and centrifuged at 10,000 × g for 10 min. The phenolic compounds obtained using this technique were designated bound phenolic compounds using base hydrolysis (BP-base). The supernatant was stored at –18°C until further analysis. The residue remaining after extraction was hydrolyzed in dilute acid solution (25 mL, pH 2.0, 0.1 N HCl) for 12 h at 25°C in a water bath with shaking to extract bound phenolic compounds. The bound phenolic compounds in the acidified lyophilized supernatant were extracted with 25 mL of methanol for 1 h at 24°C in a water bath and centrifuged at 10,000 × g for 10 min. The supernatant was stored at –18°C until further analysis. The phenolic compounds obtained by this technique were designated bound phenolic compounds using acid hydrolysis (BP-acid).

Identification and quantification of individual phenolic compounds using liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS)

Ten milliliters of the free and bound phenolic extracts with the highest phenolic content were evaporated under a stream of nitrogen, dissolved in 1 mL of methanol and stored at –18ºC until further analysis. Standard curves were prepared using a stock standard solution prepared by dissolving 5 mg of each phenolic standard in 50 mL of methanol. The white wheat flour protein-fraction extracts were analyzed by LC-MS/MS according to the method described by Obied et al.^[22] using an Agilent 1100 chromatography system (Agilent 1100, Agilent Technologies, Wilmington, DE, USA) equipped with a diode-array ultraviolet (UV) detector. The samples were injected onto a Thermo C₁₈ reversed-phase column (pore size 5 µm, 250 × 4.6 mm i.d. Thermo Fisher Scientific, San Jose, CA, USA).

The analysis was performed using a gradient solvent system with aqueous formic acid (1%) as solvent (A) and methanol/acetonitrile/formic acid mixture (89.5/9.5/1 v/v/v) as solvent (B). A seven-step linear gradient elution with a total run time of 65 min was employed as follows: 0–10 min, 90–70% solvent A and 10–30% solvent B; 10–15 min isocratic; 15–25 min, 60% solvent A and 40% solvent B; 25–40 min, 50% solvent A and 50% solvent B; 40–50 min, 100% solvent B; 50–55 min, 90% solvent A and 10% solvent B; and 55–65 min isocratic. An injection volume of 15 µL at a constant flow rate of 0.75 mL/min was used for each analysis. The entire flow from the high-performance liquid chromatography (HPLC) was directed into a triple-quadrupole mass spectrometer (API 3200; MDS Sciex, Concord, ON, Canada). The mass spectral data were acquired in negative ion mode with a capillary voltage of 4000 V, an atmospheric pressure chemical ionization (APCI) ion source, a cone voltage of 70 V, a collision energy of 10 eV, a drying temperature of 350ºC, N₂ as the drying gas with a flow rate of 4.0 L/min, helium as the nebulizer gas with a flow rate of 40 psi, and Analyst software version 3.5.1. A diode-array UV detector was used to scan between 200 and 400 nm to evaluate the contents of individual phenolic compounds, and the eluted samples and standards were detected at 280 nm. A mixture of external standards (p-hydroxybenzoic acid, gallic acid, caffeic acid, ferulic acid, vanillic acid, tyrosol, hydroxytyrosol, p-coumaric acid, syringic acid, apigenin, hesperidin, luteolin, quercetin, rutin, and sinapic acid) were used to quantify the individual phenolic compounds.

Determination of total phenolic contents

The Folin–Ciocalteu spectrophotometric method was used to determine the total phenolic content of each extract as described by Alu’datt et al.^[21] A 100-µL extract sample was added to 8.4 mL of distilled water, and 0.5 mL of Folin–Ciocalteu reagent was added and mixed by vortexing (Reamix 2789, 50 Hz) for 4 min; next, 1 mL of 5% sodium carbonate solution (Na₂CO₃) was added to the mixture, followed by mixing by vortexing. The absorbance was measured 1 h later at 725 nm using a spectrophotometer (UV 1800, 50Hz, UK). The total phenolic content was expressed as milligrams of gallic acid equivalents (GAE) per gram of dry matter (mg of GAE.g⁻¹). The gallic acid stock solution was prepared at concentrations of 0, 0.25, 0.50, 0.75, and 1.0 (mg.mL⁻¹). The analysis was conducted in duplicate for each sample.

Determination of antioxidant activity

The antioxidant activity of wheat protein fraction samples was determined using the 1,1-diphenyl-2-picryl-hydrazyl (DPPH) method as described by Brand-Williams et al.^[23] A 0.1-mL sample of extract was mixed vigorously with 3.9 mL of 6×10^–5 M DPPH solution (2.4 mg of DPPH in 100 mL of methanol) and incubated in the dark for 30 min at room temperature. The absorbance (A) was determined in a spectrophotometer at 515 nm at 0 and 30 min using methanol as a blank. Antioxidant activity was calculated according to the following equation:

where A is the absorbance of the sample at 30 min and B is the absorbance of the control at 0 min. The analysis was conducted in duplicate for each sample.

Measurement of carbohydrate digestion enzyme activity (α-amylase inhibition)

The α-amylase inhibitory activity of the white wheat flour protein fraction samples was determined by the method of McCue et al.^[24] with modifications. A 0.03% (w/v) porcine pancreatic α-amylase (10080, Sigma Chemical Co, USA) solution was prepared in 100 mL of distilled water. Then, 100 µL of sample, 500 µL of α-amylase solution, and 500 µL of phosphate buffer (pH 7) were mixed and incubated at 25°C for 10 min; 100 µL of water was used as a control. Next, 500 µL of starch solution (0.125 g of starch powder in 25 mL of phosphate buffer at (pH 6.9) and incubated at 65°C for 20 min) was added and mixed well, followed by incubation at 25°C for 10 min in a water bath. One milliliter of colorimetric reagent (19.8 g of sodium hydroxide, 10.6 g of 3,5-dinitrosalicyclic acid (DNS), 7.6 g of phenol, 3.06 g of sodium potassium tartrate, and 8.3 g of sodium metabisulfite in 1416 mL of distilled water) was added, heated in a water bath at 95ºC for 5 min and cooled to room temperature in an ice bath. The mixture was diluted to 10 mL with distilled water. This colorimetric method was used to evaluate liberated maltose by measuring the absorbance at 540 nm. Maltose stock solutions were prepared at concentrations of 0, 0.5, 1.0, 1.5, 2.0, and 2.5 (mg.mL⁻¹). The inhibitory activity of α-amylase was calculated according to the following equation:

where AB_C is the absorbance of the control at 540 nm and AB_s is the absorbance of the sample at 540 nm.

ACE inhibitory activity

The inhibition of ACE by the white wheat flour protein fractions was determined according to the method of Cushman and Cheung^[25] with modifications. Hippuryl-histidyl-leucine (HHL) solution was prepared by dissolving of 0.3 g of HHL in 100 mL of distilled water and mixed with 50 mM HEPES-HCl buffer containing 300 mM sodium chloride (pH 8.3) at 37°C. Next, 0.33 U of ACE was diluted in 1 mL of distilled water, and 200 µL of HHL was mixed with 100 µL of the phenolic extract of wheat flour. A 50-µL aliquot of diluted ACE was added, and the mixture was incubated at 37°C for 15 min. The enzymatic reaction was stopped by adding 0.25 mL of HCl (1.0 M), and 2 mL of ethyl acetate was added and mixed well to extract the liberated hippuric acid. One milliliter of the ethyl acetate layer was separated by centrifugation at 3000 rpm for 3 min. The supernatant was removed by boiling in a water bath for 15 min, and the residue was diluted with 3 mL of distilled water. The amount of liberated hippuric acid was determined by measuring the absorbance at 228 nm. A blank was prepared by adding 200 μL of HHL and 50 μL of ACE to 100 μL of distilled water instead of sample. The inhibitory activity of ACE was calculated using the following equation:

where AB_C is the control absorbance at 228 nm and AB_s is the sample absorbance at 228 nm.

Determination of reduction of allergenicity using the enzyme-linked immunosorbent assay (ELISA)

ELISA (ELISA System Pty Ltd Queensland-Australia) was performed according to the method of Tezcucano^[26] to detect the presence of protein after dephosphorylation and hydrolysis via a double antibody (sandwich) ELISA in microwells coated with specific antibodies (Anti-Gliadin (wheat) antibody 20 µg.mL⁻¹) at 100 µL.mL⁻¹ overnight. The wells were thoroughly washed five times with phosphate-buffered saline (PBS-T buffer), and 100 µL of the extracted test sample was added. The wells were mixed by moving the strip holder gently sideways for 10 s, followed by incubation for 45 min at 37°C. The wells were washed thoroughly five times with wash buffer PBS-T and tapped firmly onto an absorbent paper towel. A 100-µL aliquot of the enzyme conjugate (Anti-Gliadin [wheat]-Peroxidase antibody 5 µg.mL⁻¹) was added to each well, mixed for 10 s and incubated for 45 min at 37°C, followed by five washes with wash buffer PBS-T. Next, 100 µL of stabilized tetramethylbenzidine (TMP) substrate was added to each well, mixed for 10 s, and incubated for 15 min in the dark. Finally, 100 µL of sulfuric acid (1 M) was added to stop the reaction. The results were read in a microplate reader (Biotek instruments, ELX800, SN 219962) at 450 and 630 nm, and the raw data were processed using microplate manager V5.2 software (ELISA Systems Pty Ltd. Protocol, 2005). The absorbance reading was obtained by subtracting the absorbance at 630 nm from the absorbance at 450 nm.

The results were expressed as the % reduction of allergenicity using the following equation:

where A is the protein after the removal of phenolic compounds and B is the protein before the removal of phenolic compounds.

Statistical analysis

Statistical analysis was performed using the general linear model (GLM) of the SAS software package (Version 9.1 SAS 2002 Institute Inc., NC, USA). Means separation was performed using the least significant difference (LSD) multiple-range test procedure at an alpha level of 0.05.

Results and discussion

Effect of removal of phenolics on the biological properties of wheat protein fractions

The total aromatic compounds, antioxidant activities (%), ACE inhibitory activity (% of inhibition), and α-amylase inhibitory activity (% of inhibition) of the wheat flour protein fractions before and after removal of free and bound phenolic compounds are presented in Table 1. The aromatic compound content of the globulin protein fraction differed significantly (p < 0.05) before and after the removal of phenolic compounds (1.13 and 1.05 mg/g, respectively). As determined by the radical-scavenging assay, the antioxidant capacity of wheat flour was highest before the removal of phenolic compounds, 1.88%. After the removal of phenolic compounds, the prolamin and globulin fractions exhibited the highest antioxidant capacity, with values of 1.51 and 1.60%, respectively; these values were not significantly different from the values for the prolamin and globulin fractions before the removal of phenolic compounds.

Table 1. Total aromatic compound content (mg/g), antioxidant activities (%), ACE inhibitory activity (% inhibition), and α-amylase inhibitory activity (% inhibition) of protein fractions before and after removal of free and bound phenolic compounds extracted from wheat flour.¹

Protein fraction	Total aromatic compounds (mg/g)	Antioxidant activity (%)	ACE inhibition (%)	α-amylase inhibition (%)
Before removal of phenolic compounds
Wheat flour	0.49^b	1.88^a	40.62^c	89.81^ab
Albumin	0.49^b	0.25^c	22.83^d	67.54^c
Glutelin-1	0.38^b	1.13^ab	73.17^a	96.41^a
Glutelin-2	0.43^b	1.00^bc	24.39^d	60.53^c
Prolamin	0.54^b	1.63^ab	11.92^e	90.65^ab
Globulin	1.13^a	1.77^ab	47.22^b	88.84^b
SE	0.05	0.22	1.42	2.07
After removal of phenolic compounds
Wheat flour	0.48^cd	1.04^ab	64.29^b	89.27^a
Albumin	0.43^d	0.38^b	78.69^a	61.09^c
Glutelin-1	0.64^bc	0.94^ab	21.34^d	64.96^c
Glutelin-2	0.45^d	0.47^b	15.67^d	79.57^b
Prolamin	0.76^b	1.51^a	22.52^d	91.23^a
Globulin	1.05^a	1.60^a	53.40^c	95.17^a
SE	0.05	0.29	2.45	2.71

¹Mean values (n = 2) with the same superscript in a column are not significantly different (p > 0.05).

SE: standard error.

Glutenin-1 exhibited the highest ACE inhibitory activity before the removal of phenolic compounds, 73.17%, and this activity differed significant from that in all other fractions (p < 0.05). After the removal of phenolic compounds, albumin had the highest ACE inhibitory activity, with a value of 78.69%. Before the removal of phenolic compounds, glutenin-1 exhibited the highest α-amylase inhibitory activity, with a value of 96.41%, but after the removal of phenolic compounds, wheat flour, prolamin, and globulin exhibited the highest activities, with values of 89.27, 91.23, and 95.17%, respectively.

In plants, phenolic compounds are classified as secondary metabolites and are mainly composed of hydroxylated aromatic rings.^[27] There is a high correlation between total phenolic content and antioxidant activity.^[28] Moreover, antioxidant activity is strongly correlated with amino acid content, hydrophobicity, and conformation.^[29] Therefore, the structure and amino acid composition of a protein affects its antioxidant activity.^[30] Notably, the bioactive properties of the protein fractions were higher than those of the raw extract.^[31,32] The biological properties of the extracted peptides are consistent with antioxidant activity, including free radical-scavenging activity, suggesting potential applications in functional foods and pharmaceuticals.^[33]

LC-MS/MS analysis of free and bound phenolic compounds

Table 2 shows the phenolic profile identified by LC-MS/MS for the free phenolic extracts of white wheat flour. The LC-MS/MS profile of the wheat flour FP-30°C extract revealed p-hydroxybenzoic acid, gallic acid, vanillic acid, apigenin, p-coumaric, syringic acid, quercetin, luteolin, rosmarinic acid, and rutin. The predominant individual phenolic compounds in the FP-30°C extract were quercetin and apigenin (26.96 and 19.00%, respectively). The FP-50°C contained gallic acid, ferulic acid, chlorogenic acid, syringic acid, luteolin, quercetin, rutin, and sinapic acid. The albumin fraction profile of the FP-30°C extract revealed the presence of gallic acid, chlorogenic acid, quercetin and rutin. The predominant individual phenolic compounds in the FP-30°C extract were chlorogenic acid and gallic acid (47.69 and 25.84%, respectively). The FP-50°C extract contained chlorogenic acid, quercetin, rutin, and sinapic acid.

Table 2. Free phenolic compound content measured in wheat flour protein fractions by RP-HPLC (%)*.

Phenolic compounds	Retention timeime (min)	Wheat		Albumin		Glutelin-1		Glutelin-2		Prolamin		Globulin
		FP-25°C extract	FP-50°C extract	FP-25°C extract	FP-50°C extract	FP-25°C extract	FP-50°C extract	FP-25°C extract	FP-50°C extract	FP-25°C extract	FP-50°C extract	FP-25°C extract	FP-50°C extract
p-Hydroxybenzoic acid	11.9	8.31^d	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND
Gallic acid	4.4	13.48^c	2.25^b	25.84^b	ND	ND	4.22^cd	ND	ND	ND	ND	ND	ND
Caffeic acid	15.6	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND
Ferulic acid	21.3	ND	92.33^a	ND	ND	97.32^a	23.88^b	ND	ND	ND	ND	ND	ND
Vanillic acid	14.7	11.61^cd	ND	ND	ND	ND	ND	91.99^a	82.17^a	ND	ND	ND	ND
Epicatechin	16.4	ND	ND	ND	ND	ND	ND	ND	0.94^d	ND	ND	ND	ND
Chlorogenic acid	14.7	ND	1.76^bc	47.69^a	4.99^b	0.22^de	6.18^c	ND	ND	14.02^bc	55.32^a	ND	ND
p-coumaric acid	18.7	3.69^e	ND	ND	ND	ND	2.31^de	ND	ND	ND	ND	ND	ND
Naringin	38.5	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND
Syringic acid	16.6	2.96^e	1.37^c	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND
Apigenin	43.2	19.00^b	ND	ND	ND	0.83^b	ND	5.22^b	ND	48.91^a	ND	98.31^a	98.88^a
Diosmetin	32.1	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND
Hesperidin	26.7	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND
Luteolin	38.8	0.72^e	0.08^d	0.90^c	ND	0.03^e	0.14^e	ND	ND	ND	ND	1.70^b	1.12^b
Quercetin	36.9	26.96^a	1.43^bc	11.44^c	1.45^c	0.58^bc	3.88^cd	2.8^b	3.31^c	6.88^c	44.68^a	ND	ND
Rutin	26.9	0.46^e	0.37^d	10.71^c	2.36^c	0.33^cd	2.40^de	ND	ND	ND	ND	ND	ND
Sinapic acid	20.8	ND	0.47^d	ND	1.55^c	ND	ND	ND	2.87^c	24.14^b	ND	ND	ND
Rosmarinic acid	30.5	3.75^e	ND	ND	ND	0.16^de	ND	ND	ND	ND	ND	ND	ND
Non-identified peak*	33.1	10.55^cd	ND	3.43^c	89.65^a	0.53^c	56.99^a	ND	10.71^b	6.05^c	ND	ND	ND
SE	11.9	1.29	0.26	3.94	0.58	0.09	1.02	1.97	0.50	3.552	3.023	0.51	0.30

^aMean value of two replicates. Percentage of total phenolic content based on peak area. Column values with the same letters are not significantly different (p < 0.05).

SE: standard error. ND: not detected.

*Calculated based on peak area of oleuropein at a similar retention time.

The glutelin-1 profile of the FP-30°C extract showed the presence of ferulic acid, chlorogenic acid, apigenin, luteolin, quercetin, rutin, and rosmarinic acid. The predominant individual phenolic compound in the FP-30°C extract was ferulic acid (97.32%). The FP-50°C extract included gallic acid, ferulic acid, chlorogenic acid, p-coumaric acid, luteolin, quercetin, and rutin. In the glutelin-2 profile of the FP-30°C extract, vanillic acid, apigenin, and quercetin were observed. The predominant individual phenolic compound in the FP-30°C extract was vanillic acid (91.99%). For the FP-50°C extract, the results showed the presence of vanillic acid, epicatechin, quercetin, rutin, and sinapic acid. The predominant individual phenolic compound in the FP-50°C extract was vanillic acid (56.99%). The prolamin fraction of the FP-30°C extract showed the presence of chlorogenic acid, apigenin, quercetin and sinapic acid. The predominant individual phenolic compound in the FP-30°C extract was apigenin (48.91%. For the FP-50°C extract, the results showed the presence of chlorogenic acid and quercetin. The predominant individual phenolic compound in the FP-50°C extract was chlorogenic acid (55.32%). The LC-MS/MS profile of the BP-base extract showed the presence of chlorogenic acid, quercetin, and sinapic acid. Sinapic acid was the predominant individual phenolic compound in the BP-base extract (50.61%). The globulin profile of the FP-30°C and FP-50°C extract showed the presence of apigenin and luteolin. The predominant individual phenolic compound in the FP-30°C and FP-50°C extracts was apigenin (98.31 and 98.88%, respectively).

In flaxseed, the free and bound phenolic compounds are vanillic, syringic, ferulic, sinapic, p-hydroxybenzoic, protocatechuic, caffeic, and coumaric acids.^[34–39] The free phenolic compounds determined by Alu’datt et al.^[40] were gallic acid, protocatechuic acid, p-hydroxybenzoic acid, caffeic acid, syringic acid, ferulic acid, and p-coumaric acid, but the free phenolic compounds isolated from whole and defatted soybean were syringic acid, sinapic acid, ferulic acid, p-coumaric acid, gallic acid, p-hydroxybenzoic acid, quercetin, caffeic acid, and hesperidin. The free phenolic acids in soy protein isolate are genistic, syringic, o-coumaric, p-coumaric, and ferulic acid.^[36] Table 3 shows the phenolic profile identified by LC-MS/MS in the bound phenolic extracts of white wheat flour. The wheat flour profile of the BP-base extract showed the presence of gallic acid, ferulic acid, chlorogenic acid, syringic acid, quercetin, and rutin. Ferulic acid was the predominant individual phenolic compound in both the FP-50°C and BP-base extracts (92.33 and 84.38%, respectively). The BP-acid extract contained p-hydroxybenzoic acid, chlorogenic acid, p-coumaric, syringic acid, quercetin, and rutin, and the main individual phenolic compounds were syringic acid and chlorogenic acid (34.37 and 26.08%, respectively). The albumin profile of the BP-base extract showed the presence of ferulic acid, chlorogenic acid, syringic acid, luteolin, quercetin, and rutin. Ferulic acid was the predominant individual phenolic compound in the BP-base extract (64.96%). The BP-acid extract contained chlorogenic acid, ferulic acid, hesperidin, quercetin, rutin, and sinapic acid, and the main individual phenolic compound in the BP-acid extract was ferulic acid (97.76%). The glutelin-1 profile of the BP-base extract showed the presence of ferulic acid, chlorogenic acid, epicatechin, apigenin, luteolin, quercetin, and sinapic acid, and ferulic acid was the predominant individual phenolic compound in the BP-base extract (87.87%). The BP-acid extract contained p-hydroxybenzoic acid, gallic acid, ferulic acid, chlorogenic acid, quercetin, and sinapic acid. The main individual phenolic compound in the BP-acid extract was ferulic acid (50.08%). The glutelin-2 profile of the BP-base extract showed vanillic acid, quercetin, and sinapic acid, and the BP-acid extract contained chlorogenic acid, apigenin, quercetin and sinapic acid. The main individual phenolic compound in the BP-acid extract was chlorogenic acid (39.86%). Prolamin in the BP-base extract showed the presence of chlorogenic acid, quercetin, and sinapic acid, which was the predominant individual phenolic compound in the BP-base extract (50.61%). The BP-acid extract contained apigenin and quercetin. The main individual phenolic compound in the BP-acid extract was apigenin (66.72%).

Table 3. Bound phenolic compound content in wheat flour protein fractions measured by RP-HPLC (%).

Phenolic compounds	Retention time (min)	Wheat		Albumin		Glutelin-1		Glutelin-2		Prolamin		Globulin
		BP-base extract	BP-acid extract	BP-base extract	BP-acid extract	BP-base extract	BP-acid extract	BP-base extract	BP-acid extract	BP-base extract	BP-acid extract	BP-base extract	BP-acid extract
p-hydroxybenzoic acid	11.9	ND	9.97^cd	ND	ND	ND	3.79^bc	ND	ND	ND	ND	ND	ND
Gallic acid	4.4	7.61^b	ND	ND	ND	ND	26.10^ab	ND	ND	ND	ND	ND	ND
Caffeic acid	15.6	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND
Ferulic acid	21.3	84.38^a	ND	64.96^a	97.76^a	87.87^a	50.08^a	ND	ND	ND	ND	ND	ND
Vanillic acid	14.7	ND	ND	ND	ND	ND	ND	17.09^b	ND	ND	ND	72.72^a	ND
Epicatechin	16.4	ND	ND	ND	ND	0.25^d	ND	ND	ND	ND	ND	ND	ND
Chlorogenic acid	14.7	3.83^c	26.08^b	13.33^b	0.66^b	1.55^cd	1.74^c	ND	39.86^a	24.87^a	ND	5.40^c	ND
p-coumaric acid	18.7	ND	5.92^de	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND
Naringin	38.5	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	0.74^c	ND
Syringic acid	16.6	2.53^cd	34.37^a	9.93^b	ND	ND	ND	ND	ND	ND	ND	ND	ND
Apigenin	43.2	ND	ND	ND	ND	7.33^b	ND	ND	14.35^bc	ND	66.72^a	ND	ND
Diosmetin	32.1	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND
Hesperidin	26.7	ND	ND	ND	0.39^b	ND	ND	ND	ND	ND	ND	17.84^b	100^a
Luteolin	38.8	ND	ND	0.3395^b	ND	0.04^d	ND	ND	ND	ND	ND	ND	ND
Quercetin	36.9	0.96^de	7.66^d	5.50^b	0.39^b	0.54^cd	2.54^c	1.83^c	11.52^bc	24.52^a	25.69^ab	ND	ND
Rutin	26.9	0.70^e	13.27^c	4.06^b	0.35^b	ND	ND	ND	ND	ND	ND	ND	ND
Sinapic acid	20.8	ND	ND	ND	0.45^b	1.10^c	8.38^bc	4.44^c	23.60^b	50.61^a	ND	ND	ND
Rosmarinic acid	30.5	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND
Non-identified peak*	33.1	ND	2.72^e	1.89^b	ND	0.46^d	6.48^bc	76.65^a	10.67^c	ND	7.59^b	3.30^c	ND
SE		0.50	1.23	4.83	0.12	0.46	7.09	1.43	3.36	6.857	11.019	1.86	0.00

^aMean value of two replicates. Percentage of total phenolic content based on peak areas. Column values with the same letters are not significantly different (p < 0.05).

SE: standard error; ND: not detected.

*Calculated based on peak area of oleuropein at a similar retention time.

The globulin profile of the BP-base extract showed the presence of vanillic acid, chlorogenic acid, naringin, and hesperidin. Vanillic acid was the predominant individual phenolic compound in the BP-base extract (72.72%). The main individual phenolic compound in the BP-acid extract was hesperidin (100%). The phenolic compounds in soybean and flaxseed (p-coumaric acid, ferulic acid, and p-hydroxybenzoic acid) interact with proteins. Gallic acid, syringic acid, ferulic acid, and p-coumaric acid are found in the bound form, but the bound phenolic compounds of alkaline-hydrolyzed soybean protein isolate are p-coumaric acid, hesperidin, and syringic acid.^[40] The predominant free and bound phenolic compounds in soybean are coumaric, p-hydroxybenzoic, protocatechuic, vanillic, syringic, ferulic, caffeic, and sinapic acids.^[34,37,39] Uddin et al.^[41] reported that the predominant identified phenoic compound in traditional wheat flour was rutin.

Correlation coefficients (R) for the biological properties of extracted phenolic compounds for Osborne protein fractions from wheat flour

As shown in Table 4, there are positive correlations between the biological properties of the wheat flour and protein fractions (albumin, glutelin-1, glutelin-2, prolamin, and globulin) and total phenolics, ACE inhibitory activity, α-amylase inhibitory activity, and antioxidant activity. There are significant correlations between the antioxidant activity of phenolic compounds and the total phenolic content in the albumin protein fraction, with a correlation coefficient of 0.746 and a strong positive linear correlation (0.858) between the antioxidant activity and α-amylase inhibitory activity of the phenolic compounds. Thus, there are strong correlations between total phenolic content and antioxidant activity.^[28] Antioxidant activity exhibited a strong negative correlation with phenolic content (–0.804) in the glutelin-1 protein fractions. By contrast, in olive oil, vegetables, fruit, and grain, there are positive correlations between phenolic content and antioxidant activity.^[42,43] In glutelin-2, the α-amylase inhibitory activity showed a strong negative correlation with total phenolic content, and ACE inhibitory activity also showed a negative correlation with antioxidant activity, with values of –0.819 and –0.772, respectively. By contrast, the reduction of allergenicity showed a strong positive correlation with antioxidant activity with a value of (0.824). Hence, total phenolic compounds are highly correlated with α-amylase inhibitory activity.^[44] A strong positive linear correlation (0.860) was observed between total phenolic content and antioxidant activity in prolamin. The total phenolic content of globulin showed a positive correlation with ACE inhibitory activity but a strong negative correlation with the reduction of allergenicity, with correlation values of 0.719 and –0.8999, respectively. However, many different relationships were observed for the biological properties of whole white wheat flour, including a strong positive correlation between ACE inhibitory activity and total phenolic contents and between reduction of allergenicity and antioxidant activity, with values of 0.887 and 0.799, respectively, and a strong negative correlation between α-amylase inhibitory activity and antioxidant activity, with a value of –0.844. Blair and Reichert^[45] reported high correlation coefficients between ACE inhibitory activity and antioxidant activity, α-amylase inhibitory activity, and total phenolic content. Alu’datt et al.^[46] reported that the antioxidant activity was strongly positively correlated with phenolics contents in Zingiber officinale.

Table 4. Correlation coefficients (r) between total phenol, antioxidant activity, ACE inhibitory activity, α-amylase inhibitory activity and reduction of allerginicity of extracted phenolic compounds for Osborne protein fractions from wheat flour.

Fraction	Variable	Antioxidant	Alpha amylase	ACE inhibition	Reduction of allergenicity
Albumin	Total phenolics	0.746*	0.4823	–0.5491	0.2163
	Antioxidant		0.8581**	–0.1593	–0.302
	Alpha amylase			0.3441	–0.4974
	ACE				–0.5169
Glutelin-1	Total phenolics	–0.8041*	0.1105	–0.4396	–0.0296
	Antioxidant		–0.4848	0.4727	–0.4933
	Alpha amylase			0.4284	0.2686
	ACE				–0.5564
Glutelin-2	Total phenolics	–0.4193	–0.819*	0.1803	–0.4793
	Antioxidant		0.0209	–0.7721*	0.8239*
	Alpha amylase			0.3039	0.3625
	ACE				–0.3678
Prolamin	Total phenolics	0.8596**	–0.0599	–0.0781	0.5607
	Antioxidant		0.0192	–0.5688	0.1608
	Alpha amylase			–0.2374	–0.0522
	ACE				0.5782
Globulin	Total phenolics	–0.5614	0.0248	0.7186*	–0.8999**
	Antioxidant		–0.204	–0.6863	0.5907
	Alpha amylase			–0.1574	–0.3248
	ACE				–0.6655
Wheat flour	Total phenolics	–0.0671	0.2926	0.8873**	0.4581
	Antioxidant		–0.8436**	–0.4346	0.7988*
	Alpha amylase			0.6862	–0.439
	ACE				0.1676

*Correlation factors with superscripts in a column are significantly different (p < 0.05).

**Correlation factors with superscripts in a column are significantly different (p < 0.01).

Conclusion

The presence of phenolic compounds affects the various biological properties of wheat protein fractions, including antioxidant activity, ACE inhibitory activity, and α-amylase inhibitory activity. These effects arise through interactions between the compounds and protein that lead to structural and conformational changes. RP-HPLC analysis revealed that for protein isolates from white wheat flour, the phenolic compounds differed between the free phenolic group and bound phenolic group. The removal of phenolic compounds from the protein fractions of white wheat flour reduced their allergenicity. These compounds may be good sources for the development of foods with enhanced functional, chemical, nutritional, and biological properties, including anti-hypertensive, anti-diabetic, and antioxidant effects. Wheat flour and all wheat protein fractions exhibited significant correlation coefficients with total phenols, antioxidant, α-amylase, and ACE activities, and reduction of allergenicity.

Funding

The authors acknowledge the Jordan University of Science and Technology for research funding. The authors also extend their appreciation to the Deanship of Scientific Research at King Saud University for funding the work through the research group project no. RGP-VPP-193.

Additional information

Funding

The authors acknowledge the Jordan University of Science and Technology for research funding. The authors also extend their appreciation to the Deanship of Scientific Research at King Saud University for funding the work through the research group project no. RGP-VPP-193.