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A Generalized Method for Determining Free Soluble Phenolic Acid Composition and Antioxidant Capacity of Cereals and Legumes

Published: June 10th, 2022



1Department of Food and Human Nutritional Sciences, University of Manitoba, 2Richardson Centre for Functional Foods and Nutraceuticals, University of Manitoba

Phenolic acids are important phytochemicals that are present in whole grains. They possess bioactive properties such as antioxidant protective functions. This work aimed at reporting on a generalized method for the HPLC identification, total phenolic content estimation, and determination of the antioxidant capacity of phenolic acids in cereals and legumes.

Phenolic acids are a class of organic compounds that bear both a phenolic group, and a carboxylic group. They are found in grains and concentrate in the bran of cereals or seed coat of legumes. They possess antioxidant properties that have generated much research interest in recent years, about their potential antioxidant protective health functions. This work presents a generalized method for the extraction of free soluble phenolic acids from whole grains and analysis of their antioxidant capacity. Five whole grain samples comprising two cereals (wheat and yellow corn) and three legumes (cowpea bean, kidney bean, and soybean), were used. The grains were milled into flour and their free soluble phenolic acids extracted using aqueous methanol. The compounds were then identified using a high-pressure liquid chromatograph (HPLC). The Folin-Ciocalteu method was used to determine their total phenolic content while their antioxidant capacities were determined using the DPPH radical scavenging capacity, Trolox equivalent antioxidant capacity (TEAC) and oxygen radical absorbance capacity (ORAC) assays. The phenolic acids identified included vanillic, caffeic, p-coumaric and ferulic acids. Vanillic acid was identified only in cowpea while caffeic acid was identified only in kidney bean. p-Coumaric acid was identified in yellow corn, cowpea, and soybean, while ferulic acid was identified in all the samples. Ferulic acid was the predominant phenolic acid identified. The total concentration of phenolic acids in the samples decreased in the following order: soybean > cowpea bean > yellow corn = kidney bean > wheat. The total antioxidant capacity (sum of values of DPPH, TEAC and ORAC assays) decreased as follows: soybean > kidney bean > yellow corn = cowpea bean > wheat. This study concluded that HPLC analysis as well as DPPH, TEAC, and ORAC assays provide useful information about the phenolic acid composition and antioxidant properties of whole grains.

Phenolic acids are among the most important phytochemicals studied in plants due to the vital role they play in plant defense against herbivory and fungal infection, as well as maintaining structural support and integrity in plant tissues1,2. They are abundant in the bran of cereals and seed coat of legumes3. Structurally, they are divided into two groups: the hydroxybenzoic acids (Figure 1) and hydroxycinnamic acids (Figure 2). The common hydroxybenzoic acids in cereals and legumes include gallic, p-hydroxybenzoic, 2,....

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1. Type of samples

  1. Use five whole grain samples, comprising two cereals (e.g., durum wheat and yellow corn) and three legumes (e.g., Blackeye cowpea bean, soybean, and red kidney bean) for this study.
  2. Mill 50 g of each grain in triplicates into flour, using a coffee grinder, and pass them through a 500 µm sieve.
  3. Store them at -20 °C.

2. Sample preparation

  1. Determination of dry matter content a.......

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Table 2 shows the phenolic acids that were identified in the cereal and legume grains. Based on available authentic standards, four phenolic acids were identified in the samples and they are: vanillic, caffeic, p-coumaric, and ferulic acids. Vanillic acid is a hydroxybenzoic acid while the other three are hydroxycinnamic acids. Vanillic acid was identified only in Blackeye cowpea bean while caffeic acid was identified only in kidney bean. p-Coumaric acid was identified in yellow corn, c.......

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The whole grains were selected as representative cereal grains and legumes that find wide food applications worldwide. While variations may exist among cultivars of each grain, the focus of this study was to demonstrate a generalized method for free phenolic acid extraction and analysis for whole grains. The extraction method was modified by substantially reducing the amounts of samples and solvents, in order to reduce the amount of chemicals that would be released into the environment when such experiments are conducted.......

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The authors gratefully acknowledge the technical support of Ms. Alison Ser and Ms. Hannah Oduro-Obeng, as well as the video editing support by Ms. Janice Fajardo and Mr. Miguel del Rosario.


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Name Company Catalog Number Comments
15 mL Falcon conical centrifuge tubes Fisher Scientific 05-527-90
2 mL Amber glass ID Surestop vial Thermo Scientific C5000-2W
2 mL Amber microcentrifuge tubes VWR 20170-084
2,2′-Azobis(2-amidinopropane) dihydrochloride (AAPH) Sigma-Aldrich 440914-100G
2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) (C18H18N4O6S4) ≥98%, Sigma Aldrich A1888-2G
2,2-Diphenyl-1pikrylhydrazyl (DPPH) (C18H12N5O6) Sigma Aldrich D913-2
6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) (C14H18O4), ≥98% Fluka Chemika 56510
9 mm Autosampler Vial Screw Thread Caps Thermo Scientific 60180-670
96 well flat bottom plates Fisher Scientific 12565501
Agilent BioTek ELx800 microplate reader Fisher Scientific BT-ELX800NB
Agilent BioTek Precision 2000 96/384 Automated Microplate Pipetting System Fisher Scientific N/A
Agilent BioTek FLx800 Microplate Fluorescence Reader Fisher Scientific N/A
Analytical balance SI-114 Denver Instrument SI-114.1
Autosampler, Waters 717 Plus Waters WAT078900
BD 3 mL syringe Luer-Lok Tip BD 309657
Bransonic ultrasonic cleaner, Branson 5510 Millipore Sigma Z245143
Corning LSE Vortex Mixer Corning 6775
Durapore Filter (0.45 µm PVDF Membrane) Merck Millipore Ltd HVLP04700 
Durapore Membrane Filters (0.45 µm HV) Merck Millipore Ltd HVHP04700
Eppendorf Research plus, 0.5-10 µL Eppendorf 3123000020
Eppendorf Research plus, 0.5-5 mL Eppendorf 3123000071
Eppendorf Research plus, 100-1000 µL Eppendorf 3123000063
Eppendorf Research plus, 10-100 µL Eppendorf 3123000047
Ethyl acetate, HPLC grade Fisher Chemical E195-4
Ferulic acid standard Sigma Aldrich 128708-5G
Fluorescein Fisher Scientific AC119245000
Folin & Ciocalteu phenol reagent Sigma Aldrich F9252
Formic acid, 99% Acros Organics, Janssen Pharmaceuticalaan 3a 27048-0010
Gallic acid standard Sigma G7384
High performance liquid chromatograph (HPLC), Waters 2695 Waters 960402
Methanol, HPLC grade Fisher Chemical A452-4
Micro pipet tips, 0.5-10 µL Fisherbrand 21-197-2F
Microcentrifuge Sorvall Legend Micro 21 centrifuge Thermo Scientific 75002435
Multichannel micropipette, Proline Plus, 30-300 µL Sartorius 728240
Photodiode array detector, Waters 2996 Waters 720000350EN
Pipet tips, 1000 µL VWR 83007-382
Pipet tips, 1-5 mL VWR 82018-840
Potassium persulfate (K2S2O8), ≥99.0% Sigma Aldrich 216224-100G
Potassium phosphate dibasic anhydrous (K2HPO4) Fisher Scientific P288-500
Potassium phosphate monobasic (KH2PO4) Fisher Scientific P285-500
PYREX 250 mL Short Neck Boiling Flask, Round Bottom Corning 4321-250
Reversed phase C18 Analytical Column (100 x 3 mm) Accucore aQ Thermo Scientific 17326-103030
Roto evaporator, IKA RV 10 IKA  0010005185
Sodium carbonate (NaCO3) anhydrous Fisher Chemical S263-1
Sodium chloride (NaCl) Mallinckrodt AR® 7581
Sodium phosphate dibasic anhydrous (Na2HPO4) Fisher Scientific BP332-500
Sodium phosphate monobasic anhydrous (NaH2PO4) Fisher bioreagents BP329-500
Standardization pipet tips 0-200µL Fisherbrand 02-681-134
Syringe Driven Filter unit (0.22 µm)  Millex®-GV SLGVR04NL
Target micro-serts vial insert (400 µL) Thermo Scientific C4011-631
Ultrapure water (Direct Q-3 UV system with pump) Millipore ZRQSVP030

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