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

Summary

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.

Abstract

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.

Introduction

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 tissues^1,2. 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,....

Protocol

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 and expression of dry weight basis
   NOTE: Determine the dry matter content of each powdered sample according to the method of AOAC (2000....

Results

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.......

Discussion

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.......

Materials

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