CyTA - Journal of Food

ISSN: 1947-6337 (Print) 1947-6345 (Online) Journal homepage: https://www.tandfonline.com/loi/tcyt20

Effect of simulated gastrointestinal digestion

on the phenolic compound content and in vitro
antioxidant capacity of processed Cowpea (V.
unguiculata) cultivars

Mlungisi Mtolo, Abe Gerrano & John Mellem

To cite this article: Mlungisi Mtolo, Abe Gerrano & John Mellem (2017) Effect of simulated
gastrointestinal digestion on the phenolic compound content and in�vitro antioxidant capacity
of processed Cowpea (V.�unguiculata) cultivars, CyTA - Journal of Food, 15:3, 391-399, DOI:
10.1080/19476337.2017.1285816

To link to this article: https://doi.org/10.1080/19476337.2017.1285816

© 2017 The Author(s). Published by Informa

UK Limited, trading as Taylor & Francis
Group.

Published online: 29 Mar 2017.

Submit your article to this journal

Article views: 1144

View Crossmark data

Citing articles: 1 View citing articles

Full Terms & Conditions of access and use can be found at

https://www.tandfonline.com/action/journalInformation?journalCode=tcyt20
CYTA – JOURNAL OF FOOD, 2017
VOL. 15, NO. 3, 391–399
https://doi.org/10.1080/19476337.2017.1285816

Effect of simulated gastrointestinal digestion on the phenolic compound content

and in vitro antioxidant capacity of processed Cowpea (V. unguiculata) cultivars
Mlungisi Mtoloa, Abe Gerranob and John Mellem a

a
Department of Biotechnology and Food Technology, Durban University of Technology, Durban, South Africa; bAgricultural Research Council,
Vegetable and Ornamental Plant Institute, Pretoria, South Africa

ABSTRACT ARTICLE HISTORY

The aim of this study was to analyze the phenolic content and antioxidant activity of five cowpea Received 7 November 2016
cultivars after processing and in vitro digestion. Raw cowpea samples showed a significant decrease in Accepted 19 January 2017
total phenolic content (TPC) when compared with the processed samples, however an increase was KEYWORDS
subsequently observed in digested samples. The antioxidant activity determined using, DPPH, ABTS, Simulated gastric digestion;
ferric reducing antioxidant power (FRAP) assay, and total peroxyl radical-trapping antioxidant parameter FRAP; DPPH; TRAP; Cowpea
showed a similar trend to the phenolic content with a significant decrease in activity upon processing
and an increase after digestion. In conclusion, all cowpea cultivars showed a high TPC content as well as PALABRAS CLAVE
an increased antioxidant activity after digestion indicating the potential health benefits which cowpea digestión gástrica estimu-
could provide to consumers. Therefore, this study shows that in vitro digestion improves the digestion lada; FRAP; DPPH; TRAP;
judía de careta
and absorption of beneficial components of processed cowpea at the intestinal level.

Efecto de la digestión gastrointestinal estimulada en el contenido de compuestos

fenólicos y la capacidad antioxidante in-vitro de cultivos de judía de careta procesada
(V. unguiculata)
RESUMEN
El objetivo de este estudio fue analizar el contenido fenólico y la actividad antioxidante de cinco
cultivos de judía de careta después del procesado y la digestión in vitro. Las muestras crudas de
judías de careta mostraron un descenso significativo en el contenido total de fenoles (TPC) cuando
se comparó con las muestras procesadas, sin embargo se observó posteriormente un aumento en
las muestras digeridas. La actividad antioxidante determinada utilizando DPPH, ABTS, el ensayo
FRAP y el parámetro antioxidante total capturador de radicales peroxilo mostró una tendencia
similar al contenido fenólico con una disminución significativa en la actividad frente al procesado y
un aumento después de la digestión. En conclusión, todos los cultivos de judías de careta mostraron
un alto contenido en TPC, como también un aumento en la actividad antioxidante después de la
digestión indicando los beneficios potenciales para la salud que las judías de careta podrían aportar
a los consumidores. Por lo tanto, este estudio muestra que la digestión in vitro mejora la digestión y
la absorción de los componentes beneficiosos de las judías de careta procesadas a nivel intestinal.

Introduction anti-nutritional factors. According to Knekt et al. (2002),

this legume is a good source of phenolic compounds
The world today is encountering concerns that relate to
and has the potential to protect the body against
poor nourishment and particularly the coexistence of
chronic diseases. This is also in agreement with the
under- and overnutrition. This leads to a dual problem
results from a study by Wang, Melnyk, Tsao, and
regarding health that includes infectious and noncom-
Marcone (2011) where the majority of plant-based
municable diseases. Amongst the many food sources that
foods (including cowpea) were identified as having a
may possibly be consumed, based on traditional knowl-
high content of phytochemical antioxidants and exhibit
edge, in order to establish improved health is cowpea
cardio-health promotion properties. The awareness of
(Vigna unguiculata). Cowpea is one of the most nutritive,
consumers with regard to the health benefits associated
versatile, and widely adapted of the grain legumes and
with legumes has increased in recent years resulting in
has been classified as an indigenous and underutilized
an increase in the demand for convenient, ready-to-eat
food security crop in South Africa. The Agricultural
whole grain cereal, and leguminous products. However,
Research Council has characterized and selected different
processing methods have been shown to affect both the
cowpea genotypes for yield and yield-related traits to
quality and quantity of available phytochemicals.
improve the genetic potential of this crop that will
Previous studies have reported a decrease in the antho-
improve the food security and increase the income of
cyanin and total phenolic content (TPC) in thermally
the communities within rural areas. Bressani (1985) noted
processed corn, but the thermal processing may have
that the nutritional profile of cowpea is similar to that of
been responsible for the release of bound phenolic
common beans; however, cowpea has lower levels of

CONTACT John Mellem [email protected] Department of Biotechnology and Food Technology, Durban University of Technology, PO Box 1334, Durban
4000, South Africa
© 2017 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.
392 M. MTOLO ET AL.

acids (Dewanto, Wu, Adom, & Liu, 2002). Other studies Simulated gastrointestinal digestion model
have also shown thermal processing to significantly
Simulated gastrointestinal digestion was carried out accord-
decrease the TPC, anthocyanin content, and antioxidant
ing to methods defined by Gil-Izquierdo, Zafrilla, and Tomás-
activity (Hiemori, Koh, & Mitchell, 2009). TPC compounds
Barberán (2002) with minor modifications. The process
are known to act as antioxidants, delaying the formation
involves three consecutive phases: the first two phases are
of free radicals or reducing reactive oxygen species,
saliva and gastric digestion, to mimic the mouth and the
which may lead to deterioration of biological molecules
gastric conditions, respectively, followed by digestion with
(Fereidoon Shahidi, 1997). Bermúdez-Soto, Tomás-
bile salts and pancreatin, which mimics the intestinal diges-
Barberán, and García-Conesa (2007) found that digestion
tion process. For the first two digestion phases, 10 g of
might be responsible for an alteration to the composi-
cowpea sample (raw and processed, respectively) was
tion and levels of TPC. This amplifies the importance to
mixed with 6 ml of synthetic saliva comprising KCl (89.6 g/
investigate the effect of digestion on the antioxidant
L), KSCN (20 g/L), NaH2PO4 (88.8 g/L), Na2SO4 (57 g/L), NaCl
capacity of cowpea. The effect of simulated gastrointest-
(175.3 g/L), NaHCO3 (84.7 g/L), urea (25 g/L), and 290 mg of
inal digestion on TPC and antioxidant capacity of cooked
α-amylase [Sigma-Aldrich (5 kU)]. The pH of this solution was
cowpea varieties was investigated by Hachibamba,
adjusted to 6.8 with 0.1 N NaOH. The cowpea and synthetic
Dykes, Awika, Minnaar, and Duodu (2013), who found
saliva mixture were then placed in stomacher bags contain-
that the TPC and radical scavenging property of cowpea
ing 40 ml of distilled water and homogenized using a sto-
was reduced upon cooking, but increased with simulated
macher for 30 s. To this mixture, 0.5 g of pepsin [Sigma-
enzyme digestion. From previous literature, it can be
Aldrich (250 U/mg)] dissolved in 25 mL of 0.1 N HCl was
concluded that in vitro digestion affects TPC and antiox-
added. The mixture was then adjusted to a pH of 2 using 6 N
idant activity. In a study by Faller, Fialho, and Liu (2012),
HCl, and incubated in a 37°C orbital shaker at 250 rpm for
the TPC and flavonoid content in feijoada (beef and pork
2 h. Following this procedure the intestinal digestion phase
stewed with beans) were unaffected by digestion, how-
was simulated. The pH was increased to 6.5 using 0.5 N
ever the antioxidant activity was higher before digestion
NaHCO3, and then 5 mL of (1:1; v/v) pancreatin (8 mg/mL)
than afterwards. In vitro digestion of white-bread sam-
[Sigma-Aldrich] and bile salts (50 mg/mL), dissolved in 20 mL
ples was also found to increase antiradical activity
of water, were added and incubated in a 37°C orbital shaker
(Gawlik-Dziki et al., 2013). In the present study, an in
at 250 rpm for 2 h.
vitro model of the gastrointestinal tract was used to
simulate the digestion process to assess any changes in
the antioxidant activity of extracts from raw and pro-
cessed cowpea samples, in order to evaluate the impact Determination of total phenolic content (TPC)
of digestion on the TPC and antioxidant capacity.
TPC was determined using the Folin–Ciocalteu assay as
noted by Hachibamba et al. (2013), briefly 1 ml of the sample
(1, 20, 40, 60, 80, 100, 250, and 500 μg/ml respective sample
Materials and methods concentrations) was reacted with 0.4 ml Folin–Ciocalteu
reagent and 0.9 ml of 0.5 M ethanolamine for 20 min at
Sample preparation
25°C. The absorbance was measured at 765 nm against a
Samples of five cowpea cultivars (Veg Cowpea 2, Veg reagent blank (70% aqueous acetone). A calibration curve
Cowpea 3, Makhathini, Embu buff, and Glenda) were was generated using a standard gallic acid solution
obtained from the Agricultural Research Council- (R2 = 0.9913). The TPC values were expressed as milligrams
Vegetable and Ornamental Plant Institute (ARC-VOPI), of gallic acid equivalents (GAE)/g of cowpea sample (dry
Pretoria, South Africa. Plants were grown at the Research basis).
Farm of ARC-VOPI [25.604S 28.345E], during the 2014/2015
cropping seasons at an altitude of 1168 m above sea level.
The location received approximately 610 mm of rain dur-
ing the growing period with a minimum and maximum Determination of antioxidant capacity
recorded temperature of 9.11°C and 36.37°C, respectively, Free radical scavenging capacity (DPPH)
during the growth period. Legumes from each of the The DPPH (1,1-diphenyl-2-picryl-hydrazyl) free radical
cultivars were subjected to two different processing meth- scavenging capacity was determined by using a method by
ods viz. boiling and pressure-cooking. Pretreatment Oboh (2006). 1 ml aliquots of samples (1, 20, 40, 60, 80, 100,
involved soaking 50 g of each of the respective cultivars 250, and 500 μg/ml) were mixed with 1 ml, 0.3 mM metha-
for 24 h at 25°C in a ratio of 1:10 w/v, the water was then nol solution containing 1.5 mM DPPH solution. The mixture
decanted and the cowpea samples subjected to the dif- was left in the dark at 25°C for 30 min before determining
ferent processing techniques according to Sagratini et al. the absorbance at 516 nm. Rutin (1 mM) was used as a
(2013); Boiling: Pretreated cowpea samples were weighed positive control.
and boiled for 30 min in distilled water (1:20 w/v) in a pot. DPPH scavenging capacity (%) = (absorbance sample/
The cooking liquids and cowpea were separated by filtra- absorbance control) × 100.
tion for simulated gastric digestion and pressure-cooking: The scavenging effect of the samples was expressed as
Pretreated cowpea samples (1:5 w/v) were subjected to 50% effective concentration (EC50), which represented the
autoclaving for 20 min at 120°C. The cooking liquid and concentration of sample having 50% DPPH radical scaven-
sample were separated by filtration for simulated gastric ging effect.
digestion.
 CYTA – JOURNAL OF FOOD 393

ABTS (2,2’-azinobis-(3-ethylbenzothiazoline-6-sulfonic Results and discussion

acid) free radical scavenging assay
An in vitro gastrointestinal model was used in this study
The total antioxidant activity of cowpea extracts was mea-
to mimic the in vivo physiological environment. TPC was
sured by the ABTS radical cation decolorization assay
found to be high in the raw samples of all the V. ungui-
containing preformed ABTS radical cation, according to
culata cultivars, however after processing a significant
methods by Siddhuraju (2006). ABTS was generated by
reduction was observed. This may be attributed to the
mixing 1000 ml of 1.7 mM ABTS with 18 ml of 47 mM
fact that the raw cowpea samples had the full variety of
potassium per sulfate in the dark at 25°C for 16 h. The
the phenolic compounds found in the cultivars as noted in
solution was diluted to a ratio of 1:15 (deionized water)
a study by Nderitu, Dykes, Awika, Minnaar, and Duodu
and the absorbance was measured at 734 nm. The ABTS
(2013). The reduction in TPC observed in the cowpea
radical cation scavenging activity of the samples was then
cultivars after processing may be due to the leaching of
assessed by mixing 3 ml ABTS solution (absorbance of
phenolic compounds into the cooking water which was
0.7 ± 0.05) with 1 ml of sample (1, 20, 40, 60, 80, 100,
subsequently discarded. In a study by Siddhuraju and
250, and 500 μg/ml). Ascorbic acid (1 μg/ml) was used as
Manian (2007), it was found that during processing phe-
a positive control. Scavenging capacity was calculated by
nolic compounds had the ability to form complexes with
the following formula: % of scavenging = ((Ao−A1)/
proteins as well as carbohydrates consequently becoming
Ao) × 100, where Ao is absorbance of control and A1 is
unextractable. Oxidation of other phenolic compounds
absorbance of sample.
during cooking may have also occurred since different
compounds have different levels of susceptibility to oxida-
Ferric reducing antioxidant power (FRAP) assay tion due to their chemistry. There was no significant dif-
The ferric reducing antioxidant power (FRAP) assay was ference observed in the TPC of boiled against the
performed as described by Benzie and Szeto (1999). The pressure-cooked samples across all the cultivars. With
FRAP reagent was prepared as follows: A – Acetate buffer regard to samples that underwent simulated digestion,
300 mM (pH 3.6); B – TPTZ (2,4,6-tripyridyl-s-triazine) 10 mM the TPC of all digested samples was increased in both
in 40 mM HCl; and C – FeCl3 6H2O (M.W. 270.30) 20 mM. The the processed samples and those subjected to simulated
working FRAP reagent was prepared by mixing solutions A, digestion (Figure 1). However, the difference was only
B, and C in the ratio of 10:1:1 at the time of use. To measure significant in pressure-cooked vs. pressure-cooked
the FRAP, 1 ml of the sample (1, 20, 40, 60, 80, 100, 250, and digested samples (p ≤ 0.05). There was no significant
500 μg/ml) was mixed with 3 ml of working FRAP reagent difference that was observed in TPC between pressure-
and the absorbance (593 nm) was measured at 0 min after cooked samples and those subjected to simulated diges-
mixing. Thereafter, a sample was placed at 37°C in water tion across all the cultivars with the exception of Veg
bath for 4 min and the absorbance was again measured. Cowpea 3. The conditions generated in the course of
Ascorbic acid (1000 μM) was used as a positive control. simulated gastrointestinal digestion due to the enzymes
used as well as the pH conditions encourage macromole-
Total peroxyl radical-trapping antioxidant parameter cules to undergo hydrolysis, these macromolecules include
(TRAP) proteins, which may be bound to phenolic compounds.
The radical-trapping antioxidant parameter (total TRAP) was This binding of phenolic compounds with macromolecules
determined according to the methods by Cao, Alessio, and has the ability to be liberated therefore becoming more
Cutler (1993) and Ioannone et al. (2015). Briefly, the final extractable in the enzyme digestion (Hachibamba et al.,
reaction mixture of 100 ml for the assay contained 0.43 mg 2013). Components of the food matrix may also contribute
R-phycoerythrin (target probe) and 0.3 g AAPH (peroxyl significantly to improvement of the stability of the phe-
radicals) in 50 mM phosphate buffer (pH = 7.4). A final nolic compounds in various pH conditions of simulated
volume of 2 ml was used with 1 ml diluted sample (1, 20, gastrointestinal digestion. In a study by Ortega, Macià,
40, 60, 80, 100, 250, and 500 μg/ml of cowpea sample). Once Romero, Reguant, and Motilva (2011) on carob flour, it
the sample was mixed, the reaction mixture was incubated was reported that the soluble dietary fiber as well as the
at 37°C for 5 min. Fluorescence was measured in a quartz lipids, were found to bring about an improvement in the
cuvette at the emission of 565 nm and excitation of 540 nm stability of phenolic compounds in the course of in vitro
using a fluorescence spectrophotometer [Varian (Cary duodenal digestion. Overall, there was a trend in TPC,
Eclipse) Fluorescence Spectrophotometer]. Ascorbic acid which displayed a directly proportional result with regard
was used to develop a standard curve and TRAP values to the antioxidant assays of all the cultivars. Results show
were calculated using a calibration curve obtained from that readings were high on the raw samples and subse-
increasing concentrations of Trolox. TRAP results were quently decreased upon processing, however an increase
expressed as μmol of Trolox equivalents per g (dry weight). was observed after samples were subjected to simulated
digestion. This trend may be attributed to the fact that
phenolic compounds are mainly responsible for the anti-
Statistical analysis oxidant activity of the samples, therefore the higher the
TPC, the higher the antioxidant activity (Brewer, 2011;
All experiments were carried out in triplicate and data
Shahidi, 2006).
expressed as mean ± standard deviation (SD). Experimental
The free radical scavenging capacity values were
data were analyzed using two-way analysis of variance and
found to be significantly high for all the raw samples
Tukey’s multiple comparison tests of means. A level of
(Figure 2(a)). The percentage DPPH values in the raw
p ≤ 0.05 was considered significant. Statistical computations
samples of ranged from concentrations 60–500 µg/ml
and analyses were carried out using GraphPad Prism.
394 M. MTOLO ET AL.

Figure 1. Total phenol content (TPC) Vigna unguiculata cultivars before processing, after processing, and processed and subjected to simulated digestion (a –
Raw, b – boiled, c – pressure-cooked, d – boiled + digested, and e – pressure-cooked + digested). All values are expressed in micrograms of gallic acid
equivalents per gram (μg GAE/g). Bars denote mean ± standard deviation (n = 3) [* = p ≤ 0.05; ** = p ≤ 0.01].

Figura 1. Contenido total de fenoles (TPC) de cultivos de Vigna unguiculata antes del procesado, después del procesado, procesadas y sujetas a digestión
estimulada (a – cruda, b – hervida, c – cocida a presión, d -hervida + digerida y e – cocida a presión + digerida). Todos los valores se expresan en microgramos
de ácido gálico equivalentes por gramo (μg GAE/g). Las barras denotan promedio ± desviación estándar (n = 3) [* = p ≤ 0,05; ** = p ≤ 0,01].

showing no significant difference between cultivars or cooked vs. pressure-cooked (digested) and boiled vs.
concentrations, however at concentrations 1–40 µg/ml boiled (digested) showed a significant difference
there was a significant difference between both the cul- (p ≤ 0.05). The DPPH values of pressure-cooked
tivars and the concentrations (p ≤ 0.05). The percentage (digested) and boiled (digested) were found not to be
radical scavenging capacity of processed samples significantly different (p ≤ 0.0001) (Figure 2(d and e)).
(Figure 2(b and c)) was reduced when compared with However, these values were increased after the samples
the raw samples (Figure 2(a)). Although there was no were subjected to simulated digestion post processing.
significant difference that was found between the pres- When comparing processed and raw samples, the unpro-
sure-cooked and boiled samples across all the cultivars, cessed samples had a higher free radical scavenging
there was a significant difference between the raw and capacity which may be attributed to a high amount of
pressure-cooked samples (p ≤ 0.05) apart from Veg free radical reducing phenolic compound present which
Cowpea 2. However, only two cultivars (Glenda and are found not to be stable upon cooking due to the heat
Makathini) displayed significant difference between raw treatment i.e. pressure-cooking as well boiling. The DPPH
and boiled samples (p ≤ 0.05). With respect to the sam- values in the raw samples amongst different concentra-
ples that underwent simulated digestion, the percentage tions (60–500 µg/ml) had high values and did not vary
radical scavenging capacity of all digested samples significantly for concentrations ranging from 1–40 µg/ml.
increased in both pressure-cooked and boiled samples This lack of variation may be due to raw samples having
as shown in Figure 2(d and e). The increase in the a high amount of reactive compounds that react with
DPPH values of digested samples was such that there DPPH radical even in low concentrations, resulting in
was no significant difference from those found in the high readings. In previous studies, compound such as
raw samples across all the cultivars. Both pressure- carotenoids, have been found to interfere with test
 CYTA – JOURNAL OF FOOD 395

Figure 2. DPPH radical scavenging capacity of Vigna unguiculata cultivars at concentrations 1–500 µg/mL before processing, after processing and processed and
subjected to simulated digestion (a – Raw, b – boiled, c – pressure-cooked, d – boiled + digested, and e – pressure-cooked + digested). Bars denote mean ±
standard deviation (n = 3) with Rutin (1 mM) serving as a positive control [* = p ≤ 0.05; ** = p ≤ 0.01].

Figura 2. Capacidad de barrido de radicales DPPH de los cultivos de Vigna unguiculata a concentraciones de 1–500 µg/mL antes del procesado, después del
procesado, procesadas y sujetas a digestión estimulada (a – cruda, b – hervida, c – cocida a presión, d – hervida + digerida y e – cocida a presión + digerida).
Las barras denotan promedio ± desviación estándar (n = 3) con Rutina (1 mM) como control positivo [* = p ≤ 0,05; ** = p ≤ 0,01].

results (Rajamanikandan et al., 2011). The percentage conditions), since small molecules are found to have
DPPH values of processed samples (Figure 2(b and c)) better access to the radical site of DPPH.
were reduced when compared with the raw samples FRAP of cowpea samples were found to be significantly
(Figure 2(a)). This may be attributed to a decrease in high for all the raw samples as shown in Figure 3(a).
reducing agents that had high availability of atoms, Across the concentration range (1–500 µg/ml), Veg
which can donate electrons therefore reacting with Cowpea 3 was observed to have the highest percentage
DPPH free radicals and consequently causing a conver- FRAP values in contrast to Veg Cowpea 2 which had the
sion of these free radicals into stable compounds that lowest. The percentage FRAP values of processed samples
will end the radical chain activity (Rajamanikandan et al., (Figure 3(b) and C) were found to be reduced when
2011). After simulated digestion, the percentage DPPH compared with the raw samples (Figure 3(a)). Although
values of all digested samples increased in both pres- there was no significant difference found between pres-
sure-cooked and boiled samples exposed to simulated sure-cooked and boiled samples across all the cultivars,
digestion (Figure 2(d and e)). This may be attributed to there was however a significant difference between raw
the DPPH color being lost through radical reaction or and processed samples (pressure-cooked samples and
reduction, as well as other unrelated reactions that boiled samples) (p ≤ 0.05). With regard to samples that
might have also occurred. Simulated digestion may also underwent simulated digestion, FRAP values of all
have increased the steric accessibility for the DPPH to digested samples were increased in both pressure-cooked
react with the compounds (Pyrzynska & Pękal, 2013). This vs. pressure-cooked and digested; as well as boiled vs.
could have occurred by decreasing the size of molecules boiled and digested as shown in Figure 3(d and e). The
into smaller portions (due to enzymatic action or pH increase in FRAP values of digested samples was such that
396 M. MTOLO ET AL.

Figure 3. Results of FRAP of Vigna unguiculata cultivars at concentrations 1–500 µg/mL before processing, after processing, and processed and subjected to
simulated digestion (a – Raw, b – boiled, c – pressure-cooked, d – boiled + digested, and e – pressure-cooked + digested). Bars denote mean ± standard
deviation (n = 3) with ascorbic acid (10 mM µg/mL) serving as a positive control [* = p ≤ 0.05; ** = p ≤ 0.01].

Figura 3. Resultados de FRAP de cultivos de Vigna unguiculata a concentraciones de 1–500 µg/mL antes del procesado, después del procesado, procesadas y
sujetas a digestión estimulada (a – cruda, b – hervida, c – cocida a presión, d – hervida + digerida y e – cocida a presión + digerida). Las barras denotan
promedio ± desviación estándar (n = 3) con ácido ascórbico (10 mM µg/mL) como control positivo [* = p ≤ 0,05; ** = p ≤ 0,01].

there was no significant difference from those found in samples as well as raw vs. boiled cooked samples
raw samples across cultivars. However, FRAP values were (p ≤ 0.05). With regard to samples that underwent simu-
observed to increase after the samples were subjected to lated digestion, the ABTS values of all digested samples
simulated digestion post processing. The FRAP values in were found to increase in both pressure-cooked vs. pres-
the raw samples of Glenda amongst different concentra- sure (digested) and boiled cooked vs. boiled (digested) as
tions (1–500 µg/ml) were observed to be higher in com- shown in Figure 4(d and e). The increase in ABTS values of
parison to raw samples of other cultivars. In previous digested samples was such that they were not signifi-
studies, it was found that lipid-soluble antioxidants were cantly different from those found in raw samples across
responsible for total protection to the target probe (R- all the cultivars. Both pressure-cooked vs. pressure
phycoerythrin), which may have caused the other cultivars (digested) and boiled cooked vs. boiled (digested) were
to have a lower reading when compared to Glenda (Cao significantly different (p ≤ 0.05). The ABTS values of pres-
et al., 1993). sure (digested) and boiled (digested) were found not to
The capacity of cowpea samples to scavenge ABTS free be significantly different from each other as shown in
radicals was found to be significantly high for all the raw Figure 4(d and e). However, ABTS values were observed
samples as shown in Figure 4(a). The percentage ABTS to increase after the samples were subjected to simulated
values of processed samples (Figure 4(b and c)) were digestion post processing. The change in pH conditions
reduced when compared with the raw samples. A similar during simulated digestion could be the main reason for
trend to the DPPH assay results was seen with no signifi- an increase in the antioxidant capability in this test due to
cant difference found between pressure-cooked vs. boiled the electron transfer being facilitated at an acidic pH in
cooked samples across all the cultivars. There was how- this assay. Thermodynamics may also have had an influ-
ever a significant difference in raw vs. pressure-cooked ence since a compound could reduce ABTS if it possesses
 CYTA – JOURNAL OF FOOD 397

Figure 4. ABTS radical action of Vigna unguiculata cultivars at concentrations 1–500 µg/mL before processing, after processing, and processed and subjected to
simulated digestion (a – Raw, b – boiled, c – pressure-cooked, d – boiled + digested, and e – pressure-cooked + digested). Bars denote mean ± standard
deviation (n = 3) with ascorbic acid (10 mM µg/mL) serving as a positive control [* = p ≤ 0.05; ** = p ≤ 0.01].

Figura 4. Acción de radical ABTS de cultivos de Vigna unguiculata a concentraciones de 1–500 µg/mL antes del procesado, después del procesado, procesadas y
sujetas a digestión estimulada (a – cruda, b – hervida, c – cocida a presión, d – hervida + digerida y e – cocida a presión + digerida). Las barras denotan
promedio ± desviación estándar (n = 3) con ácido ascórbico (10 mM µg/mL) como control positivo [* = p ≤ 0,05; ** = p ≤ 0,01].

a redox potential, which is below that of ABTS (0.68 V). (digested) as shown in Figure 5(d and e). In both pres-
Most phenolic compounds were found to have a redox sure-cooked vs. pressure (digested) and boiled cooked vs.
potential, which is lower than that of ABTS; therefore can boiled (digested), there was a significant difference.
be involved in a reaction with ABTS (Rajamanikandan Overall, the trend was similar with the observations
et al., 2011). made in the TPC determination results, FRAP assay,
The potential of cowpea samples to restrict the reac- DPPH assay as well as the ABTS assay; the TRAP values
tion between peroxyl radicals generated by AAPH and a of all the cultivars were high for raw samples and then
target probe (R-phycoerythrin) was expressed in terms of decreased upon processing. However, these values were
TRAP values as shown in Figure 5. The values were found observed to increase after the samples were subjected to
to be significantly high for all the raw samples as shown simulated digestion post processing. The reduction of
in Figure 5(a) with values for the raw samples of Glenda at TRAP values upon processing may possibly be explained
concentrations 1–500 µg/ml observed to be higher in by the decrease of flavanols as well as proanthocyanidins
comparison to other cultivars. This is similar to finding (Ioannone et al., 2015). With regard to the samples that
for the ABTS assay where no significant difference was underwent simulated digestion, the TRAP values of all
found in pressure-cooked vs. boiled cooked samples digested samples increased in both pressure-cooked vs.
across all the cultivars, however there was a significant pressure (digested) and boiled cooked vs. boiled
difference in raw vs. pressure-cooked as well as raw vs. (digested). The increase of TRAP values observed in the
boiled cooked samples. With regards to the samples that digested samples may be attributed to the development
underwent simulated digestion, the TRAP values of all of proanthocyanidins, which possess high molecular
digested samples increased in both pressure-cooked vs. weight as well as higher reducing power (Di Mattia
pressure (digested) and boiled cooked vs. boiled et al., 2013).
398 M. MTOLO ET AL.

Figure 5. Total Radical-Trapping Antioxidant Parameter (TRAP) value of Vigna unguiculata cultivars at concentrations 1–500 µg/mL before processing, after
processing, and processed and subjected to simulated digestion (a – Raw, b – boiled, c – pressure-cooked, d – boiled + digested, and e – pressure-cooked +
digested). Bars denote mean ± standard deviation (n = 3) with ascorbic acid (10 µg/mL) serving as a positive control [* = p ≤ 0.05; ** = p ≤ 0.01].

Figura 5. Valor del Parámetro antioxidante total capturador de radicales (TRAP) de cultivos de Vigna unguiculata a concentraciones de 1–500 µg/mL antes del
procesado, después del procesado, procesadas y sujetas a digestión estimulada (a – cruda, b – hervida, c – cocida a presión, d – hervida + digerida y e – cocida a
presión + digerida). Las barras denotan promedio ± desviación estándar (n = 3) con ácido ascórbico (10 µg/mL) como control positivo [* = p ≤ 0,05; ** = p ≤ 0,01].

Conclusion Disclosure statement

In conclusion, processing and simulated digestion were found to No potential conflict of interest was reported by the authors.
significantly affect the TPC and the antioxidant activity for the V.
unguiculata cultivars. The changes in the overall antioxidant
activity of processed cowpea may be attributed to cumulative Funding
factors, such as the leaching of water-soluble antioxidant com-
This work was supported by the the National Research Foundation of
pounds and the formation or breakdown of antioxidants during South Africa [Grant Number 93988].
processes. Processing reduced the TPC and antioxidant proper-
ties of the cowpea cultivars, however these parameters were
found to increase upon simulated digestion. This implies that ORCID
products of enzyme digestion, such as phenolics, may possess John Mellem http://orcid.org/0000-0002-4784-699X
bioactivity and have the potential to scavenge reactive oxygen
species thereby potentially defending the human body against
chronical diseases as a result of excessive free radicals formed. References
Benzie, I.F., & Szeto, Y. (1999). Total antioxidant capacity of teas by the
Acknowledgments ferric reducing/antioxidant power assay. Journal of Agricultural and
Food Chemistry, 47(2), 633–636. doi:10.1021/jf9807768
The authors acknowledge the Agricultural Research Council-Vegetable Bermúdez-Soto, M.-J., Tomás-Barberán, F.-A., & García-Conesa, M.-T.
and Ornamental Plant Institute (ARC-VOPI), Pretoria, South Africa for (2007). Stability of polyphenols in chokeberry (Aronia melanocarpa)
providing the cowpea samples and the National Research Foundation subjected to in vitro gastric and pancreatic digestion. Food Chemistry,
of South Africa [grant number 93988] for support. 102(3), 865–874. doi:10.1016/j.foodchem.2006.06.025
 CYTA – JOURNAL OF FOOD 399

Bressani, R. (1985). Nutritive value of cowpea. In S.R. Singh & K.O. Rachie Knekt, P., Kumpulainen, J., Järvinen, R., Rissanen, H., Heliövaara, M.,
(Eds.), Cowpea research, procuction and utilization (pp. 353–359). New Reunanen, A., et al. (2002). Flavonoid intake and risk of chronic
York: Wiley. diseases. The American Journal of Clinical Nutrition, 76(3), 560–568.
Brewer, M. (2011). Natural antioxidants: Sources, compounds, mechan- Nderitu, A.M., Dykes, L., Awika, J.M., Minnaar, A., & Duodu, K.G. (2013).
isms of action, and potential applications. Comprehensive Reviews in Phenolic composition and inhibitory effect against oxidative DNA
Food Science and Food Safety, 10(4), 221–247. doi:10.1111/ damage of cooked cowpeas as affected by simulated in vitro gastro-
crf3.2011.10.issue-4 intestinal digestion. Food Chemistry, 141(3), 1763–1771. doi:10.1016/j.
Cao, G., Alessio, H.M., & Cutler, R.G. (1993). Oxygen-radical absorbance foodchem.2013.05.001
capacity assay for antioxidants. Free Radical Biology and Medicine, 14 Oboh, G. (2006). Antioxidant properties of some commonly consumed
(3), 303–311. doi:10.1016/0891-5849(93)90027-R and underutilized tropical legumes. European Food Research and
Dewanto, V., Wu, X., Adom, K.K., & Liu, R.H. (2002). Thermal processing Technology, 224(1), 61–65. doi:10.1007/s00217-006-0289-x
enhances the nutritional value of tomatoes by increasing total anti- Ortega, N., Macià, A., Romero, M.-P., Reguant, J., & Motilva, M.-J. (2011).
oxidant activity. Journal of Agricultural and Food Chemistry, 50(10), Matrix composition effect on the digestibility of carob flour phenols
3010–3014. doi:10.1021/jf0115589 by an in-vitro digestion model. Food Chemistry, 124(1), 65–71.
Di Mattia, C., Martuscelli, M., Sacchetti, G., Scheirlinck, I., Beheydt, B., doi:10.1016/j.foodchem.2010.05.105
Mastrocola, D., et al. (2013). Effect of fermentation and drying on Pyrzynska, K., & Pękal, A. (2013). Application of free radical diphenylpicryl-
procyanidins, antiradical activity and reducing properties of cocoa hydrazyl (DPPH) to estimate the antioxidant capacity of food samples.
beans. Food and Bioprocess Technology, 6(12), 3420–3432. Analytical Methods, 5(17), 4288–4295. doi:10.1039/c3ay40367j
doi:10.1007/s11947-012-1028-x Rajamanikandan, S., Sindhu, T., Durgapriya, D., Sophia, D., Ragavendran, P.,
Faller, A.L.K., Fialho, E., & Liu, R.H. (2012). Cellular antioxidant activity of & Gopalakrishnan, V. (2011). Radical scavenging and antioxidant activity
feijoada whole meal coupled with an in vitro digestion. Journal of of ethanolic extract of Mollugo nudicaulis by in vitro assays. Indian
Agricultural and Food Chemistry, 60(19), 4826–4832. doi:10.1021/ Journal of Pharmaceutical Education and Research, 45(4), 310–316.
jf300602w Sagratini, G., Caprioli, G., Maggi, F., Font, G., Giardinà, D., Mañes, J., et al.
Gawlik-Dziki, U., Świeca, M., Dziki, D., Baraniak, B., Tomiło, J., & Czyż, J. (2013). Determination of soyasaponins I and βg in raw and cooked
(2013). Quality and antioxidant properties of breads enriched with legumes by solid phase extraction (SPE) coupled to liquid chromato-
dry onion (Allium cepa L.) skin. Food Chemistry, 138(2), 1621–1628. graphy (LC)–mass spectrometry (MS) and assessment of their bioac-
doi:10.1016/j.foodchem.2012.09.151 cessibility by an in vitro digestion model. Journal of Agricultural and
Gil-Izquierdo, A., Zafrilla, P., & Tomás-Barberán, F.A. (2002). An in vitro Food Chemistry, 61(8), 1702–1709. doi:10.1021/jf304136g
method to simulate phenolic compound release from the food Shahidi, F. (1997). Natural antioxidants: Chemistry, health effects, and appli-
matrix in the gastrointestinal tract. European Food Research and cations. Champaign, IL: The American Oil Chemists Society. AOCS Press.
Technology, 214(2), 155–159. doi:10.1007/s00217-001-0428-3 Shahidi, F. (2006). Functional foods: Their role in health promotion and
Hachibamba, T., Dykes, L., Awika, J., Minnaar, A., & Duodu, K.G. (2013). disease prevention. Journal of Food Science, 69(5), R146–R149.
Effect of simulated gastrointestinal digestion on phenolic composi- doi:10.1111/j.1365-2621.2004.tb10727.x
tion and antioxidant capacity of cooked cowpea (Vigna unguiculata) Siddhuraju, P. (2006). The antioxidant activity and free radical-scaven-
varieties. International Journal of Food Science & Technology, 48(12), ging capacity of phenolics of raw and dry heated moth bean (Vigna
2638–2649. doi:10.1111/ijfs.12260 aconitifolia)(Jacq.) Marechal seed extracts. Food Chemistry, 99(1), 149–
Hiemori, M., Koh, E., & Mitchell, A.E. (2009). Influence of cooking on 157. doi:10.1016/j.foodchem.2005.07.029
anthocyanins in black rice (Oryza sativa L. japonica var. SBR). Journal Siddhuraju, P., & Manian, S. (2007). The antioxidant activity and free
of Agricultural and Food Chemistry, 57(5), 1908–1914. doi:10.1021/ radical-scavenging capacity of dietary phenolic extracts from horse
jf803153z gram (Macrotyloma uniflorum (Lam.) Verdc.) seeds. Food Chemistry,
Ioannone, F., Di Mattia, C.D., De Gregorio, M., Sergi, M., Serafini, M., & 105(3), 950–958. doi:10.1016/j.foodchem.2007.04.040
Sacchetti, G. (2015). Flavanols, proanthocyanidins and antioxidant Wang, S., Melnyk, J.P., Tsao, R., & Marcone, M.F. (2011). How natural
activity changes during cocoa (Theobroma cacao L.) roasting as dietary antioxidants in fruits, vegetables and legumes promote vas-
affected by temperature and time of processing. Food Chemistry, cular health. Food Research International, 44(1), 14–22. doi:10.1016/j.
174, 256–262. doi:10.1016/j.foodchem.2014.11.019 foodres.2010.09.028