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British Journal of Healthcare and Medical Research - Vol. 9, No. 2
Publication Date: April, 25, 2022
DOI:10.14738/jbemi.92.11955. Mbaiogaou, A., Mbaihougadobe, S., Madjitoloum-Betoloum, S., Naitormbaide, M., & Djadda, M. A. (2022). Phenolic Compound
Content of a Plant of the Genus Vigna From Chad: Vigna Unguiculata (Fabaceae). British Journal of Healthcare and Medical
Research, 9(2). 98-107.
Services for Science and Education – United Kingdom
Phenolic Compound Content of a Plant of the Genus Vigna From
Chad: Vigna Unguiculata (Fabaceae)
Abel MBAIOGAOU
Laboratory of Research on Natural Substances, Faculty of
Pure and Applied Sciences (F.P.A.S.), University of N'Djamena, Chad
Severin MBAIHOUGADOBE
Laboratory of Research on Natural Substances, Faculty of
Pure and Applied Sciences (F.P.A.S.), University of N'Djamena, Chad
Salmon MADJITOLOUM BETOLOUM
Laboratory of Research on Natural Substances, Faculty of
Pure and Applied Sciences (F.P.A.S.), University of N'Djamena, Chad
Michel NAITORMBAIDE
Chadian Institute of Agronomic Research for
Development (CIARD), Bebedja, Chad
DJADDA Mouta Adji
Laboratory of Research on Natural Substances, Faculty of
Pure and Applied Sciences (F.P.A.S.), University of N'Djamena, Chad
ABSTRACT
Nine (09) varieties of Vigna unguiculata provided by the CIARD of Bebedja city in
Chad were studied for their antioxidant activities and their contents in total
polyphenols, total anthocyanins and total flavonoids. The measurement of the
antioxidant capacity of the total extracts was estimated by the DPPH test. The
contents of total polyphenols, total flavonoids and total anthocyanins in the seed
extracts were determined by the Folin Ciocalteu, aluminum trichloride (AlCl3) and
pH-differential methods respectively. From this study, three (3) varieties rich in
antioxidants were found: NDJAM-2, TN5-78 and NDJAM-1 with respective contents
of 3.14 mg QE/g, 3.113 mg QE/g and 3.07 mg QE/g of dry seeds. These varieties were
also rich in total polyphenols with contents of 4.457 mg GAE/g, 4.095 mg GAE/g and
3.875 mg GAE/g of dry seeds respectively. The varieties TN5-78, VYA, IT99K573-1-
1 are the best in total flavonoids with values of 0.744 mg QE/g, 0.712 mg QE/g and
0.579 mg QE/g of dry seeds respectively. Total anthocyanins are generally low in
most varieties except TN5-78 and N'DJAM-2 which have 0.161 mg/g and 0.133 mg/g
respectively.
Keywords: Vigna unguiculata, Total polyphenols, Total flavonoids, Total anthocyanins,
Total antioxidant activity, Chad.
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Mbaiogaou, A., Mbaihougadobe, S., Madjitoloum-Betoloum, S., Naitormbaide, M., & Djadda, M. A. (2022). Phenolic Compound Content of a Plant
of the Genus Vigna From Chad: Vigna Unguiculata (Fabaceae). British Journal of Healthcare and Medical Research, 9(2). 98-107.
URL: http://dx.doi.org/10.14738/jbemi.92.11955
INTRODUCTION
Vigna unguiculata commonly called the black-eyed bean or cowpea in french, loubia in local
Arabic, ngala in Kanembou language, mindji in Ngambaye language, is a species of plants of the
family Fabaceae and of the genus Vigna native to tropical Africa, of which several sub-species
are cultivated as food plants for their seeds or for their pods. It is the main food legume in
tropical Africa, providing an economical source of high protein.
Other legumes of the same genus contain secondaries metabolits that are responsible for the
colors observed in the seeds. These metabolits have antioxidant properties and are very
abundant in fruits and vegetables. They are involved in the prevention of cancer, cardiovascular
diseases and other degenerative diseases related to oxidative stress [1]. Oxidative stress can
cause the oxidation of a large number of biomolecules such as lipids, proteins and DNA, causing
a high risk of inflammatory and cardiovascular diseases, Alzheimer's disease, cataracts [2,3].
To prevent or reduce oxidative stress induced by free radicals, it is necessary to consume a
sufficient amount of antioxidants. Fruits, vegetables and seeds contain antioxidant compounds,
such as polyphenols and carotenoids.
Nowadays, the therapeutic virtues of plants are undergoing a revival thanks to improved
extractive techniques and advances in structural analysis methods for the discovery of new
active principles. It is estimated that two thirds of the current drugs have a natural origin or by
modification of a natural product [4].
Polyphenols are probably the most widespread natural compounds in nature and therefore are
elements that are part of the animal diet. They also have anti-ulcer, anti-carcinogenic, anti- mutagenic and anti-inflammatory activities [5].
Polyphenols play a great role in the nutritional and hygienic quality of food, some of them have
vitamin properties used by the pharmaceutical industry. They are also involved in the
digestibility of food, in the physiological use of proteins (with which tannins combine).
In Africa, where legumes of the genus Vigna, particularly V. unguiculata, are widely consumed,
it is important to have a database on their antioxidant content in order to manage the above- mentioned diseases. Indeed, these data on the contents of total antioxidants constitute
additional nutritional information which will facilitate a better integration and valorization of
V. unguiculata among the foods with functional character following the example of other fruits
and vegetables [6].
Do the seeds of Vigna unguiculata contain polyphenolic compounds and what are their
contents?
This work consists in valorizing nine (9) varieties of raw V. unguiculata collected in Chad as a
source of antioxidants.
MATERIALS AND METHODS
Plant material
The study concerns nine (9) varieties of Vigna unguiculata harvested at the end of October
2020, seven (7) of which were supplied by the Chadian Institute of Agronomic Research for
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British Journal of Healthcare and Medical Research (BJHMR) Vol 9, Issue 2, April - 2022
Services for Science and Education – United Kingdom
Development (CIARD) of Bebedja city in Chad and two (2) of which were purchased at the millet
market in N'Djaména, Chad.
Extraction
The dry seeds of the different varieties of V. unguiculata were crushed. Then, 3 g of powder of
each variety were extracted with 15 mL of methanol-water-acetic acid system (70 : 29.5 : 0.5)
by maceration for 24 hours at 4 °C. The extracts are filtered and the residues are extracted again
twice with 10 mL of solvent for 24 hours. The filtrates are stored in a refrigerator at 4 °C for
determination of total polyphenol and total antioxidant contents [7].
METHODOLOGIES
Measurement of antioxidant capacity of extracts by DPPH-method
To evaluate the antioxidant capacities, we used the DPPH method. The commercial DPPH
radical is dissolved in methanol at a concentration of 0.04 mg/mL and kept at 4 °C protected
from light. To each extract sample (1.5 mL) are added 2 mL of DPPH solution and the
absorbance was read after 10 min at 517 nm. The results are expressed as mg QE/g dry material
[8].
Determination of polyphenolic compounds
The contents of polyphenolic compounds in V. unguiculata extracts were determined by the
method of Folin - Ciocalteu [9]. It consists in reacting 1.05 mL of the studied sample with 5 mL
of Folin's reagent (diluted 10 times). After 8 min, 4 mL of 7.5% (w/v) sodium carbonate are
added. After 30 min of incubation, the absorbance was read at 765 nm. Blanks were prepared
for each variety by replacing Folin's reagent with distilled water. Gallic acid was used as the
standard (Table 1). Results are expressed as mg gallic acid equivalents (GAE)/g dry material.
Determination of total flavonoids contents
The flavonoids in the extracts are estimated by the aluminum trichloride (AlCl3) method, which
is based on the formation of a flavonoid-aluminum complex. The quantification of flavonoids
was done using a calibration curve performed by a standard (quercetin) at different
concentrations under the same conditions as the sample (Table 1). Results are expressed as mg
quercetin equivalent per gram (mg GAE/g) of dry material [10].
4 mL of 2% aluminum trichloride (AlCl3) is added to 4 mL of the sample. The mixture is left to
react for 15 min at room temperature and protected from light. The reading is made at 430 nm.
All experiments were performed in triplicate [10].
Determination of total anthocyanins
The total anthocyanin contents of the extracts were estimated by the pH-differential method
using two buffer systems: potassium chloride (KCl) solution, pH 1.0 (0.025 M) and sodium
acetate (CH3COONa) solution, pH 4.5 (0.4 M).
To 1.2 mL of the extract, 10.8 mL of the corresponding buffers were added and the absorbance
was read against the blank at 510 nm and at 700 nm 15 minutes later. The absorbance A was
calculated as follows:
� = (�!"# − �$##)%&".# − (�!"# − �$##)%&(.! (1)
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Mbaiogaou, A., Mbaihougadobe, S., Madjitoloum-Betoloum, S., Naitormbaide, M., & Djadda, M. A. (2022). Phenolic Compound Content of a Plant
of the Genus Vigna From Chad: Vigna Unguiculata (Fabaceae). British Journal of Healthcare and Medical Research, 9(2). 98-107.
URL: http://dx.doi.org/10.14738/jbemi.92.11955
The monomeric concentration of anthocyanin dyes in the extract was calculated as cyanidin-3-
glucoside [11] :
)*
+ = (
,×./×01×"###
2×3 ) (2)
where A: Absorbance; MW: Molecular Weight (449.2); DF: dilution factor; ε: molar absorptivity
(26900). Total anthocyanin contents are expressed as micrograms of cyanidin-3-glucoside per
gram of dry material.
RESULTS AND DISCUSSION
Standard curves
Table 1. Establishment of standard curves
Calibration
curves
Standard Equations Correlation coefficients
RFC Gallic Acid Y = 36.233x + 0.2911
(3)
R2 = 0.9956
DPPH Quercetin
Quercetin
Y = -25.45x + 0.670 (4) R2 = 0.9950
TFT Y = 14.185x + 0.4992
(5)
R2 = 0.9883
The contents of antioxidants, total polyphenols, total flavonoids and total anthocyanins are
recorded below.
Table 2. Antioxidant (TAO), total polyphenol (TPP), total flavonoid and total anthocyanin (TAT)
contents in the 9 varieties of V. unguiculata seeds by DPPH method
N° Varieties
TAO (mg QE/g) TPP (mg GAE/g) TFT (mg QE/g) TAT (mg/g)
DPPH RFC AlCl3 pH – diff
1 NDJAM-2 3.14±0.006 4.457±0.001 0.037±0.0013 0.133±0.001
2 TN5-78 3.113±0.017 4.095±0.003 0.744±0.003 0.161±0.001
3 NDJAM-1 3.07±0.019 3.875±0.003 0.0036±0.001 0.093±0.001
4 VYA 2.664±0.090 2.457±0.004 0.712±0.002 0.043±0.0006
5 IT99K573-1-1 2.525±0.006 2.105±0.002 0.579±0.026 0.0053±0.0002
6 VITA-5 2.383±0.011 2.033±0.017 0.026±0.002 0.033±0.002
7 IT81D-994 2.357±0.005 1.928±0.012 0.012±0.001 0.026±0.001
8 IT99K573-1-2 2.317±0.0004 1.74±0.004 0.004±0.0013 0.0012±0.00017
9 IT81D-985 2.265±0.0013 1.648±0.012 0.008±0.0006 0.016±0.00011
The contents of total antioxidants (TAO), total polyphenols (TPP), total anthocyanins (TAT),
total flavonoids (TFT) of 9 varieties of V. unguiculata were systematically determined, six of
which are white and three colored.
The differences between the antioxidant, flavonoid, anthocyanin and total polyphenol contents
of these different varieties of V. unguiculata are often very large. This variation in content is a
function of several factors.
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British Journal of Healthcare and Medical Research (BJHMR) Vol 9, Issue 2, April - 2022
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Total antioxidant levels (TAO)
Total antioxidant levels (TAO) were determined by the DPPH method. The results of the
quantitative analysis of total antioxidant levels are shown in Table 2 and Figure 1. The following
three (3) varieties DJAM-1, TN5-78 and NDJAM-2 have an average content of 3.107 mg of TE/g
of dry seeds. They are followed by the varieties VYA, IT99K573-1-1, VITA-5, IT81D-994,
IT99K573-1-2, and IT81D-985 with an average content of 2.418 mg QE/g dry seeds (Table 2,
Figure 1). By this method the richest varieties NDJAM-2, TN5-78 and DJAM-1, all colored. The
nine (9) varieties of V. unguiculata seeds studied have higher antioxidant contents than the
seeds of V subterranea varieties from Chad and Burkina Faso determined by the same method
by Abel en al in 2015 [5].
Figure 1. TAO of different varieties of V. unguiculata seeds (DPPH)
Total polyphenol contents (TPP)
Many previous works have shown that the antioxidant activity of plant extracts is largely due
to the presence of polyphenols.
The results of quantitative analysis of polyphenol contents presented in Table 2 showed that
the seed varieties DJAM-1, TN5-78 and NDJAM-2 are the richest in total polyphenols with values
of 3.875, 4.09 and 4.457 mg TE/g of dry seeds respectively. The above mentioned varieties are
VYA, IT99K573-1-1 and VITA-5 with an average total polyphenol content of 2.198 mg GAE/g
dry seed. The least rich are IT81D-994, IT99K573-1-2 and IT81D-985 with an average
polyphenol content of 1.772 mg GAE/g dry seed.
0
0.5
1
1.5
2
2.5
3
3.5
NDJAM-2
TN5-78
NDJAM-1
VYA
IT99K573-1-1
VITA-5
IT81D-994
IT99K573-1-2
IT81D-985
TAO (mg/g)
DPPH_Quercetin