INTRODUCTION
Cereals can be defined as classes of grass planted
and harvested for food purposes [1]. Different types
of cereals are cultivated all over the world and occupy
an area of about 60%. They have notable benefits
contributing to human health due to nutrients and
biologically active substances in their composition [2–4].
In the present study, sorghum was of our interest.
Sorghum is also called Jowar in India, Guinea corn
in West Africa, and Kaoliang in China. In Nigeria, it
is called Oka by the Yorubas, Dawa by the Hausas,
and Sorghum by the Igbos. There are also other
nomenclatures for sorghum and its role in the food chain
is well documented by Sarwar et al. [5].
Varoquax et al. reported that sorghum is highly
resistant towards drought and heat, which allows
it to flourish and thrive even under hot and arid
environmental conditions [6]. Sorghum is known to
be able to boost blood level. This is of tremendous
importance to human health. For instance, women who
suffer from myoma have anemia due to the excessive
blood losses, especially during menstrual period.
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Sorghum, with its high nutraceutical value, could help
these women [7].
In Africa, where diverse species or cultivars of
sorghum are cultivated, this cereal is served as an
important food crop. In Nigeria, sorghum is classified
into three cultivars depending on the nature and color
of their seminal glands and endosperm, namely Guines,
Kaura, and Farafara [8]. Nonetheless, small-scale
farmers prefer Farafara to Kaura due to the fact that the
former is known to have a better storage behaviour and
attributes.
Sorghum is classified as a tall grass that often grows
to as high as two to eight feet, occasionally being as high
as fifteen feet. Generally, a whitish wax coating covers
the stalks and leaves of sorghum; while specifically,
stalk’ piths of some species are juicy and sweet [9, 10].
A well manured sorghum leaf is around 76 cm long and
5 cm wide. Panicle portion of sorghum is responsible for
the production of tiny flowers which can be from loose
to dense, with clusters containing 800–3000 kernels.
The diversity of species is identifiable by the coloration,
shape, and size of the seeds, which are smaller than
wheat seeds [10].
Statistics in 2016 showed that Nigeria provided 23%
of the total sorghum production in African, which made
Nigeria the largest producer of sorghum in Africa [11].
Mathur et al. documented the emergence of Sorghum
bicolor as a viable option for producing lignocellulosic
biofuel [12]. Vanamala et al. reported that sorghum
contains bioactive compounds that play a crucial role in
its pharmacological potential and immune modulatory
properties [13]. Hassan et al. studied the effect of
ultrasonic waves and microwaves on extraction of the
lipid fraction from sorghum. They revealed that these
techniques increased the oil yield [14].
Since sorghum seed oil and its defatted extracts
are widely used in Africa, the aim of this study was to
evaluate the nutraceutical potential, phytochemical
components, and secondary metabolites of sorghum
from Ota (Nigeria).
STUDY OBJECTS AND METHODS
Sample collection and preparation. Sorghum seeds
were purchased from a local market in Ota, Nigeria.
The seeds were washed, air-dried, and finally dried in
a Thermofisher vacuum oven until constant weight was
achieved. The seeds then were finely powdered with a
mechanical blender. Prior to extraction, the powdered
seeds were protected from sunlight, dust, as well as
other particulate matter to avoid oxidation and microbial
contamination.
Oil extraction. 200 g of the powdered seeds of
sorghum was weighed, carefully wrapped in Whatman
filter paper, and mounted up on a Soxhlet extractor. One
liter of petroleum ether was transferred into a round
bottom flask connected with a thimble with the sample
in. When the extraction process completed, the petether
solvent was removed with an IKA® RV 10 rotary
evaporator, and the sample was stored in a refrigerator.
GC-MS analysis condition. Agilent 7890B
GC/5977 MS was utilized for the GC-MS analysis of
the extract using the given conditions: column – HP 5
capillary (60 m×0.25 mm×0.25 μm); oven temperature
program – the column was held initially at 50°C for
1 min after injection, then ramped to 300°C at 7°C
per minute and held for 14 min; injector temperature –
250°C; detector (MS) temperature – 275°C; carrier gas –
helium; inlet pressure – 40.65 psi; linear gas velocity –
39 cm/s; column flow rate – 2.7 mL/min; split ratio –
10:1; injection volume – 1 μL. The components were
identified by retention time determination on the
capillary column as well as by matching mass spectra
with the data of the NIST mass spectral library.
Phytochemical tests. Terpenoids. 0.30 g of the seed
powder was carefully transferred into a 250 mL beaker
and extracted with 30 mL of chloroform for 2 h. 2 mL
of trichloromethane and 3 mL of concentrated sulphuric
acid were added to 5 mL of the extract, thereby forming
a layer. Reddish brown color at the interface confirmed
the presence of terpenoids [15].
Cardiac glycosides were determined by two
methods. According to the Raymond method, 50%
C2H5OH was gradually added to the extract in a
test-tube, followed by 0.10 mL of 1% ethanolic
m-dinitrobenzene. The resulting mixture then was
titrated with 20% NaOH. Violet coloration confirmed
the presence of active methylene group. According to
the Killer Killiani method, the extract was solubilized
in 1% FeSO4 in 5% glacial acetic acid, followed by the
addition of concentrated H2SO4. The development of
blue coloration indicated the presence of deoxy sugar.
Quinones. Diluted NaOH was added to 1 mL of the
sorghum extracted. Blue green or red coloration implied
the presence of quinones [16].
Saponins. The extract sample was vigorously mixed
with 5 mL of distilled H2O. The frothing was mixed
with few drops of olive oil and shaken vigorously. The
appearance of foam demonstrated the presence of
saponins.
Steroids. 2 mL of acetic anhydride was introduced
into 0.5 mL of the extract, followed by the addition of
2 mL of sulphuric acid. Change in the extract coloration
from violet to blue or green indicated the presence of
steroids [16].
Tannins. 10 mL of bromine water was introduced
into 0.5 g of the extract sample. Decolorization of
Br2/H2O showed the presence of tannins.
Proximate determination. Proximate analysis
was carried out using combination of techniques and
methodologies earlier reported. For instance, crude
protein and moisture contents were determined using the
method by Ajani et al., carbohydrate by Owoeye et al.,
Molisch’s test by Gangwal et al., Biuret test by Suneetha
et al., and total ash by Abdulkadir et al. [2, 3, 17–19].
RESULTS AND DISCUSSION
Sorghum has been identified and rated as the fifth
cereal crop of greatest significance globally [20, 21].
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Sorghum is used in food and feed production, in
wallboards, fences, biodegradable packing material,
as well as for ethanol production [22, 23]. According to
the data in [24], before the outbreak of COVID-19, the
trend in the world sorghum production from 2012 to
2019 fluctuated between 57 to 59 million tons (Fig. 1).
Based on the world sorghum production data, there was
a drastic increase in 2015 which was as a result of the
increaseв sorghum usage by the Chinese in livestock
feed meal. This made them to purchase large volumes of
sorghum from the USA.
In our previous works we studied seed oils and their
extracts, namely from Caryota mitis L., Adenanthera
pavoninalinn L., and sandbox tree (Hura crepitans L.)
[2, 3, 25]. Continuing our research, this work featured
the phytochemical screening, proximate determination,
and GC-MS analysis of extract from sorghum from
Ota (Nigeria) in order to investigate its nutraceutical
potential and add more secondary metabolites to the
organic structure database.
Seeds of sorghum were harvested from the plant. It
was crushed and mounted on Soxhlet extractor to obtain
the oil while the remaining defatted component was
herein referred to as the crude extract. The processing
stages of the sorghum to identify the secondary
metabolites is demonstrated in Figure 2.
Phytochemical screening. The phytochemical
screening of the sorghum extract under study was
performed by using standard methods as reported in
our previous work [25]. The qualitative phytochemical
constituent composition was determined in the sorghum
seed oil obtained with the help of two different solvents:
petroleum ether and ethanol (Table 1). Saponin
availability in the sorghum extracts was established with
foam test and Froth test. In the extract obtained with
petroleum ether saponins were not detected, while the
extract obtained with ethanol contained saponins.
Saponins contain an agent with surface activity
due to the sugar units which are very soluble in water.
Although, the sapogenin units in saponins have high
Figure 1 Statistics of world sorghum production from 2012 to 2019 [24]
Figure 2 Stages of sorghum processing to identify secondary metabolites
50
53
56
59
62
65
68
2012-13 2013-14 2014-15 2015-16 2016-17 2017-18 2018-19
Quantity Produced (MMT)
Year of Production
World Sorghum Production in MMT
5.00 10.00 15.00 2000000
4000000
6000000
8000000
1e+07
1.2e+07
1.4e+07
1.6e+07
1.8e+07
2e+07
2.2e+07
2.4e+07
2.6e+07
2.8e+07
Time-->
Table 1 Phytochemical screening of sorghum extract
Phytochemicals Type of test Petroleum
ether extract
Ethanol
extract
Steroids Salkowski – +
Saponins Foam test/Froth – +
Tannins Ferric chloride – –
Terpenoids Acidified
chloroform
+ +
Alkaloids Dragendroff + –
Cardiac
glycosides
Killer Killiani + +
Phenol Ferric chloride – –
Oxalates Acid digestion – –
Quinone Dilute NaOH + +
The symbol (–) represents absence, while symbol (+) represents
presence
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Ajani O.O. et al. Foods and Raw Materials, 2021, vol. 9, no. 2, pp. 371–378
lipophilicity, which make them soluble in fat [26]. DE
Bruijn found that a wide variety of leguminous plants
contains diverse saponins; for instance, five classes
of saponins were reported in soya beans [27]. This
explained why the extracts obtained with ethanol had
positive saponin test. Saponins play a significant role
in the reduction of plasma cholesterol as a result of the
effective inhibition of cholesterol absorbing capacity
in the intestinal tract of experimentally investigated
animals [28].
Cardiac glycosides are present in the both
petroleum ether and ethanol extracts. These valuable
secondary metabolites are able to enhance myocardial
contraction, treating thereby congestive heart
failure [29]. Cardiac glycosides also indirectly effect
on vascular resistance [30]. Thus, the presence of
cardiac glycosides in sorghum could be exploited for
their medicinal potential. The presence of cardiac
glycosides in the sorghum extracts was detected
by Raymond and Killer Killiani tests; the latter
effectively transformed 2-deoxy-sugars of cardiac
glycoses by distinctive coloration, which made the
qualitative and quantitative monitoring easier [29].
Molecules of cardiac glycosides are capable of inhibiting
Na+/K+- ATPase [31].
Both petroleum ether and ethanol extracts of
sorghum contained terpenoids. Terpenoids form a group
of compounds, the majority of which occur in the plant
kingdom. Simpler mono- and sesquiterpenes are the
main constituents of essential oils [32]. Because of their
sweet smell, these essential oils are used in perfumery
in cosmetic chemistry [33]. Quinone was also present
in the tested sorghum extracts. Naturally, quinone plays
an important role in transduction and accumulation
of energy, which is necessary in such processes as
respiration and photosynthesis [34]. Alkaloids were
found in the sorghum extracts, while tannins, phenol,
and oxalates were not detected in the both ethanol and
petroleum ether samples.
Proximate and physico-chemical analyses. The
proximate analysis results are presented in Table 2. The
importance of oil in human dietary intake cannot be
overestimated. Its biological availability and fatty acid
profile depends in most cases on environmental, crop,
and genetic conditions [35]. The Soxhlet extraction
with n-hexane as a solvent revealed the crude fat of the
extract of 11.00 ± 0.34%. This was a higher yield than
the fat content (9.32%) reported from the distiller dried
grain (DDG) sorghum [36].
The ash content in the sorghum extracts under
study was 3.05 ± 0.11%, which is in accordance with
the results obtained by Mohammed et al. who studied
nutritional composition of three commonly consumed
varieties of sorghum [8]. The ash content may be
affected by the nature and amount of ions in the soil
from which plants draw nutrients. In our work, the
ash content is within acceptable limit (< 5%) [37]. Soil
composition has a partial but direct effect on ash
content [38], as the ash content of seeds may partially
be a function of the soil composition on which the plants
grow [39].
The protein content was determined to be 5.54 ±
0.15%, which was within the range of earlier reported
values, namely 4.82 ± 2.39% for white sorghum and
6.06 ± 0.40% for red sorghum. The moisture content of
the sorghum tested was 5.94 ± 0.18%, which indicated
moderate shelf-life of sorghum [19].
In addition, the investigated sorghum herein had the
fiber content of 0.20 ± 0.07%. Dietary fiber is valuable
in digestion, hormone production, and cardiovascular
health. This also assists in the reduction of low-density
lipoprotein cholesterol due to its bile reabsorbed
reduction capability in the intestinal tract. Fibers in food
prevent excess starch in the body and regulate metabolic
conditions such as diabetes and hypercholesterinemia
[40].
The carbohydrate content, which generally referred
to the readily digested carbohydrates like sugar, starch,
as well as organic acids, amounted for 74.27 ± 0.85%.
High carbohydrate content supplies energy for the
metabolic process, thus stabilizing health status of the
consumers [41]. The proximate analysis showed that the
studied sorghum extract contained 91.01 ± 0.93% of total
organic matter.
The physicochemical parameters of the sorghum
extract included its refractive index, density, and cloud
point (Table 2). According to the refractive index
value, the oil was of a good quality, so it can be used
for homogeneous binary mixture formation. In their
research, Ospina et al. reported that the above mentioned
parameters are characteristics for fast and cheap testing
of the purity of essential oils [42]. The density of the
seed oil of sorghum was 0.81 g/cm3, which was lower
than that of Caryota mitis (0.93 g/cm3) and Adenanthera
pavonina (0.85 g/cm3) in our previous works [2, 3]. This
also implies that the oil of sorghum was less viscous
than that of Adenanthera pavonina and Caryota mitis.
Table 2 Physicochemical and proximate determination
of sorghum extract
Parameters Obtained values
Proximate determination parameters
Moisture content 5.94 ± 0.18%
Ash content 3.05 ± 0.11%
Crude fiber 0.20 ± 0.07%
Protein 5.54 ± 0.15%
Crude fat 11.00 ± 0.34%
Carbohydrates 74.27 ± 0.85%
Organic matter 91.01 ± 0.93%
Selected physicochemical parameters
Refractive index 1.46
Density 0.81
Cloudy 0.40°C
Values are mean ± SD for triplicate measurement
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GC-MS analysis. Based on the GC-MS analysis,
Figure 3 demonstrates the chromatogram of the
sorghum extracts under study. The chromatogram
allows us to compare spectra of each composition and
the NIST library data. The molecular structures of the
identified constituents are shown in Figure 4, with
9,12-Octadecadienoic acid, methyl ester bring being the
predominant fatty acid.
The mass spectrum chromatography assay showed
that the major constituents of the sorghum included
organic acids, esters, sterols, tocopherols, and fatty
aldehyde. Organic acids alone accounted for about 72%
of the sorghum composition. Overall, the most abundant
compounds are 9,12-Octadecadienoic acid (Z,Z)-,
Stigmasterol, 8-Dodecen-1-ol, acetate, (Z)-, vitamin E,
Linoleic acid ethyl ester and 9,12-Octadecadienoic acid,
and methyl ester, which accounted for about 85% of the
sorghum composition.
Among the organic acids, 9,12-Octadecadienoic acid
(Z,Z)-, Linoleic acid ethyl ester and hexadecenoic acids
were contained in high concentrations. Hexadecanoic
acids had three isomers, namely hexadecanoic acid,
methyl ester; hexadecanoic acid, ethyl ester; and
n-Hexadecanoic acid, making up about 5% of the
composition.
9,12-Octadecadienoic acid (Z,Z)-, was the
predominant fatty acid. It had two double bonds
(C=C), which qualified it as an unsaturated fatty acid.
9,12-Octadecadienoic acid (Z,Z)-, occurs as glycosides
in plants, which corroborates the presence of cardiac
glycosides in their phyto-constituents. This essential
fatty acid is a functional component of human food
Figure 3 GC-MS chromatogram of sorghum extract
O
OH
9,12-Octadecadienoic acid (Z,Z)-
Linoelaidic acid
O
O
Ethyl tetracosanoate
O
9,17-Octadecadienal, (Z)-
O
OH
Vitamin E
O
O
8-Dodecen-1-ol, acetate, (Z)-
O
9,17-Octadecadienal, (Z)- O
Cyclopropaneoctanal, 2-octyl-
N
N
NH2
1H-Imidazole-5-ethanamine,
1-methyl- Cyclononene
O
H
H
(1S,15S)-Bicyclo[13.1.0]hexadecan-2-one
O
O
Hexadecanoic acid, methyl ester
Figure 4 Structural identification of sorghum extract
components analyzed chromatographically
Table 3 Identification of sorghum constituents (GC-MS)
Sample № Retention time Area Pct Library (ID) Molecular formula (molecular weight)
1 12.4708 0.0443 12-Heptadecyn-1-ol C17H32O (252.44)
2 13.0716 0.0063 1H-Imidazole-5-ethanamine, 1-methyl- C6H11N3 (125.17)
3 13.4607 0.0376 Cyclononene C9H16 (124.22)
4 14.0329 0.018 (1S,15S)-Bicyclo[13.1.0]hexadecan-2-one C16H28O (236.39)
5 14.1874 0.9545 Hexadecanoic acid, methyl ester C17H34O2 (270.45)
6 14.7424 1.5527 Hexadecanoic acid, ethyl ester C18H36O2 (284.48)
7 15.3032 2.3082 n-Hexadecanoic acid C16H32O2 (256.42)
8 15.5549 4.1409 9,12-Octadecadienoic acid, methyl ester C19H34O2 (294.47)
9 16.1042 5.17 Linoleic acid ethyl ester C20H36O2 (308.50)
10 16.5391 – 31.8454 55.7397 9,12-Octadecadienoic acid (Z,Z)- C18H32O2 (280.45)
11 19.9093 2.4792 Tetracosanoic acid, methyl ester C25H50O2 (382.66)
12 20.3042 3.3967 Ethyl tetracosanoate C26H52O2 (396.69)
13 20.8649
26.4667
3.3824 9,17-Octadecadienal, (Z)- C20H38O (294.52)
14 22.2496 5.6524 Vitamin E C29H50O2 (430.71)
15 23.2738 8.2181 Stigmasterol C17H32O (252.44)
16 24.4526 5.866 8-Dodecen-1-ol, acetate, (Z)- C14H26O2 (226.36)
17 33.5963 1.0329 Cyclopropaneoctanal, 2-octyl- C19H36O (280.49)
2017-18 2018-19
5.00 10.00 15.00 20.00 25.00 30.00
2000000
4000000
6000000
8000000
1e+07
1.2e+07
1.4e+07
1.6e+07
1.8e+07
2e+07
2.2e+07
2.4e+07
2.6e+07
2.8e+07
Time-->
Abundance
T IC : A D D M 1 .D \ d a ta .m s
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Ajani O.O. et al. Foods and Raw Materials, 2021, vol. 9, no. 2, pp. 371–378
which takes a part in biosynthesis of prostaglandins
and cell membranes. Other polyunsaturated fatty acids
have recently been reported to have implication on
inflammatory thrombotic condition like COVID-19 [43].
Prominent esters included ethyl tetracosanoate
and 8-Dodecen-1-ol, acetate, (Z)-, which accounted
for about 9% of the sorghum composition. Sterols
(stigmasterol), tocopherols (vitamin E) and fatty
aldehyde (9,17-Octadecadienal, (Z)-, linoleate group &
Cyclopropaneoctanal, 2-octyl-) were contained in
less quantities, accounting for about 8%, 6%, and 4%,
respectively.
CONCLUSION
The sorghum oil extract was analyzed for
identification of phytoconstituents, proximate compositions,
and physicochemical parameters. It was also
characterized spectroscopically for the nature and
structures of its secondary metabolites using GC-MS.
Proximate determination showed that the sorghum
sample contained beneficial amounts of nutrients, while
phytochemical screening revealed the presence of
bioactive essential phytochemicals.
Thus, sorghum and sorghum-based food could
be of high benefit to the population with nutritional
deficiencies, for example, to developing countries. This
work provided a base for a comparison of nutritional
value and therapeutic potential of sorghum extract with
other natural food cereal sources. Sorghum requires
further research on fortification and functionalization
of food with sorghum extract to decrease nutraceutical
shortage in the population.
CONTRIBUTION
Olayinka O. Ajani designed the work and wrote
the original draft. Taiwo F. Owoeye collected
and pretreated the sample, as well as carried out
phytochemical screening. Kehinde D. Akinlabu carried
out phytochemical screening. Oladotun P. Bolade ran
and discussed the GC-MS analysis. Oluwatimilehin
E. Aribisala carried out sample pretreatment, Soxhlet
extraction, and formal laboratory analysis. Bamidele
M. Durodola carried out laboratory testing and editing
of the manuscript. All authors read and approved the
final manuscript before submission and they are equally
responsible for plagiarism.
CONFLICT OF INTEREST
The authors state that there is no conflict of interests
related to the publication of this article.
ACKNOWLEDGEMENT
The authors gratefully acknowledge Covenant
University for the support for this work.

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