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Nutritional and phenolic proles of Hibiscus cannabinus L.: Food and feed
industries prospect
Tlou Christopher Kujoana
a
, Monnye Mabelebele
a
, William James Weeks
b
,
Freddy Manyeula
a
, Nthabiseng Amenda Sebola
a,*
a
Department of Agriculture and Animal Health, College of Agriculture and Environmental Sciences, University of South Africa, Florida, South Africa
b
Agricultural Research Services, North-West Agricultural Development Institute, Potchefstroom, South Africa
ARTICLE INFO
Keywords:
Phytochemical properties
Kenaf
Crude protein
Energy
Amino acids
Kenaf leaves
ABSTRACT
Hibiscus cannabinus L. (kenaf) plant of the Malvaceae family is primarily a bre crop with high in dietary pro-
teins, bre and bioactive phytochemicals of signicant nutritional and therapeutic benets making it a suitable
alternative to the current expensive and unavailable conventional crops including soyabean. Hence, this study
aimed at evaluating the nutritional and phenolic proles of the kenaf’s selected organs and their applications in
food and feed industries. Five selected organs of kenaf were analysed for nutritional and phenolic compositions.
Data were analysed using a Statistical Analysis System (SAS). Descriptive statistics were applied to the phenolic
data, and the results were presented as they were. Tender leaves (TL) constituted higher (p<0.05) crude protein
and energy, while roots had higher (p<0.05) crude bre. Stems had higher (p<0.05) ash, while TL, mature
leaves (ML) and late mature leaves (LML) recorded higher (p<0.05) calcium and phosphorus. Stems also
recorded higher magnesium, whereas ML recorded the highest amounts of iron. Except for methionine, amino
acids varied signicantly throughout the selected organs where lysine and aspartic acid were highest (p<0.05)
in kenaf mature leaves. The ML were abundant with phenolics, followed by TL, except for LML, having most of its
phenolics different from other selected organs. Therefore, current ndings indicated that kenaf has high key
nutrients and phenolics, implying that it has considerable health and nutritional benets for both humans and
animals, making them critical to the food, feed and health industries.
1. Introduction
Globally, the commercial production of conventional crops that offer
important nutrients and health benets is threatened by unfavourable
climatic conditions and environmental shocks (Gonzalez, 2011), leading
to the by-products of these crops being pricey, making them inaccessible
to some food, feed producers and livestock farmers and meeting the high
demand of functional foods (Tripathi et al., 2019). This puts great
pressure on the agricultural sector to produce enough food by any means
possible to meet the demand (Wallace, 2000). Therefore, it is necessary
to explore efforts to look into alternative food and feed sources that can
provide critical nutrients and medicinal benets for both humans and
animals (Birch &Bonwick, 2019). With extensive research, several
traditional crops have been discovered that may be employed as viable
food and feed sources that can deliver adequate critical nutrients for
both human consumption and animal feeding (Henchion et al., 2017).
Hibiscus cannabinus L. (kenaf) is one of many crops readily available
and abundant in primary and secondary metabolites of the nutritional
and pharmaceutical type such as amino acids (AAs), carbohydrates,
minerals, fatty acids, vitamins, bres phenolics, avonoids, steroids and
saponins (Bhadane et al., 2015). These metabolites make the plant more
acceptable for human and animal consumption, thereby improving the
immune system (Birhanie et al., 2021). It is a multipurpose plant in the
Malvaceae family that is widely recognized for its ability to easily adapt
to any environmental conditions, climates and soil types (Vayabari
et al., 2023;Hassan et al., 2023). Under favourable conditions, kenaf
plants are therefore able to grow up to 20 feet height with period of
maturity up to 5 months (Sullivan, 2002;Kamal, 2014). Furthermore,
with the most recent biotechnological approaches to kenaf plant culti-
vation, such as in vitro propagation techniques, plant production yield
grows quickly (’Aizat Norhisham et al., 2023;Vayabari et al., 2023).
Indeed, Vayabari et al. (2023) indicated that a shorter period is taken on
in vitro propagation, and this method demands less labour and requires
no land, and kenaf cultivation can take place in all seasons as compared
* Correspondence author.
E-mail address: sebolan@ul.ac.za (N.A. Sebola).
Contents lists available at ScienceDirect
Applied Food Research
journal homepage: www.elsevier.com/locate/afres
https://doi.org/10.1016/j.afres.2024.100689
Received 22 December 2024; Received in revised form 25 December 2024; Accepted 31 December 2024
Applied Food Research 5 (2025) 100689
Available online 1 January 2025
2772-5022/© 2025 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ ).
to the traditional way. With this characteristics and newest cultivation
technology, this plant show great potential in the food and feed in-
dustries, especially in countries or areas with not fertile soil for plants to
grow (Hasnain et al., 2022;Kujoana et al., 2023a).
Traditionally, kenaf has been used as a vegetable (Morogo) for
human consumption in South Africa, other parts of Africa, and Asia
through its palatable soft tender leaves, shoots, and owers (Manyelo et
al., 2022). Although the quality of the leaves deteriorates as they
mature, they can still be used as forage and are included in animal diets
to improve feed quality (Giwa Ibrahim et al., 2019). The seeds meal with
increased protein content have been used as supplemental feed for an-
imal rations after being oil pressed with increased protein content as
kenaf seed cake and the oil has been considered edible for human con-
sumption and can further be processed into cooking oil, gum Arabic,
coffee creamer, food appetizer and mayonnaise (Cheng et al., 2016).
Kenaf seed meal can further be processed into our with high nutritional
content and mixed with other plant-based our for human consumption
(Chan et al., 2013). However, antinutritional substances contained un-
processed kenaf seed such as tannins, oxalates, phytates, trypsin, in-
hibitors, saponins and nitrates have the potential to impair the digestion
of kenaf seed meal-based diets (Olawepo et al., 2014), thereby reduces
animal output.
With the recognizable impact of different maturation levels on the
nutritional qualities and secondary metabolites of different medicinal
plant leaves, there have not been enough investigations on kenaf leaves
as potential animal feed and human food. Therefore, the current study
aimed at evaluating the nutritional and phenolic proles of the kenaf’s
selected organs and their applications in food and feed industries. It was
hypothesised that the kenaf organs will not exhibit varying levels of
nutritional and phenolic compounds.
2. Material and methods
2.1. Harvesting and processing of plant material
Samples were sourced from Hibiscus cannabinus L. (kenaf) plant
population trial with four treatments viz. control (4489 plants/ha), low
plant population (83 831 plants/ha), medium plant population (167 000
plants/ha) and high plant population (333 500 plants/ha) replicated
ve times within a randomised complete block design following
methods described by Roy et al. (2010) and Shrestha (2015) . The trial
took place at the Vulimehlo Majara Farm Project (27◦18
′
56
″
" S 25◦59
′
41
″″
E±1.3 m) in the Makwassie Hills Municipal District of the
North-West Province, South Africa. The sampling site temperatures
ranged between 14 and 30 ◦C during the summer season and between
0 and 22 ◦C during the winter.
The eld trial was aimed at determining the impact of agronomical
practices on seed and biomass for primary bre production cultivars
(Tainung 2). The trial establishment was on the 15th of November 2022
during the austral summer. Tainung 2 is a ‘late-maturing’photosensitive
cultivar that initiates reproductive growth when the day length drops
below 12.5 h. The optimal period for vegetative growth in South Africa
may be considered between the second week of October and the second
week of March. Early leaf harvesting for tender leaves (TL) was done on
the 17th of January 2023. Mature leaf (ML), harvesting was done on the
15th of February 2023 at which rst owering had been noticed. The ML
harvesting also coincided with stem and root harvesting. Late mature
leaf (LML) harvesting was done at plant senescence and seed ripening
during the austral autumn on the 4th of May 2023 (Table 1). The har-
vested leaves (Fig. 1A), roots (Fig. 1C) and stems (Fig. 1D) samples were
dried at room temperature of 25 ◦C in a plant science laboratory (Uni-
versity of South Africa) with additional ventilation system from two
electrical fans. Samples were hammer-milled in ne powder (e.g.,
Fig. 1B) to pass through a 1 mm sieve using Restch Cross Beater Mill SK
100, Monitoring and Control laboratories (Pty) Ltd, Parkhurst, South
Africa. The samples were then stored separately in plastic zip bags that
had been properly dried and labelled in a storeroom at room tempera-
ture of 25 ◦C. They were then taken to the University of Pretoria and
Stellenbosch University in South Africa for further nutritional and
phenolic proling, respectively.
2.2. Proximate and mineral analysis
The standardized methods of the AOAC (2012) were followed during
the proximate analysis of dry matter (method number 930.15), ash
(method number 924.05), and crude bres (ANKOM Technology, NY),
crude protein (method number 978.04) and energy. The 5 g samples of
kenaf organs such as roots, stems and leaves were oven-dried at 105 ◦C
for four hours in order to assess the dry matter. After the samples were
taken out and allowed to cool in desiccators, they were dried and
weighed again. The percentage dry matter was calculated as follows:
Five grams of samples were burned for four hours at 550 ◦C in a
mufe furnace, cooled in desiccators, and weighed until the weight
remained constant in order to measure the amount of ash. The Ash
percentage was calculated through the following:
Ash (%) = Weight of ash
Initial weight of sample x 100
Crude bre was assessed by inserting a 10 g sample of each selected
kenaf organ into the extraction device, adding 150 mL of hot 0.2 N
H
2
SO
4
, and digesting for 30 min. The acid was then drained, and the
sample was washed for an hour with hot deionised water. The crucibles
were removed, and oven dried at 105 ◦C overnight, then chilled,
weighed and heated in a mufe furnace at 550 ◦C overnight before being
reweighed after cooling. Percentage extracted ber was calculated as:
Table 1
Kenaf leaf sampling related to growth duration and governing day length.
Sample Days after
sowing
Days after
gemination
Daylength and
increment
Tender leaves (TL) 63 55 13:40
′
(−0
′
97
″
)
Mature leaves (ML) 92 84 13:03
′
(−1
′
71
″
)
Late mature leaves
(LML)
170 162 11:01
′
(−1
′
57
″
)
Dry mater (%) = initial weight of dried samples −weight of oven dried samples
initial weight of dried samples x 100
T.C. Kujoana et al. Applied Food Research 5 (2025) 100689
2
The contents of neutral detergent bre, acid detergent bre, and acid
detergent lignin, which represent the cell wall components of the
selected kenaf organs, were determined using the ANKOM 200 Fibre
Analyser (ANKOM Technology, 2008) in accordance with Van Soest
et al. (1991) techniques.
A micro-kjeldahl was used to determine crude protein content. About
2 g of selected kenaf organ samples were weighed in triplicates. Each
sample was then subjected to a mixture of 2.5 g of CuSO
4
, K
2
SO
4
, and
TiO
2
along with 10 mL of concentrated H
2
SO
4
and digested in a Kjeldahl
digestion ask (KDN-20C, China) at 380 oC for six hours until the
mixture turned clear. After ltering the digest into a 500 mL volumetric
ask, 100 mL of deionized water was added to make it up to the mark,
and the ask was connected for distillation. After an hour of steam
distillation, 20 mL of a 40 % NaOH solution were added to the ammonia.
In a 250 mL conical ask with 20 mL of 0.2 N H
2
SO
4
and methyl red
indicator, 200 mL of the distillate were collected. The excess acid in the
ask was calculated by back titrating the ammonia that distilled into the
receiving conical ask with 0.2 N H
2
SO
4
after the colour changed from
red to yellow. Twenty millilitres of 0.1 N NaOH were used to titrate the
distillate. Colorimetric techniques were used to determine the sample’s
total nitrogen content (Nielson, 2010). The nitrogen content was
multiplied by factor 6.25, or N ×6.25, to determine the crude protein
concentration (James, 1995).
Mineral elements were determined using a slightly modied version
of the Shahidi et al. (1999) method. The samples were prepared by
drying them to ash and using an atomic absorption spectrophotometer
(ASS) to measure the mineral content in diluted acid (Horwitz, 2000).
Porcelain crucibles were heated to 550 ◦C for 2 h before being placed in
a desiccator to cool to room temperature. Each sample was weighed and
dried in porcelain crucibles at 550 ◦C for 2 h before being dissolved in
5.0 mL of HNO
3
/HCL/H
2
O in a 1:2:3 ratio and heated on a hot plate
until brown fumes disappeared. The contents of the crucibles were
ltered into 100 mL volumetric asks with Whatman No 1 lter paper.
The solutions were then lled to the mark in 100 mL volumetric asks
and utilised for mineral analysis with an Atomic Absorption Spectro-
photometer. The energy value of the samples was analysed using a
Gallenkamp ballistic bomb calorimeter (Cam Metric Ltd, Cambridge,
UK) in comparison to a thermos-chemical grade benzoic acid standard.
2.3. Amino acid proling
Amino acids were detected and separated using a photodiode array
(PDA) detector, the Water Acquity Ultra Performance Liquid Chroma-
tography (UPLC) (Agilent 1220, Agilent Technologies). The reagent, a
vial containing 3 mg of AQV, was prepared by adding 1 mL of dry
acetonitrile. The vial was then heated, vortexed, and sonicated to
Fig. 1. Depicting kenaf dried leaves (A), leaf meal(B), roots (C) and stems (D).
Crude fibre (%) = weight of digested sample −Weight of ashed sample
Weight of samples x 100
T.C. Kujoana et al. Applied Food Research 5 (2025) 100689
3
guarantee the reagent was fully dissolved. In order to transfer derivat-
ized amino acids onto a Waters UltraTax C 18 column (2.1 ×50 mm ×
1.7 µm) kept at 60 ◦C, 1 µL of sample or standard solution had to be
introduced into the mobile phase. A gradient was used to elute the
analytes from the column. Individual amino acids emerged from the
column at distinct retention durations, allowing the PDA detector to
detect analytes eluting from the column. as described by Ye et al. (2020)
and Manyelo et al. (2022).
2.4. Characterization of phenolic compounds
An extraction method modied by Agbor et al. (2005) was employed.
For each kenaf organ (leaves, stems, and roots), 2 g of each dry sample
was prepared with 20 mL of 80 % methanol (MeOH), and the mixture
was maintained for two hours in an ultrasonic bath (Branson 3510) at
room temperature (25 ◦C). The samples were then centrifuged for 15
min at 4000 xg a centrifuge (Labofuge 200, Heraeussepatech), the su-
pernatant was removed, and the extraction was repeated three times.
The collected supernatant was evaporated using a rotary evaporator
(Eyela SB-1300, Shanghai Eyela Co. Ltd., Shanghai, China) at 40 ◦C and
then restored with 2 mL of MeOH. Lastly, 0.45
μ
m syringe lters were
used to lter the extracts and were stored at −18 ◦C for further analysis.
2.4.1. Liquid chromatography-mass spectrometry analysis
High-resolution ultra-performance liquid chromatograph –mass
spectrometer (UPLC-MS) analysis was performed using a Waters Synapt
G2 Quadrupole time-of-ight (QTOF) MS and a Waters Acquity UPLC
(Waters, Milford). The column eluate is routed via a Photodiode Array
(PDA) detector before being collected by the mass spectrometer. This
allows for simultaneous capture of UV and MS spectra. Electrospray
ionisation was utilised in negative mode with a 15 V cone voltage, 275
◦C desolvation temperature, 650 L/h desolvation gas, and optimised MS
parameters for resolution and sensitivity. The data was collected by
scanning from 150 to 1500 m/z in both resolution and MSE modes. In
MSE mode, two channels of MS data were acquired: one at a low colli-
sion energy (4 V) and another utilising a collision energy ramp (40 - 100
V) to get fragmentation data. The lock mass (reference mass) was leucine
enkapha lin, and the device was calibrated with sodium formate to
ensure accurate mass measurement. A Waters HSS T3 column (2.1 ×100
mm, 1.7
μ
m) was used to separate the samples. The injection volume
was 2
μ
L, and the mobile phase comprised of 0.1 % formic acid (solvent
A) and acetonitrile with 0.1 % formic acid (solvent B). The gradient was
linear, beginning at 100 % solvent A for 1 min and decreasing to 28 % B
after 22 min. The process included a 50-second increase to 40 % B, a 1.5-
minute wash at 100 % B, and a 4-minute return to the initial settings.
The ow rate was 0.3 mL/min, with a column temperature of 55 ◦C.
Compounds were measured relative to a calibration curve using catechin
standards ranging from 0.5 to 100 mg/L. The data was analysed using
MSDIAL and MSFINDER (RIKEN Centre for Sustainable Resource Sci-
ence: Metabolome Informatics Research Team, Kanagawa, Japan)
(Tsugawa et al., 2015;Manyelo et al., 2020).
2.5. Statistical analysis
The collected data were captured using an Excel spreadsheet and
analysed using Statistical Analysis System (SAS, 2010) version 9.2.1.
Proximate and mineral analysis and amino acid proling data were
analysed using one-way analysis of variance (ANOVA) followed by
Duncan’s multiple range test for multiple mean comparisons at a 5 %
level of probability. Descriptive statistics were applied to the phenolic
compound data, and the results are presented as they are. The following
general linear model was employed:
Yij =
μ
+Ti+eij;
where Y
ij
is the observation of the dependent variable ij (proximate
analysis composition),µis the xed effect of the population mean for the
variable, T
i
is the effect of the harvested kenaf plant organs (i=5; tender
and mature leaves, stems, seed meal, and root), and eij is the random
error associated with the observation of ij, which is considered to be
normally and independently distributed. Furthermore, Principal
Component Analysis (PCA) was employed to examine the relationship
between phenolic compounds and the proximate and mineral
compositions.
3. Results
3.1. Proximate analysis of various selected organs of the kenaf plant
Signicant differences in the nutritional composition of the selected
organs of the kenaf plant are shown in Table 2. Roots, stems, tender
leaves (TL), and mature leaves (ML) had higher (p<0.05) dry matter
(DM) content than late-matured leaves (LML). The TL had the highest (p
<0.05) crude protein (CP) content at 26.94 %, whereas roots had
signicantly higher (p<0.05) CF, NDF, ADF and ADL. The TL had a
signicantly (p<0.05) higher energy content at 18.07 %, followed by
ML and LML, and stems had the signicantly highest (p<0.05) ash
content at 11.27 %.
3.2. Mineral composition
The results presented in Table 3 revealed signicant differences in
both macro- and trace-mineral composition analysis comparisons be-
tween selected plant organs. Potassium (K) and manganese (Mn) were
the exception with no signicant differences. The signicantly highest (p
<0.05) calcium (Ca) level was found in tender leaves (TL). Similar
concentrations of Phosphorus (P) were found in matured leaves (ML)
Table 2
Proximate analysis composition of various selected organs of the kenaf plant (%).
Nutrient Plant organs
1
SEM P
-value
Roots Stems TL ML LML
Dry matter 94.03
a
94.42
a
95.03
a
94.50
a
92.32
b
0.30 0.0009
Crude protein 6.98
e
11.72
d
26.94
a
26.35
b
22.06
c
0.12 <0.0001
Crude bre 40.60
a
31.67
b
12.31
cd
10.30
d
13.08
c
0.58 <0.0001
NDF 53.22
a
40.89
b
16.93
d
13.85
e
29.95
c
0.65 <0.0001
ADF 42.59
a
33.47
b
12.70
c
10.88
d
13.84
c
0.38 <0.0001
ADL 4.80
a
2.77
b
3.03
b
2.23
b
2.23
b
0.21 <0.0001
Energy 16.23
c
15.11
d
18.07
a
17.33
ab
17.07
bc
0.22 <0.0001
Ash 8.62
c
11.27
a
8.83
c
8.60
c
10.06
b
0.08 <0.0001
*abc: Mean on the same row with different superscripts differ signicantly (p<0.05).
NDF: neutral detergent bre.
ADF: acid detergent bre.
ADL: acid detergent lignin.
1
Standard error of the mean.
T.C. Kujoana et al. Applied Food Research 5 (2025) 100689
4
and late mature leaves (LML), which were signicantly (p<0.05) above
concentrations found in roots and stems. Stems had the highest (p<
0.05) magnesium content, but with similar amounts of sodium in com-
parison to roots, TL, and ML. Roots, stems and ML had the signicantly
highest (p<0.05) copper content, with ML having the signicantly
highest (p<0.05) iron content. Zinc was signicantly (p<0.05) higher
in LML but remained undetected in roots.
3.3. Amino acids proling
The results in Table 4 reveal signicant differences (p<0.05) in the
composition of both essential and non-essential amino acids (AAs)
extracted from selected kenaf plant organs (roots, stems, tender, mature,
and late mature leaves), as shown in Fig. 2, with methionine being the
exception. Kenaf mature leaves had the signicantly highest (p<0.05)
concentrations of essential and non-essential AAs, followed by tender
and late mature leaves.
3.4. Characterization of phenolic compounds
As shown in Table 5, approximately fty-three (53) phenolic com-
pounds were present in kenaf roots, stems, TL, and ML, except for LML,
where only eight (8) similar compounds were detected, with twenty-six
(26) phenolic compounds detected in LML and not in roots, stems, TL,
and ML. The matrix analysis and chromatograms of various organs of the
kenaf plant are shown in Figs. 3 and 4, respectively. Phenolic com-
pounds were characterized as phenolic acids, avonoids and diverse
compounds belonging to different metabolite classes. Their derivatives
were tentatively characterised based on the information generated by
the Liquid Chromatography-Mass Spectrometry Analysis (LC-MS). The
ML recorded the highest concentration of phenolic compounds followed
by TL in panasenoside, kaempferitrin and quercetin 3-galactoside,
daucic acid, 3-caffeoylquinic acid, chlorogenic acid and 5-caffeoylquinic
acid, whereas roots were high in quercitrin and moupinamide and stems
high in syringic acid deriv. Uniquely, 9 phenolic acids such as neo-
chlorogenic acid (601.00 mg/kg), cryptochlorogenic acid (126.70 mg/
kg), chlorogenic acid B, C, D, E, F and G (565.90, 340.40, 128.90,
Table 3
The mineral content of various selected organs of the kenaf plant (%).
Nutrients Plant organs
1
SEM P-
value
Roots Stems TL ML LML
Macro-minerals
Calcium 0.39
d
1.06
b
1.13
a
1.08
b
1.01
c
0.008 <0.0001
Phosphorus 0.11
c
0.15
b
0.18
a
0.18
a
0.18
a
0.003 <0.0001
Magnesium 0.07
d
0.17
a
0.11
c
0.11
c
0.12
b
0.000 <0.0001
Potassium 0.04 0.04 0.04 0.04 0.04 0.000 Nd
Sodium 0.03
a
0.03
a
0.03
a
0.02
b
0.03
a
0.001 0.0009
Trace minerals
Copper 0.69
ab
0.71
a
0.67
b
0.69
ab
0.54
c
0.005 <0.0001
Iron 0.25
b
0.20
d
0.23
c
0.27
a
0.18
e
0.003 <0.0001
Manganese 0.10 0.09 0.44 0.12 0.07 0.149 0.4040
Zinc Nd 0.02
c
0.03
b
0.01
d
0.05
a
0.000 <0.0001
*abc: Mean on the same row with different superscripts differ signicantly (p<0.05)
NDF: neutral detergent bre.
ADF: acid detergent bre.
1
Standard error of the mean.
Nd: not detected.
Table 4
Amino acid composition of various selected kenaf plant organs (% g/100 g CP).
Amino acids Plant organs
1
SEM P
-value
Roots Stems TL ML LML
Essential Amino Acids
Histidine 0.12
b
0.16
b
0.58
a
0.66
a
0.58
a
0.03 <0.0001
Arginine 0.26
b
0.56
b
1.34
a
1.58
a
1.21
a
0.13 0.0001
Threonine 0.16
c
0.24
c
0.69
b
1.26
a
0.76
b
0.07 <0.0001
Lysine 0.09
d
0.24
c
1.06
b
1.31
a
1.00
b
0.03 <0.0001
Isoleucine 1.67
d
0.20
d
0.86
b
1.12
a
0.72
c
0.02 <0.0001
Phenylalanine 0.20
d
0.29
d
1.36
b
1.58
a
0.96
c
0.03 <0.0001
Methionine 0.12 0.17 0.15 0.21 0.19 0.02 0.1018
Valine 0.22
c
0.28
c
1.15
b
1.54
a
1.06
b
0.05 <0.0001
Leucine 0.29
d
0.33
d
1.65
b
2.06
a
1.38
c
0.04 <0.0001
Non-Essential Amino Acids
Serine 0.22
c
0.28
c
0.95
a
1.10
a
0.72
b
0.04 <0.0001
Glycine 0.22
e
0.35
d
1.57
b
1.43
a
0.91
c
0.02 <0.0001
Aspartic acid 0.61
d
0.89
c
3.01
a
2.96
a
2.06
b
0.04 <0.0001
Glutamine 0.54
c
0.63
c
2.57
b
3.13
a
2.30
b
0.08 <0.0001
Alanine 0.22
e
0.38
d
1.26
b
1.55
a
1.02
c
0.03 <0.0001
Proline 1.09
c
1.66
b
3.18
a
3.32
a
3.05
a
0.09 <0.0001
Tyrosine 0.16
d
0.23
d
1.09
b
1.27
a
0.68
c
0.03 <0.0001
*abc: Mean on the same row with different superscripts are signicantly different at p<0.05.
1
Standard error of the mean.
TL: tender leaves.
ML: matured leaves.
LML: late matured leaves.
T.C. Kujoana et al. Applied Food Research 5 (2025) 100689
5
758.70, 178.00 and 301.40 mg/kg, respectively) and 3-O-cCaffeoyl-4-O-
methylquinic acid (143.6 mg/kg) and 19 avonoids and other com-
pounds such as cucumegastigmane II;(+)-cucumegastigmane II (255.5
mg/kg), rupestrin B;(+)-rupestrin B (124.60 mg/kg), pisumionoside
(743.70 mg/kg), orthosiphol J;(-)-orthosiphol J (181.20 mg/kg), rutin
(300.30 mg/kg), liriodendrin (62.9 mg/kg), neocarlinoside (112.80 mg/
kg), quercetin 3-[rhamnosyl-(1->2)-alpha-l-arabinopyranoside]
(186.20 mg/kg), Apiin A, B, and C (254.00, 278.00 and 166.70 mg/kg,
respectively), quercitrin A and B (273.60 and 141.70 mg/kg, respec-
tively), kaempferol 3-alpha-l-arabinopyranoside (95.30 mg/kg), genis-
tein (323.10 mg/kg), 6-Methoxyluteolin (124.2 mg/kg), 7-glucuronide
(290.70 mg/kg) and 13-O-Methylvernojalcanolide 8-O-acetate (39.00
mg/kg) were detected in LML. Neochlorogenic acid and chlorogenic B
and F were observed in high quantities among the phenolic acids and
pisumionoside was the highest among the avonoids and diverse
compounds.
3.5. Principal component analysis
To further explore potential differences between a few selected or-
gans of the kenaf plant, principal component analysis (PCA) was per-
formed; the ndings are shown in Fig. 5. The analysis’s ndings indicate
that the PC1
′
s phenolic content, with a variance of 50.3 %, was domi-
nated by mature (ML) and tender leaves (TL). The ML and TL contrib-
uted high component loadings of PC1, while the stems, roots, and late
mature leaves contributed negatively to loading plots. According to the
PCA, the majority of the phenolic compounds in kenaf are concentrated
in the plant’s leaves.
3.6. Heat map analysis of phenolic compounds
A heatmap and hierarchical clustering diagram shown in Fig. 6, were
constructed for the quantitative analysis of the phenolic compounds in
the selected organs of the kenaf plant by High-resolution ultra-perfor-
mance liquid chromatograph –mass spectrometer (UPLC-MS).
Approximately twenty-ve (25) phenolic compounds categorised as
phenolic acids (10) and avonoids and other compounds (15), were
identied. The phenolic components of the kenaf roots, stems, tender
(TL), mature (ML) and late mature leaves (LML) were created in a hi-
erarchically clustered heat map. The samples and the phenolic com-
pounds were present at the axis of the map. The branching pattern
showed their similarity, and each branch point showed a divergence.
The dark brown colour had higher content, while the dark blue colour
included less. Hence, the colour distinction indicated the difference
between selected organs of kenaf plant. The phenolic compounds were
divided into four groups including PC-1, PC-2, PC-3, and PC-4. Kenaf
leaves (TL and ML) has shown high phenolic acids and avonoids and
other compounds in the heat map followed by the stems. Roots were
noted for their high kelampayoside A, oxyisocyclointegrin, suberic acid
and sibiricose A3, whereas LML showed to be poorly concentrated in the
phenolic compounds.
4. Discussion
4.1. Proximate and mineral composition
Various climatic and environmental factors have a considerable
impact on the genotype, maturation stage and growing conditions of
different plants, inuencing biomass yield and nutritional quality (Ayadi
et al., 2017). The CP of Hibiscus cannabinus L. (kenaf) leaves which are
made up of tender (TL), matured (ML) and late matured leaves (LML)
deteriorated from 26.94, 26.35 and 22.06 % CP, respectively with the
advancement in growth, however, remained beyond 12 %, in the current
study. This was also explained by Crozier et al. (2009) that if plants left
to grow unhindered, their leaf quality deteriorates with maturation and
ageing. As a result, the current ndings suggest that kenaf leaves can be
harvested and incorporated into the animal diet as a reliable source of
protein at any stage of the plant’s maturity and growth to improve
quality, palatability and feed intake, potentially improving overall ani-
mal performance (Kujoana et al., 2023b). These ndings demonstrate
that kenaf leaves can be harvested and consumed as vegetable for
human consumption and also be incorporated into the animal diet as a
reliable source of protein at any stage of the plant’s growth to improve
quality, palatability and feed intake, potentially improving overall ani-
mal performance (Kujoana et al., 2023b). This was also highlighted by
Phillip et al. (2002) that due to their palatability and high in protein
content, kenaf leaves can be used as in livestock feeding. However, to
our knowledge, there is no documentation on the inclusion of kenaf leaf
meal in animal diet to enhance the quality and overall livestock
performance.
The CF, NDF, ADF and ADL were signicantly higher in the roots
than in the stems and leaves. To our knowledge, results similar to the
Fig. 2. Stacked bar graph of Amino acids composition in various parts of kenaf plant, Hibiscus cannabinus: TL-Tender leaves; ML-Matured leaves; LML-Late
matured leaves.
T.C. Kujoana et al. Applied Food Research 5 (2025) 100689
6
current ones have not been previously documented comparing CF, NDF,
ADF, and ADL in various parts of kenaf plants. However, the highest
bre content was expected to be detected in stems since they are the
bre-yielding parts of the plant and have traditionally been used to
produce ropes, matting and baskets (Sahu et al., 2013). The dietary bre
content accumulated in the roots may be attributable to the increased
lignin, cellulose and hemicellulose contents, which are known to in-
crease the retention time of the digesta hence impairing the bioavail-
ability of the nutrients (Manyelo et al., 2022). Furthermore, it can be
emphasised that kenaf roots could alternatively be regarded as a source
of bre to maintain normal physiological functions in the healthy animal
digestive system (Lindberg, 2014). According to Tamminga (1996), it is
often expected that the energy content increases when the protein
content of a fodder plant deteriorates during the dry season. However,
the current results suggest that the energy levels of kenaf leaves dete-
riorated from 18.07 to 17.03 %, with a decrease in protein levels as the
plant leaves matured. The higher quantities of ash in kenaf stems may
indicate that there are more minerals overall since ash content is a
measure of total mineral content. However, stems were only high in
magnesium (Mg), sodium and copper in this study. These minerals helps
in to maintain normal nerve, muscle function, keeps the heartbeat
steady, maintain a balance of body uids (Rehrer, 2001;Faryadi, 2012),
formation the body red blood cells, and keep the nerve cells healthy
(Osredkar and Sustar, 2011). The Mg is also high in mineral is crucially
used for bodily chemical reactions and absorption of zinc in the in-
testines (Muhammad et al., 2011). The observed increased concentra-
tions of Ca and P in kenaf leaves suggest that these leaves can be
responsible for the normal growth, activities of muscles and healthy
skeletal development (Tu et al., 2016), the synthesis of protein for
growth, maintenance and repair of cells and tissues, while increasing the
use of carbohydrates and fats in the body (Ciosek et al., 2021).
Furthermore, these minerals are available in the largest quantity in the
structural formation of the body and bones (Serna &Bergwitz, 2020).
The increase ion content in ML, suggest kenaf leaf meal could be
necessary in formation of haemoglobin, a protein in the blood respon-
sible for the transportation of oxygen and cellular processes of growth
and division, as described by Sebola (2015). Therefore, it is evident that
kenaf plants contain adequate minerals for animals to thrive. As previ-
ously stated, shortages in essential nutrients and minerals affect animal
growth, health, and overall performance. Furthermore, because of its
availability and capacity to grow in biomass even through harsh climatic
conditions, the kenaf plant could be an excellent answer for the food and
feed industries, beneting disadvantaged rural populations and
under-resourced animal farmers (Benjamin &Van Weenen, 2000). This
is congruent with the South African National Development Plan estab-
lished in 2011 and Sustainable Development Goals agenda adopted in
2015, which emphasised the need of ending poverty and addressing
global hunger and malnutrition challenges by 2030 (Okello et al., 2021).
4.2. Amino acid proling and phenolic compounds of various organs of
the kenaf plant
The current results further demonstrate the signicant differences in
the Amino acids (AAs) composition in various parts of the kenaf plant
which could be due to the nature of the selected organs of the plant
(Sandstr¨
om et al., 1994). Amongst the selected organs of kenaf, leaves
dominated in both essential and non-essential AAs concentrations in
kenaf leaves than in roots and stems. This may be because of the high
concentration of CP in the leaves. Lysine and aspartic acid (AA), both
essential and non-essential AAs, seem to be the centres of crucial AAs.
Lysine is essential for normal growth and plays a crucial role in the
creation of carnitine, a nutrient responsible for turning fatty acids into
energy and helping decrease cholesterol, whilst non-essential AAs are
important for the generation and release of hormones in the body as well
as the regular operation of the neurological system (Manyelo et al.,
2022). Therefore, these results show that at any given stage of maturity
Table 5
Composition of various phenolic compounds contained in various selected or-
gans of kenaf plants (mg/kg).
Plant organs
No Phenolic
compounds
Roots Stems TL ML LML
Phenolic acids
1 Daucic acid 175.1 345.7 1 953.9 2 562.0 0.00
2 Methylisocitric acid A 94.2 168.3 220.7 228.8 0.00
3 Methylisocitric acid B 333.3 655.4 663.5 846.5 0.00
4 Cis-Aconitic acid 111.8 206.4 257.9 270.4 0.00
5 3-caffeoylquinic acid 302.3 729.6 1619.8 1662.9 0.00
6 Chlorogenic acid deriv 6.5 33.4 1094.3 1229.4 108.3
7 Salicylic acid beta-d-
glucoside
117.1 101.3 32.8 33.6 0.00
8 Chlorogenic acid 8.7 11.1 725.1 736.1 0.00
9 3-O-p-
Coumaroylquinic acid
23.8 163.9 421.5 400.2 0.00
10 5-Caffeoylquinic acid 562.0 846.3 1470.5 1587.1 231.7
11 2-Feruloylquinic acid 13.30 47.6 580.7 403.5 0.00
12 2-O-
Caffeoylhydroxycitric
acid
735.5 320.1 77.8 142.7 0.00
13 Cis-Coumaric acid 8.3 132.2 83.2 68.1 0.00
14 Coumaroylquinic acid 5.0 88.5 106.7 65.5 0.00
15 Caffeoylmalic acid 42.1 251.5 82.7 97.2 0.00
16 Syringic acid deriv 130.6 1104.8 507.5 393.0 0.00
17 3-Feruloylquinic acid 18.2 51.7 337.1 337.6 0.00
18 Coumaroylquinic acid 14.5 120.4 552.2 629.6 0.00
19 Coumaric acid 25.8 15.9 14.8 7.6 0.00
20 Suberic acid 616.6 63.7 21.0 24.2 0.00
Flavonoids and other compounds
21 Reiniose A;(-)-Reiniose
A
6.6 6.2 26.3 15.1 0.00
23 Oxyisocyclointegrin 158.9 19.6 1.4 1.6 0.00
24 L-Phenylalanine 6.8 17.4 503.1 479.9 201.8
25 Laccaridione A 2.6 1.6 0.4 0.9 0.00
26 Veranisatin C 2.4 10.7 232.8 243.2 0.00
27 Phenethyl rutinoside 2.4 10.7 232.8 243.2 0.00
28 4-demethylwyosine 47.0 150.5 28.9 25.3 0.00
29 Lentztrehalose 0.8 0.8 2.5 1.0 0.00
30 Lilaline 115.0 66.9 100.5 119.9 0.00
31 Sibiricose A3;
(+)-Sibiricose A3
161.6 73.7 13.5 3.8 0.00
32 Ebenone 36.2 80.7 60.2 37.1 0.00
33 Diaspanoside B 52.3 61.2 677.0 514.5 0.00
34 Cynaroside A 160.2 104.8 154.0 98.2 0.00
35 Feruloylglucose 27.8 108.3 410.1 171.6 0.00
36 Coumaroyl-glucose 5.6 47.9 419.1 404.5 0.00
37 Zizybeoside I 1.6 0.2 0.7 1.7 0.00
38 Bergenin 20.1 39.5 311.9 89.6 231.7
39 Kelampayoside A 31.0 9.2 0.4 1.1 0.00
40 Rupestrin B 1.5 8.7 218.9 293.3 0.00
41 Benzyl-Beta-Prime
Veroside
23.2 26.9 353.2 281.2 0.00
42 Echimidine 305.4 410.5 261.6 230.2 112.8
43 Panasenoside 179.2 501.9 845.9 838.0 0.00
44 Astragalin 7-
rhamnoside
1.6 8.2 328.7 315.6 153.3
45 Neocarlinoside 1.8 22.2 59.8 33.4 112.8
46 Echimidine 603.4 332.4 73.9 89.7 0.00
47 Quercetin 3-
galactoside
25.4 19.7 791.7 783.9 300.3
48 Ankorine 131.9 235.5 76.4 60.6 0.00
49 Kaempferitrin 601.5 530.5 939.1 793.1 389.9
50 Quercitrin 5217.4 3480.9 653.9 597.9 0.00
51 Phenethyl rutinoside 7.8 12.6 353.6 286.5 88.8
52 Kaempferol
rhamnoside
13.5 21.7 757.8 734.4 0.00
53 Lancerin 14.4 55.4 678.8 468.4 0.00
54 Moupinamide 2485.9 1083.8 2065.4 957.6 0.00
TL: tender leaves.
ML: matured leaves.
LML: late matured leaves.
Nd: Not detected.
T.C. Kujoana et al. Applied Food Research 5 (2025) 100689
7
Fig. 3. Matrix analysis of phenolic compounds composition in various parts of kenaf plant, Hibiscus cannabinus: TL-Tender leaves; ML-Matured leaves; LML-Late
matured leave.
Fig. 4. Chromatograms of different parts of kenaf, Hibiscus cannabinus L.: Stems (A); Tender leaves (B); Roots (C), Matured leaves (D); Late matured leaves (D).
T.C. Kujoana et al. Applied Food Research 5 (2025) 100689
8
Fig. 5. The Principal components of the scatter plot of the phenolic compounds found in the selected organs (roots, stems, tender leaves (TL), mature leaves (ML)and
late mature leaves) of the kenaf plant.
Fig. 6. Heatmap showing phenolic cmpounds of ve selected organs of the kenaf plant. Dark brown boxes mean concentrations are higher among selected organs,
while dark blue boxes mean lower concentrations. PC 1–4: phenolic compound clusters.
T.C. Kujoana et al. Applied Food Research 5 (2025) 100689
9
of the kenaf plant, the spectrum of amino acids remains unaffected by
maturity, which indicates that harvested kenaf leaves can successfully
supply adequate AAs, both essential and non-essential, as this will be of
great importance and benecial to both the food and feed industries.
Moreover, the current results further showed that proline is crucially
involved in metabolism processes, wound healing, antioxidative pro-
cesses and immunological responses similar to most phenolic com-
pounds (Shetty &Wahlqvist, 2004) because many secondary phenolic
compounds are the derivatives of amino acids and have a similar
structure (Russell &Duthie, 2011). The kenaf plant is widely recognized
for its potential use in traditional medicine due to the presence of
pharmacological properties (Sim &Nyam, 2021), which are the plant’s
secondary metabolites produced in shikimic acid of the plant and
pentose phosphate through phenylpropanoid metabolization (Saltveit,
2017).
Sim and Nyam (2021) showed kenaf leaves to have been used as
traditionally to treat anaemia, fatigue and guinea worms in Africa and
subsequently improve human health. It is indeed evident with current
investigations as kenaf leaves (TL and ML) showed to be more concen-
trated with phenolic compounds mainly the phenolic acids with daucic
acid being the most abundant, followed by caffeoylquinic acids (3 and 5
CQA) and chlorogenic acid deriv. This is evident as it is also observed in
the ndings by Park et al. (2020) where kenaf leaves contained higher
levels of total phenolic compounds as compared to other selected or-
gans. These chemicals crucially work as an intermediary in lignin pro-
duction (Alc´
azar Maga˜
na et al., 2021), preventing weight gain,
inhibiting the development of hepatic steatosis and blocking insulin
resistance induced by high-fat diet (Ma et al., 2015). However, there is a
paucity of knowledge about the roles and signicance of daucic acid,
which is abundant in both TL and ML. The kenaf plant is extremely
concentrated in avonoids through its roots, with quercitrin being even
more concentrated than the concentrations of presented phenolic acids
followed by moupinamide. Among avonoids, quercitrin is an essential
antioxidant that scavenges the free radicals in the body, which are
known to damage cell membranes by interfering with the deoxy-
ribonucleic acid (DNA) and even causing cell death (Faddah et al.,
2013). This primarily occurs in humans or animals experiencing heat
stress. Moupinamide is an alkaloid that appears to block the major
protease enzymes, as well as having anti-inammatory and antibacterial
properties (Elkaeed et al., 2022).
Although roots, stems, TL and ML contain similar phenolic com-
pounds in varying quantities (g/kg), LML contains more compounds that
are distinct from the rest of the kenaf organs, particularly TL and ML.
This could be attributed to the timing of harvest, as they were harvested
late, when seeds had reached their potential maturity stage, and were
only partially harvested. As a result, at this stage, LML would be in a
senescent stage, in which most physiological machinery of the plant cells
ages and permanently stops proliferating but does not die, hence there
are fewer compounds at the harvest stage (Paul et al., 2005). This
demonstrates that late-harvested kenaf leaves can be put to good use
because they are abundant in important phenolic compounds (Kho et al.,
2019), such as neochlorogenic acid classied as phenolic acid and
pisumionoside classied as a avonoid. The neochlorogenic acid frac-
tion may have chemopreventative and chemotherapeutic properties
lowering the risk of cancer by protecting and scavenging reactive oxy-
gen species, thus enhancing DNA repair and detoxication while also
changing carcinogen uptake and metabolism (Thurow, 2012). Pisu-
mionoside applications are unknown; nevertheless, avonoids such as
Genistein protect the body against osteoporosis and lower the risk of
cardiovascular disease, alleviate postmenopausal symptoms and have
anticancer properties (Thangavel et al., 2019). This compound directly
inuences male fertility by inhibiting testicular spermatogenesis and
steroidogenesis (Kohara et al., 2014). To the best of our knowledge, this
is the rst study to investigate phenolic compounds in LML. As a result,
further research is needed into the analysis of the available secondary
metabolites of various medicinal plants’leaves harvested at the seed
maturity stage, as well as their importance. Overall, with the increased
concentrations of phenolics in kenaf ML in recent results, suggest that
the can be processed into health promoting beverages and functional
food additives to improve the quality in bakery products (Lim et al.,
2020).
5. Conclusions
The study results show that this kenaf is not only climate-adaptable
but also has signicant nutritional and medicinal benets, regardless of
the selected part. Despite this, kenaf remains an underutilized crop with
the potential to be an alternative to the existing expensive commercial
protein ingredients (soybean and lucerne) and synthetic medications
(antibiotics). This study undoubtedly demonstrated that kenaf leaves are
an excellent source of proteins, energy, bre, minerals, amino acids, and
important phenolic compounds. With these characteristics, kenaf leaves
could be used as superior food, feed, and therapeutic source for both
human and animal production, health and reproduction. Indeed, the
harvesting period for leaves inuenced their nutritional content and
quality, as well as the composition of secondary metabolites. With the
recognizable impact of different maturation levels on the nutritional
qualities and secondary metabolites of different medicinal plant leaves,
there have been not enough investigations on kenaf leaves as potential
animal feed and human food. Hence, this necessitates food and feed-
producing companies to prioritise this plant as a potential replacement
for conventional crops and synthetic drugs in both animal and human
diets.
Ethical statement
Study requires no ethical clearance as there were no humans or an-
imals involved.
Funding sources
This work was supported by the National Research Foundation
[Grant number: 142,077] and UNISA-Women in Research.
CRediT authorship contribution statement
Tlou Christopher Kujoana: Writing –original draft, Visualization,
Methodology, Investigation, Funding acquisition, Formal analysis, Data
curation, Conceptualization. Monnye Mabelebele: Writing –review &
editing, Supervision, Funding acquisition. William James Weeks:
Writing –review &editing, Data curation. Freddy Manyeula: Writing –
review &editing, Methodology, Formal analysis, Data curation. Ntha-
biseng Amenda Sebola: Writing –review &editing, Supervision, Re-
sources, Project administration, Funding acquisition, Data curation.
Declaration of competing interest
The authors declare that they have no competing interests or per-
sonal relationships that could have appeared to inuence the reported
work reported in this paper.
Data Availability
The data supporting this study will be made available upon reason-
able request to the corresponding author: sebolan@unisa.ac.za.
Acknowledgements
The authors are grateful to the National Research Foundation and
UNISA-Women in Research grants for their nancial support, Agricul-
tural Research Services of the Northwest Department of Agriculture
Conservation and Environment, Potchefstroom for supplying the
T.C. Kujoana et al. Applied Food Research 5 (2025) 100689
10
Hibiscus cannabinus L. samples cultivated at the Vulimehlo Majara
Project Farm and the CAF LCMS at the Stellenbosch University for
assistance with the LC-MS analysis.
Supplementary materials
Supplementary material associated with this article can be found, in
the online version, at doi:10.1016/j.afres.2024.100689.
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