Phytochemicals and antioxidant activity in the kenaf plant ( Hibiscus cannabinus L.)

Abstract

Chemical compounds from four different tissues of the kenaf plant (Hibiscus cannabinus), a valuable medicinal crop originating from Africa, were examined to determine its potential for use as a new drug material. Leaves, bark, flowers, and seeds were harvested to identify phytochemical compounds and measure antioxidant activities. Gas chromatography mass spectrometry analyses identified 22 different phytocompounds in hexane extracts of the different parts of the kenaf plant. The most abundant volatile compounds were E-phytol (32.4%), linolenic acid (47.3%), trisiloxane-1,1,1,5,5,5-hexamethyl- 3,3-bis[(trimethylsilyl)oxy] (16.4%), and linoleic acid (46.4%) in leaves, bark, flowers, and seeds, respectively. Ultra-high performance liquid chromatography identified the major compounds in the different parts of the kenaf plant as kaemperitrin, caffeic acid, myricetin glycoside, and p-hydroxybenzoic acid in leaves, bark, flowers, and seeds, respectively. Water extracts of flowers, leaves, and seeds exhibited the greatest DPPH radical scavenging activity and SOD activity. Our analyses suggest that water is the optimal solvent, as it extracted the greatest quantity of functional compounds with the highest levels of antioxidant activity. These results provide valuable information for the development of environmentally friendly natural products for the pharmaceutical industry.

Full text

J. Ryu
･
S.-J. Kwon
･
J.-W. Ahn
･
Y. D. Jo
･
S. H. Kim
･
M. K.
Lee
･
J.-B. Kim
･
S.-Y. Kang (

)
Advanced Radiation Technology Institute, Korea Atomic Energy
Research Institute, Jeongup, Jeonbuk 56212, Korea
e-mail: sykang@kaeri.re.kr
S. W. Jeong
Jangheung Research Institute for Mushroom Industry, Jangheung
59338, Korea
Phytochemicals and antioxidant activity in the kenaf plant (Hibiscus
cannabinus L.)
Jai hy un k Ry u ･Soon-Jae Kwon ･Joon-Woo Ahn ･Yeong Deuk Jo ･Sang Hoon Kim ･Sang Wook Jeong ･
Min Ky u Lee ･Ji n-B aek K i m ･Si-Yong Kang
Received: 29 May 2017 / Revised: 20 June 2017 / Accepted: 20 June 2017
ⓒKorean Society for Plant Biotechnology
This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0)
which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract Chemical compounds from four different tissues
of the kenaf plant (Hibiscus cannabinus), a valuable medicinal
crop originating from Africa, were examined to determine its
potential for use as a new drug material. Leaves, bark, flowers,
and seeds were harvested to identify phytochemical compounds
and measure antioxidant activities. Gas chromatography mass
spectrometry analyses identified 22 different phytocompounds
in hexane extracts of the different parts of the kenaf plant. The
most abundant volatile compounds were E-phytol (32.4%),
linolenic acid (47.3%), trisiloxane-1,1,1,5,5,5-hexamethyl-
3,3-bis[(trimethylsilyl)oxy] (16.4%), and linoleic acid (46.4%)
in leaves, bark, flowers, and seeds, respectively. Ultra-high
performance liquid chromatography identified the major
compounds in the different parts of the kenaf plant as kaemperitrin,
caffeic acid, myricetin glycoside, and p-hydroxybenzoic acid
in leaves, bark, flowers, and seeds, respectively. Water extracts
of flowers, leaves, and seeds exhibited the greatest DPPH
radical scavenging activity and SOD activity. Our analyses
suggest that water is the optimal solvent, as it extracted the
greatest quantity of functional compounds with the highest
levels of antioxidant activity. These results provide valuable
information for the development of environmentally friendly
natural products for the pharmaceutical industry.
Keywords Kenaf, Functional compounds, Antioxidant acti vities,
Drug materials
Introduction
Phytochemicals are biologically active plant chemicals that
provide various health benefits (Khare, 2007; Jin et al. 2013).
They are naturally occurring bioactive substances that provide
plants with particular defense mechanisms and protect them
from various diseases. An important category of phyto-
chemicals commonly present in plants has high antioxidant
activity (Yusri et al. 2012; Chen et al. 2014). Phytochemical
analyses are of paramount importance for the identification
of new sources of therapeutically and industrially valuable
compounds with medicinal significance and for the best and
most judicious use of naturally available materials (Hossain
et al. 2011).
Flavonoid and phenolic compounds, which exhibit a broad
range of biological activities, are known to be more abundant
than flavonoid monomers in plants (Wang et al. 2008; Jin
et al. 2013). The wide use of plants as food, food additives
and drug has increased the number of researches investigating
the phytochemicals and biological activity of these sources
(Rauter et al. 2002; Nyam et al. 2009). The medicinal plants
were used for health care because they contain aromatic
components of high therapeutic potential. Especially, essential
oils and fatty acids are widely used in medicine, as flavoring
additives in the food industry, and as fragrances in the
cosmetic industry (Kobaisy et al. 2001; Ryu et al. 2013).
Kenaf, an annual herbaceous crop of the Malvaceae family,
is a short-day plant. Kenaf, a valuable medicinal crop
originated from Africa, is contained various functional
compounds. The kenaf leaf was applied to Guinea worms
and the stem bark has been used for anaemia in Africa
(Alexopoulou et al. 2013). In ayurvedic medicine, the
kenaf leaves are used for bilious, blood, diabetes, coughs
and throat disorders (Khare, 2007; Alexopoulou et al. 2013;
Jin et al. 2013). The flower juice is used for biliousness
(Alexopoulou et al. 2013). The seeds are also consumed
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DOI:https://doi.org/10.5010/JPB.2017.44.2.191
Research Article
ISSN 1229-2818 (Print)
ISSN 2384-1397 (Online)

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Fig . 1 Profiles of the different parts of the kenaf plant. A: leaf; B: stem bark; C: flower; D: seeds
to weight increase and bruises (Kubmarawa et al. 2009;
Alexopoulou et al. 2013). The kenaf leaf and seed contains
a variety of different compounds, including phenolic com-
pounds, flavonoids, essential oils, and fatty acids (Mohamed
et al. 1995; Jin et al. 2013; Ryu et al. 2013; Nandagopalan
et al. 2015; Obouayeba et al. 2015). Kenaf has been reported
to exhibit properties associated with anodynes, aperitifs,
aphrodisiacs, anti-inflammatory medications, and antioxidants
for leaf and seed. It has also been related to weight gain,
anemia, and fatigue (Kobaisy et al. 2001; Khare, 2007;
Kubmarawa et al. 2009; Alexopoulou et al. 2013). Phenyl-
propanoids, which are abundant in the kenaf leaf, are im-
portant for these beneficial health effects (Jin et al. 2013;
Ryu et al. 2016). Various biologically active compounds
have been reported in the kenaf seed, including omega-3
fatty acids, phenolic compounds, and sterols (Alexopoulou
et al. 2013; Ryu et al. 2013). However, studies on the
phytochemical of different parts were little known.
A selection of the best solvents for the extraction of
chemical compounds from plant materials is important to
improving the efficiency of extraction yield (Sultana et al.
2007). Therefore, it is essential to determine the solvents
on the extraction of functional compounds and antioxidant
properties of kenaf to select an optimal solvent for the
extraction of the bioactive compounds from the different
parts of the kenaf plant.
This study analyzed the nutritional properties, functional
compounds, and antioxidant activities in the leaves, stem
bark, flowers, and seeds of kenaf. This is the first report
on the phytochemical composition of the different parts of
the kenaf plant. Additionally, the differences in antioxidant
activity and major components that were a result of the
different solvents were investigated to identify novel, poten-
tially environmentally friendly natural products that would
be useful in the food industry.
Materials and Me thods
Plant materials and extraction
The ‘Auxu’ cultivars were studied. The leaves, bark, flowers,
and seed of the kenaf plant were harvested (Fig. 1) and
freeze drying. Ten gram of each samples were extracted
for 24 hour with 50 ml of methanol, water, ethanol and
chloroform. Extract samples used for determine total poly-
phenol content, flavonoid content, perform ultra-high per-
formance liquid chromatography (UPLC) and antioxidant
analysis.
Gas chromatography mass spectrometry (GC-MS) analysis
Plant extraction for GC-MS analysis was determined by
previously study (Ryu et al. 2013), with the following modi-
fications. The powdered material of the leaf, stem bark,
flower, and seed (10 g) was extracted in 50 mL hexane
for 2 h, and 500 µL 2 N potassium hydroxide in methanol
was added. From this, 2 µL of the extracts from the different
parts of the kenaf plant was analyzed by GC-MS (Plus-2010,
Shimadzu, Kyoto, Japan). The chemical composition of the
different parts of the kenaf plant were analyzed using a
GC-MS instrument equipped with an HP-88 capillary column
(60 m × 0.25 mm × 0.25 m, J&W Scientific, C.L. USA)
under the following conditions: ionization voltage, 70 eV;
mass scan range, 50–450 mass units; injector temperature,
230°C; detector temperature, 230°C; inject volume, 1 µL;
split ratio, 1:30; carrier gas, helium; and flow rate, 1.7
mL/min. The column temperature program specified an
isothermal temperature of 40°C for 5 min followed by an
increase to 180°C at a rate of 5°C/min and a subsequent
increase to 28°C at a rate of 1°C/min. We identified the
substances present in the extracts by retention time (RT)
and mass spectra database (Nist. 62 Library).

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Total phenolic content
The total phenolic content (TPC) was determined with the
Folin–Ciocalteau colorimetric method (Jin et al. 2013). A
small quantity (0.2 mL) of each extract and 1.5 mL of Folin-
Ciocalteau reagent (20% v/v) were mixed thoroughly. After
4 mL Na2CO3 (7%) was added, and fill up to 10 mL with
water. The mixture was allowed to dark exposure with room
temperature at 90 min. The absorbance was measured at
760 nm using a UV-spectrophotometer (UV-1800, Shimazuda,
Kyoto, Japan). TPC was calculated using a calibration curve
of tannic acid.
Total flavonoid content
The total flavonoid content (TFC) content in different part
of kenaf was determined by Zhishen et al., 1999. Each extract
samples (0.2 ml) was added to 4 mL double-distilled water
and 0.3 mL of 5% NaNO2 to the flask. The samples were
maintained for 5 min, and 0.3 mL of 10% AlCl3 was added.
After 6 min, 2 mL NaOH and fill double-distilled water
up to 10 mL. The absorbance was measured at 510 nm.
TFC was calculated using a calibration curve of quercetin
equivalents.
Ultra-high performance liquid chromatography (UPLC)
analysis
Functional compounds were analyzed with a UPLC system
(CBM-20A, Shimadzu Co., Kyoto, Japan) with two gradient
pump systems (LC-30AD, Shimadzu, Kyoto, Japan), a UV
detector (SPD-M30A, Shimadzu), an auto sample injector
(SIL-30AC, Shimadzu), and a column oven (CTO-30A,
Shimadzu). Separation was achieved on an XR-ODS column
(3.0 × 100 mm, 1.8 µm, Shimadzu, Japan) using a linear
gradient elution program with a mobile phase containing
solvent A [0.1% trifluoroacetic acid (v/v) in distilled deionized
water] and solvent B [0.1% trifluoroacetic acid (v/v) in
acetonitrile]. For UPLC analysis, ground samples (5 g) were
extracted in 5 mL of each solvent (methanol, ethanol, water,
and chloroform) for 60 min and filtered through a 0.45-µm
membrane filter. Flavonoids and phenolic acids were sep-
arated using the following gradient: 0–5 min, 10–15% B;
5–10 min, 15–20% B; 10–15 min, 20–30% B; 15–20 min,
30–50% B; 20–25 min, 50–75% B; 25–30 min, 75–100%
B; 30–32 min, 100–5% B; and 32–35 min, 5–0% B. Fla-
vonoids, phenolic acids, and anthocyanins were detected at
280, 350, and 520 nm, respectively.
Determination of antioxidant activity
The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging
activity DPPH radical scavenging activity was measured
by Jin et al. 2013. Each solvent (methanol, ethanol, water,
and chloroform) extract added to 0.15 mM DPPH, and after
30 min, the remaining DPPH radicals were quantified
using a plate reader (Benchmark Plus; Bio-Rad, Hercules,
CA, USA) at 517 nm. The super oxide dismutase (SOD)
activity was measured by SOD assay kit (Dojindo Molecular
Technologies, USA) manual. The DPPH and SOD activity
effect was calculated as follows:
[(1-As/Ac) × 100%]
As: absorbance value with sample; Ac: absorbance value
without extract solution.
Statistical analysis
The chemical analysis data were subjected to analysis of
variance using a multiple comparisons method with the
statistical software package SPSS, Version 12 (SPSS Institute,
USA). Differences were determined to be significant at p <
0.05. When the treatment effect was significant, the means
were separated using Duncan’s multiple range tests.
Res ults
GC-MS analysis
The compounds identified in the different parts of the kenaf
plant, along with their RT, molecular formula, molecular
weight and percentage, are shown in Table 1 and Fig. S1.
The leaf extract showed 13 phytocompounds, including
3,7,11,15-Tetramethyl-2-hexadecen (4.4%), Tetradecanoic acid
(0.8%), 6,10,14-trimethyl-pentadecan-2-ol (0.9%), 6,10,14-
Trimethyl-2-pentadecanone (3.4%), Hexadecanoic acid (14.3%),
9-Octadecenoic acid (2.9%), Phytol acetate (2.3%), 9,12-
Octadecadienoic acid (6.8%), Phytol (32.4%), 9,12,15-Octade-
catrienoic acid (n=6, 0.7%), 9,12,15-Octadecatrienoic acid
(n=3, 27.6%), 2-5-oxohexyl-cyclopentanone (1.4%), and
2,6,10,14,18,22-Tetracosahexaene (2.1%). Eight different
phytocomponents were identified in the stem bark extract
of kenaf: Hexadecanoic acid (25.4%), 9-Hexadecenoic acid
(3.0%), Octadecanoic acid (3.1%), 9-Octadecenoic acid
(1.2), Phytol acetate (0.7%), 9,12-Octadecadienoic acid
(10.7%), Phytol (8.7%), and 9,12,15-Octadecatrienoic acid

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Table 1 Volatile constituents in four different kenaf tissue types
No. RT(min)* Name of the compound Molecular
formula MW Total
%
Leaf
1 12.6 3,7,11,15 tetramethyl-2 hexadecen-1-ol (Z-phytol) C20H40O 296 4.4
2 13.9 Tetradecanoic acid C15H30O2242 0.8
3 15.1 6,10,14-Trimethyl-pentadecan-2-ol C18H38O 270 0.9
4 15.3 2-Pentadecanone, 6,10,14-trimethyl C18H36O 268 3.4
5 15.5 Hexadecanoic acid C17H34O2270 14.3
6 18.2 9-Octadecenoic acid C17H32O2268 2.9
7 18.8 Phytol, acetate C22H42O2338 2.3
8 19.4 9,12-Octadecadienoic acid C19H34O2296 6.8
9 20.0 E-Phytol C20H40O 296 32.4
10 20.7 9,12,15-Octadecatrienoic acid (n=6) C19H32O2292 0.7
11 21.0 9,12,15-Octadecatrienoic acid (n=3) C19H32O2292 27.6
12 22.4 Cyclopentanone, 2-(5-oxohexyl) C11H18O2182 1.4
13 26.2 2,6,10,14,18,22-Tetracosahexaene C30H50 410 2.1
Bark
1 15.4 Hexadecanoic acid C17H34O2270 25.4
2 15.9 9-Hexadecenoic acid C17H32O2268 3.0
3 17.5 Octadecanoic acid C19H38O2298 3.1
4 18.2 9-Octadecenoic acid C19H38O2296 1.2
5 18.5 Phytol, acetate C22H42O2338 0.7
6 19.3 9,12-Octadecadienoic acid C19H34O2294 10.7
7 20.3 E-Phytol C20H40O 296 8.7
8 20.9 9,12,15-Octadecatrienoic acid (n=3) C19H32O2292 47.3
Flower
1 13.5 Trisiloxane,1,1,1,5,5,5-hexamethyl-3,3-bis[(trimethylsilyl)oxy] C12H36O2384 16.4
2 15.1 3-Isopropoxy-1,1,1,7,7,7-hexamethyl-3,5,5-tris(trimethylsiloxy)tetrasiloxane C18H52O7576 10.3
3 15.3 Hexadecanoic acid C17H34O2270 16.1
4 16.8 15-methylhexadecanoic acid C17H34O2274 1.6
5 17.3 Octadecanoic acid C19H38O2298 5.3
6 17.7 Octasiloxane, 1,1,3,3,5,5,7,7,9,9,11,11,13,13,15,15-hexadecamethyl C16H50O7578 8.6
7 18.0 9-Octadecenoic acid C19H36O2296 12.5
8 19.1 9,12-Octadecadienoic acid C19H34O2294 4.5
9 20.7 9,12,15-Octadecatrienoic acid (n=3) C19H32O2292 7.2
10 21.7 Hexasiloxane, tetradecamethyl C19H42O5458 8.9
11 27.6 Heptasiloxane, hexadecamethyl C16H48O6532 8.6
Seed
1 13.9 Tetradecanoic acid C15H30O2242 0.1
2 15.4 Hexadecanoic acid C17H34O2270 20.9
3 16.0 9-Hexadecenoic acid C17H32O2268 0.6
4 17.0 cis-10-Heptadecenoic acid C18H34O2282 0.3
5 17.5 Octadecanoic acid C19H38O2298 2.2
6 17.9 9-Octadecadienoic acid (trans) C19H36O2296 1.2
7 18.2 9-Octadecenoic acid (cis) C19H36O2296 27.4
8 19.4 9,12-Octadecadienoic acid C19H34O2294 46.4
9 20.2 Nonadecanoic acid C21H42O2326 0.3
10 20.9 9,12,15-Octadecatrienoic acid (n=3) C19H32O2292 0.6
*RT: Retention time
(n=3, 47.3%). Eleven phytocompounds were identified in
the flower extract: Trisiloxane,1,1,1,5,5,5-hexamethyl-3,3-
bis[(trimethylsilyl)oxy] (16.4%), 3-Isopropoxy-1,1,1,7,7,7-
hexamethyl-3,5,5-tris(trimethylsiloxy)tetrasiloxane (10.3%),
Hexadecanoic acid (16.1%), 15-Methylhexadecanoic acid
(1.6%), Octadecanoic acid (5.3%), Octasiloxane, 1,1,3,3,5,

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Fig . 2 Total phenolic and flavonoid content of different kenaf
tissues. A: total phenolic content; B: total flavonoid content.
Superscript letters indicate significant differences at the 5% level
(Duncan’s multiple range tests, n=3)
5,7,7,9,9,11,11,13,13,15,15-hexadecamethyl (8.6%), 9-octa-
decenoic acid (12.5%), 9,12-Octadecadienoic acid (4.5%),
9,12,15-Octadecatrienoic acid (n=3, 7.2%), Hexasiloxane
tetradecamethyl (8.9%), and Heptasiloxane hexadecamethyl
(8.6%). Ten different phytocomponents were identified in
the seed extract: Tetradecanoic acid (0.1%), Hexadecanoic
acid (20.9%), 9-Hexadecenoic acid (0.6%), cis-10-Heptadecenoic
acid (0.3%), Octadecanoic acid (2.2%), trans-9-Octadecadienoic
acid (1.2%), cis-9-Octadecadienoic acid (27.4%), 9,12-Octa-
decadienoic acid (46.4%), Nonadecanoic acid (0.3%), and
9,12,15-Octadecatrienoic acid (n=3, 0.6%).
TPC and TFC analysis
TPC and TFC analysis of the extracts of the different parts
of the kenaf plant are presented in Fig. 2. TPC obtained
with the different solvents varied significantly, with the
exception of the flower, in which the water and methanol
extractions did not differ significantly. TPC and TFC in-
creased in the extracts with increasing solvent polarity.
Among all the extracts, water resulted in the highest TPC
for all the different parts of the kenaf plant. The water
extract of the leaf had the highest TPC (555.9 mg/100 g),
followed by the water extract of the flower (308.5 mg/100
g), the methanol extract of the flower (298.6 mg/100 g),
the ethanol extract of the flower (183.4 mg/100 g), the
methanol extract of the leaf (178.1 mg/100 g), the water
extract of the seed (162.7 mg/100 g), the ethanol extract
of the leaf (119.1 mg/100 g), the chloroform extract of the
seed (107.0 mg/100 g), the water extract of the stem bark
(84.2 mg/100 g), the ethanol extract of the seed (83.7
mg/100 g), the methanol extract of the seed (52.6 mg/100
g), the chloroform extract of the leaf (28.1 mg/100 g), the
ethanol extract of the stem bark (24.8 mg/100 g), the
methanol extract of the stem bark (23.2 mg/100 g), the
chloroform extract of the stem bark (5.6 mg/100 g), and
the chloroform extract of the flower (2.9 mg/100 g).
The results revealed that there was a significant dif-
ference (p < 0.05) in the TFC obtained with the different
solvents. The water extract exhibited the highest significant
TFC for all the different parts of the kenaf plant. The water
extract of the flower had the highest TFC (755.2 mg/100 g);
this was followed by the methanol extract of the flower
(620.0 mg/100 g); the water extracts of the leaf (563.7
mg/100 g), the seed (331.1 mg/100 g), and the stem bark
(299.3 mg/100 g); the chloroform extract of the seed
(154.2 mg/100 g); the methanol extract of the leaf (126.7
mg/100 g); the ethanol extracts of the flower (126.4
mg/100 g) and the seed (98.9 mg/100 g); the methanol
extract of the stem bark (70.2 mg/100 g); the ethanol
extracts of the leaf (40.2 mg/100 g) and the stem bark
(38.0 mg/100 g); the methanol extract of the seed (17.5
mg/100 g); and the chloroform extracts of the flower, the
stem bark (8.7 mg/100 g), and the leaf (4.8 mg/100 g).
UPLC analysis
The representative UPLC fingerprint chromatograms of the
different parts of the kenaf plant are shown in Table 2 and
Fig. S2. The measurement of phenolic compounds was not
possible for the chloroform extracts of the leaf, stem bark
and flower. Six compounds were detected for the water
extract of the leaf: kaempferitrin (178.2 mg/100 g), kaem-
pferol glycoside (59.2 mg/100 g), caffeic acid (76.4 mg/100
g), afzelin (38.5 mg/100 g), chlorogenic acid (23.4 mg/100
g), and isoquercitrin (18.0 mg/100 g). Six compounds were
detected for the methanol extract of the leaf: kaempferitrin
(29.6 mg/100 g), caffeic acid (7.9 mg/100 g), kaempferol
glycoside (7.0 mg/100 g), afzelin (5.9 mg/100 g), chlorogenic
acid (2.6 mg/100 g), and isoquercitrin (2.5 mg/100 g). Six
compounds were detected for the chloroform extract of the
leaf: chlorogenic acid (0.5 mg/100 g), kaemperitrin (0.1
mg/100 g). Nine compounds were detected for the ethanol
extract of the leaf: kaemperitrin (10.0 mg/100 g), an unknown
compound (8.0 mg/100 g), naringin (7.6 mg/100 g), naringin
isomer (5.1 mg/100 g), kaempferol glycoside (2.2 mg/100

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Table 2 Phytochemical constituents in different kenaf tissues in various solvents by UPLC (mg/100 g)
No. Name of the compound Methanol Water Ethanol Chloroform
Leaf
1 Chlorogenic acid 2.6b 23.4a 0.4c 0.5c
2 Caffeic acid 7.9b 76.4a 1.3c -d
3 Kaempferol glycoside 7.0b 59.2a 2.2c -d
4 Afzelin 5.9b 38.5a 1.9b -d
5 Isoquercitrin 2.5b 18.0a 0.8c -d
6 Kaempferitrin 29.6b 178.2a 10.0c 0.1d
7 Unknown -b -b 8.0a -b
8 Naringin -b -b 7.6a -b
9 Naringin isomer -b -b 5.1a -b
Bark
1 Gallic acid 1.5b 4.7a -c -c
2p-Hydroxybenzoic acid 1.0b 15.9a -c -c
3 Vanillin 2.1b 19.9a -c -c
4 Caffeic acid isomer 1.5a 18.9a -c -c
5 Caffeic acid 5.2a 51.3a -c -c
6 Kaempferol glycoside 2.9b 10.0a 4.2b -c
7 Afzelin 4.9b 13.7a 4.5b -c
8 Isoquercitrin 2.8b 9.5a 1.1b -c
9 Kaempferitrin 17.5b 25.0a 24.1c -d
Flower
1p-Hydroxybenzoic acid 9.1b 36.0a -c -c
2 Anthocyanin (520 nm) 14.8b 41.4a 1.5c -
3 Caffeic acid 5.2b 17.7a 3.5b -c
4 Unknown compound 3.8b 11.8a 2.5b -c
5 Myricetin glycoside 162.5a 142.5b 44.7c -d
Seed
1 Gallic acid 1.1b 6.7a 1.3b -c
2p-Hydroxybenzoic acid 3.0b 95.5a -c -c
3 syringic acid 0.8b 13.5a -c -c
4 p-coumaric acid -b 26.7a -b -b
5 Vanillin 0.6b 44.3a -c -c
6 Tannic acid 1.3c -d 9.7b 14.0a
7 Tannic acid isomer 1.5c -d 7.7b 11.1a
8 Proanthocyanidin -c -c 8.0b 13.2a
9 Proanthocyanidin isomer -c -c 8.7b 12.4a
10 Unknown -b -b 5.1a 5.8a
11 Naringin -b -b 4.3a 6.5a
12 Naringin isomer -b -b 4.1a 6.7a
a,b,c,dDifferent letters indicate a significant difference at the 5% level (Duncan’s multiple range tests, n=3).
g), afzelin (1.9 mg/100 g), caffeic acid (1.3 mg/100 g),
isoquercitrin (0.8 mg/100 g), and chlorogenic acid (0.4
mg/100 g).
Nine compounds were detected in the water and methanol
extracts of the stem bark: caffeic acid (water: 51.3 mg/100
g; methanol: 5.2 mg/100 g), kaempferitrin (water: 25.0 mg/100
g; methanol: 17.5 mg/100 g), vanillic acid (water: 19.9 mg/100
g; methanol: 2.1 mg/100 g), caffeic acid isomer (water: 18.9
mg/100 g; methanol: 1.5 mg/100 g), p-hydroxybenzoic
acid (water: 15.9 mg/100 g; methanol: 1.0 mg/100 g),
afzelin (water: 13.7 mg/100 g; methanol: 4.9 mg/100 g),
kaempferol glycoside (water: 10.0 mg/100 g; methanol: 2.9
mg/100 g), isoquercitrin (water: 9.5 mg/100 g; methanol:
2.8 mg/100 g), and gallic acid (water: 4.7 mg/100 g;
methanol: 1.5 mg/100 g). Four compounds were detected
in the ethanol extract of the stem bark: kaempferitrin (24.1
mg/100 g), afzelin (4.5 mg/100 g), kaempferol glycoside
(4.2 mg/100 g), and isoquercitrin (1.1 mg/100 g).
Five compounds were detected in the water and methanol
extracts of the flower: myricetin glycoside (water: 142.5
mg/100 g; methanol: 162.5 mg/100 g), anthocyanin (water:
41.4 mg/100 g; methanol: 14.8 mg/100 g), p-hydroxybenzoic

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Fig . 3 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging
activity (A) and superoxide dismutase (SOD) activity (B) for
the different kenaf tissues. Superscript letters indicate significant
differences at the 5% level (Duncan’s multiple range tests, n=3)
acid (water: 36.0 mg/100 g; methanol: 9.1 mg/100 g), caffeic
acid (water: 17.7 mg/100 g; methanol: 5.2 mg/100 g), and
an unknown compound (water: 11.8 mg/100 g; methanol:
3.8 mg/100 g)., Four compounds were detected in the ethanol
of the flower: myricetin glycoside (44.7 mg/100 g), antho-
cyanin (1.5 mg/100 g), caffeic acid (3.5 mg/100 g), and
an unknown compound (2.5 mg/100 g)
Five compounds were detected in the water extract of
the seed: p-hydroxybenzoic acid (95.5 mg/100 g), vanillin
(44.3 mg/100 g), p-coumaric acid (26.7 mg/100 g), syringic
acid (13.5 mg/100 g), and gallic acid (6.7 mg/100 g). Six
compounds were detected in the methanol extract of the
seed: p-hydroxybenzoic acid (3.0 mg/100 g), tannic acid
(1.3 mg/100 g), tannic acid isomer (1.5 mg/100 g), gallic
acid (1.1 mg/100 g), and syringic acid (0.8 mg/100 g).
Eight compounds were detected in the ethanol extract of
the seed: tannic acid (9.7 mg/100 g), tannic acid isomer
(7.7 mg/100 g), proanthocyanidin (8.0 mg/100 g), proantho-
cyanidin isomer (8.7 mg/100 g), an unknown compound
(5.1 mg/100 g), naringin (4.3 mg/100 g), naringin isomer
(4.1 mg/100 g), and gallic acid (1.3 mg/100 g). Seven com-
pounds were detected in the chloroform extract of the seed:
tannic acid (14.0 mg/100 g), tannic acid isomer (11.1 mg/100
g), proanthocyanidin (13.2 mg/100 g), proanthocyanidin isomer
(12.4 mg/100g), an unknown compound (5.8 mg/100 g),
naringin (6.5 mg/100 g), and naringin isomer (6.7 mg/100 g).
Antioxidant activity
The results of the DPPH radical scavenging activity of the
different solvent extracts of the parts of the kenaf plant are
shown in Fig. 3-A. The water extracts of the flower, leaf,
and seed (80.6%, 73.2%, and 70.4%, respectively), and the
methanol extract of the flower (71.84%) exhibited the
greatest DPPH radical scavenging activity, this was followed
by the methanol extract of the leaf (62.1%), the ethanol
extract of the leaf (37.7), the water extract of the stem bark
(29.1%), the ethanol extracts of the seed and stem bark
(21.1% and 15.4%, respectively), the methanol extracts of
the stem bark and seed (15.2% and 5.1%, respectively), the
chloroform extracts of the leaf, flower, and stem bark
(4.0%, 2.0%, and 1.0%, respectively). The results of the
SOD activity of the different solvent extracts of the parts
of the kenaf plant are shown in Fig. 3-B. The water extract
of the leaf, seed and flower and methanol extract of the
leaf and flower give the maximum SOD activity among
the other extracts.
Discussion
The results of this study revealed the presence of phyto-
compounds in the hexane extracts of the different parts of
the kenaf plant by GC-MS analysis. The leaf extract contained
13 phytocomponds, of which E-phytol and 9,12,15-octade-
catrienoic acid were the most predominant. Phytol and
linolenic acid have medicinal properties. Phytol is an
oxygenated diterpene that is a precursor for vitamins E
and K1 and is used with simple sugar in candies (Ebije et
al. 2014). Phytol exhibits antibacterial activity against
Staphylococcous aureus, Colletotrichum fragariae, Colletotrichum
gloeosporioides, and Colletotrichum accutatum by causing
damage to cell membranes, which results in a leakage of
potassium ions from bacterial cells (Kobaisy et al. 2001;
Inoue et al. 2005; Nandagopalan et al. 2015). Extracts of
the stem bark identified eight phytocompounds, primarily
fatty acids, of which hexadecanoic acid and 9,12,15-
octadecatrienoic acid were the most abundant. Rauter et al.
(2002) reported that the major compounds identified in the
petroleum extract of the stem bark of kenaf were fatty
acids (hexadecanoic acid and 9,12-octadecadienoic acid).

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The main components of the flower extract were identified
as hydrocarbons (52.8%) and fatty acids (47.2%). Similarly,
Ebije et al. (2014) reported that the major compound identified
in the Hibiscus sabdariffa flower was hexadecanoic acid.
Hydrocarbon compounds have been shown to exhibit anti-
microbial activity (Kobaisy et al. 2001; Ebije et al. 2014).
Kenaf seeds oil is used for margarine, and it is thus safe
for human and animals (Mohamed et al. 1995; Ghafar et
al. 2012; Alexopoulou et al. 2013; Ghafar et al. 2013; Ryu
et al. 2016).
The phenolic and flavonoid compounds present in the
plant body suggest its medicinal importance (Kobaisy et
al. 2001; Nyam et al. 2009; Chen et al. 2014). The functional
groups in phenolic and flavonoid compounds present in
the kenaf plant exhibit antioxidant properties and inhibit
the angiotensin I-converting enzyme and lipid peroxidation
(Jin et al. 2013; Ghafar et al. 2013). In this study, the TPC
and TFC of the kenaf leaf and flower were promising. Among
the solvents studied, the highest content of phenylpropanoid
and phenolic compounds were observed in the water extract.
The major compounds identified in the different parts of
the kenaf plant were kaemperitrin in the leaf, caffeic acid
in the stem bark, myricetin glycoside in the flower, and
p-hydroxybenzoic acid in the seed for water extract. In the
present study, extraction using a binary water solvent was
adopted after considering health and safety (Sultana et al.
2007; Yusri et al. 2012). Our experimental results are
consistent with previous reports, suggesting that a binary
solvent system is superior to a solvent system in which
phenolic compounds are extracted according to their relative
polarity (Wang et al. 2008). Water is the best solvent for
the extraction of functional compounds from the kenaf
plant, with the exceptions of myricetin glycoside, tannic
acid, proanthocyanidin, and naringin. Furthermore, we saw
high levels of antioxidant activity, which may have been
due to the increase in kaempferitrin, anthocyanin, p-hydroxy-
benzoic acid, or other minor compounds. Similarly, water
has been suggested to be the most effective solvent for the
extraction of functional compounds from the fresh leaves,
stem barks, and seeds of Calotropis procera (Abegunde
and Ayodele-Oduola, 2013). Kaempferitrin was found to
have an acute lowering effect on blood glucose levels in
diabetic rats and showed decreased lipid peroxidation and
antimicrobial and anti-inflammatory effects in macrophage
cells (Jorge et al. 2004; Jin et al. 2013; Zhao et al. 2014).
Caffeic acid is a well-known polyphenolic compound with
strong antioxidant activity (Chen et al. 2014). de Freitas
et al. (2013) reported that the water extract from the stem
bark of Scutia buxifolia revealed a high concentration of
caffeic acid. The compounds identified in the flowers of
the genus Hibiscus include anthocyanins, flavonoids, and
polyphenols (Khare, 2007; Salem et al. 2014; Obouayeba
et al. 2015). Rakhimkhanov et al. (1970) reported that the
kenaf flower contained the anthocyanin, delphinidin 3-0-fl-
D-glycosyl-fl-D-xyloside, which we have called cannabinidin.
The kenaf flowers are a potential
source of antioxidants,
including anthocyanins and phenolic compounds (Rakhimkhanov
et al. 1970; Khare, 2007; Salem et al. 2014). The kenaf
flowers have been relegated to the status of little-known
functional food sources. However, their potential as a new
source of edible flowers and natural food colorants may
elevate their status. The phytoconstituents present in the
different parts of the kenaf plant may explain their medicinal
characteristics. In the future, the isolation and purification
of these phytocompounds may be useful in the preparation
of novel drugs for the treatment of disease.
The potential of the kenaf plant as an important source
of functional compounds in the tropics is highlighted in
the present study. Our results showed that the extraction
solvents significantly altered the TPC, TAC and antioxidant
activity of the different parts of the plant, and water was
the optimal solvent for the extraction of functional com-
pounds and antioxidant activity. These results agree with
Jin et al. (2013) who reported that TPC, TAC plays an
important role in the antioxidant activities in kenaf. The
results of phytochemical analysis and antioxidant in different
part of kenaf are suitable for source of functional food and
drugs.
Acknowledgement
This work was supported by a grant from the Korea Atomic
Energy Research Institute (KAERI), Republic of Korea.
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Fig . S1 Gas chromatography-mass spectrometry (GC-MS) chromatogram of volatile compounds from different kenaf tissues. A: leaf;
B: bark; C: flower; D: seed

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Fig . S2 Ultra-high performance liquid chromatography (UPLC) chromatograms for different kenaf tissues. A: methanol extract of
leaves; B: water extract of leaves; C: ethanol extract of leaves; D: chloroform extract of leaves; E: methanol extract of bark; F: water
extract of bark; G: ethanol extract of bark; H: chloroform extract of bark; I: methanol extract of flowers; J: water extract of flowers;
K: ethanol extract of flowers; L: chloroform extract of flowers; M: methanol extract of seeds; N: water extract of seeds; O: ethanol
extract of seeds; P: chloroform extract of seeds. Peak numbers are listed in Table 2

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Fig . S2 Continued.