全 文 ：Kayode et al. / J Zhejiang Univ-Sci B (Biomed & Biotechnol) 2015 16(8):680-689
680
Cytotoxicity and effect of extraction methods on the chemical
composition of essential oils of Moringa oleifera seeds
Rowland Monday Ojo KAYODE, Anthony Jide AFOLAYAN‡
(Medicinal Plant and Economic Development Research Centre, Department of Botany,
University of Fort Hare, Private Bag X1314, Alice 5700, South Africa)
E-mail: kayodermosnr@gmail.com; AAfolayan@ufh.ac.za
Received Nov. 11, 2014; Revision accepted Apr. 30, 2015; Crosschecked July 8, 2015
Abstract: Renewed interest in natural materials as food flavors and preservatives has led to the search for suitable
essential oils. Moringa oleifera seed essential oil was extracted by solvent-free microwave and hydrodistillation. This
study assessed its chemical constituents. Cytotoxicity of the oils was investigated using hatchability and lethality tests
on brine shrimps. A total of 16 and 26 compounds were isolated from the hydrodistillation extraction (HDE) and
solvent-free microwave extraction (SME) oils, respectively, which accounted for 97.515% and 97.816% of total iden-
tifiable constituents, respectively. At 24 h when the most eggs had hatched, values of the SME (56.7%) and HDE
(60.0%) oils were significantly different (P<0.05) from those of sea water (63.3%) and chloramphenicol (15.0%). Larva
lethality was different significantly (P<0.05) between HDE and SME oils at different concentrations and incubation
periods. The median lethal concentration (LC50) of the oils was >1000 mg/ml recommended as an index for non-toxicity,
which gives the oil advantage over some antioxidant, antimicrobial, therapeutic, and preservative chemicals.
Key words: Moringa oleifera seed, Extraction methods, Essential oil, Cytotoxicity
doi:10.1631/jzus.B1400303 Document code: A CLC number: Q946
1 Introduction
Moringa oleifera Lamarch is one of the most
widely distributed and naturalized species of the
monogeneric family Moringaceae (Ramachandran
et al., 1980). The plant is known for its nutritional and
medicinal value. It contains some phytochemicals,
which make it a good source of antioxidant and anti-
microbial substances. The leaves, pod, and seed are
now being used as a food commodity in some tropical
countries where protein malnutrition exists. Moringa
leaves are reported to be a rich source of β-carotene,
protein, vitamin C, calcium, and potassium, which
makes it a good source of natural antioxidants and
thus may enhance the shelf-life of fat-containing
foods due to the presence of various types of antioxi-
dant compounds such as ascorbic acids, flavonoids,
phenolics, and carotenoids (Dillard and German, 2000;
Siddhuraju and Becker, 2003). The ethanolic extract
of the seed has shown the presence of some bioactive
compounds such as benzyl carbamate, benzyl isothio-
cyanate, niazimicin, sitosterol, and niazirin (Guevara
et al., 1999). In recent times, there has been growing
interest in Moringa plant propagation for industrial
application in developing countries like Nigeria.
Essential oils are aromatic and volatile com-
pounds found in most parts of plant materials such as
the leaves, seed, flower, bark, fruit, and peel (Sánchez
et al., 2010). The chemical compositions of essential
oils are secondary metabolites, which play important
roles in a plant’s defense against microbial attacks
and have been added to foods as spices for decades
(Hyldgaard et al., 2012). The active compounds in
essential oils have been broadly divided into four
Journal of Zhejiang University-SCIENCE B (Biomedicine & Biotechnology)
ISSN 1673-1581 (Print); ISSN 1862-1783 (Online)
www.zju.edu.cn/jzus; www.springerlink.com
E-mail: jzus@zju.edu.cn
‡ Corresponding author
ORCID: Rowland Monday Ojo KAYODE, http://orcid.org/0000-
0002-2828-2519
© Zhejiang University and Springer-Verlag Berlin Heidelberg 2015
Kayode et al. / J Zhejiang Univ-Sci B (Biomed & Biotechnol) 2015 16(8):680-689
681
groups according to their chemical structures. The first
group is terpenes such as limonene, p-cymene, sab-
inene, terpinene, and pinene. Terpenes are hydro-
carbons produced from a combination of several iso-
prene units (C5H8), and are synthesized in the cyto-
plasm of plant cells; they have a hydrocarbon back-
bone, which can be rearranged into cyclic structures
by cyclases, thus forming monocyclic or bicyclic
structures (Caballero et al., 2003). The main terpenes
are monoterpenes (C10H16) and sesquiterpenes (C15H24),
although other chains such as diterpenes (C20H32),
triterpenes (C30H40), and even longer chains exist
(Hyldgaard et al., 2012). The second group is terpe-
noids such as thymol, citronellal, piperitone, car-
vacrol, linalyl acetate, and menthol. Terpenoids are
terpenes that undergo biochemical modification via
enzymes that incorporate oxygen molecules and shift
or remove methyl groups (Caballero et al., 2003).
According to Caballero et al. (2003) and Hyldgaard
et al. (2012), terpenoids are subdivided into alcohols,
aldehydes, ketones, esters, ethers, epoxides, and
phenols. The third group is phenylpropenes, which
constitute a subfamily among the various groups of
organic compounds called phenylpropanoids that are
synthesized from the amino acid precursor phenyl-
alanines in plants. Phenylpropanoids have their names
from the six-carbon aromatic phenol group and the
three-carbon propene tail of cinnamic acid, produced
in the first step of phenylpropanoid biosynthesis
(Hyldgaard et al., 2012). In addition, there are essen-
tial oils, which contain a number of different degra-
dation products originating from unsaturated fatty
acids, lactones, terpenes, glycosides, and compounds
that contain either sulfur or nitrogen (Caballero et al.,
2003; Hyldgaard et al., 2012). The chemical composi-
tion of a specific essential oil may vary depending on
the season of harvest and the methods used to extract
the oil (Pereira and Meireles, 2010; Sánchez et al.,
2010; Demuner et al., 2011; Hyldgaard et al., 2012).
Essential oils from plant sources have been isolated
by traditional distillation using water, steam, water
and steam, organic solvents such as alcohol, ether,
hexane, or their mixtures at different concentrations.
Essential oils have also been isolated by hydrodistil-
lation (Khajeh et al., 2004; Okoh and Afolayan, 2011)
and supercritical carbon dioxide extraction (Khajeh
et al., 2004). Steam and solvent distillation has resulted
in degradation and loss of several volatile compounds
in addition to the formation of new compounds due to
the prolonged extraction period (Okoh and Afolayan,
2011). The degradation of unsaturated components of
oils through thermal and/or hydrolytic processes is a
disadvantage of these methods (Khajeh et al., 2004).
Brenes and Roura (2010) reported the use of essential
oil as a good antioxidant substance in diets. Scientist’s
keen interest in essential oils and their application in
food preservation have been geared in recent years by
the continuous increase of negative consumer per-
ception of most synthetic preservatives (Hyldgaard
et al., 2012).
Presently, about 3000 kinds of essential oils have
been identified, out of which only 300 are being
commercially used in the flavor and fragrance market
(Burt, 2004). The food industry primarily uses essen-
tial oils as aroma and color substances in food and as
such, detailed knowledge of their chemical properties,
which may give information on their effect on food
matrix components, is important. Hence, this work is
designed to assess the cytotoxicity potentials and
effect of extraction methods on the chemical con-
stituents of the essential oil of Moringa oleifera seeds.
Knowledge gained from the study may provide in-
formation on a suitable method of extraction of the
essential oil for industrial application, and may facili-
tate implementation of the essential oil as a natural
source of plant and animal food additives.
2 Materials and methods
2.1 Sources and preparation of Moringa oleifera
seeds
The seeds (1 kg) of matured Moringa oleifera
plant were collected in clean polythene bags at the
University of Ilorin Moringa Plantation Farm, Ilorin,
Kwara State, Nigeria. The seeds were taken to the
Department of Plant Biology, Faculty of Science,
University of Ilorin, Ilorin, Kwara State, Nigeria for
authentication. The Moringa seeds were carefully
crushed using a pestle and mortar to de-hull the seeds.
Thereafter, the seeds were manually cleaned by sep-
aration from the hulls. The cleaned seeds were
packaged into a clean and ice-packed plastic con-
tainer before transporting to the Medicinal Plant and
Economic Research Centre, University of Fort Hare,
Republic of South Africa for further analysis.
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2.2 Solvent-free microwave extraction (SME) of
the essential oil of Moringa oleifera seeds
Two hundred grams of uncoated Moringa seeds
were weighed into the reactor of an automatic mill-
stone Dry DIST microwave apparatus. The multi-
mode microwave reactor has a twin magnetron (2800 W,
2450 MHz) with a maximum delivered power of
1000 W in 10 W increments. A rotating microwave
diffuser ensures homogenous microwave distribution
throughout the plasma-coated polytetrafluoroethylene
(PTFE) cavity (35 cm×35 cm×35 cm). The tempera-
ture was monitored by a shielded thermocouple
(ATC-300) inserted directly into the corresponding
container. Temperature was controlled by a feedback
to the microwave power regulator, which indicated
that the initial temperature was 20 °C and increased at
7.5 °C/min up to 100 °C before commencement of
essential oil extraction for 10 min. After complete
extraction of the oil, the temperature decreased at
7.0 °C/min until 30 °C was reached. The total running
time of the extraction process was 30 min. The ex-
tracted essential oil was retained in 1.0 ml n-hexane
used as solvent phase in the oil collector column of
the equipment. Afterwards, the essential oil was
carefully separated into a glass sample vial and kept
in the refrigerator at 4 °C for further analysis.
2.3 Hydrodistillation extraction (HDE) of the es-
sential oil of Moringa oleifera seeds
Two hundred grams of uncoated Moringa seeds
were weighed into a 5-L capacity of a glass HDE
apparatus containing 3.0 L of sterile distilled water.
The extracted essential oil was retained in 1.0 ml
n-hexane used as solvent phase in the oil collector
column of the hydrodistillation apparatus. Thereafter,
the essential oil was carefully separated into a glass
sample vial and kept in the refrigerator at 4 °C for
chemical analysis. The essential oil yield was calcu-
lated using the formula below: oil yield (%)=(weight
of oil extracts)/(weight of sample)×100%.
2.4 Determination of chemical composition of the
essential oil of Moringa oleifera seeds
The seed oil of Moringa oleifera was analyzed
with an Agilent 6890N Network gas chromatographic
(GC) system connected to an Agilent 5973 Network
mass selective detector. The machine was equipped
with a mass spectroscopy (HP-5MS) column (30 m×
0.25 mm 5% phenyl methylpolysiloxane capillary
column, film thickness (0.25 μm); injector tempera-
ture 250 °C and transfer line temperature 240 °C).
The oven temperature was programmed as follows:
initial temperature was 50 °C for 15 min, and then
increased at 2 °C/min up to 150 °C, which was
maintained for 10 min before a further increase at
2 °C/min up to 220 °C which was maintained for
20 min (total run time is 130 min). Hydrogen was
used as the carrier gas at 2 ml/min. The amount of
sample injected into the machine was 5 μl (split ratio
1:20), while ionization energy was 70 eV. Qualitative
identification of the different constituents was per-
formed by comparison of their relative retention time
(RT) and mass spectra with those of standard refer-
ence compounds or by comparison of their retention
indices and mass spectra with those shown in the
literature (Adams, 1995). Probability merge search
software and the NIST MS spectra search program
were used. The relative amount (RA) of an individual
component of the essential oil was expressed as a
percentage of the peak area relative to the total peak
area. The method of Kováts (1958) modified by
IUPAC (1997) was adopted in the calculation of
Kováts indices (KI) of the components relative to the
RT of a series of n-alkanes with linear interpolation
on the HP-5MS column.
2.5 Hatchability test of brine shrimps
This was carried out to assess the effect of
Moringa oleifera essential oil extracted by different
methods on the hatchability of brine shrimp eggs.
Twenty shrimp eggs were introduced into a 40-ml
capacity sterile petri-dish each containing freshly
prepared mixture of the essential oil and sea water at
different concentration gradients of 250, 500, 750,
1000, 1500, 2000, 4000, and 6000 μg/ml. A control
sample consisting of 0.1% dimethyl sulphoxide
(DMSO; 0.1 ml DMSO in 100 ml sea water) without
the addition of oil was prepared. Each treatment and
control sample was distributed in triplicate into the
sterile petri-dish before incubation at 28 °C under
constant illumination in a digital incubator (MRC
Laboratory Equipment, Model LE-509). Thereafter,
the petri-dishes were examined with the aid of a hand
lens against a white background that allows the
moving larvae (naupili) to be separated from shells
and counted at every 12 h for 72 h. The percentage
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hatchability was calculated by comparing the number
of hatched naupili with the total number of brine
shrimp eggs stocked (Carballo et al., 2002).
2.6 Toxicity test on brine shrimps
This was performed in order to predict the toxicity
level of the essential oil of Moringa oleifera seeds
extracted by different methods. The test was con-
ducted according to the methods of Okoh and
Afolayan (2011) and Nkya et al. (2014). The brine
shrimp eggs were sourced from Ocean Star Interna-
tional, Inc. (USA) by the Medicinal Plant and Eco-
nomics Development Unit, Department of Botany,
University of Fort Hare, South Africa. Shrimp eggs
were placed into natural sea water in a 40-ml capacity
sterile petri-dish and incubated at 28 °C under con-
stant illumination in a digital incubator (Model
BOD-150) for 48 h to hatch. The hatched shrimps
(naupili) in the petri-dishes were attracted with a light
source to one side and then harvested. Stock solution
of the oil containing 100 mg/ml DMSO was prepared
by dissolving 100 mg of the essential oil in 1.0 ml of
DMSO. From the stock solution, 100 ml of different
concentrations of 250, 500, 750, 1000, 1500, 2000,
4000, and 6000 μg/ml of the essential oils in natural
sea water were prepared. A control sample without
essential oil consisting of 0.1% DMSO in sea water
was prepared. The control and each of the treatment
samples were distributed in triplicate petri-dishes.
Thereafter, 15 harvested brine shrimp larvae were
added to each of the petri-dishes before incubation
at 28 °C in a digital incubator (MRC Laboratory
Equipment, Model LE-509). The petri-dishes were
examined with the aid of a lens against a white
background for mortality at every 12 h for 72 h.
To ensure that larvae (naupili) mortality is at-
tributed to the bioactive compounds inherent in the oil
and not as a result of starvation or contacts with
DMSO, the number of dead larvae in the control was
compared with the number of dead larvae in each of
the treatment samples. The percentage of larva mor-
tality (LM) was calculated according to this formula:
LM (%)=[(N−NT)−(N−NC)]/N×100%, where N is the
number of larvae used and constant for treatments and
control, NT is the number of survived larvae in each
treatment, and NC is the number of survived larvae in
the control.
2.7 Design and statistical analysis
Completely randomized design was used in the
experiment. Data obtained for mortality in every
treatment and positive control were used to construct
a dose against response graph. The best-fit line from
the linear regression analysis of the percentage mor-
tality versus concentration was used to determine
median lethal concentration (LC50) values. Treat-
ments having values of LC50 greater than 1000 μg/ml
were considered nontoxic (Gupta et al., 1996). The
analysis was performed on MINITAB Version 12 for
Windows. One-way analysis of variance followed by
Fisher’s least significant difference post-hoc analysis
was used to test for the effect of concentration and
time of exposure of the essential oil.
3 Results
3.1 Chemical composition of the essential oil
The yield, retention indices, and chemical con-
stituents of the essential oil of Moringa oleifera seeds
processed by different methods are indicated in
Table 1. The yields of a faint yellow-colored volatile
oil of the SME and HDE processes were 6.694 and
6.982 g/kg, respectively. From the GC-MS analysis, a
total of 16 and 26 chemical compounds were isolated
from the HDE and SME volatile oils, which ac-
counted for 97.515% and 97.816% of the total iden-
tifiable constituents, respectively. According to Chuang
et al. (2007), GC-MS analysis has revealed 44 dif-
ferent chemical compounds in Moringa oleifera leaf
extracts. Cyclopentane (51.493%) was the predomi-
nant compound in the oil processed by SME, followed
by n-hexadecanoic acid (11.143%), 2-(E)-decenal
(4.369%), eicosane (3.102%), and 1,5-dimethyl-2-
pyrrolecarbonitrile (2.003%). Other compounds ranged
between 0.122% (tetracosane) and 1.924% (1-nonanol).
The oil extracted by HDE showed the presence of
tetracosane (34.259%) as the most abundant com-
pound, followed by heptadecane (22.202%), eicosane
(19.583%), n-hexadecanoic acid (8.454%), phenan-
threnecarboxylic acid (3.784%), and cyclopentane
(3.598%), while other compounds ranged between
0.215% (phthalic acid) and 1.718% (acetamide). The
oxygenated compounds of the SME and HDE pro-
cessed oils indicated 27.678% and 15.979%, while
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684
the non-oxygenated compounds showed 72.138% and
81.967%, respectively.
Fourteen chemical isolates consisting of 8 oxy-
genated and 6 non-oxygenated compounds were
concurrently detected in both the HDE and SME
volatile oil samples as presented in Table 1.
3.2 Effect of the oil on hatchability of brine
shrimp eggs
Fig. 1 shows the percentage hatchability of brine
shrimp eggs at different incubation periods in the
volatile oil. At the end of 12 h, not less than 1.7% and
3.3% of the eggs incubated in the SME and HDE oil
treated samples were hatched and rapidly increased to
maximum values of 66.7% and 68.3%, respectively,
in 48 h. The values obtained in the SME (56.7%) and
HDE (60.0%) oil-treated samples were significantly
different (P<0.05) compared with the chloramphenicol-
treated sample (15.0%) and the natural sea water
(63.3%) at 24 h when the most eggs hatched. Fig. 2
presents the percentage hatchability of the brine shrimp
eggs at different concentrations of the essential oil.
Table 1 Chemical compounds of the essential oil of Moringa oleifera seeds extracted using solvent-free microwave
and hydrodistillation methods
No. Chemical constituent KI
Relative peak area (%)
SME HDE
1 Cyclopentane 816 51.493 3.598
2 Nonanal 1252 1.737 0.284
3 Azulene 1262 0.224
4 2-(E)-decenal 1314 4.369
5 1-Nonanol 1324 1.924 0.431
6 1,2-Dimethyl-4-(2-propenyl)benzene 1356 8.734
7 Tridecane 1409 0.937 0.256
8 1,6-Dibromo-silane 1457 0.468 0.239
9 Heptadecane 1495 1.648 22.202
10 Propanoic acid 1523 0.945
11 Butanoic acid 1533 1.324
12 2,4-Diphenyl-4-methyl-2-(E)-pentene 1554 1.378 0.837
13 Docosane 1557 0.864 0.570
14 Nonadecane 1572 0.793 0.491
15 Isamoxole 1596 0.626
16 Phenanthrenecarboxylic acid 1599 3.784
17 n-Hexadecanoic acid 1607 11.143 8.454
18 8-Dimethyl-2-isopropylphenanthrene 1615 0.937
19 Tetracosane 1626 0.122 34.259
20 2,2-Bipyridine-3,3-diol 1721 1.239
21 Phthalic acid 1917 0.494 0.215
22 Diethyldithiophosphoric acid 1941 0.374
23 Propanenitrile 1947 1.498 1.025
24 Cyclohexane 1948 0.252
25 1,5-Dimethyl-2-pyrrolecarbonitrile 1977 2.003
26 Acetamide 1967 1.718
27 Phenol 1984 1.188
28 Eicosane 2000 3.102 19.583
29 Unidentified compound* 2.184 2.485
Sum of peak areas of the identified compounds (%) 97.816 97.515
Extraction time (min) 30 180
Essential oil yield (g oil/kg seed weight) 9.694 6.982
Total peak areas of oxygenated compounds (%) 27.678 15.979
Total peak areas of non-oxygenated compounds (%) 72.138 81.967
KI: Kováts index; SME: solvent-free microwave extraction; HDE: hydrodistillation extraction. * No matches found in the
C:\Database\NIST05; DB-5MS non-polar column relative to C3 to C24 n-alkanes
Kayode et al. / J Zhejiang Univ-Sci B (Biomed & Biotechnol) 2015 16(8):680-689
685
Hatchability was significantly different (P<0.05) among
oil-treated samples, sea water, and positive control at
all levels. The value recorded in the chloramphenicol-
treated sample was significantly lower (P<0.05) at all
treatment levels compared with the oil-treated sam-
ples and sea water.
3.3 Effect of the oil on lethality of brine shrimp larvae
The mortality of brine shrimp larvae incubated
in different concentrations of the essential oil of
Moringa oleifera seeds is presented in Fig. 3. The
values obtained for mortality of the brine shrimp
larvae were significantly higher (P<0.05) at all levels
in the chloramphenicol-treated sample compared with
essential oil-treated samples. Mortality of the brine
shrimp larvae at different periods of incubation in the
essential oil extracted by different methods is illus-
trated in Fig. 4. Naupili mortality was first observed
after 24 h in the chloramphenicol-treated sample and
was in acute status from 24 to 36 h when it became
completely logarithmic after 48 h in all the samples
except natural sea water. The shrimp mortality was
significantly different (P<0.05) in the HDE and SME
oils at 48 h until experiment was terminated. The
value recorded at every period of assessment was
significantly lower (P<0.05) in the sea water com-
pared to the positive control and the essential oil-
treated samples. This study revealed LC50 of the SME
and HDE oils at 2906.83 and 3495.82 µg/ml, respec-
tively, as presented in Table 2. These values were
greater than 2716.71 µg/ml recorded for chloram-
phenicol used as a positive control.
Fig. 2 Hatchability of brine shrimp eggs incubated in
different concentrations of the essential oil of Moringa
oleifera seeds extracted by different methods
Fig. 3 Mortality of brine shrimp larvae incubated in
different concentrations of the essential oil of Moringa
oleifera seeds extracted by different methods
Fig. 4 Mortality of brine shrimp larvae at different
periods of incubation in the essential oil Moringa oleifera
seeds extracted by different methods
Fig. 1 Hatchability success of brine shrimp eggs at dif-
ferent periods of incubation in the essential oil of Moringa
oleifera seeds extracted by different methods
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4 Discussion
The pattern of occurrence of the oxygenated and
non-oxygenated compound obtained in the extracted
oils is in agreement with the findings of Okoh et al.
(2010) who similarly reported higher value of oxy-
genated (28.63%) compounds compared with the
non-oxygenated (26.98%) constituents of the essen-
tial oil of Rosmarinus officinalis extracted by solvent-
free microwave technique. Okoh et al. (2010) at-
tributed this phenomenon to diminution of the ther-
mal and hydrolytic properties of SME compared with
the hydrodistillation method which required a large
quantity of water for extraction. The absence of most
chemical compounds in the oil extracted by the hy-
drodistillation process may be attributed to the water
solubility potential and/or the thermal instability of
the various compounds inherent in Moringa oleifera
seeds. Thus, the polar property of water makes it a
good solvent for the acceleration of many reactions
via carbocation intermediates (Lucchesi et al., 2004).
Consequently, the emergence of new compounds in
the oil extracts of both techniques may be linked to
the partial or complete degradation of some unsatu-
rated components during oil extraction from the seeds.
Khajeh et al. (2004) earlier reported that the degra-
dation of unsaturated fats in oil through thermal
and/or hydrolytic process is disadvantage of steam
and solvent extraction methods. The microwave irra-
diation highly accelerated the extraction process
without causing considerable changes in the volatile
oil composition, a phenomenon already described by
Pare and Belanger (1997) and Okoh et al. (2010).
The presence of n-hexadecanoic acid (palmitic
acid) in the oils collected by both processing methods
is in agreement with the suggestions of Lyman et al.
(1990) that hydrolysis should not be an important
factor that may influence the fate of the compound
during processing, since it lacks functional groups
that could hydrolyze under environmental conditions.
Meylan and Howard (1993) reported that palmitic
acid exhibited negligible direct photo-degradation in
sea water. Palmitic acid is one of the more common
fatty acids found in natural fats and oils (Verschueren,
1996), and has been detected in matured grains of
brown rice and in the natural constituents of many
plants (Taira et al., 1988). Fatty acids are an important
part of the normal daily diet of mammals, birds, and
invertebrates (Anneken et al., 2006).
Cyclopentane, which appeared to be the most
abundant compound in the SME volatile oil, was
detected in the smaller quantity of 3.598% in the
essential oil extracted by the HDE method. This
phenomenon is expected and also in conformity with
New Jersey Department of Health and Senior Ser-
vices (1999) who reported that cyclopentane had a
low boiling point of 49.5 °C and was also strongly
soluble in water; perhaps the compound became un-
recoverable in the condensate or merely dissolved in
the still water during a prolonged hydrodistillation
process of the essential oil from the seeds. From the
results obtained in Fig. 2, it could be deduced that the
volatile oil of Moringa oleifera seeds extracted by
both HDE and SME had no potential to completely
inhibit hatching of the brine shrimp eggs at the con-
centrations used in this study. Increased percentage
mortality was noticed as the concentrations of the
essential oil increased (Fig. 3). This phenomenon
seems to have depended on the length of incubation
period as earlier reported by some authors (Pour and
Sasidharan, 2011; Otang et al., 2013). It may be
suggested that the larvae had reached their instar
phase and exhibited greater sensitivity to the test
compound (Lewis, 1995), which led to a maximum
mortality at a longer exposure time. An incubation
period greater than 60 h had no significant changes on
Table 2 Mortality of brine shrimp larvae as indicated by LC50 in differently extracted Moringa oleifera seed
essential oils
Treatment LC50 (µg/ml) Regression equation R2 (%) P-value
SME 2906.83 Y=0.0172X+0.0025 97.50 0.000
HDE 3495.82 Y=0.0143X+0.0097 98.30 0.000
Chloramphenicol 2716.71 Y=0.0184X+0.0125 84.30 0.003
SME: solvent-free microwave extraction; HDE: hydrodistillation extraction. R2: coefficient of determination of the regression equation;
P-value: significance level of the regression equation, which is significant when P<0.05
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the mortality of larvae in the oil treated samples
compared with the control (sea water). Similar ob-
servations were made when the naupili of brine
shrimps were exposed to the methanol extracts of
different parts of Lantana camara (Pour and Sasi-
dharan, 2011) and hexane and acetone extracts of
three South African plants (Otang et al., 2013).
The suspected cytotoxic chemical constituents
isolated from the Moringa oleifera seed essential oils
include cyclopentane, n-hexadecanoic acid, 2-(E)-
decenal, eicosane, 1,5-dimethyl-2-pyrrolecarbonitrile,
1-nonanol, tetracosane, heptadecane, phenanthrene-
carboxylic acid, phthalic acid, diethyldithiophos-
phoric acid, and acetamide. Previous studies had elu-
cidated the presence of n-hexadecanoic and phthalic
acids in lemon, acetamide and propanoic acid in gar-
lic, and 1,5-dimethyl-2-pyrrolecarbonitrile and phe-
nol in turmeric (Nyaitondi, 2007). Diethyldithio-
phosphoric acid has been identified as a useful com-
pound for the production of organophosphate insec-
ticides such as parathion and parathion-methyl
(Okuniewski and Becker, 2011). The crude meth-
anolic plant extract containing most of the suspected
chemical compounds has demonstrated antibacterial
activities on both Gram-positive and Gram-negative
bacteria (Nyaitondi, 2007). Polyunsaturated fatty
acids are known to be essential for the maintenance of
good health, but are subject to peroxidation when in
contact with atmospheric oxygen leading to frag-
mentation and reactive decomposition. A prominent
autoxidation product of either arachidonic acid or
trilinolein has been reported to be trans-4,5-epoxy-
2(E)-decenal (Lin et al., 2001), which was isolated
from the oil. This compound has been reported reac-
tive with nucleophiles on proteins, leading to loss of
cell function and viability (Zamora and Hidalgo,
1994). Therefore, it could be suggested that the
presence of 2-(E)-decenal in the essential oil ex-
tracted by SME has contributed to the increase in
brine shrimp mortality over the treatment containing
the HDE-extracted oil in addition to increased oil
concentrations. Trans-4,5-epoxy-2(E)-decenal has
also been found to be a useful tool in elucidating the
effects of peroxidative damage in experimental mod-
els (Zamora and Hidalgo, 1994). The derivatives of
2-pyrrolecarbonitrile are important constituents in the
synthesis of porphobilinogen and the main building
block used in the biosynthesis of pigments in plant
(Santos and Ribeiro da Silva, 2012). Another deriva-
tive such as tetrapyrrolic compounds has been applied
in photodynamic therapy for the treatment of cancer.
Potent activity of synthesized 2-pyrrolecarbonitrile
has been demonstrated on the progesterone receptor
antagonist, which can be a potential contraceptive
agent (Santos and Ribeiro da Silva, 2012). The vola-
tile oils of Moringa oleifera seeds obtained by HDE
and SME methods contain aromatic hydrocarbons,
phenols, ketones, organic acids, and different terpenes,
which may have impacted inhibitory effects on the
shrimp larvae at different concentrations over time.
The varied occurrences of these compounds in the oil
samples may indicate that their inhibitory effects on
the shrimp larvae were not the direct effect of a
compound or single group, but rather that the com-
pounds possibly acted in synergy to bring about a
toxic effect especially at higher doses. The non-toxicity
effect of both the SME and HDE essential oils at
1000 µg/ml is in agreement with Meyer’s index for
non-toxic oil (Meyer et al., 1982; Gupta et al., 1996).
This may be an advantage for the utilization of the
essential oil of Moringa oleifera seeds over some
antioxidant and antimicrobial chemical substances
commonly used as therapeutic agents, preservatives
or as carriers of other useful additives in cosmetics
and processed foods.
5 Conclusions
The renewed interest of the pharmaceutical in-
dustries and food processors in essential oils as pos-
sible natural substances to replace synthetic drugs and
preservatives has redirected the focus of many re-
searchers. This study revealed the chemical compo-
sition of the volatile oils of Moringa oleifera seeds
extracted by different methods. The chemical com-
positions of the volatile oils of the HDE and SME
varied considerably. Thus, SME-extracted volatile oil
possessed a greater number of chemical compounds,
some of which were not detected in the HDE oil. This
observation is in agreement with the report of Okoh
et al. (2010). The volatile oils were toxicologically
screened using the brine shrimp egg hatchability test
and larva mortality at different concentrations and
durations of incubation. It was revealed that the shrimp
eggs and larvae were more sensitive to the SME oil
Kayode et al. / J Zhejiang Univ-Sci B (Biomed & Biotechnol) 2015 16(8):680-689
688
than to the HDE oil, and were both significantly
different (P<0.05) when compared with the natural
sea water control samples, although the oil-treated
samples were found to be significantly lower (P<0.05)
compared with chloramphenicol-treated sample (posi-
tive control). The predominant activities of the SME
volatile oil over the HDE volatile oil may partly be
due to the greater number of oxygenated compounds
present in the SME volatile oil, many of which have
been proved to be strongly active antimicrobial agents
(Nyaitondi, 2007; Sandri et al., 2007). The SME and
HDE volatile oils of Moringa oleifera seeds exhibited
low brine shrimp larva toxicity and possessed LC50
values of 2908.23 and 3473.63 µg/ml, respectively.
These values were greater than 1000 µg/ml recom-
mended as an index for non-toxic oil (Meyer et al.,
1982). As such, the oil may be explored for the de-
velopment of useful plant-based pharmaceuticals,
food preservatives and antioxidant agents or as car-
riers of other additives such as flavor in processed
foods and fragrance in cosmetic production.
Acknowledgements
We greatly acknowledged the University of Fort Hare,
Alice, South Africa for granting one of the authors the oppor-
tunity to serve as a research visitor in the institution and her
financial support through Govan Mbeki Research and Devel-
opment Centre in the publication of these research findings.
Compliance with ethics guidelines
Rowland Monday Ojo KAYODE and Anthony Jide
AFOLAYAN declare that they have no conflict of interest.
This article does not contain any studies with human or
animal subjects performed by any of the authors.
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中文概要
题 目：辣木籽精油的细胞毒性及其提取方法对化学成分
的影响
目 的：研究水蒸汽蒸馏法（HDE）和无溶剂微波萃取法
（SME）对辣木籽精油化学组成的影响，并评估
精油的细胞毒性。
创新点：比较了不同的提取精油方法对精油的化学成分及
其细胞毒性的影响。
方 法：分别采用 HDE 和 SME 两种方法提取辣木籽精油，
然后使用气相色谱-质谱法分析其化学成分。通过
比较在不同浓度和孵化时间下盐水虾卵的孵化
率和幼虾的死亡率来检测精油的细胞毒性。
结 论：HDE 和 SME 两种方法提取的辣木籽精油分别含
有 16 和 26 种化学成分，各自占总可识别成分的
97.515%和 97.816%。因此，SME 提取法含有更
多的成分。盐水虾卵分别在含有两种精油、海水
和氯霉素的样品中孵化 24 h 后，孵化率分别为
60.0%（HDE）、56.7%（SME）、63.3%（海水）
和 15.0%（氯霉素），精油中的孵化率远高于含
有氯霉素的样品的孵化率（P<0.05）。同时，SME
和HDE精油对幼虾的半致死量分别为 2908.23和
3473.63 µg/ml（大于 1000 µg/ml 被认为无毒）。
因此，两种方法提取的精油可广泛适用于药物、
食品防腐剂、抗氧化剂和香料等添加剂。
关键词：辣木籽；提取方法；精油；细胞毒性