Journal of Oleo Science

Copyright ©2022 by Japan Oil Chemists’ Society

doi : 10.5650/jos.ess22095
J. Oleo Sci. 71, (9) 00000-00000 (2022)

Composition and Characterization of Cold Pressed

Moringa oleifera Seed Oil –Beneficial Potentials of
Moringa oleifera Seed Oil–
Karima Gharsallah1,2＊, Leila Rezig3,4, Fatma B’chir5, Soumaya Bourgou6,
Nahed Ben Achour3, Chaima Jlassi7, Abdellah Chalh8, and Taoufik Soltani1
1
 hysics laboratory of Soft Matter and Electromagnetic Modeling, LR99ES16, Faculty of Science of Tunis, Tunis El Manar University, 2092 Tunis,
P
TUNISIA
2
Process engineering department, Higher Institute of Technological Studies of Zaghouan, General Direction of Technological Studies, 1121
Tunis, TUNISIA
3
High Institute of Food Industries, 58 Alain Savary Street, El Khadra City, 1003 Tunis, TUNISIA
4
University of Carthage, National Institute of Applied Sciences and Technology, LR11ES26, LIP-MB ‘Laboratory of Protein Engineering and
Bioactive Molecules’, Tunis, TUNISIA
5
Laboratory of Natural Substances, National Institute of Research and Physico-chemical analyses, Sidi Thabet Technology Center, 2020, Sidi
Thabet, TUNISIA
6
Laboratory of Aromatic and Medicinal Plants, Biotechnology Center of Borj-Cedria Technopole, BP. 901, Hammam-Lif 2050, TUNISIA
7
University of Manouba, ISBST, BVBGR-LR11ES31, Biotechpole Sidi Thabet, 2020, Ariana, TUNISIA
8
Laboratoire des Interactions Plante Sol Environnement (LR21ES01), Faculty of Science of Tunis, Tunis El Manar University, 2092 Tunis,
TUNISIA

Abstract: The present study aims to investigate the volatile compound and the triacylglycerol profiles of
Tunisian cold pressed Moringa oleifera seed oil (MoSO) and to assess its thermal properties and its
biological activities. GC-MS analysis identified thirty six phyto-compounds amounting to 98.99% of the
total oil. These compounds were classified into eleven groups among which the fatty acid one exhibited the
highest intensity (91.63%). Cis, 6-octadecenoic acid was the most abundant compound (70.68%). The
triacylglycerol composition of MoSO was characterized by the predominance of the glycerol trilinoleate
(LLL) associated with glycerol–oleate–linoleate-linolenate (OLLn) (14.42±0.12%). Thermogravimetric
analysis of MoSO showed that the oil possess an interesting thermal stability with a highly Onset
temperatures (Tonset) of 390.72°C and 357.47°C, respectively in nitrogen and air atmospheres. By using the
ABTS assay, MoSO exhibited an interesting antioxidant capacity of 3.65 mM TEAC/mg of oil. The oil was
also endowed with a relatively strong anti-inflammatory activity since its treatment at the different
concentrations tested (75, 150 and 300 μg/mL). However, no antimicrobial activity was observed. On the
basis of the obtained results, MoSO could be used in diverse industrial applications such as pharmaceutical,
cosmetic, and food fields thanks to its thermal stability and interesting biological activities.

Key words: Moringa oleifera seed oil, GC-MS analysis, TG/DTG, anti-inflammatory activity, antioxidant capacity

1 Introduction
Moringa oleifera, or Ben oil tree1）is a fast-growing tree
belonging to the Moringaceae family. Moringa oleifera is
native to the sub-Himalayan areas of Northern India;
however, it is cultivated all over the world due to its multi-
ple utilities2）. All parts of Moringa oleifera such as seeds,
leaves, roots and even flower are fit for consumption as
vegetable3）. The root extract was found to have an antimi-
crobial activity4）. The extracts from the flowers were also
reported to possess hepatoprotective effect5）. Moreover,
the seed cake is a natural coagulant and can be effectively
utilized for treatment and purification of the highly turbid
water6）.
Several investigations proved that Moringa oleifera
seed kernels possess significant oil content（up to 40％）
with a high proportion of fatty acids（oleic acid＞70％）7−11）

＊
Correspondence to: Karima Gharsallah, Physics laboratory of Soft Matter and Electromagnetic Modeling, LR99ES16, Faculty of
Science of Tunis, Tunis El Manar University, 2092 Tunis, TUNISIA
E-mail: karima.gharsallah@fst.utm.tn
Accepted April 28, 2022 (received for review March 15, 2022)
Journal of Oleo Science ISSN 1345-8957 print / ISSN 1347-3352 online
http://www.jstage.jst.go.jp/browse/jos/  http://mc.manusriptcentral.com/jjocs

000　22095Gharsallah.indd 1 2022/07/29 10:09:20

 K. Gharsallah, L. Rezig, F. B’chir et al.
and a remarkable resistance to thermal and oxidative deg-
radation7, 11）. Fair enough, this rich oil profile makes the
Moringa seeds ideal for human ingestion and commercial
utilization12）.
A large number of medicinal and nutritional properties
have also been ascribed to various parts of the plant
kingdom and in particular its seed oil9）. Moringa oleifera
seeds are known for its antioxidant, antimicrobial, anti-in-
flammatory, hypocholesterolemic and anti-asthmatic activi-
ty2, 13）. However, these activities have largely been studied
using its extracts and no data is available on Moringa seed
oil’s anti-inflammatory activities, especially that originating
from North African countries. Given these facts, the main
purpose of this work is to study the volatile compound and
the triacyglycerol profiles of Tunisian cold pressed
Moringa oleifera seed oil （MoSO） and to assess its biologi-
cal activities and its thermal stability by using the thermo-
gravimetric analysis. Scanning Electron Microscope（SEM）
was also used to examine the structural morphology of
Moringa oleifera seed, seed flour, and defatted flour.
2 Materials and Methods
2.1 Seed material and lipid extraction
Moringa oleifera seeds were collected from a plant
nursery located in the town of Beni Khiar （36° 29'02.3"N 10°
48'24.2"E）in Nabeul, Tunisia. The seeds were isolated and
washed to eliminate impurities before being air-dried in the
shade. The seeds’shells were manually removed. Cold
pressed Moringa oleifera seed oil （MoSO）was obtained by
using a Komet DD 85 G vegetable oil screw press（ IBG
MonfortsOekotec GmbH & Co. KG, Mönchengladbach,
Germany）. The oil extraction was carried out when the
Moringa seeds（ 2 kg）were ground and pressed by the
conical screw rotation. The oil was driven through a perfo-
rated tube. The meal was then evacuated at the end of the
shaft by a calibrated orifice that often interchangeably acts
as a barrier to the flow of the meal（residual fat, nutritional
value, etc.）. The remaining oil flowed into the centrifuge
（1248-Gyrozen）for 15 min at 5000 rpm in order to filter
the oil and remove plant debris. This phase was automati-
cally followed by further filtration. The seed oil was then
（−20℃）
stored in a freezer at for further analysis.
2.2 GC-MS analysis
One hundred microliters of MoSO were dissolved in 200
µL hexane. In order to convert the mixture to its corre-
sponding volatile trimethylsilyl derivatives, 100 µL of pyri-
dine and 200 µL of Bis（trimethylsilyl）trifluoroacetamide
（BSTFA）were added and incubated for 1 hour at 70℃. The
sample was then analyzed by Hewlett Packard HP 6890
series gas chromatography equipped with a Hewlett
Packard 5973 mass spectroscopy detector （GC-MS）, using
2
J. Oleo Sci. 71, (9) 00000-00000 (2022)
000　22095Gharsallah.indd 2 2022/07/29
a HP-5 MS capillary column（Agilent 19091S-433, 30 m×
250 µm internal diameter×0.25 µm film thickness）. The
GC oven temperature was kept at 45℃ and programmed to
250℃ at a rate of 5℃/min then kept constant at 250℃ for
48 min. High purity helium was used as the carrier gas at a
constant flow rate of 1 mL/min. Split ratio and split flow
were adjusted to 20:1 and of 20 mL/min, respectively.
The injection volume of the oil mixture was of 1 µL. The
temperature of the ion source was set to 200℃ while the
interface was set to 250℃. Electron impact（EI）ionization
mode was 70 ev and the linear velocity of the column was
36 cm/sec. Mass range was between m/z 30 to 700.
Identification of individual components was archived
using library search software from The Wiley/NBS Registry
Mass Spectral Data and in-house“BASER Library of Fatty
Acid Constituents”.
2.3 Triacylglycerol analysis
High-performance liquid chromatography（ HPLC）
（Agilent 1100, Santa Clara, CA, USA） with an auto-injector
and refractive index detector was used to obtain the triac-
ylglycerol（TAG）profile. TAGs were isolated on an RP-18
column（250×4 mm）with a particle size of 5 m and eluted
from the column with a 25:75 mixture of acetonitrile and
acetone at 1 mL/min. Ten microlitres of the mixture（0.05 g
oil diluted in 1 mL acetone）was injected into the HPLC
column, with a total run time of 1 h. TAG peaks of MoSO
sample mixture detected by the high-performance liquid
chromatography were identified by comparison with the
retention times of standard TAG peaks and those of other
vegetable oils such as olive, sunflower, soybean and corn
oils under similar analytical conditions, as previously de-
scribed.
2.4 Microscopic observations
A Quanta 200（ （ESEM-EDAX）, Germany）scanning elec-
tron microscope（SEM） was used to examine the structural
morphology of Moringa oleifera seed, seed flour, and de-
fatted flour. Surface images of all solid materials can be ob-
tained at sizes ranging from a magnifying glass（×10）to an
electron microscope in transmission（×500,000 or more）.
2.5 FT-IR analysis
The Nicolet 380 Fourier transform infrared spectrometer
was used for the FT-IR spectra acquisition. The sample did
not require any previous preparation. FTIR spectrum was
measured in the frequency range of 4000 cm−1 to 400 cm−1
with a 32 scans and 4 cm−1 resolution. The data were pro-
cessed with Spectrum software. Analyses were carried out
in triplicate and average spectra were used.
2.6 Thermogravimetric analysis
Thermogravimetric（ TG）curves were obtained with a
TGA-50 TG analyzer（Shimadzu）. Thermogravimetric anal-
10:09:20
 Beneficial Potentials of Moringa oleifera Seed Oil
yses was performed in a 25 to 600℃ temperature range
with heating rate of 10℃/min in atmospheres of nitrogen
and air（100 mL/min）. Approximately 5 mg of sample was
put in alumina crucibles. Shimadzu TA-60WS（2.20）soft-
ware was used to analyze the data from 3 independent
measurements in order to obtain the TG and 1st derivative
TG（DTG） curves.
2.7 Antioxidant activity of MoSO
The ABTS assay was carried out according to Re et al.14）
with some modifications. ABTS radical cation（ABTS.＋） was
formed by reacting 7 mM ABTS aqueous solution with 140
mM potassium persulfate and allowing the mixture to stand
in dark at room temperature for 12 to 16 h before use. The
ABTS.＋ solution was diluted with methanol to achieve an
absorbance of 0.70（±0.02）at 734 nm. In the other hand,
the Trolox standard curve was prepared with different
concentrations（40-400 mM）. Absorbance readings were
taken after applying an appropriate MoSO volume’ s sample,
methanol as a blank, or Trolox standard to 1 mL of diluted
ABTS.＋ solution after 2 minutes of incubation at 30℃ in a
glass cuvette. Decrease in absorbance was obtained at 734
nm. All measurements were in triplicate. The antioxidant
ability of MoSO sample was measured in mM of Trolox
equivalent antioxidant capacity/mg MoSO（mM TEAC/mg
MoSO）.
2.8 Antimicrobial bioassay
The antibacterial activity of MoSO was determined using
the disk diffusion method of Bauer-Kirby according to
（Staphylococcus
Gortzi et al.15）. Four Gram positive strains
aureus ATCC 25923, Bacillus cereus ATCC 11778, Staph-
ylococcus aureus ATCC 6538 and Enterococcus feacium
19434） and four Gram negative strains （Listeria monocyto-
genes ATCC 15313, Escherichia coli ATCC 25922, Pseu-
domonas aeruginosa ATCC 27853 and Klebsiella pneu-
monia ATCC 35657）were tested. The antifungal activity
was determined against one fungal strain（Candida albi-
cans ATCC 10231）. A suspension of the tested microor-
ganisms was spread on the appropriate solid media plates
and incubated overnight at 37℃. After 1 day, 4-5 loops of
pure colonies were transferred to saline solution in a test
tube for each bacterial strain and adjusted to the 0.5 Mc-
Farland turbidity standards（∼108 cells/mL） . Sterile cotton
dipped into the bacterial suspension and the agar plates
were streaked three times, each time turning the plate at a
60°angle. Sterile paper discs（ Glass Microfibre filters,
Whatman; 6 mm in diameter） were placed onto inoculated
plates and impregnated with 10 μL MoSO diluted in Di-
methylsulfoxide （DMSO） （100, 200, 400, 500 and 1000 mg/
mL） . Ampicillin（10 μg/disc） and Nystatin（100 μg/disc）were
used as positive control for strains bacteria and fungi, re-
spectively. Inoculated plates with discs were placed in a
37℃ incubator. After 24 hours of incubation, the results
J. Oleo Sci. 71, (9) 00000-00000 (2022)
000　22095Gharsallah.indd 3 2022/07/29
were recorded by measuring the zones of growth inhibition
surrounding the disc. Clear inhibition zones around the
discs indicated the presence of antimicrobial and antifungal
activities. The test was run in triplicate.
2.9 Anti-inflammatory activity
The anti-inflammatory activity of MoSO was evaluated
on the murine macrophage RAW 264.7 cell line（ATCC）
through the accumulation of nitric oxide（NO）. Cells were
grown in 24-well plates at a concentration of 2×105 cells/
mL for 24 h at 37℃. Cells were then treated with various
concentrations of MoSO dissolved in the DMSO or the posi-
tive control（L-NAME） for 1 hour. In order to avoid solvent
toxicity, the final DMSO concentration in the culture
medium mustn’ t exceed 0.1％ （v/v） . To check the cytotox-
icity of samples on cells, the resazurin test was performed
for tested MoSO concentrations（75, 150 and 300 µg/mL） .
Lipopolysaccharide（LPS） （100 µg/mL）was added to the
treatment group of plates, while medium or LPS alone was
added to the control group. After 24 h of LPS stimulation
at 37℃ in a 5％ CO2, the cell-free supernatants were col-
（NO）
lected and assayed for nitric oxide levels using Griess’s
reagent（1％ sulfanilamide, 5％ phosphoric acid and 0.1％
N-（1-naphthyl）-ethylenediamine dihydrochloride）16）. The
absorbance was measured at 540 nm using an automated
96-well Varioskan Ascent plate reader（Thermo Scientific
354）. Nitric oxide（NO） levels, produced by murine macro-
phage-like RAW 264.7 cells, were determined by compari-
son with a sodium nitrite（ NaNO 2）standard curve. All
samples were analyzed in triplicate.
2.10 Analytical methods
Analysis was carried out in triplicate. The values of dif-
ferent parameters were expressed as the mean±standard
deviation（X̅ ±SD）.
3 Results and Discussion
3.1 Identification of volatile compounds by GC-MS analy-
sis
Table 1 illustrates the MoSO volatile compound profile
analyzed by GC-MS. Thirty six phyto-compounds amount-
ing to 98.99％ of the total oil were identified and classified
into eleven groups including fatty acids（91.63％）, phenolic
compounds（1.02％）, alcohols（0.72％）, sugars（1.09％）,
alkanes and their derivatives（ 0.67％）, organic acids
（0.58％）, aldehyde（0.1％）, ester（0.1％）, ketone（1.55％）,
amino acid（0.15％）and amine（0.69％）. Another unclassi-
fied group was also present including trisiloxane, octa-
methyl（0.46％）, Hexamethyl-Disilathiane（0.1％）, and Si-
lanamine, N,N'-methanetetraylbis［ 1,1,1］-trimethyl
（0.13％）.
Such a result was close to that reported by Adegbe et
3
10:09:21
 K. Gharsallah, L. Rezig, F. B’chir et al.

Table 1　Compounds present in MoSO cold pressed using GC-MS analysis.

Chemical Abstracts Retention time Molecular
Marker Number Compound name Peak intensity
Service (CAS) (min) formula
Fatty acids
1 Myristic acid 544-63-8 30.76 C14H28O2 0.16
2 Palmitoleic acid 373-9-9 34.22 C16H30O2 1.44
3 Hexadecanoic acid 57-10-3 34.65 C16H32O2 7.31
4 Hepthadecenoic acid C17H32O2 0.06
5 Heptadecanoic acid 506-12-7 36.4 C17H34O2 0.12
6 cis, 6-Octadecenoic acid 593-39-5 37.6 C18H34O2 69.98
7 trans, 6-Octadecenoic acid C18H34O2 0.011
8 cis-9,cis-12-Octadecadienoic acid 60-33-3 C18H32O2 1.08
9 9,12,15-Octadecatrienoic acid 463-40-1 C18H30O2 0.12
10 Octadecanoic acid 57-11-4 38.2 C18H36O2 5.33
11 11-Eicosenoic acid 5561-99-9 41.04 C20H38O2 1.78
12 Arachidic acid 506-30-9 41.47 C20H40O2 2.34
13 Docosanoic acid 112-85-6 45.07 C22H44O2 1.32
14 Lignoceric acid C24H48O2 0.58
Aldehydes
15 3-Pyridinecarboxaldehyde 500-22-1 7.27 C6H5NO 0.1
Ester
16 benzoic acid, 4-methyl-, methyl ester 99-75-2 7.044 C9H10O2 0.1
Ketones
17 Trifluoramethyl methyl ketone 421-50-1 7.97 C3H3F3O 1.55
Alcohols
18 Glycerol 56-81-5 17.65 C3H8O3 0.48
19 1,11-Undecanediol 765-04-8 40.9 C11H2O2 0.13
20 2-cis-9-Octadecenyloxyethanol 5353-25-3 41.91 C20H40O2 0.11
Organic acids
21 Semicarbazide 57-56-7 9.21 CH5N3O 0.1
22 Pyruvic acid 127-17-3 10.6 C3H4O3 0.48
Sugars
23 beta-D-glucopyranose 492-61-5 32.27 C6H12O6 0.10
24 6-O-alpha-D-Galactopyranosyl-D-glucopyranose 585-99-9 34.09 C12H22O11 0.24
25 methyl alpha-D-glucopyranoside 97-30-3 46.65 C7H14O6 0.75
Amine
26 Ethylamine 75-04-7 8.05 C2H7N 0.69
Amino acids
27 Glycine 56-40-6 13.29 C2H5NO2 0.15
Alkanes and Their
Derivatives
28 2,2-Dimethyltetradecane 544-76-3 15.77 C16H34 0.26
29 2-pentadecane 629-62-9 40.42 C15H32 0.12
30 1-Dodecyne 765-03-7 43.16 C12H22 0.29
Phenolic
compound
31 3-amino-5-methylthio-1,2,4-triazole 45534-08-5 11.07 C3H6N4S 0.6
32 alpha-(4-t-Butylphenyl) propanoic acid 40150-91-2 16.96 C13H18O2 0.26
33 1,3-Benzodioxol-5-amine 14268-66-7 19.26 C7H7NO2 0.16
Others
34 Trisiloxane, octamethyl 107-51-7 11.58 C8H24O2Si3 0.46
35 Hexamethyl-Disilathiane 3385-94-2 8.14 C6H18SSI2 0.1
Silanamine, N,N'-methanetetraylbis
36 1000-70-0 8.47 C7H18N2Si2 0.13
[1,1,1]-trimethyl
Total 98.99

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 Beneficial Potentials of Moringa oleifera Seed Oil

al.17） on Moringa seed oil extracted by n-hexane using a physical and functional properties is of a great interest by
soxhlet apparatus. In fact, among 24 identified compounds, determining the type as well as quantities of TAG species
the major constituents found in the fixed oil were oleic acid these oils. Table 2 depicts the distribution of TAGs in cold
（22.51％）, palmitic acid（10.64％）, stearic acid（6.07％）, pressed MoSO and their respective equivalent carbon
9-octadecenal（12.76％）and phenyl but-3-1-yne（5.79％）. number. Eleven TAGs were detected. Among these TAGs,
Other noticeable constituents found in the oil were o-Eth- glycerol trilinoleate（LLL）associated with glycerol–oleate–
yltoulene（4.64％）, m-Propyltoulene（3.56％）, 4-methylin- linoleate-linolenate（OLLn）were the most abundant （14.42
din（2.35％）, 2-phenyl-2-pentane（2.47％）, p-mentha -1, 3, ±0.12％）followed by glycerol–palmitate–linoleate–oleate
8-triene（2.36％） （2.21％）
and arachidic acid . （PLO）, glycerol–dioleate–linolenate（ OOLn）, glycerol–
Cis, 6-octadecenoic acid was the most abundant oleate–dilinoleate（OLL） , glycerol–trioleate（OOO） and glyc-
compound（70.68％）followed by hexadecanoic acid erol–stearate–dioleate（ SOO）with respective values of
（7.41％）and octadecanoic acid（5.63％）. The high amount 11.22±0.03％, 10.97±0.05％, 10.12±0.06％, 9.92±
of oleic acid is in the same range than those reported by 0.02％, and 8.89±0.02％. Such a result is in compliance
Ruttarattanamongkol and Petrasch18） and Pereira et al.19） with those reported by Abdulkarim et al.7） and Salama et
on cold pressed Moringa seed oil with respective amounts al.23）on Malaysian and Egyptian MOSO extracted by petro-
of 71.87％ and 70.2％. Such a finding categorizes the oil leum ether. In fact, the latter found that trioleate（OOO）
into the high-oleic acid oil group. It is worth noting that was the predominant TAG with a content varying between
high amount of oleic acid make MoSO stable during frying20, 21）. 33.75％ and 36.70％.
The high monounsaturated fatty acids（MUFA）content of It was reported that the thermal behavior of TAG is de-
MoSO suggests its incorporation in a MUFA-rich diet for pendent on its fatty acid composition24）. In terms of oxida-
lowering blood cholesterol, modulating immune function, tive stability of the oil, Neff et al.25） found that OLL, POL,
reducing susceptibility to LDL oxidation, and improving OOO, OOL, SOL and POO are the main TAG forms respon-
HDL fluidity. However, it should be noted that according to sible for increasing the oil stability. Such a finding proves
WHO22）, the ideal ratio recommended in a diet between that MoSO, rich in OLL and OOO forms, is endowed with a
saturated, unsaturated and polyunsaturated fatty acids is 1: higher oxidative stability which could be of a great interest
1.5: 1. in food industrial applications.
According to Abdulkarim et al. 7）, the oil extraction
3.2 Triacylglycerol composition process did not affect the nature and the distribution of
Fatty acid composition of vegetable oils is useful either MoSO TAGs.
in analyzing their stability toward oxidation or in studying
their nutritional aspect. Furthermore, the study of their 3.3 Scanning electron microscope
Scanning electron micrographs were processed to reveal
the location of lipid cells. For this, the surface appearance
Table 2　Triacylglycerol（TAG）composition of MoSO cold
of shelled seeds and the cellular structure of ground seeds
pressed.
before and after extraction were examined. Figure 1 illus-
Relative trates the scanning electron micrographs of Moringa oleif-
ECN TAGa
composition (%) era shelled seeds（a and b）, of Moringa oleifera ground
42 (14.42±0.12%) LLL＋OLLn 14.42±0.12 seeds（b）, and Moringa oleifera seeds waste after cold
pressing（d） .
44 (21.09%) OLL 10.12±0.06
Moringa oleifera shelled seed reveals the presence of a
OOLn 10.97±0.05 thin layer surrounding the spongy endosperm （Figs. 1a and
46 (19.94%) LOO 8.72±0.02 1b）. The latter presented a non-uniform filamentous
PLO 11.22±0.03 aspect with an irregular structure of the surface. Such ob-
48 (17.66%) OOO 9.92±0.02 servations were also reported by Tavengwa et al.26） who
confirm the presence of a matrix representing the endo-
POO 6.66±0.01
sperm and containing the seed reserves in terms of miner-
SLO 1.08±0.01 als, proteins, and lipids.
50 (10.44%) SOO 8.89±0.02 Moringa oleifera ground seeds showed swollen and
POS 1.55±0.01 bulging lipid cells on the surface of the cell tissues（Fig.
1c） . It is worth noting that after cold pressing, these cells
52 (5.31%) SOS 5.31±0.02
become defatted and flattened（Fig. 1d） .
Others － 11.14±0.03 Such a feature is mainly due to the depletion of lipid re-
a
L, linoleate; Ln, linolenate ; O, oleate; S, stearate; P, serves in the ground seeds without affecting the external
palmitate; S, stearate, ECN, equivalent carbon number. morphological structure of the cell tissue which remains
5
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 K. Gharsallah, L. Rezig, F. B’chir et al.

Fig. 1　Scanning electron micrographs of Moringa oleifera（a）shelled seeds（13×）

,（b）shelled seeds（1500×）,（c）ground
（3000×）
seeds （d）
and seeds waste after cold pressing（3000×）.

Fig. 2　FT-IR spectra of cold pressed MoSO.

smooth and intact. tional groups present in the sample27）.

The FTIR characteristics peaks of the oil sample were
3.4 FT-IR spectra detected in the regions 3010-2800, 1750-100, and 1000-550
Figure 2 illustrates the Fourier-transform infrared cm−1. Such a finding is consistent with previous study con-
（FT-IR）spectrum of cold pressed MoSO. The peaks and ducted by Duarte et al. 28） on Brazilian mechanically
shoulders provide information about structure and func- pressed MoSO.
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 Beneficial Potentials of Moringa oleifera Seed Oil

A peak was marked at 3002 cm −1 indicating C＝C-H

stretching（asymmetry）cis olefinic groups. Furthermore,
peaks at 2920 and 2851 cm−1 were attributed to asymmet-
ric and symmetric stretching vibration of the aliphatic CH2
groups, respectively.
Peaks at 1743 and 1463 cm−1 were attributed to the
ester carbonyl stretching（C＝O）of fatty acids and C＝H
bending （scissoring） of CH2 and CH3 groups, respectively.
In addition, the peak at around 1377 cm−1（1377.15 cm−1）
was attributed to C-H bending of CH2 group, whereas peaks
around 1236 cm−1 （1236.41 cm−1） and 1162 cm−1（1158 cm−1）
−1 −1
and those around 1117 cm （1117.58 cm ） and 1098 cm−1
−1
（1097.09 cm ）could represent the stretching and the
stretching vibration of the C-O ester group.
Minor peaks were also detected at 664.56 cm −1 and
583.71 cm−1. The peak corresponding to 721 cm−1 could be
attributed to the C-H rocking of the CH2 group.

3.5 Thermogravimetric analysis

Oil thermal stability is an aspect of great importance
since its exposition to high temperatures causes its deterio-
ration. According to Chacón-Fernándeza et al.27） thermal
stability of vegetable oils is enhanced by its richness in
natural bioactive compounds.
The thermal gravity analysis（TG）and differential ther-
mogravimetry（DTG）data of MoSO, in nitrogen and air at-
mospheres, are illustrated in Fig. 3.
In both, nitrogen and air atmospheres, thermal gravity
TG/DTG curves of MoSO showed only one mass-loss stage.
The latter corresponded to the volatilization and/or com-
bustion of triacylglycerols. Szabo et al. 29） and Santos et
al.30） reported that the decomposition of vegetable oil is
mainly represented by the degradation of triacylglycerol.
In fact, during heating, triglycerides, representing 96 to
98％ of edible oils, generate low molar mass molecular vol-
atile compounds that are continuously removed by the
produced vapor.
The cold pressed Tunisian MoSO’ s thermal behavior was
different to that observed by Duarte et al.28） in Brazilian
MoSO extracted mechanically and analyzed under air at-
mosphere. In fact, three mass loss events were observed in
which the first, the second, and the mass losses were due
respectively to polyunsaturated, monounsaturated, and
saturated fatty acids decompositions.
In nitrogen atmosphere （Fig. 3a） the Tonset and the Tend of
the thermal mass loss transition MoSO were of 390.72℃
and 461.1℃, respectively with a peak occurring at
425.91℃. Such a result was consistent with previous study
conducted by Sharma et al.31）on moringa seed oil extract-
ed by hexane in a soxhlet apparatus analyzed in the same
conditions.
Figure 3b shows the TG and DTG curves of the oil
sample in air atmosphere. The decomposition process was
characterized by a Tonset of 357.47℃, a peak occurring at
J. Oleo Sci. 71, (9) 00000-00000 (2022)
Fig. 3　TG and DTG curves of MoSO in nitrogen atmosphere
（a）and in air atmosphere（b）.
421.96℃ and a Tend of 485.48℃. It is interesting to note
that decomposition in air atmosphere begins at a lower
temperature when compared to that observed in nitrogen
one. According to Santos et al.30）, heating in the air atmo-
sphere accelerates the reactions in which oxygen absorp-
tion are involved such as oxidative stability. When compar-
ing the Tonset of commercial olive,（299.41℃）and canola oils
（298.21℃）, and Moringa oleifera seed oil mechanically
pressed （304.69℃）obtained in air atmosphere by Duarte et
al.28）, Tunisian cold pressed MoSO exhibited the highest
thermal stability. Such a result suggests that MoSO is ther-
mally stable and can be used safely at high-temperatures.
3.6 ABTS Radical scavenging assay
Chu et al.32）reported that scavenging of ABTS radical is
due to scavenging of proton radicals induced through do-
nation of electrons. The antioxidant capacity of MoSO was
of 3.65 mM TEAC/mg of oil. To the best of our knowledge,
no studies were undertaken previously on the antioxidant
capacity of MoSO using the ABTS radical scavenging assay.
Adebayo et al.33） studied the antioxidant activity of the
polar fractions of Moringa oleifera seeds（aqueous residue,
butanol fraction, crude ethanol extract, and crude water
7

000　22095Gharsallah.indd 7 2022/07/29 10:09:22

 K. Gharsallah, L. Rezig, F. B’chir et al.
extract）. The ABTS antioxidant activity test revealed that
the crude water extract has the highest antioxidant
capacity（29 µM TEAC/mg sample）while aqueous residue
has the least（7 µM TEAC/mg）. Our result proved that
MoSO is endowed with an interesting antioxidant capacity
which could be attributed to its richness in bioactive anti-
oxidant compounds（polyphenols, sterols, tocopherols） .
3.7 Antimicrobial activity
MoSO wasn’ t active against all bacterial and fungal
strains tested. Such a result was in accordance with those
of Spiliotis et al.34）and Ruttarattanamongkol & Petrasch18）
on MoSO extracted by simple hydraulic hand press and
soxhlet apparatus using petroleum ether as solvent, re-
spectively. However, our findings are inconsistent with the
work of Lalas et al.35）who proved that Moringa peregrina
seed oil originating from Saudi Arabia and extracted by
soxhlet extractors using n-hexane as solvent had activity
toward Gram＋ （Staphylococcus aureus ATCC 25923 and
Staphylococcus epidermidis ATCC 12228）and Gram –
（Pseudomonas aeruginosa ATCC 27853, Enterobacter
cloacae ATCC 13047, Klebsiella pneumonia ATCC 13883
and Escherichia coli ATCC 25922）bacteria, as well as
against human pathogenic fungi（Candida glabrata ATCC
28838, Candida tropicalis ATCC 13801 and Candida al-
bicans ATCC 10231）. It is worth mentioning that the
minimal inhibitory concentration（MIC）of the seed oil for
bacteria strains varied between 3.35 mg/mL for Staphylo-
coccus epidermidis and 4.95 mg/mL for Escherichia coli.
For fungal strains, MIC was of 3.25 mg/mL for Candida
glabrata, 3.30 mg/mL for Candida tropicalis, and 5.70
mg/mL for Candida albicans. Variation in antimicrobial
activities could be attributed to variations in Moringa
species and the oil extraction procedure18）.
3.8 Anti-inflammatory activity
The anti-inflammatory effect of MoSO was expressed as
the amount of NO produced in LPS-stimulated RAW 264.7
macrophages. NO is among the major macrophage prod-
ucts and is produced from L-arginine by inducible nitric
oxide synthase （iNOS） . It has been shown to play an impor-
tant role in inflammatory reaction36）. Excess NO may lead
to tissue injury and causes inflammatory diseases. There-
fore, NO development inhibition could be an interesting
technique for treating different inflammatory disorders37）.
The anti-inflammatory activity of MoSO cold pressed was
illustrated in Fig. 4. Firstly, it should be noted that no cyto-
toxic effect was observed for all tested concentrations （75,
150 and 300 μg/mL, data not shown） . Based on these data,
subsequent assays were performed to elucidate the anti-in-
flammatory effect of this oil using LPS-stimulated RAW
264.7 macrophages. Our findings showed that treatment
with LPS induced a substantial accumulation of nitrite in
control cells. Interestingly, treatment with MoSO at the dif-
8
J. Oleo Sci. 71, (9) 00000-00000 (2022)
000　22095Gharsallah.indd 8 2022/07/29
Fig. 4　Effect of MoSO cold pressed on LPS-induced NO
production in RAW 264.7 macrophages. Different
letters on the top of data bars indicate significant
differences（p＜0.05）between mean values（±SD,
n＝3）.
ferent concentrations tested（75, 150 and 300 μg/mL）sig-
nificantly inhibited NO output. In fact, the RAW 264.7 mac-
rophages treated with MoSO inhibition of NO released in
LPS were 31±2.81％, 36±3.82％ and 45±3.48％ for re-
spective concentrations of 75, 150 and 300 μg/mL, com-
pared with control. Muangnoi et al.38） reported that pre-
treated cells with the M. oleifera pod extract at different
concentrations（31–250 μg/mL）inhibited LPS induced NO
production （31％ for 31 µg/mL vs 95％ for 250 µg/mL） .
For olive oil phenolic fraction compounds, it was proved
that the latter down-regulated anti-inflammatory enzyme
activities, inducing a NO production, after incubation with
inflammatory cells. According to Aparicio-soto et al.39）and
Cardon et al.40）, the anti-inflammatory activity may be at-
tributed to the bioactive compounds, mainly phenolics and
sterols. Such hypothesis was consolidated by Moreno41）
and Montserrat-de la Paz et al. 42） who reported that
β-sitosterol decreased NO release in activated macro-
phages, which was associated with iNOS activity impair-
ment.
4 Conclusion
The present study was conducted to investigate the
thermal stability, the biological activities, the volatile com-
pounds, and the triacylglycerol composition of Tunisian
cold pressed Moringa oleifera seed oil（MoSO） .
Thermogravimetric analyses in nitrogen and air atmo-
spheres, suggest the higher thermal stability of MoSO, indi-
cating that the oil can be used safely at high temperatures.
Furthermore, our findings showed that MoSO is endowed
with an interesting anti-inflammatory activity. The antioxi-
dant capacity of MoSO was of 3.65 mM TEAC/mg. The in-
teresting capacity of MoSO in scavenging the free radical
ABTS.＋ could be attributed to the richness of this uncon-
10:09:22
 Beneficial Potentials of Moringa oleifera Seed Oil

ventional oil source in bioactive compounds. However,
MoSO didn’ t exhibit an antimicrobial activity against four
Gram positive strains, four Gram negative strains, and one
fungal strain.
GC-MS analysis revealed the presence of thirty six com-
pounds classified in eleven groups. Oleic acid was the pre-
dominant phyto-compound among the major fatty acid
group which represented 91.63％ of the total volatile com-
ponents identified. This fact is of a great economic interest
due to several possible applications of this component in
the food, cosmetic and medical industries.
On the light of the obtained results, Tunisian cold
pressed MoSO has good prospect for use by the food, phar-
maceutical and medical factories. Nevertheless, it should
be noted that the use of Moringa seed oil for industrial ap-
plications could necessitate its exposure to high thermal
treatments, which would have a negative impact on its
quality. It is therefore relevant that further studies are
needed to investigate the effects of thermo-oxidation on
the physicochemical properties and bioactive activities of
such unconventional oil source.
Conflict of Interest
The authors hereby declare that there are no conflicts of
interest.
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