Journal of Oleo Science

Copyright ©2022 by Japan Oil Chemists’ Society

doi : 10.5650/jos.ess22095
J. Oleo Sci. 71, (9) 1263-1273 (2022)

Composition and Characterization of Cold Pressed

Moringa oleifera Seed Oil
Karima Gharsallah1,2,3＊, Leila Rezig4,5, Fatma B’chir6, Soumaya Bourgou7,
Nahed Ben Achour4,8, Chaima Jlassi9, Taoufik Soltani1, and Abdellah Chalh2
1
 hysics laboratory of Soft Matter and Electromagnetic Modeling, LR99ES16, Faculty of Science of Tunis, Tunis El Manar University, 2092 Tunis,
P
TUNISIA
2
Laboratoire des Interactions Plante Sol Environnement, LR21ES01, Faculty of Science of Tunis, Tunis El Manar University, 2092 Tunis,
TUNISIA
3
Process engineering department, Higher Institute of Technological Studies of Zaghouan, General Direction of Technological Studies, 1121
Tunis, TUNISIA
4
University of Carthage, National Institute of Applied Sciences and Technology, LR11ES26, LIP-MB ‘Laboratory of Protein Engineering and
Bioactive Molecules’, 1080 Tunis, TUNISIA
5
High Institute of Food Industries, 58 Alain Savary Street, El Khadra City, 1003 Tunis, TUNISIA
6
Laboratory of Natural Substances, National Institute of Research and Physico-chemical analyses, Sidi Thabet Technology Center, 2020, Sidi
Thabet, TUNISIA
7
Laboratory of Aromatic and Medicinal Plants, Biotechnology Center of Borj-Cedria Technopole, BP. 901, Hammam-Lif 2050, TUNISIA
8
University of Jendouba, High Institute of Biotechnology of Beja, BP. 382, Jendouba 9000, TUNISIA
9
University of Manouba, ISBST, BVBGR-LR11ES31, Biotechpole Sidi Thabet, 2020, Ariana, 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 trioleate (OOO)
(32.42±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 365 μM
TEAC. 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 the seed cake is a natural coagulant and can be effectively

Moringa oleifera, or Ben oil tree1 is a fast-growing tree utilized for treatment and purification of the highly turbid
belonging to the Moringaceae family. Moringa oleifera is water6 .
native to the sub-Himalayan areas of Northern India; Several investigations proved that Moringa oleifera
however, it is cultivated all over the world due to its multi- seed kernels possess significant oil content up to 40
ple utilities2 . All parts of Moringa oleifera such as seeds, with a high proportion of fatty acidsoleic acid70 711

leaves, roots and even flower are fit for consumption as and a remarkable resistance to thermal and oxidative deg-
vegetable3 . The root extract was found to have an antimi- radation7, 11 . Fair enough, this rich oil profile makes the
crobial activity4 . The extracts from the flowers were also Moringa seeds ideal for human ingestion and commercial
reported to possess hepatoprotective effect5 . Moreover, utilization12 .

＊
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: [Link]@[Link] ORCID ID: [Link]
Accepted April 28, 2022 (received for review March 15, 2022)
Journal of Oleo Science ISSN 1345-8957 print / ISSN 1347-3352 online
[Link] [Link]

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 K. Gharsallah, L. Rezig, F. B’chir et al.

A large number of medicinal and nutritional properties 48 min. High purity helium was used as the carrier gas at a
have also been ascribed to various parts of the plant constant flow rate of 1 mL/min. Split ratio and split flow
kingdom and in particular its seed oil9 . Moringa oleifera were adjusted to 20:1 and of 20 mL/min, respectively.
seeds are known for its antioxidant, antimicrobial, anti-in- The injection volume of the oil mixture was of 1 µL. The
flammatory, hypocholesterolemic and anti-asthmatic activi- temperature of the ion source was set to 200 while the
ty2, 13 . However, these activities have largely been studied interface was set to 250 . Electron impact EI ionization
using its extracts and no data is available on Moringa seed mode was 70 ev and the linear velocity of the column was
oil s anti-inflammatory activities, especially that originating 36 cm/sec. Mass range was between m/z 30 to 700.
from North African countries. Given these facts, the main Identification of individual components was archived
purpose of this work is to study the volatile compound and using library search software from The Wiley/NBS Registry
the triacyglycerol profiles of Tunisian cold pressed Mass Spectral Data and in-houseBASER Library of Fatty
Moringa oleifera seed oil MoSO and to assess its biologi- Acid Constituents .
cal activities and its thermal stability by using the thermo-
gravimetric analysis. Scanning Electron Microscope SEM 2.3 Triacylglycerol analysis
was also used to examine the structural morphology of High-performance liquid chromatography HPLC
Moringa oleifera seed, seed flour, and defatted flour. 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
column2504 mm with a particle size of 5 m and eluted
2 Materials and Methods from the column with a 25:75 mixture of acetonitrile and
2.1 Seed material and lipid extraction acetone at 1 mL/min. Ten microlitres of the mixture 0.05 g
Moringa oleifera seeds were collected from a plant oil diluted in 1 mL acetone was injected into the HPLC
nursery located in the town of Beni Khiar 36 29'02.3"N 10 column, with a total run time of 1 h. TAG peaks of MoSO
48'24.2"E in Nabeul, Tunisia. The seeds were isolated and sample mixture detected by the high-performance liquid
washed to eliminate impurities before being air-dried in the chromatography were identified by comparison with the
shade. The seeds shells were manually removed. Cold retention times of standard TAG peaks and those of other
pressed Moringa oleifera seed oil MoSO was obtained by vegetable oils such as olive, sunflower, soybean and corn
using a Komet DD 85 G vegetable oil screw press IBG oils under similar analytical conditions, as previously de-
MonfortsOekotec GmbH & Co. KG, Mönchengladbach, scribed.
Germany . The oil extraction was carried out when the
Moringa seeds 2 kg were ground and pressed by the 2.4 Microscopic observations
conical screw rotation. The oil was driven through a perfo- A Quanta 200 ESEM-EDAX , Germany scanning elec-
rated tube. The meal was then evacuated at the end of the tron microscopeSEM was used to examine the structural
shaft by a calibrated orifice that often interchangeably acts morphology of Moringa oleifera seed, seed flour, and de-
as a barrier to the flow of the mealresidual fat, nutritional fatted flour. Surface images of all solid materials can be ob-
value, etc. . The remaining oil flowed into the centrifuge tained at sizes ranging from a magnifying glass10 to an
1248-Gyrozen for 15 min at 5000 rpm in order to filter electron microscope in transmission500,000 or more .
the oil and remove plant debris. This phase was automati-
cally followed by further filtration. The seed oil was then 2.5 FT-IR analysis
stored in a freezer at
20 for further analysis. The Nicolet 380 Fourier transform infrared spectrometer
was used for the FT-IR spectra acquisition. The sample did
2.2 GC-MS analysis not require any previous preparation. FTIR spectrum was
One hundred microliters of MoSO were dissolved in 200 measured in the frequency range of 4000 cm1 to 400 cm1
µL hexane. In order to convert the mixture to its corre- with a 32 scans and 4 cm1 resolution. The data were pro-
sponding volatile trimethylsilyl derivatives, 100 µL of pyri- cessed with Spectrum software. Analyses were carried out
dine and 200 µL of Bis trimethylsilyl trifluoroacetamide in triplicate and average spectra were used.
BSTFA were added and incubated for 1 hour at 70 . The
sample was then analyzed by Hewlett Packard HP 6890 2.6 Thermogravimetric analysis
series gas chromatography equipped with a Hewlett Thermogravimetric TG curves were obtained with a
Packard 5973 mass spectroscopy detector GC-MS , using TGA-50 TG analyzerShimadzu . Thermogravimetric anal-
a HP-5 MS capillary column Agilent 19091S-433, 30 m yses was performed in a 25 to 600 temperature range
250 µm internal diameter0.25 µm film thickness . The with heating rate of 10 /min in atmospheres of nitrogen
GC oven temperature was kept at 45 and programmed to and air 100 mL/min . Approximately 5 mg of sample was
250 at a rate of 5/min then kept constant at 250 for put in alumina crucibles. Shimadzu TA-60WS 2.20 soft-
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J. Oleo Sci. 71, (9) 1263-1273 (2022)
 Beneficial Potentials of Moringa oleifera Seed Oil

ware was used to analyze the data from 3 independent 2.9 Anti-inflammatory activity
measurements in order to obtain the TG and 1st derivative The anti-inflammatory activity of MoSO was evaluated
TGDTG curves. on the murine macrophage RAW 264.7 cell line ATCC
through the accumulation of nitric oxide NO . Cells were
2.7 Antioxidant activity of MoSO grown in 24-well plates at a concentration of 210 5 cells/
The ABTS assay was carried out according to Re et al.14 mL for 24 h at 37 . Cells were then treated with various
with some modifications. ABTS radical cationABTS . was concentrations of MoSO dissolved in the DMSO or the posi-
formed by reacting 7 mM ABTS aqueous solution with 140 tive controlL-NAME for 1 hour. In order to avoid solvent
mM potassium persulfate and allowing the mixture to stand toxicity, the final DMSO concentration in the culture
in dark at room temperature for 12 to 16 h before use. The medium mustn t exceed 0.1 v/v . To check the cytotox-
ABTS. solution was diluted with methanol to achieve an icity of samples on cells, the resazurin test was performed
absorbance of 0.70 0.02 at 734 nm. In the other hand, for tested MoSO concentrations75, 150 and 300 µg/mL .
the Trolox standard curve was prepared with different Lipopolysaccharide LPS 100 µg/mL was added to the
concentrations 40-400 μM . Absorbance readings were treatment group of plates, while medium or LPS alone was
taken after applying an appropriate MoSO volume s sample, added to the control group. After 24 h of LPS stimulation
methanol as a blank, or Trolox standard to 1 mL of diluted at 37 in a 5 CO2, the cell-free supernatants were col-
ABTS. solution after 2 minutes of incubation at 30 in a lected and assayed for nitric oxide NO levels using Griess s
glass cuvette. Decrease in absorbance was obtained at 734 reagent 1 sulfanilamide, 5 phosphoric acid and 0.1
nm. All measurements were in triplicate. The antioxidant N- 1-naphthyl -ethylenediamine dihydrochloride 16 . The
ability of MoSO sample was measured in μM of Trolox absorbance was measured at 540 nm using an automated
μM TEAC .
equivalent antioxidant capacity 96-well Varioskan Ascent plate reader Thermo Scientific
354 . Nitric oxideNO levels, produced by murine macro-
2.8 Antimicrobial bioassay phage-like RAW 264.7 cells, were determined by compari-
The antibacterial activity of MoSO was determined using son with a sodium nitrite NaNO 2 standard curve. All
the disk diffusion method of Bauer-Kirby according to samples were analyzed in triplicate.
Gortzi et al.15 . Four Gram positive strainsStaphylococcus
aureus ATCC 25923, Bacillus cereus ATCC 11778, Staph- 2.10 Analytical methods
ylococcus aureus ATCC 6538 and Enterococcus feacium Analysis was carried out in triplicate. The values of dif-
19434 and four Gram negative strains Listeria monocyto - ferent parameters were expressed as the meanstandard
genes ATCC 15313, Escherichia coli ATCC 25922, Pseu- deviationX SD .
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- 3 Results and Discussion
ganisms was spread on the appropriate solid media plates 3.1 Identification of volatile compounds by GC-MS analy-
and incubated overnight at 37 . After 1 day, 4-5 loops of sis
pure colonies were transferred to saline solution in a test Table 1 illustrates the MoSO volatile compound profile
tube for each bacterial strain and adjusted to the 0.5 Mc- analyzed by GC-MS. Thirty six phyto-compounds amount-
8
Farland turbidity standards10 cells/mL . Sterile cotton ing to 98.99 of the total oil were identified and classified
dipped into the bacterial suspension and the agar plates into eleven groups including fatty acids91.63 , phenolic
were streaked three times, each time turning the plate at a compounds 1.02 , alcohols 0.72 , sugars 1.09 ,
60 angle. Sterile paper discs Glass Microfibre filters, alkanes and their derivatives 0.67 , organic acids
Whatman; 6 mm in diameter were placed onto inoculated 0.58 , aldehyde 0.1 , ester 0.1 , ketone 1.55 ,
plates and impregnated with 10 μL MoSO diluted in Di- amino acid 0.15 and amine 0.69 . Another unclassi-
methylsulfoxide DMSO 100, 200, 400, 500 and 1000 mg/ fied group was also present including trisiloxane, octa-
mL . Ampicillin 10 μg/disc and Nystatin 100 μg/disc were methyl 0.46 , Hexamethyl-Disilathiane 0.1 , and Si-
used as positive control for strains bacteria and fungi, re- lanamine, N,N'-methanetetraylbis 1,1,1 -trimethyl
spectively. Inoculated plates with discs were placed in a 0.13 .
37 incubator. After 24 hours of incubation, the results Such a result was close to that reported by Adegbe et
were recorded by measuring the zones of growth inhibition al.17 on Moringa seed oil extracted by n-hexane using a
surrounding the disc. Clear inhibition zones around the soxhlet apparatus. In fact, among 24 identified compounds,
discs indicated the presence of antimicrobial and antifungal the major constituents found in the fixed oil were oleic acid
activities. The test was run in triplicate. 22.51 , palmitic acid 10.64 , stearic acid 6.07 ,
9-octadecenal 12.76 and phenyl but-3-1-yne 5.79 .
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 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

Other noticeable constituents found in the oil were o-Eth- erol palmitate-dioleate POO , the glycerol stearate-diole-
yltoulene 4.64 , m-Propyltoulene 3.56 , 4-methylin- ateSOO , and the glycerol stearate-linoleate-oleateSLO
din 2.35 , 2-phenyl-2-pentane 2.47 , p-mentha -1, 3, with respective values of 14.120.06 , 11.970.05 ,
8-triene2.36 and arachidic acid
2.21 . and 9.720.02. The predominance of the glycerol triole -
Cis, 6-octadecenoic acid was the most abundant ate, the glycerol palmitate-dioleatePOO , and the glycerol
compound 70.68 followed by hexadecanoic acid stearate-dioleate SOO is in accordance with previous
7.41 and octadecanoic acid 5.63 . The high amount studies conducted by Abdulkarim et al. 7 and Salama et
of oleic acid is in the same range than those reported by al.23 on Malysian and Egyptian MoSO extracted by petro-
Ruttarattanamongkol and Petrasch18 and Pereira et al.19 leum ether, respectively. In fact, the latter found that trio-
on cold pressed Moringa seed oil with respective amounts leate OOO was the predominant TAG with a content
of 71.87 and 70.2 . Such a finding categorizes the oil varying between 33.75 and 36.70.
into the high-oleic acid oil group. It is worth noting that It was reported that the thermal behavior of TAG is de-
high amount of oleic acid make MoSO stable during frying20, 21 . pendent on its fatty acid composition24 . In terms of oxida-
The high monounsaturated fatty acids MUFA content of tive stability of the oil, Neff et al.25 found that OLL, POL,
MoSO suggests its incorporation in a MUFA-rich diet for OOO, OOL, SOL and POO are the main TAG forms respon-
lowering blood cholesterol, modulating immune function, sible for increasing the oil stability. Such a finding proves
reducing susceptibility to LDL oxidation, and improving that MoSO, rich in OLL and OOO forms, is endowed with a
HDL fluidity. However, it should be noted that according to higher oxidative stability which could be of a great interest
WHO22 , the ideal ratio recommended in a diet between in food industrial applications.
saturated, unsaturated and polyunsaturated fatty acids is 1: According to Abdulkarim et al. 7 , the oil extraction
1.5: 1. process did not affect the nature and the distribution of
MoSO TAGs.
3.2 Triacylglycerol composition
Fatty acid composition of vegetable oils is useful either 3.3 Scanning electron microscope
in analyzing their stability toward oxidation or in studying Scanning electron micrographs were processed to reveal
their nutritional aspect. Furthermore, the study of their the location of lipid cells. For this, the surface appearance
physical and functional properties is of a great interest by of shelled seeds and the cellular structure of ground seeds
determining the type as well as quantities of TAG species before and after extraction were examined. Figure 1 illus-
these oils. Table 2 depicts the TAG distribution of the cold trates the scanning electron micrographs of Moringa oleif-
pressed MoSO. Eleven TAGs were detected. Among these era shelled seeds a and b , of Moringa oleifera ground
TAGs, the glycerol trioleate OOO was the most abundant seeds b , and Moringa oleifera seeds waste after cold
32.420.12 followed in descending order by the glyc- pressingd .
Moringa oleifera shelled seed reveals the presence of a
thin layer surrounding the spongy endosperm Figs. 1a and
Table 2 Triacylglycerol TAG composition of MoSO
1b . The latter presented a non-uniform filamentous
cold pressed.
aspect with an irregular structure of the surface. Such ob-
TAGa Composition (%) servations were also reported by Tavengwa et al.26 who
OOO 32.42±0.12 confirm the presence of a matrix representing the endo-
sperm and containing the seed reserves in terms of miner-
POO 14.12±0.06
als, proteins, and lipids.
SOO 11.97±0.05 Moringa oleifera ground seeds showed swollen and
SLO 9.72±0.02 bulging lipid cells on the surface of the cell tissues Fig.
SOS 2.31±0.02 1c . It is worth noting that after cold pressing, these cells
LLL 2.92±0.02 become defatted and flattened Fig. 1d .
Such a feature is mainly due to the depletion of lipid re-
PLO 2.72±0.03
serves in the ground seeds without affecting the external
PLS 1.89±0.02 morphological structure of the cell tissue which remains
POP 1.66±0.01 smooth and intact.
POS 1.55±0.01
3.4 FT-IR spectra
PLP 1.08±0.01
Figure 2 illustrates the Fourier-transform infrared
Others 17.64±0.03 FT-IR spectrum of cold pressed MoSO. The peaks and
a
L: linoleic acid, O: oleic acid, P: palmitic acid, S: stearic shoulders provide information about structure and func-
acid tional groups present in the sample27 .
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 K. Gharsallah, L. Rezig, F. B’chir et al.

(a) (b)

(c) (d)

Fig. 1 Scanning electron micrographs of Moringa oleiferaa shelled seeds13 ,b shelled seeds1500 ,c ground
seeds
3000 and
d seeds waste after cold pressing3000 .

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

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 Beneficial Potentials of Moringa oleifera Seed Oil

The FTIR characteristics peaks of the oil sample were

detected in the regions 3010-2800, 1750-100, and 1000-550
cm1 . Such a finding is consistent with previous study con-
ducted by Duarte et al. 28 on Brazilian mechanically
pressed MoSO.
A peak was marked at 3002 cm 1 indicating CC-H
stretching asymmetry cis olefinic groups. Furthermore,
peaks at 2920 and 2851 cm1 were attributed to asymmet-
ric and symmetric stretching vibration of the aliphatic CH2
groups, respectively.
Peaks at 1743 and 1463 cm1 were attributed to the
ester carbonyl stretching CO of fatty acids and CH
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
and those around 1117 cm1 1117.58 cm 1 and 1098 cm1
1097.09 cm 1 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 cm1 . The peak corresponding to 721 cm1 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 Fig. 3 TG and DTG curves of MoSO in nitrogen atmosphere
stability of vegetable oils is enhanced by its richness in a and in air atmosphereb .
natural bioactive compounds.
The thermal gravity analysis TG and differential ther- ed by hexane in a soxhlet apparatus analyzed in the same
mogravimetry DTG data of MoSO, in nitrogen and air at- conditions.
mospheres, are illustrated in Fig. 3. Figure 3b shows the TG and DTG curves of the oil
In both, nitrogen and air atmospheres, thermal gravity sample in air atmosphere. The decomposition process was
TG/DTG curves of MoSO showed only one mass-loss stage. characterized by a Tonset of 357.47 , a peak occurring at
The latter corresponded to the volatilization and/or com- 421.96 and a Tend of 485.48 . It is interesting to note
bustion of triacylglycerols. Szabo et al. 29 and Santos et that decomposition in air atmosphere begins at a lower
al.30 reported that the decomposition of vegetable oil is temperature when compared to that observed in nitrogen
mainly represented by the degradation of triacylglycerol. one. According to Santos et al.30 , heating in the air atmo-
In fact, during heating, triglycerides, representing 96 to sphere accelerates the reactions in which oxygen absorp-
98 of edible oils, generate low molar mass molecular vol - tion are involved such as oxidative stability. When compar-
atile compounds that are continuously removed by the ing the Tonset of commercial olive,299.41 and canola oils
produced vapor. 298.21 , and Moringa oleifera seed oil mechanically
The cold pressed Tunisian MoSO s thermal behavior was pressed 304.69 obtained in air atmosphere by Duarte et
different to that observed by Duarte et al.28 in Brazilian al.28 , Tunisian cold pressed MoSO exhibited the highest
MoSO extracted mechanically and analyzed under air at- thermal stability. Such a result suggests that MoSO is ther-
mosphere. In fact, three mass loss events were observed in mally stable and can be used safely at high-temperatures.
which the first, the second, and the mass losses were due
respectively to polyunsaturated, monounsaturated, and 3.6 ABTS Radical scavenging assay
saturated fatty acids decompositions. Chu et al.32 reported that scavenging of ABTS radical is
In nitrogen atmosphere Fig. 3a the Tonset and the Tend of due to scavenging of proton radicals induced through do-
the thermal mass loss transition MoSO were of 390.72 nation of electrons. The antioxidant capacity of MoSO was
and 461.1 , respectively with a peak occurring at of 365 μM TEAC. To the best of our knowledge, no studies
425.91. Such a result was consistent with previous study were undertaken previously on the antioxidant capacity of
conducted by Sharma et al.31 on moringa seed oil extract- MoSO using the ABTS radical scavenging assay. Adebayo et
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 K. Gharsallah, L. Rezig, F. B’chir et al.

al.33 studied the antioxidant activity of the polar fractions

of Moringa oleifera seeds aqueous residue, butanol frac-
tion, crude ethanol extract, and crude water 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 at-
tributed to its richness in bioactive antioxidant compounds
polyphenols, sterols, tocopherols .

3.7 Antimicrobial activity

MoSO wasn t active against all bacterial and fungal Fig. 4 Effect of MoSO cold pressed on LPS-induced NO
strains tested. Such a result was in accordance with those production in RAW 264.7 macrophages. Different
of Spiliotis et al.34 and Ruttarattanamongkol & Petrasch18 letters on the top of data bars indicate significant
on MoSO extracted by simple hydraulic hand press and differences p0.05 between mean values SD,
soxhlet apparatus using petroleum ether as solvent, re- n3 .
spectively. However, our findings are inconsistent with the
work of Lalas et al.35 who proved that Moringa peregrina 264.7 macrophages. Our findings showed that treatment
seed oil originating from Saudi Arabia and extracted by with LPS induced a substantial accumulation of nitrite in
soxhlet extractors using n-hexane as solvent had activity control cells. Interestingly, treatment with MoSO at the dif-
toward Gram Staphylococcus aureus ATCC 25923 and ferent concentrations tested 75, 150 and 300 μg/mL sig-
Staphylococcus epidermidis ATCC 12228 and Gram – nificantly inhibited NO output. In fact, the RAW 264.7 mac-
Pseudomonas aeruginosa ATCC 27853, Enterobacter rophages treated with MoSO inhibition of NO released in
cloacae ATCC 13047, Klebsiella pneumonia ATCC 13883 LPS were 312.81 , 363.82 and 453.48 for re-
and Escherichia coli ATCC 25922 bacteria, as well as spective concentrations of 75, 150 and 300 μg/mL, com-
against human pathogenic fungiCandida glabrata ATCC pared with control. Muangnoi et al.38 reported that pre-
28838, Candida tropicalis ATCC 13801 and Candida al- treated cells with the M. oleifera pod extract at different
bicans ATCC 10231 . It is worth mentioning that the concentrations 31–250 μg/mL inhibited LPS induced NO
minimal inhibitory concentration MIC of the seed oil for production 31 for 31 µg/mL vs 95 for 250 µg/mL .
bacteria strains varied between 3.35 mg/mL for Staphylo- For olive oil phenolic fraction compounds, it was proved
coccus epidermidis and 4.95 mg/mL for Escherichia coli. that the latter down-regulated anti-inflammatory enzyme
For fungal strains, MIC was of 3.25 mg/mL for Candida activities, inducing a NO production, after incubation with
glabrata, 3.30 mg/mL for Candida tropicalis, and 5.70 inflammatory cells. According to Aparicio-soto et al.39 and
mg/mL for Candida albicans. Variation in antimicrobial Cardon et al.40 , the anti-inflammatory activity may be at-
activities could be attributed to variations in Moringa tributed to the bioactive compounds, mainly phenolics and
species and the oil extraction procedure18 . sterols. Such hypothesis was consolidated by Moreno41
and Montserrat-de la Paz et al. 42 who reported that
3.8 Anti-inflammatory activity β-sitosterol decreased NO release in activated macro-
The anti-inflammatory effect of MoSO was expressed as phages, which was associated with iNOS activity impair-
the amount of NO produced in LPS-stimulated RAW 264.7 ment.
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 4 Conclusion
to tissue injury and causes inflammatory diseases. There- The present study was conducted to investigate the
fore, NO development inhibition could be an interesting thermal stability, the biological activities, the volatile com-
technique for treating different inflammatory disorders37 . pounds, and the triacylglycerol composition of Tunisian
The anti-inflammatory activity of MoSO cold pressed was cold pressed Moringa oleifera seed oilMoSO .
illustrated in Fig. 4. Firstly, it should be noted that no cyto- Thermogravimetric analyses in nitrogen and air atmo-
toxic effect was observed for all tested concentrations 75, spheres, suggest the higher thermal stability of MoSO, indi-
150 and 300 μg/mL, data not shown . Based on these data, cating that the oil can be used safely at high temperatures.
subsequent assays were performed to elucidate the anti-in- Furthermore, our findings showed that MoSO is endowed
flammatory effect of this oil using LPS-stimulated RAW with an interesting anti-inflammatory activity. The antioxi-
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J. Oleo Sci. 71, (9) 1263-1273 (2022)
 Beneficial Potentials of Moringa oleifera Seed Oil

dant capacity of MoSO was of 3.65 mM TEAC/mg. The in- era seeds using a response surface methodology. Sep.
teresting capacity of MoSO in scavenging the free radical Purif. Technol. 113, 9-17 2013 .
ABTS. could be attributed to the richness of this uncon- 7 Abdulkarim, S.M.; Long, K.; Lai, O.M.; Muhammad,
ventional oil source in bioactive compounds. However, S.K.S.; Ghazali, H.M. Some physicochemical properties
MoSO didn t exhibit an antimicrobial activity against four of Moringa oleifera seed oil extracted using solvent
Gram positive strains, four Gram negative strains, and one and aqueous enzymatic methods. Food Chem. 93,
fungal strain. 253-2632005 .
GC-MS analysis revealed the presence of thirty six com- 8 Anwar, F.; Rashid, U. Physico-chemical characteristics
pounds classified in eleven groups. Oleic acid was the pre- of Moringa oleifera seeds and seed oil from a wild
dominant phyto-compound among the major fatty acid provenance of Pakistan. Pak. J. Bot. 39, 1443-1453
group which represented 91.63 of the total volatile com - 2007 .
ponents identified. This fact is of a great economic interest 9 Leone, A.; Spada, A.; Battezzati, A.; Schiraldi, A.; Aris-
due to several possible applications of this component in til, J.; Bertoli, S. Moringa oleifera seeds and oil: Char-
the food, cosmetic and medical industries. acteristics and uses for human health. Int. J. Mol. Sci.
On the light of the obtained results, Tunisian cold 17, 21-412016 .
pressed MoSO has good prospect for use by the food, phar- 10 Bhutada, P.R.; Jadhav, A.J.; Pinjari, D.V.; Nemade, P.R.;
maceutical and medical factories. Nevertheless, it should Jain, R.D. Solvent assisted extraction of oil from Mor-
be noted that the use of Moringa seed oil for industrial ap- inga oleifera Lam. seeds. Ind. Crop Prod. 82, 74-80
plications could necessitate its exposure to high thermal 2016 .
treatments, which would have a negative impact on its 11 Gharsallah, K.; Rezig, L.; Msaada, K.; Chalh, A.; Soltani,
quality. It is therefore relevant that further studies are T. Chemical composition and profile characterization
needed to investigate the effects of thermo-oxidation on of Moringa oleifera seed oil. S. Afr. J. Bot. 137, 475-
the physicochemical properties and bioactive activities of 4822021 .
such unconventional oil source. 12 Anwar, F.; Ashraf, M.; Bhanger, M.I. Interprovenance
variation in the composition of Moringa oleifera oil
seeds from pakistan. J. Am. Oil Chem. Soc. 82, 45-51
2005 .
Conflict of Interest 13 Nepolean, P.; Anitha, J.; Renitta, E. Isolation, analysis
The authors hereby declare that there are no conflicts of and identification of phytochemicals of antimicrobial
interest. activity of Moringa oleifera Lam. Current Biotica 3,
33-392009 .
14 Re, R.; Pellegrini, N.; Proteggente, A.; Pannalla, A.;
Yang, M.; Rice-Evan, C. Antioxidant activity applying
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 Beneficial Potentials of Moringa oleifera Seed Oil

42 Montserrat-de la Paz, S.; Fernández-Arche, A.; Angel- CC BY 4.0 Attribution 4.0 International . This
Martín, M.; García- Giménez, M.D. The sterols isolated license allows users to share and adapt an article,
even commercially, as long as appropriate credit is
from evening primrose oil modulate the release of pro-
given. That is, this license lets others copy, distrib-
inflammatory mediators. Phytomedicine 19, 1072-
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