South African Journal of Botany 137 (2021) 475
482

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South African Journal of Botany

journal homepage: www.elsevier.com/locate/sajb

Chemical compositionand profile characterization of moringa oleifera

seed oil
Karima Gharsallaha,*, Leila Rezigb,c, Kamel Msaadad, Abdellah Chalhe, Taoufik Soltania
a
Physics laboratory of Soft Matter and Electromagnetic Modeling, LR99ES16, Faculty of Science of Tunis, Tunis El Manar University, 2092 Tunis, Tunisia
b
High Institute of Food Industries, 58 Alain Savary Street, El Khadra City, 1003 Tunis, Tunisia
c
University of Carthage, National Institute of Applied Sciences and Technology, LR11ES26, Laboratory of Protein Engineering and Bioactive Molecules’, BP. 676,
1080 Tunis, Tunisia
d
Laboratory of Aromatic and Medicinal Plants, Biotechnology Center in Borj-Cedria Technopole, BP. 901, 2050 Hammam-Lif, Tunisia
e
Unit of plant ecology, UR13ES25, Faculty of Science of Tunis, Tunis El Manar University, 2092 Tunis, Tunisia

A R T I C L E I N F O A B S T R A C T

Article History: The purpose of the current study was to investigate the chemical composition and the oil properties of the
Received 2 July 2020 seeds of Moringa oleifera from a cultivated origin of Tunisia. Expressed on dry weight basis, the seeds exhib-
Revised 16 October 2020 ited a moisture content of 7.78 § 0.62%, whereas contents of proteins, fiber, ash, fat, and total sugars were
Accepted 17 November 2020
33.39 § 3.16%, 3.9 § 0.32%, 3.1 § 0.02%, 41.7 § 3.71%, and 10.13 § 2.32% respectively. Oleic acid was the
Available online xxx
main fatty acid (C18:1) (73.36 § 0.22%). The determination of total phenolic content exhibited
Edited by AR Ndhlala 102 § 2.41 mg gallic acid equivalents per kg. HPLC analysis confirmed the presence of four phenolic acids
(gallic, caffeic, vanillic, ferrulic) with a predominance of ferulic acid coupled with two flavonoids (Apigenin
Keywords:
and Naringenin). The seed oil was also found to contain high rates of tocopherols with a predominance of the
Moringa oleifera seed oil
a-tocopherol (51%). The sterol marker b-Sitosterol, accounted for 47.24% of the total sterol content. The dif-
Fatty acids
ferent seed oil analyses proved that M. oleifera seed oil could potentially be utilised for different industrial,
Phenolic acids
Sterols cosmetic, and pharmaceutical applications.
Tocopherols © 2020 SAAB. Published by Elsevier B.V. All rights reserved.
Rheological behavior

1. Introduction various parts of this extremely useful tree. Almost every part of the M.
oleifera tree has been used for traditional medicine purposes. The flow-
Moringa oleifera is a fast-growing, soft wood tree of the Moringaceae ers, leaves, roots and seeds have for instance been associated with the
family, native to the sub-Himalayan areas of Northern India treatment of a number of inflammatory, infectious, cardiovascular, gas-
(Bhatnagar and Gopala Krishna, 2013). One species among other recog- trointestinal, hematological and hepatorenal diseases and/or disorders
nised 13 species, Moringa oleifera is primarily grown in African, Asian (Govardhan Singh et al., 2013).
and in most Middle Eastern countries (Leone et al., 2016). By virtue of In order to ensure a top-quality Moringa seed oil, highly effective
its drought tolerance, the M. oleifera tree was spread to other areas extraction techniques are hence required. It is widely accepted that
including tropical and subtropical regions. M. oleifera is also deemed as organic solvents are conventionally used for seed oil extraction
a highly polytropic plant. The seeds, leaves, roots and even flowers of (Rezig et al., 2018). In oil extraction, n-hexane is commonly used for
this plant are fit for both human and animal consumption.The leaves different plant materials because of its advantageous oil recovery,
are,in particular, a good source of protein, vitamins, minerals, b-caro- boiling point and solubilising features (Rezig et al., 2018). However,
tene and antioxidants and have ever been utilized for dietary and using hexane as a solvent has led to adverse reactions from a number
medicinal practices (Leone et al., 2016).Yet most importantly, the seeds of governmental bodies and paragovernmental organisations. This
have gained much scientific attention as the M. oleifera seed kernels rejection was basically due to the fact that hexane induces air pollu-
possess a significant oil content (up to 40%), a high proportion of fatty tion and toxicity and that it may endanger human health (Rezig et al.,
acids (oleic acid > 70%) and a remarkable resistance to oxidative degra- 2018). It was nonetheless feasible to overcome such environmental
dation. Fair enough, this rich oil profile makes the Moringa seeds ideal issues by using organic solvents that would enable a healthy and an
for human ingestion and commercial utilisation alike (Anwar et al., eco-friendly oil extraction (Mitra et al., 2009).
2005). A number of medicinal properties have also been ascribed to For that reason, a number of researchers have incessantly been
concerned with elaborating new extraction techniques that would
* Corresponding author. replace the use of organic solvents and that would eventually
E-mail address: gharsallah.karima@yahoo.fr (K. Gharsallah).

https://doi.org/10.1016/j.sajb.2020.11.014
0254-6299/© 2020 SAAB. Published by Elsevier B.V. All rights reserved.
K. Gharsallah, L. Rezig, K. Msaada et al. South African Journal of Botany 137 (2021) 475
482

guarantee the safety of human health. Cold-pressed extraction is Table 1

undoubtably one of the methods that best complies with the regula- Chemical composition (dry basis) of
M. oleifera seeds (% w/w).
tions and that would never undermine the quality of the extracted
products (Tsaknis et al., 1999). Constituents
We might now claim that scientific literature on the subject of
Moisture content 7.78 § 0.62
cold pressed Moringa seed oil native to North Africa and to Tunisia, in Crude protein 33.39 § 3.16
particular, is scarce. An attempt at precisely delineating the composi- Crude oil 41.7 § 3.71
tion and the physicochemical properties would significantly pave the Total fiber 4.23 § 0.32
Total ash 3.1 § 0.02
way for the potential uses of M.oleifera seed oil for cosmetic, pharma-
Total sugar 17.58 § 2.32
ceutical and food applications.
This study was performed to identify the physicochemical and Values are means § SD of three
determinations.
rheological properties and the oxidative stability of the Tunisian
cold-pressed M.oleifera seed oil. Results would demonstrate the
inventive utilization of Moringa seed oil in the industrial field. 2 mL of heptane with 0.2 mL of 2 N methanolic KOH. The FAMEs
were then analysed by gas chromatography (Agilent, HP 6890 series)
2. Materials and methods equipped with flame ionization detector (FID), using a fused-silica
capillary column (CP-Sil 8850 Agilent, 50 m x 0.25 mm internal
2.1. Samples diameter £ 0.2 mm film thickness). Both the injector and the detector
temperatures were held at 250 °C. The initial oven temperature was
M. oleifera seeds were bought from a plant nursery located in the set to 165 °C and it was later raised up to 210 °C at a rate of 2 °C/min.
town of Beni Khiar (36°290 02.300 N 10°480 24.200 E) in Nabeul, Tunisia. The injected volume of the FAMEs was 1 mL and helium was used as
The seeds were directly isolated, washed to remove impurities, then the carrier gas at 1.5 mL/min. Fatty acids were identified by compar-
air-dried in the shade. ing retention times with those of standard compounds.

2.2. Proximate analysis of moringa oleifera seeds 2.6. Determination of total phenolic content (TPC)

Moisture was determined according to (AOAC, 1990). The protein Phenolic compounds were isolated using a double extraction proce-
content was determined by Kjeldahl’s method (Rezig et al., 2012) and dure of an oil solution in hexane (5 g of oil into 10 ml of hexane) with a
was calculated for nitrogen content as follows: N (%) £ 6.25. Ash con- 10 ml of methanol/water mixture (80:20, v/v). Five hundred microlitres
tent was obtained after mineralization of sample seeds at 600 °C for of the Folin-Ciocalteau was added to a suitable aliquot of the combined
8 h. The fat content was measured by a Soxhelt extraction apparatus extracts, and in 3 min, a 1 ml of saturated sodium carbonate solution
using hexane as a solvent for 8 h. Crude fiber was determined accord- (35%, w/v) was added to the reaction mixture. The solution was then
ing to AOCS Ba 6a-05 (2017). Total sugars (TS) were calculated as fol- reduced to a 10 ml water dilute and in 1 h the absorbance at 725 nm
lows: TS = 100 - (%moisture +%protein +%ash +%fiber +%fat). was measured, against a blank solution, with a UV
visible spectropho-
tometer. The values were measured as mg gallic acid equivalents (GAE)
2.3. Oil extraction per a kg of oil (Vazquez-Roncero et al., 1973; Gutfinger, 1981).

Moringa seed oil was obtained by cold pressing the seeds using a 2.7. Analysis of phenolic compounds
Komet DD 85 G vegetable oil screw press (IBG Monforts Oekotec
GmbH & Co. KG,Mo €nchengladbach, Germany). This technique allows Chromatographic analyses were performed using an Agilent series
for cold extracting the solid oil sample found in a plant hopper. Mor- 1100 HPLC system including a G1322A vacuum degasser, a G1311A
inga seeds (2 kg) were ground and pressed by a co-rotating conical pump, a G1313A auto sampler, and a G1316A column heater. For chro-
twin screw extruder. The oil was driven by means of a perforated matographic separation, a Knauer C-18 column (250 mm £ 4.6 mm,
tube. The meal was removed through a calibrated orifice. The remain- 5 mm) was used. The elution conditions were as follows: mobile phase
ing oil was swept into the centrifuge for 15 min at 5000 rpm in order A (1% acetic acid in water) and mobile phase B (1% acetic acid in metha-
to filter the oil and remove plant debris. This phase was automatically nol), with a flow rate of 400 mL/min and an operating temperature of
followed by further filtration. The seed oil was then stored in a 40 °C. The sample-injected volume was 10 mL of the mixture that con-
freezer at (-20 °C) for further investigation. tained 100 mL M. oleifera seed oil and 900 mL of ethyl acetate. The run-
ning gradient was as follows: 0
4 min., 10%
20% B; 4
24 min.,
2.4. Chemical analysis 20%
100% B; 24
30 min., 100% B; 30
36 min., 100%
20% B. The diode
array detector performed a scan ranging from 190 to 400 nm and the
The Moringa seed oil was analysed by standard methods samples were accordingly detected at 254, 280, and 330 nm. Phenolic
AOCS (1997) for the determination of free fatty acid content (method Cd compounds were identified and quantified by comparing each peak’s
3d-63), peroxide value (method Cd 8
53), iodine value (method Cd retention time with that of injected reference standards in the same
1
25), saponification value (method Cd 3
25), specific gravity (using a chromatographic conditions (Samet et al., 2014).
10 mL pycnometer at 25 °C), unsaponifiable matter (method Ca 6a-40)
and the refractive index (using Abbe  refractometer at 40 °C). Specific 2.8. Sterols analysis (ST)
absorptivity values K232 and K270 were determined with the help of an
ultraviolet (UV) spectrophotometer by measuring the absorbance of a 1% Sterol separation was performed according to the COI (2017). a-cho-
solution of seed oil in cyclohexane and a path of 1 cm at 232 and 270 nm. lestanol was used as an internal standard solution. Analyses were con-
ducted through a Gas Chromatography (Agilent, HP 6890 series)
2.5. Fatty acid composition equipped with a flame ionization detector (FID), using a DB-5 Agilent
column (5% pheynyl methyl polysiloxane, 30 m x 0.32 mm internal
Fatty acid composition was determined according to COI (2001). diameter £ 0.25 mm film thickness). Helium was used as a carrier gas at
Fatty acids were transesterified into their corresponding fatty acid a flow of 2 ml/min using the split-splitless injection. The injector/detec-
methyl esters (FAMEs) by shaking of a solution of 0.1 g of oil and tor temperatures were held at 280 and 290 °C, respectively. The column
476
K. Gharsallah, L. Rezig, K. Msaada et al. South African Journal of Botany 137 (2021) 475
482

Table 2 Table 4
Physicochemical characterization of M. oleifera seed oil. Total Phenolic Content (mg GAE/Kg) and
phenolic compounds (mg/Kg) of M. oleifera
Parameter seed oil.
Refractive index (40 °C) 1.467 § 0.03 Total phenolic content Composition
Specific gravity (25 °C) 0.916
Acid value (mg KOH/g oil) 1.5 § 0.21 102 § 2.41
Saponification value (mg KOH/g oil) 168.3 § 0.45 Phenolic compounds Composition
Iodine value (g I2/100 g oil) 67.42 § 0.21 Phenolic acids
Peroxide value (meq O2/kg oil) 7.5 § 0.03 Gallic acid 0.20 § 0.05
Unsaponifiable matter (%) 1.13 § 0.14 Caffeic acid 4.30 § 0.18
K232 1.17§ 0.02 Vanillic acid 0.20 § 0.08
K270 0.043 § 0.01 Ferulic acid 22.90 § 0.32
Oil stability index (h) 30 § 0.5 Flavonoids

Viscosite a 20 °C (mPa.s) 97.11 § 0.02 Apigenin 16.12 § 0.23
Color parameters Naringenin 20.70 § 0.25
Red 3.6 § 0.1
Values are means § standard deviations
Yellow 70.00 § 0.0
(SD) of three determinations.
Values are means § SD of three determinations.

The temperature was set to 120 °C, and the air flow was of 20 L per
temperature was set to 240 °C and it was next raised to 260 °C at a rate hour. During the oxidation process, volatile acids were trapped in the
of 4 °C /min. Sterols peaks were identified according to COI (2017) and distilled water and were conductimetrically measured. Induction
confirmed by GC
MS (NIST database, 2002) database whilst operating period was determined as the necessary time to reach the inflection
under similar conditions as to that of the GC-FID. point of the conductivity curve (Halbault et al., 1997).

2.9. Tocopherol composition 2.11. Determination of the free radical scavenging activity

Tocopherol composition was determined using high performance 2.11.1. Preparation of extracts
liquid chromatography (HPLC) according to the ISO 9936 (2012) stan- Precisely 10 g of seed oil samples were weighed, dissolved in
dard method. Sample solutions were prepared by dissolving 4 g of oil 20 ml of hexane and homogenised with 20 ml of methanol (60%) at
in 25 mL n-heptane and filtered through 0.45 mm PTFE filter prior to room temperature for 30 min in a shaker. The solvent mix was trans-
injection. The HPLC system was equipped with an HP Agilent 1200 ferred to a separating funnel. The lower fraction was washed with
(Agilent Technologies, Palo Alto, CA, USA.), coupled with an Agilent 20 ml of methanol (60%), twice homogenised for another 10 min, sep-
1100 Series fluorescence detector set at the wavelengths λ = 295 and arated from the aqueous layer and then added to the corresponding
330 nm for excitation and emission, respectively. Ten microlitres of separating funnel. The methanolic fraction was bulked and concen-
sample solutions and calibrated standard solutions (containing a-, trated to dryness under reduced pressure in a rotary evaporator at
b-, g -, d- Tocopherols with varied concentrations 3
6 mg/ mL in 40 °C yielding a dried methanolic extract. This extract was then uti-
methanol) were injected. The column was a normal phase YMC-Pack lised for preparing solutions at different concentrations so as to
SIL column (250 £ 2 mm ID, 5 mm; YMC Co., Kyoto, Japan). A volume determine the antioxidant activity (Nakbi et al., 2010).
fraction of tetrahydrofurane solution (3.85%) in n-heptane was used
as eluent at a flow rate of 1 mL/ min. 2.11.2. Free radical scavenging activity of Moringa oleifera seed oil
Using 1,1-diphenyl-2- picrylhydrazyl or DPPH, the free radical
2.10. Oxidative stability scavenging activity of the Moringa seed oil was successfully deter-
mined. The DPPH assay of seed oil extracts was determined according
A Metrohm Rancimat apparatus (model 743, Metrohm Co., Basel, to the method described by Lopes-Lutz et al. (2008) and modified by
Swizerland) was used to measure the induction periods of 3.5 g of oil. Sarwar Alam et al. (2007). 0.5 ml of 0.15 mM DPPH solution (in meth-
anol) was added to 1 mL of the extract solution (in methanol). The
contents were strongly mixed and kept at ambient temperature for
Table 3 30 min and the absorbance was measured at 517 nm. The samples’
Fatty acid (%) composition of M. oleifera
seed oil.
activity of the scavenging DPPH radicals was calculated as follows
(Loo et al., 2008):
Fatty acids Composition
Absorbance of control
Absobance of sample
% DPPH scavenging ¼  100
Palmitic (C16:0) 6.11 § 0.84 Absorbance of control
Palmitoleic (C16:1) 1.4 § 0.09
Stearic (C18:0) 5.37 § 0.45 IC50 value indicated the concentration of the required sample for
Oleic (C18:1) 73.36 § 0.22 scavenging 50% of DPPH radicals. Low IC50 is equivalent to high scav-
Linoleic (C18:2) 1.01 § 0.06
enging capacity, and it is primarily calculated by plotting percentage
Linolenic (C18:3) 0.44 § 0.22
Arachidic (C20:0) 3.26 § 0.04
inhibition against different concentrations of oil (Guergouri et al., 2017).
Gadoleic (C20:1) 2.21 § 0.25
Behenic(C22:0) 5.71 § 0.2 2.12. Rheological measurement
Lignoceric (C24:0) 0.66 § 0.27
SAFA 21.11 § 0.65
MUFA 76.97 § 0.19
Viscosity measurements were performed using a controlled-stress
PUFA 1.45 § 0.16 rheometer (AR 2000, TA Instruments, Ltd., Crawley, UK). The viscosity
SAFA: saturated fatty acids; MUFA:
of the oil was measured at a shear stress rate ranging between 10 and
monounsaturated fatty acids; PUFA: 1000 s
1 for different temperatures extending from 20 to 80 °C.
polyunsaturated fatty acids;. Throughout these measurements, temperature was maintained using
Values are means § SD of three a connected water bath Peltier system (§ 0.1 °C) (Chouaibi et al.,
determinations.
2012).
477
K. Gharsallah, L. Rezig, K. Msaada et al. South African Journal of Botany 137 (2021) 475
482

2.13. color measurements higher than those reported by Abdulkarim et al. (2007) and
Ogunsina et al. (2014). While the acid value (1.5 § 0.21 mg KOH / g oil)
The oil color was directly read with a Lovibond Tintometer (Tin- was in compliance with that found by Anwar and Rashid (2007). This
tometer Ltd., Amesbury, United Kingdom). lower value indicates that M. oleifera seed oil had a low content of free
fatty acids and might have a long shelf life (Arena et al., 2007;
2.14. Analytical methods Nyam et al., 2009). The higher acidity value is due to the presence of
Free Fatty acids (FFAs) during enzymatic hydrolysis using lipase. Note
Note that all experiments were performed in triplicate and all that FFAs are more susceptible to oxidation than the fatty acids which
data are expressed as the mean § standard deviations (SD). The data are present in the triacylglycerol molecules. The saponification value
were statistically analysed using the Statsoft statistical software of M. oleifera oil (168.3 § 0.45 mg KOH / g oil) was similar to that
package (Statistica, 1998). reported by Abdulkarim et al. (2005) for the Malaysian Moringa seed
oil extracted by petroleum ether, but lower than that reported by
Ogunsina et al. (2014) for the cold-pressed Indian M. oleifera seed oil
3. Results and discussion
(190.4 mg KOH / g oil) as well as that extracted by hexane (191.2 mg
KOH / g oil). High saponification values is an evidence of the presence
3.1. Chemical composition of Moringa seeds
of high molecular weight triacylglycerols, which can be useful for soap
production (Khan et al., 2007). The K232 and K270 specific extinction
The chemical composition of Moringa seeds is shown in Table 1.
coefficients were calculated from the absorbance value at 232 and
The oil and protein contents are higher than those found by
270 nm, respectively, and the results showed that the Moringa seed oil
Anwar and Rashid (2007) and Leone et al. (2016) for the Pakistani
contains primary (hydroperoxides) and secondary oxidation products.
and Indian Moringa seeds, respectively. Differences in oil and protein
Moringa seed oil also shows a higher unsaponifiable matter content
contents could be attributed to plant varieties, growing climates, rip-
than that reported by Anwar and Rashid (2007) (0.74 § 0.08%).
ening stages, and the extraction methods used. Essentially, the high
rate of the oil yield clearly indicates that the Moringa seeds are likely
to be utilised for certain oil industry applications. 3.3. Fatty acid composition

3.2. Chemical analysis of Moringa seed oil The fatty acid composition of Moringa seed oil is illustrated in
Table 3. Ten fatty acids were detected among which five were unsatu-
Table 2 shows the physicochemical characteristics of M. oleifera rated. The most abundant was the oleic acid (73.36 § 0.22%). These
seed oil. The specific gravitational value is (0.916) comparable to that results are relatively close to those obtained by Abdulkarim et al. (2005)
found by Ogunsina et al. (2014) for Indian M.oleifera seed oil. The and Anwar and Rashid (2007), for the Malaysian M. oleifera petroleum
iodine value (67.42 § 0.21 g I2/100 g oil) also corresponds to the iodine ether seed extract and the Pakistani M.oleifera hexane seed extract,
values of the Pakistani and Indian Moringa seed oils (68.63 g I2/100 g respectively. The detected oleic acid may have favorable nutritional
oil and 67.8 g I2/100 g oil, respectively) reported by Anouar and implications and may substantially contribute to the prevention of both
Rashid (2007) and Ogunsina et al. (2014). However, they are lower cardiovascular disease (CVD) and cancer (Oomah et al., 2000).
than that reported by Chouaibi et al. (2012) for the olive oil. This con-
firms that the Moringa seed oil contains low amounts of unsaturated
fatty acids. Note that the acid and peroxide values of the M. oleifera 3.4. Free radical scavenging activity of the Moringa seed oil
seed oil were low. These values were even lower than those deter-
mined by the Codex Alimentarius (1982), which stipulated a permitted The antiradical activity by hydrogen-donating antioxidants model
maximum acid value of no more than 10 meq of peroxide oxygen/kg has been extensively used to evaluate the antioxidant features in a
oil for vegetable oils and a permitted maximum acid value of no more relatively short time (Guergouri et al., 2017). DPPH was used as a free
than 10 mg KOH/mg oil. Yet, on the one hand, the peroxide value was radical scavenging assay. The recorded IC50value of M. oleifera seed
oil was 62 mg/ml. This antioxidant activity was higher than that
observed by Bhatnagar and Gopala Krishna (2013) for the Indian
Table 5 Moringa seed oil (IC50 value of 35.5 mg/mL). The DPPH radical scav-
Sterol (mg/100 g) and tocopherol (mg/Kg) com- enging activity resulted in the identification of two main tocochro-
position of M. oleifera seed oil.
manols (tocopherols and tocotrienols) within the seed oil. In fact,
Sterol Composition according to Go  rnas et al. (2015), there was a clear correlation
Stigmasterol 56.24 § 2.30
(r = 0.994) between the total tocochromanol contents in the seed oils
Campesterol 40.17 § 1.96 and the DPPH radical scavenging activity.
Campestanol 1.01 § 0.09
Cholesterol 0.55 § 0.04
D
5,23 Stigmastadienol 2.52 § 0.54 3.5. Oxidative stability
Clerosterol 2.52 § 0.32
b-Sitosterol 136.82 § 2.97
Cold-pressed M.oleifera seed oil exhibited an induction period of
Sitostanol 2.17 § 0.6
D 5-Avenasterol 30.61 § 2.06 30 § 0.5 h (Table 2). The oxidative stability was nearly identical with
D 5,24-Stigmastadienol 3.85 § 0.11 that reported in the literature (Tsaknis et al., 1999) for the cold
D 7-Avenasterol 5.47 § 0.13 pressed Kenyan M. oleifera seed oil (34.1 h), yet higher than that
D 7-Avenasterol 7.04 § 0.30 reported by Anwar and Rashid (2007) for the Pakistani M. oleifera
24-Methylene-cholesterol 0.41 § 0.06
Total 289.62 § 3.45
hexane seed extract (9.60 h). Note that both were obtained in the
Tocopherol Composition same operating conditions (120 °C with an air flow of 20 L/h). It is
a-Tocopherol 101.11 § 0.27 known that the induction period (IP) is a major quality parameter
g -Tocopherol 86.87 § 0.15 affecting the oil’s oxidative stability (Anwar and Bhanger, 2003).
d-Tocopherol 10.36 § 0.46
According to Delfan-Hosseini et al. (2017), an increment in oil stabil-
Total 198.34 § 0.88
ity might be attributed to elevations in total polyphenol contents and
Values are means § SD of three determinations.
antioxidant capacities.
478
K. Gharsallah, L. Rezig, K. Msaada et al. South African Journal of Botany 137 (2021) 475
482

Fig. 1. Curves of shear stress vs. shear rate of cold pressed Moringa oleifera seed oil.

3.6. Total phenolic content (TPC) catechin, epicatechin, vanillin and quercetin) in free and bound phe-
nolic extracts present in Moringa seed flour.
The total phenolic content of the examined cold-pressed M. olei-
fera seed oil was of 102 § 2.41 mg GAE per Kg (Table 4). The recorded 3.8. Phytosterols
total phenolic content was in close agreement with that reported by
Bhatnagar and Gopala Krishna (2013) for the Indian Moringa hexane Phytosterol levels in vegetable oils have long been used for the
seed oil extract. Due to their diverse chemical structures, phenolic determination of oil and fat authenticity, oil derivatives and profile
phytochemicals have the capacity to protect cellular components (De-Blas and Del-Valle, 1996). Furthermore, sterols are used in order
against oxidation (Bahloul et al., 2014). But above all, polyphenols to detect the adulteration of the more expensive oils with cheaper
have been reported to prevent neurodegenerative disorders, cardio- ones (Neđeral et al., 2006). The sterol composition of the M. oleifera
vascular diseases and cancer (Liara et al., 2013). seed oil is shown in Table 5. The major sterol markers were b-Sitos-
terol (47.24%) followed by stigmasterol (19.42%), campesterol
3.7. Phenolic compounds (13.87%), and D 5-Avenasterol (10.57%). b-Sitosterol was also the
major sterol marker in cold-pressed and hexane extracted Moringa
Phenolic compounds (PCs) are part of the unsaponifiable matter seed oil and ranged between 46.16% and 49.19% (Tsaknis et al., 1999;
and are known as minor oil constituents. These compounds play a Anwar and Rashid, 2007).
crucial role in defense against oxidative damage due to their endoge- The cholesterol content (0.19%) was almost within the same range
nous high antioxidant properties (Meddeb et al., 2017). A list of some with that reported by Tsaknis et al. (1999) for the cold-pressed Mor-
identified phenolic acids and flavonoids and their relative concentra- inga seed oil (0.17%).
tions are illustrated in Table 4.
As compared to authentic standard profiles, an HPLC analysis of 3.9. Tocopherols
the phenolic compounds present in the Moringa seed oil led to dis-
cern six phenolic compounds. There were four phenolic acids (gallic, Tocopherols are conspicuously lipid-soluble alcohols of vital antioxi-
caffeic, vanillic, and ferulic) and two flavonoids (Apigenin and Narin- dant properties (Rezig et al., 2012). Alpha-tocopherol, commonly known
genin). These phenolic compounds are, to a large extent, responsible as vitamin E, prevents body lipid oxidation including polyunsaturated
for the oil’s key sensory properties like pungency and astringency. fatty acids and lipid components of cells and organelle membranes.
They also possess some biological properties that account for their Tocopherols have also been related to preventing heart diseases and
antioxidant and antiradical scavenging activities (Ahmad et al., cancer and delaying Alzheimer's disease (Nyam et al., 2009). Table 5
2011). However, no data is available for comparative purposes con- illustrates tocopherol composition and contents in the cold-pressed
cerning Moringa seed oil’s phenolic compounds. Govardhan Singh Moringa seed oil. The seed oil only contains a-, g - and d-tocopherols.
et al. (2013) reported ten phenolic compounds (gallic acid, p-couma- The main component was a-tocopherol (51%) followed by g -tocopherol
ric acid, ferulic acid, caffeic acid, protocatechuic acid, cinnamic acid, and d-tocopherol. The a-tocopherol content (101.46 mg/kg) is within
479
K. Gharsallah, L. Rezig, K. Msaada et al. South African Journal of Botany 137 (2021) 475
482

Fig. 2. Effect of temperature on the apparent viscosity of cold pressed Moringa oleifera seed oil.

the same range with the literature finding (Tsaknis et al., 1999) for the Other researchers have already observed similar results for other
cold-pressed Kenyan Moringa seed oil. However, the latter reported a vegetable oils (Kim et al., 2010; Chouaibi et al., 2012). They noted that
higher content of d -tocopherol (75.67 mg/kg) and a lower amount of vegetable oils are high viscosity Newtonian liquids because of their long
g -tocopherol (39.54 mg/kg) in comparison to that found in the Tunisian chain molecule bonds. The Moringa seed oil viscosity ƞ at 20 °C
Moringa seed oil (10.36 mg/kg vs. 86.87 mg/kg, respectively). According (97.11 § 0.02 mPa s) (Table 1) was higher than that of the olive oil
to O’Brien (2009), the a-isomer is the most biologically active, while the (76.22 mPa s) (Chouaibi et al., 2012). According to Kim et al. (2010), the
g -Tocopherol is deemed the optimal lipid oxidation inhibitor as it stabil- oil viscosity is positively correlated with the amount of oleic acid but
izes hydroperoxy and other free radicals. Briefly, the cold-pressed Tuni- negatively correlated with the amount of linoleic acid.
sian Moringa seed oil is an ideal dietary source of total a- and Fig. 2 illustrates the viscosity vs temperature data of the Moringa
g -Tocopherols. seed oil. The apparent viscosity h shows an exponential decrease
with increasing temperature (20 to 80 °C). Decreasing of vegetable
3.10. Rheological behavior oil viscosity is due to the moving away of molecules from each other
and the intermolecular bonds weaken due to a large thermal molecu-
The flow behavior curve of the Moringa seed oil was characterised lar movement, thus facilitating flow and reducing viscosity. This phe-
by steady shear measurements at different temperature intervals (20 nomenon is in perfect agreement with previous studies (Santos et al.,

80 °C). For all the examined temperatures, there was a Newtonian 2005; Kim et al., 2010). The temperature effect on the oil viscosity
behavior at shear rates between 10 and 1000 s
1 (Fig. 1). There is a was measured using the Arrhenius temperature model, which
fundamental relationship between shear stress and shear rate, which describes the exponential decrease in viscosity as a function of tem-
is in agreement with Newton’s law of viscosity: perature (Chouaibi et al., 2012), as follows:
 
s ¼ hg Ea
h ¼ A ¢ exp

RT
where s is shear stress (Pa), g is shear rate (s
1), and h is viscosity
(Pa.s). However, the oil viscosities, at different temperatures, can be where h is the oil viscosity, A is viscosity coefficient at a reference
obtained from this curve using the slope from fits of the experimental temperature (Pa.s), Ea is the activation energy (kJ/ mol), R is the gas
shear stress vs shear rate data. constant (kJ/mol.°K), and T is the absolute temperature (°K).

480
K. Gharsallah, L. Rezig, K. Msaada et al. South African Journal of Botany 137 (2021) 475
482

Activation energy can be determined from the slope of ln h vs. 1/T Anwar, F., Bhanger, M.I., 2003. Analytical characterisationof Moringa oleifera seed oil
plot. The activation energy provides information on the sensitivity of grown in temperate regions of Pakistan. J. Agric. Food Chem. 51, 6558–6563.
https://doi.org/10.1021/jf0209894.
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highest activation energy reflects a strong correlation with the tem- of Moringa oleifera oil seeds from pakistan. J. Am. Oil Chem. Soc. 82, 45–51. https://
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AOAC, 1990. In: Firestone, D. (Ed.), Official Methods of Analysis of the Association of the Offi-
more double bonds exhibited less energy activation. Activation cial Analytical Chemists. Association of the Official Analytical Chemists, Inc., VA, USA.
energy Ea of Moringa seed oil was of 30.14 kJ/mol. This value is AOCS, 1997. Official Methods and Recommended Practices of the American Oil Chem-
higher than those found by Chouaibi et al. (2012) (21.38 kJ/mol) and ist’s Society, 5th ed. AOCS Press, Champaign, USA.
AOCS Standard Procedure Ba 6a-05, 2017. Crude Fiber Analysis in Feeds By Filter Bag
Kim et al. (2010) (26.9 kJ/mol) for olive oil. Such a result can be justi-
Technique, 7th ed. AOCS Press, Champaign, USA.
fied by a high content of saturated fatty acids when compared to that Arena, E., Campisi, S., Fallico, B., Maccarone, E., 2007. Distribution of fatty acids and
of unsaturated fatty acids (Table 3). phytosterols as a criterion to discriminate geographic origin of pistachio seeds.
Food Chem 104, 403–408. https://doi.org/10.1016/j.foodchem.2006.09.029.
Bahloul, N., Kechaou, N., Mihoubi, N.B., 2014. Comparative investigation of minerals,
3.11. color chlorophylls contents, fatty acid composition and thermal profiles of olive leaves
(Olea europeae L.) as by-product. GrasasAceites 65 (3), 3–35. http://dx.doi.org/
The M. oleifera seed oil color value (3.60 R + 70.00 Y) as illustrated in 10.3989/gya.0102141.
Bhatnagar, A.S., Gopala Krishna, A.G., 2013. Natural antioxidants of the Jaffna variety of
Table 2, was higher, in terms of redness and yellowness, than the inves- Moringa Oleífera seed oil of Indian origin as compared to other vegetable oils. Gra-
tigated M. oleifera oil values (1.00 R + 29.00 Y, 0.80 R+35.00 Y, and 1.90 R sasy Aceites 64 (5), 537–545. https://doi.org/10.3989/gya.010613.
+30.00 Y) from Pakistan (Anwar and Bhanger, 2003), India (Lalas and Cheikhyoussef, N., Kandawa-Schulz, M., Bock, R., Cheikhyoussef, A., 2020. Cold pressed
Moringa oleifera seed oil. In: Ramadan, M.F. (Ed.), Cold Pressed Oil, Green Technol-
Tsaknis, 2002), and Kenya (Tsaknis et al., 1999) respectively. Such a ogy, Bioactive Compounds, Functionality, and Applications. Academic Press, United
result obviously confirms the presence of a higher carotenoid content in Kingdom, pp. 467–473.
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tive study on physicochemical, rheological and surface tension properties of Tuni-
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vegetable oils. The content of pigments in cold pressed oils is higher doi.org/10.1515/1556-3758.2759.
than in refined oils. The major pigments in cold pressed oils are carote- Codex Alimentarius Commission, 1982. 1st ed. Recommended Internal Standards Edi-
ble Fats and Oils, Volume XI. FAO/WHO, Rome, Italy.
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4. Conclusion COI, 2017. Determination of the composition and content of sterols and triterpene dia-
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The examination of the physicochemical and antioxidant proper- chromatography differentiation among different types of olive oil: virgin, refined
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tion of the cold-pressed M.oleifera seed oil revealed that the main 10.1007/BF02517973.
Delfan-Hosseini, S., Nayebzadeh, K., Mirmoghtadaie, L., Kavosi, M., Marzieh Hosseini, S.,
fatty acid, sterol, phenol and tocopherol were oleic acid, b-sitosterol, 2017. Effect of extraction process on composition, oxidative stability and rheologi-
ferrulic acid and a-Tocopherol. The given results also revealed that cal properties of purslane seed oil. Food Chem. 222, 61–66. https://doi.org/
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Go rnas, P., Soliven, A., Seglina, D., 2015. Seed oils recovered from industrial fruit
compounds. These compounds are rich in antioxidants and are thus byproducts are a rich source of tocopherols and tocotrienols: rapid separation of
endowed with very positive protective and therapeutic properties. a/b/g /d homologues by RP-HPLC/FLD. Eur. J. Lipid Sci. Technol. 117, 773–777.
Extracting oil from M. oleifera seeds might effectively contribute https://doi.org/10.1002/ejlt.201400566.
Govardhan Singh, R.S., Negi, P.S., Radha, C., 2013. Phenolic composition, antioxidant and
to its application in the cosmetic, pharmaceutical, and medicinal antimicrobial activities of free and bound phenolic extracts of Moringa oleifera seed
industries. flour. J. Function. Food 5, 1883–1891. https://doi.org/10.1016/j.jff.2013.09.009.
Nevertheless, it should be brought to attention that the use of Mor- Guergouri, F.Z., Sobhi, W., Benboubetra, M., 2017. Antioxidant activity of Algerian
Nigella Sativa total oil and its unsaponifiable fraction. J. Phytopharmacol. 6 (4),
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high thermal treatments, which would negatively impact its quality. So, Gutfinger, T., 1981. Polyphenols in olive oil. J. Am. Oil Chem. Soc. 58 (11), 966–968.
it is essential that a further study aims the effects of thermooxidation on https://doi.org/10.1007/BF02659771.
Halbault, L., Barbe , C., Aroztegui, M., De La Torre, C., 1997. Oxidative stability of semisolid
the physicochemical properties of Moringa seed oil.
excipient mixtures with corn oil and its implication in the degradation of vitamin A.
Int. J. Pharm. 147, 31–40. https://doi.org/10.1016/S0378-5173(96)04789-8.
Declaration of Competing Interest ISO 9936:2012. Animal and vegetable fats and oils - Determination of tocopherol and
tocotrienol contents by high-performance liquid chromatography.
Kim, J., Kim, D.N., Lee, S.H., Yoo, S.H., Lee, S., 2010. Correlation of fatty acid composition
The authors hereby declare that there are no conflicts of interest. of vegetable oils with rheological behavior and oil uptake. Food Chem. 118, 398–
402. https://doi.org/10.1016/j.foodchem.2009.05.011.
Acknowledgements Khan, I., Khattak, H.U., Ullah, I., Bangash, F.K., 2007. Study of the physicochemical prop-
erties of Silybum marianum seed oil. J. Chem. Soc. 29 (6), 545–548.
Lalas, S., Tsaknis, J., 2002. Characterisationof Moringa oleifera seed oil variety Periyaku-
The authors wish to thank Mr. Anis Tounsi for proofreading and lam1. J. Food Compost Anal. 15, 65–77.
improving this manuscript. Leone, A., Spada, A., Battezzati, A., Schiraldi, A., Aristil, J., Bertoli, S., 2016. Moringa olei-
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