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The Effects of Different Extraction Methods on Antioxidant Properties,

Chemical Composition, and Thermal Behavior of Black Seed ( Nigella sativa L.)
Oil

Article  in  Evidence-based Complementary and Alternative Medicine · August 2016

DOI: 10.1155/2016/6273817

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Hindawi Publishing Corporation
Evidence-Based Complementary and Alternative Medicine
Volume 2016, Article ID 6273817, 10 pages
http://dx.doi.org/10.1155/2016/6273817

Research Article
The Effects of Different Extraction Methods on Antioxidant
Properties, Chemical Composition, and Thermal Behavior of
Black Seed (Nigella sativa L.) Oil

Nameer Khairullah Mohammed,1,2 Mohd Yazid Abd Manap,1,3 Chin Ping Tan,1
Belal J. Muhialdin,1 Amaal M. Alhelli,1 and Anis Shobirin Meor Hussin1,3
1
Faculty of Food Science and Technology, Universiti Putra Malaysia (UPM), 43400 Serdang, Selangor, Malaysia
2
Department of Food Science and Biotechnology, College of Agriculture, University of Tikrit, Tikrit, Iraq
3
Halal Products Research Institute, Universiti Putra Malaysia (UPM), 43400 Serdang, Malaysia

Correspondence should be addressed to Anis Shobirin Meor Hussin; anisshobirin@gmail.com

Received 8 June 2016; Revised 22 July 2016; Accepted 25 July 2016

Academic Editor: Yoshiji Ohta

Copyright © 2016 Nameer Khairullah Mohammed et al. This is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.

The Nigella sativa L. popularly referred to as black seeds are widely used as a form of traditional nutrition and medicine. N. sativa
seeds were used for the extraction of their oil by way of supercritical fluid extraction (SFE) and cold press (CP) to determine the
physicochemical properties, antioxidant activity, and thermal behavior. The GC-MS results showed the primary constituents in
the Nigella sativa oil (NSO) were Caryophyllene (17.47%) followed by thymoquinone (TQ) (11.80%), 1,4-Cyclohexadiene (7.17%),
longifolene (3.5%), and carvacrol (1.82%). The concentration of TQ was found to be 6.63 mg/mL for oil extracted using SFE
and 1.56 mg/mL for oil extracted by CP method. The antioxidant activity measured by DPPH and the IC50 was 1.58 mg/mL
and 2.30 mg/mL for SFE oil and cold pressed oil, respectively. The ferric reducing/antioxidant power (FRAP) activity for SFE
oil and CP oil was 538.67 mmol/100 mL and 329.00 mmol/100 mL, respectively. The total phenolic content (TPC) of SFE oil was
160.51 mg/100 mL and 94.40 mg/100 mL for CP oil presented as gallic acid equivalents (GAE). This research showed that a high
level of natural antioxidants could be derived from NSO extracted by SFE.

1. Introduction which possesses antioxidant/anti-inflammatory efficacy in

models of in vitro and in vivo investigations as well as
Black seed (Nigella sativa L.), traditionally used to treat fever, asthma, diabetes, encephalomyelitis, neurodegeneration, and
headache, anxiety, diarrhea, asthma, and stroke, is known to carcinogenesis [5, 7].
be highly anti-inflammatory [1, 2]. N. sativa seed is rich in Methods of extraction of seed oils are an effective factor
phenolic compounds used as an antioxidant agent [1] and in the properties of oils. Solvent extraction, for example, is
in essential fatty acids besides bioactive compounds such as deficient in selectivity and needs extreme heat, which could
sterols and tocols [3]. Moreover, the yellowish oil contains cause the degradation of the desired components [8]. The cold
proteins, amino acids, reducing sugars, mucilage, alkaloids, press extraction is the conventional method for oil extraction.
organic acids, tannins, resins, toxic glucoside, metarbin, bitter It involves no heat and/or chemicals and this is preferred
principles, glycosidal saponins, crude fiber, minerals, and by consumers concerned about natural and safe food [9].
vitamins [4]. Among the various oil seeds, N. sativa oil However, this method affords low yields [10], and the residual
(NSO) is particularly interesting as it may be utilized in meal contains 10–12% oil content, which can eventually limit
preparations that contain phytochemicals with strong antiox- its uses in industries processing food [11]. Supercritical fluid
idant properties and health benefits [5, 6]. Thymoquinone extraction (SFE) is currently a technique among others used
(TQ) is an active compound in the crude extracts of NSO, to extract plant oils and offers some favorable features over the
2 Evidence-Based Complementary and Alternative Medicine

traditional techniques that have been used in the oil industry Rheometer (model Stress 600, Thermo Electron Corporation,
[12]. In general, SFE has been the recommended method used Karlsruhe, Germany). The rheometer is, in essence, a dynam-
to extract antioxidant compounds from NSO and exhibits a ically managed stress instrument with a standard sensor
higher concentration of thymoquinone [13]. system and a plate (PP35Ti). Data received were subjected
This study aims to compare the properties of NSO to analysis with Haake RheoWin 3 Data Manager software.
extracted using SFE and cold press with regard to the antiox- The steel cone-plate (C40/4) measuring system was utilized
idant activity, chemical composition, TQ concentration, and to measure how viscous the samples were at a shear rate of 0
the thermal behavior of the oil. to 100 s−1 . All measurements were carried out in triplicate.

2. Experimental 2.5. GC-MS Test. GC-MS analyses were conducted in a gas

chromatograph Trace GC Ultra gas chromatograph attached
2.1. Materials and Methods. N. sativa seeds were sourced to a TSQ Quantum XLS triple quadruple mass spectrometer,
from the Serdang spice market in West Malaysia. Analytical- both from Thermo Scientific (Waltham, MA, USA). All the
grade solvents, thymoquinone (99.9% purity) and 1,1- analyses were performed with a fused silica DB-5-MS column
diphenyl-picryl-hydrazyl, came from Sigma-Aldrich (St. (30 m × 0.25 mm × 0.25 𝜇m), which was employed for all
Louis, MO, USA). the analyses. The temperature of the oven was raised to
220∘ C from 70∘ C at 4∘ C/min and maintained isothermally
2.2. Chemical Properties of Nigella sativa L. Seeds. Moisture for 15 min. The temperatures of the injector and detector
(930.04), ash (930.05), and protein (978.04) contents were temperatures were maintained at 220∘ C and 240∘ C, respec-
established based on [17]. Fat determination was done using tively, with the preparation of 10% of samples in acetonitrile.
Soxhlet extractor and hexane as the solvent. In brief, 15 g of The split mode ratio of 1 : 15 was applied for the injection of
seed were ground and fat was obtained after 8 h of extraction a 0.5 𝜇L sample. Carrier gas used was helium at 1 mL/min
and the results were presented as the amount in percent of flow rate. Other parameters were kept the same in relation
lipids in the dry matter of seed powder, while estimation of to GC analyses. EI at 70 eV provided the mass spectra with
the carbohydrate content achieved was expressed in terms of mass scanning done from 40 to 400 amu. Percentage of each
the variance of the mean values, that is, component was calculated based on GC peak areas.

[100 − (protein + lipids + ash + moisture)] . (1) 2.6. HPLC Quantification of Thymoquinone. Thymoquinone
evaluation of NSO was conducted along the lines of the
2.3. Oil Extraction approach taken by [19] with minor modification. The oils
obtained from SFE and cold press extraction methods were
2.3.1. Cold Pressing. The N. sativa L. seeds were pressed at passed through a C18 column preeluted with methanol.
room temperature (25∘ C) by mechanical pressing without any 20 𝜇L of NSO followed by 800 𝜇L (400 𝜇L × 2) methanol
heating treatment. Crushed seeds were stored for one night was added to give an eluate free from greasy and fatty
at room temperature to separate oil phase from fibers, and materials. The analysis was carried out on the oils from
then oil was filtered using Watman #4 filter paper and a glass SFE and cold press extraction methods for TQ with the use
funnel. of Agilent 1200 HPLC system (Agilent Technologies, Palo
Alto, USA) with a diode array detector (HPLC-DAD). A
2.3.2. Supercritical Fluid Extraction. Extracting essential oil Prevail C18 column (250 × 4.6 mm ID, with 5 𝜇m particle
from N. sativa L. seed was performed using the SFE instru- size, Agilent Technologies, USA) was employed with a mobile
ment (FeyeCon Development B. V., Netherland) at the super- phase of water : methanol : 2-propanol (50 : 45 : 5% v/v) which
critical fluid centre, Faculty of Food Science and Technology, was filtered using a 0.45 mm Millipore filter and 20 𝜇L was
UPM, as described by [18], with some modification. The dried the volume of the injection. Analysis of thymoquinone was
seeds were completely crushed for 3-4 min in a stainless steel detected at 254 nm at room temperature. A 1.5 mL/min flow
grinder (Waring Commercial, Torrington, CT, USA) and kept rate was used and identification was confirmed through a
in a 50-liter extractor container of the same material and comparison of the time, the standard compound was retained
tightly sealed. Supercritical fluid extractions were conducted with that of oil sample and the quantity calculations were
at pressures of 600 bar and temperatures of 40∘ C for the achieved by constructing the standard linear calibration
duration of 1 h, and liquid CO2 was injected at approximately curves.
150 L/hour and controlled by an automatic back pressure
regulator. 2.7. 1, 1-Diphenyl-2-picrylhydrazyl (DPPH) Radical Scaveng-
ing Activity Test. This assay was performed according to [20]
2.4. Physicochemical Properties of Nigella sativa Oil. Peroxide with some changes made to establish the radical scavenging
value (PV), free fatty acids content (FFA), saponification activity of NSO. The DPPH solution was prepared fresh
value (SV), and iodine value (IV) were established based as and when it was needed by diluting 2.5 mg DPPH in
on [17]. The refractive index (RI) was calculated with Abbe 100 mL methanol. Then, the mixture of 0.25 mL of NSO and
Refractometer at 20∘ C. The viscosity of NSO was extracted 1.75 mL methanolic DPPH was prepared in a 96-wall plate.
using SFE and cold press was recorded at 25∘ C with a Haake Following 30 min of incubation in the dark and at ambient
Evidence-Based Complementary and Alternative Medicine 3

temperature (25∘ C), the absorbance was measured at 515 nm, 2.11. Thermal Behavior DSC. Thermal properties of NSO
the wave length of highest absorbance of DPPH was noted extracted by SFE and cold press were examined by differential
with an ELISA reader (labomed, model UVD-2950, USA). scanning calorimeter using DSC 820e from Mettler Toledo
A blank experiment or control was done applying similar (Schwerzenbach, Switzerland). Weighing of the oil samples
steps to a solution minus the test material. The absorbance was done (8-9 mg) straight into a DSC-pan (SFI-Aluminium,
was recorded as 𝐴. The free radical scavenging activity of all TA Instrument T11024). An empty aluminium pan, hermeti-
solutions was then calculated as percentage of inhibition on cally sealed, was the reference. The samples were heated from
the basis of the following equation: 30∘ C with a speed of 5∘ C/min to 90∘ C. The same process
was repeated and recording of the DSC thermographs was
(𝐴 − 𝐵) done during the second melting. The DSC manufacturer’s
% inhibition = ∗ 100, (2) software (STARe Speciality Library) was employed to analyse
𝐴
the data of the heat flow and the exact heat of the oil samples
was calculated. Results were derived by averaging triplicate
where 𝐴 = absorbance of control and 𝐵 = absorbance of the
samples.
sample.
Antioxidant activities of test compounds were expressed
as IC50 , which is represented by the amount of antioxidant 2.12. Thermogravimetric Analysis (TGA). Thermogravimet-
compounds that caused 50% scavenging of DPPH radicals for ric method determines the thermal properties of oils as
the duration of the defined time frame. a function of temperature. Examination of the thermally
induced degradation of the oils was conducted in a thermo-
gravimetric analysis (TGA) Perkin-Elmer Thermogravimetry
2.8. FRAP Assay. The ability of the NSO antioxidant of the
Analyzer Pyris 2. The analysis was carried out on approx-
ferric reducing power was assessed with (FRAP) assay as
imately 25 mg of samples at the temperature range of 25–
indicated by [21]. Briefly, FRAP reagent was freshly prepared,
1000∘ C at a constant heating rate of 25∘ C/min in an atmo-
and 20 mM FeC𝑙3 and 10 mM TPTZ solutions were mixed in
sphere of nitrogen.
40 mM HCl and 300 mM acetate buffer (pH 3.6) in ratios
of 1 : 1 : 10 (v/v/v). Then, 10 𝜇L of sample (dissolved 100 𝜇g
in 1 mL methanol) was distributed into a 96-well plate, and 2.13. Statistical Analysis. The considerable variance of the
then 200 𝜇L of FRAP reagent was placed into the same mean values was confirmed using Tukey’s test. A probability
well containing the sample. The solution was mixed and of 𝑝 > 0.05 was deemed significant. The statistical software,
incubated for 30 min at room temperature. The absorbance MINITAB 16 (Minitab Inc., State College, PA, USA) was
was recorded at 593 nm using ELISA reader (Power Wave employed for all the analyses.
×340, BioTek Instruments, Inc., Winooski, VT, USA). Results
of FRAP were expressed in mM of ferrous equivalents, Fe (II)
per g three times and readings were taken in triplicate. 3. Results
3.1. Physicochemical Properties of Nigella sativa L. Seed. The
2.9. Total Phenolic Content. The entire amount of phenolic approximated structure of the N. sativa seed powder and
compounds (TPC) of NSO was established with the use of related literature references are presented in Table 1. The
Folin-Ciocalteu reagent (FCR) according to [22] with certain analysis of the N. sativa L. seed demonstrated its contents to
changes. There was the addition of 0.5 mL sample to 0.5 mL of be 32.37% lipids, 6.78% moisture, 19.23% protein, 6.94% ash,
Folin-Ciocalteu reagent, and 5 minutes later, 10 mL of 7% of and 35.08% carbohydrate. FFA values of NSO extracted by
aqueous Na2 CO3 sodium carbonate solution was added and SFE and CP methods were significantly different with values
mixed to react then kept for 1 hour in total darkness at room of 6.15 and 5.98% (as oleic acid), respectively (Table 2). The
temperature. The absorbance was read at 725 nm employing iodine values were 115 g of I2/100 g of oil for SFE and 104 g of
ELISA reader (Power Wave ×340, BioTek Instruments, Inc., I2/100 g of oil for CP oil (Table 2). The SFE and CP extracted
Winooski, VT, USA). A standard curve of gallic acid was oils showed the saponification values of 243.52 (mg of KOH/g
separately made from versus concentration (0-1 mg/mL). The of oil) and 238.26 (mg of KOH/g of oil), respectively (Table 2).
outcomes were presented in gallic acid equivalents (GAE) Table 2 presents the refractive index (RI) of NSO extracted by
mg/kg dry weight. All experiments were conducted three SFE and cold press 1.47813 and 1.47719, respectively. Peroxide
times and reading was obtained in triplicate. values (PV) of oils obtained by using SFE and cold pressing
extraction were 3.4 and 4.1 meq O2 /kg oil, respectively.
2.10. FTIR Spectroscopy. Fourier-transform infrared (FTIR)
spectroscopy of NSO extracted by either SFE or cold press 3.2. GC-MS Studies. The results showed significant differ-
machine was carried out on a Spectrum 100 FTIR spec- ences between the SFE and the cold press oils based on the
trometer from Perkin-Elmer Corporation, USA. FTIR for peak area (Table 3). Twenty compounds were identified in the
the samples was recorded over the range of 400–4000 cm−1 SFE oil, while the cold press oil had 19 compounds. The major
employing a sample of approximately one percent in 200 mg components of the SFE oil were Caryophyllene (17.47%)
of spectroscopic-grade potassium bromide (KBr) with 10 tons followed by thymoquinone (16.80%), 1,4-Cyclohexadiene
of pressure. (7.17%), longifolene (3.5), and carvacrol (1.82%) (Figure 1(a)).
4 Evidence-Based Complementary and Alternative Medicine

Table 1: Chemical properties of the selected Nigella sativa seed in comparison with previous studies.

Total fat Moisture Ash Protein Carbohydrate Studies

32.26 ± 0.09 6.67 ± 0.20 6.82 ± 0.10 19.19 ± 0.20 35.04 ± 0.30 Current study
31.72 ± 0.42 4.99 ± 0.29 5.29 ± 0.41 23.07 ± 1.05 34.91 ± 1.22 Solati et al., 2014 [13]
40.35 ± 0.16 — 4.41 ± 0.01 22.6 ± 0.24 32.7 ± 0.41 Cheikh-Rouhou et al., 2007 [14]
34.8 ± 1.90 7.0 ± 0.50 3.7 ± 0.70 20.8 ± 1.10 33.7 ± 0.50 Atta, 2003 [15]
37.33 ± 0.15 5.40 ± 0.13 6.72 ± 0.02 20.02 ± 0.27 30.53 Khoddami et al., 2011 [16]

Table 2: Oil physiochemical properties obtained using two extrac- the most common triglyceride molecules with certain fatty
tion methods. acids (Table 6).
Physiochemical Supercritical fluid Cold press
properties extraction extraction 3.5. Thermal Behavior of Nigella sativa L. Oil. These curves
FFA (as oleic %) 5.98 ± 0.00b 6.15 ± 0.00a of the SFE and CP oils showed a plain thermogram with
SV (mg of KOH/g of one peak possessing features such as onset temperature at
243.52 ± 0.3a 238.26 ± 0.67b
oil) 203.66∘ C and 243.03∘ C, melting enthalpy is 30.48 J/g and
IV (g of I2 /100 g of oil) 115.1 ± 0.24a 104.37 ± 0.43b 34.50 J/g, and melting peak 241.04∘ C and 264.35∘ C, respec-
PV (meq O2 /kg oil) 3.4 ± 0.05a 4.1 ± 0.15b tively (Figures 4(a) and 4(b)).
Refractive index at
The weight loss occurred in SFE oil in the percentage of
1.47 ± 0.00a 1.47 ± 0.00a 35.1 and 63.4% at 208 and 365∘ C, respectively (Figures 5(a)
(25∘ C)
and 5(b)). TGA and DTG curves for the SFE oil showed the
Viscosity (mPa s) 6.26 ± 0.07a 6.38 ± 0.08a
degradation in two steps, first point at 308.5∘ C and second
Means of triplicate measurements in the same row ± standard deviation with
degradation at 365.5∘ C, while for the CP oil had weight loss
different letters are significantly different (𝑝 < 0.05).
of 6.3 and 91.4% at 212.4 and 33.3∘ C, respectively. In addition,
degradation points have been observed in CP oil at 288.3∘ C
and 423.77∘ C, respectively.
The major components for cold press oil were 1,3,8-p-
Menthatriene (23.82%) followed by thymoquinone (16.21%),
1,4-Cyclohexadiene (7.17%), longifolene (4.49), and carvacrol 4. Discussion
(3.90%) (Figure 1(b)). The analysis of the N. sativa L. seed demonstrated that its
The HPLC analysis showed that the two oil samples (SFE contents of lipids and moisture were in the range of those
and cold pressed) contained thymoquinone TQ (Figures 2(a) found in the literature, while protein was slightly lower and
and 2(b)). The highest amount of TQ was observed in the SFE ash with carbohydrate (by difference) were higher compared
oil and was 6.63 mg/mL of oil, while the concentration of TQ to those revealed in the literature [13–16]. The chemical
was 1.56 mg/mL oil for the cold press sample (Table 4). The properties of oils are considered the most significant feature
methods of extraction showed important differences (𝑝 > to determine the quality of oil samples.
0.05) for the SFE with higher thymoquinone quantity. In this work, chemically, the properties of N. sativa L. oil
extracted using SFE and cold press were studied. Free fatty
3.3. Antioxidant Activity of Nigella sativa L. Oil. The antirad- acid and peroxide value are the commonly used indicators
ical activity of NSO to scavenge DPPH demonstrated high to monitor the quality of seed oils. In the current study,
levels with SFE oil IC50 being 1.5873 mg/mL while the CP the low FFA value demonstrated that oil extracted with the
oil IC50 was 2.3086 mg/mL (Table 5). The reducing ability SFE method had higher stability than that obtained by CP
of NSO extracted by two methods, SFE and cold press, was extraction. This finding is in agreement with the oil obtained
in the range of 329.00–538.67 mol/100 mL oil (Table 5). The through SFE method (5.83%) and Soxhlet method (6.40%)
concentration of overall phenolic compounds (TPC) in NSO in [13] and much less compared to cold pressed oil (11%)
extracted by SFE and CP was assessed using Folin-Ciocalteu [14]. In another study, the iodine value was higher than that
method presented as gallic acid equivalents as can be seen in of this study’s results according to [23] for SFE oil (127 g of
Table 5. Results in this table show a considerable difference I2 /100 g of oil) and (121 g of I2 /100 g of oil) for Soxhlet oil.
(𝑝 < 0.05) between the overall phenolic content of the SFE The iodine index shows that NSO is an extremely unsaturated
oil with a value of 160.51 mg GAE/100 mL and CP oil with a oil, suggesting that it possesses elevated levels of oleic and
value of 94.40 mg GAE/100 mL (Table 5). linoleic acids, with the lower iodine value contributing to
the high stability of the oil [14]. The saponification values
(SV) of NSO extracted by SFE and CP methods were high
3.4. FTIR Spectra of Nigella sativa L. Oil. Figure 3 reveals compared to other seed oils suggesting the presence of high
FTIR spectrum of SFE and CP oils at mid infrared region triacylglycerols content. The saponification values in this
(4,000–650 cm−1 ). In this study, the spectrum of both oils study were comparable to the values of Tunisian 211 (mg
showed very close features of absorption bands identical to of KOH/g of oil) and Iranian 218 (mg of KOH/g of oil)
Evidence-Based Complementary and Alternative Medicine 5

Table 3: GC-MS identification of chemical composition for Nigella sativa oil extracted by supercritical fluid and cold press.

Supercritical fluid Cold press

Compound name
MW RT Peak area (%) MW RT Peak area (%)
1 𝛼-Pinene — — — 136 3.19 7.10
2 3-Carene 136 3.29 2.60 136 3.29 2.21
3 Bicyclo[2.2.1]heptane 136 4.00 2.05 136 4.00 3.67
4 1,3,8-p-Menthatriene — — — 134 4.96 23.82
5 ç-Terpinene — — — 136 5.64 2.82
6 1,4-Cyclohexadiene 134 5.01 7.17 — — —
7 cis-4-Methoxy thujane 168 6.45 3.09 168 7.05 7.30
8 Cyclohexen 154 8.90 3.82 — — —
9 Limonene — — — 236 8.41 0.41
10 Thymoquinone 164 11.32 16.80 164 11.06 16.21
11 Carvacrol 150 13.51 1.82 150 13.46 3.90
12 Longifolene 204 13.70 3.50 204 15.37 4.49
13 Ylangene 204 14.50 2.99 204 13.67 0.82
14 3-Allyl-6-methoxyphenol 164 14.61 0.40 — — —
15 1-Heptatriacotanol — — — 536 20.91 0.14
16 2,3-Dihydrofarnesyl acetate — — — 266 30.91 1.54
17 Caryophyllene 204 16.03 17.45 — — —
18 Naphthalene 204 17.88 2.94 — — —
19 𝛼-Bisabolene 204 18.50 0.50 — — —
20 (−)-Spathulenol 220 22.37 0.79 — — —
21 Methyl tetradecanoate 242 24.54 0.54 — — —
22 Hexadecanoic acid 254 29.69 4.99 254 31.65 0.77
23 Ascorbic acid 652 31.75 1.12 652 34.95 2.15
24 9,12-Octadecadienoic acid (Z,Z) 294 33.70 14.05 294 35.45 7.85
25 4,8-Decadienal, 5,9-dimethyl — — — 180 33.84 8.12
26 Methyl stearate 298 34.34 0.61
27 Butyl 9,12-octadecadienoate 336 35.11 2.29 336 11.77 0.21
28 Cyclopropanebutanoic acid — — — 374 37.66 0.25
Total 89.54 93.53
MW: molecular weight.
RT: retention time.

RT: 0.00–53.51 RT: 0.00–53.48 NL:

NL:
100 16.02 100 4.96 6.57E9
1.42E10
90 11.35 TIC MS 90 TIC MS
5.03 33.68 A2-1 B2-1
80 80
Relative abundance

Relative abundance

70 70 11.06
7.05
60 29.69 60
50 50
40 18.97 40 7.05 33.83
30 30 3.19
8.91
20 20 15.36
24.54
10 10 8.90 30.91
44.10 48.81 18.48 25.09 48.98
0 0
0 5 10 15 20 25 30 35 40 45 50 0 5 10 15 20 25 30 35 40 45 50
Time (min) Time (min)
(a) (b)

Figure 1: GC-MS chromatography analysis of Nigella sativa oil extracted by (a) 474 supercritical fluid and (b) cold press.
6 Evidence-Based Complementary and Alternative Medicine

7.154
2500 900

7.149
800
2000 700
600
(mAU)

(mAU)
1500 500
400
1000
300
500 200
100
0 0
0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 14
(min) (min)
(a) (b)

7.272
2500

2000
(mAU)

1500

1000

500

0
0 2 4 6 8 10 12 14
(min)
(c)

Figure 2: Thymoquinone concentration of Nigella sativa oil as determined by HPLC: (a) 490 supercritical fluid extraction, (b) cold press
extraction, and (c) thymoquinone standard.

Table 4: Thymoquinone concentration of Nigella sativa oil extracted

by supercritical fluid and cold press as determined by HPLC.

Extraction methods Thymoquinone CP

SFE oil 6.37 ± 0.00a mg/mL

Absorbance (a.u.)

CP oil 1.78 ± 0.00b mg/mL

± Standard deviation of three replications.
ab
Different letters in the same column represent significant (𝑝 < 0.05)
SFE
differences.

extract from CP extraction with hexane reported by [14].

The result of refractive index (RI) was similar to those
reported previously, while the peroxide values (PV) of oils
were lower than cold press 13.5 meq O2 /kg oil reported by 3500 3000 2500 2000 1500 1000 500
[15]. There could be a relation between these differences −1
Wavenumber (cm )
and the dissimilarities of cultivated regions, maturity stage,
and storage circumstances. According to the data reported Figure 3: FTIR spectra of Nigella sativa L. oil scanned at 4,000–
650 cm−1 : supercritical fluid (SFE) and cold press (CP).
about the nutritional value of N. sativa seed, it is noted
that besides the high oil content it is also a rich source of
many minerals and bioactive compounds. Compounds were
recognized through comparison of their retention indices
and mass obtained in GC-MS chromatogram (Figures 1(a) peak area. Burits and Bucar’s [1] method for the extraction of
and 1(b)) with those of NIST02 library data stored in the essential oil used light petroleum followed by steam distilling
computer library and Adams libraries spectra [24, 25] and of the extract. They indicated that their essential oil possessed
enumerated along with molecular weight, retention time, and thymoquinone as the main component, while in this study
Evidence-Based Complementary and Alternative Medicine 7

Table 5: Antioxidant activities of Nigella sativa oil determined by DPPH radical scavenging and FRAP ferrous reducing, with TPC total
phenolic content.

Types of oil DPPH IC50 (mg/mL) FRAP (mmol/100 mL) TPC (mg/100 mL GAE)
Supercritical fluid oil 1.58 ± 0.07b 538.67 ± 12.58a 160.51 ± 11.43a
Cold press oil 2.30 ± 0.02a 329.00 ± 54.78b 94.40 ± 1.02b
± Standard deviation of three replications.
ab
Different letters in the same column represent significant (p < 0.05) differences.

Table 6: The main peaks in the FTIR spectrum of Nigella sativa oils extracted by supercritical fluid of and cold press with their assignment.

Oil types Peak (cm−1 ) Functional group

SFE-CP 3482 Primary amines (-NH2 groups)
SFE-CP 2922, 2856 C-H stretching vibration (aliphatic) (CH3 )
SFE-CP 1721 C=O stretching vibration (ester)
SFE-CP 1456, 1367.74 C-H bending vibration (aliphatic) (CH2 )
SFE-CP 1166.48 C-O stretching vibration (ester)
SFE-CP 935.42, 717.35 trans-CH=CH-

Caryophyllene is the major component of the SFE oil and prevalent in several species of plants. Sielicka and Samotyja
1,3,8-p-Menthatriene for cold press oil. [30] evaluated several oils for ferric reducing activity, and
The high content of thymoquinone could be because of among the tested oils, the highest activity was observed for
the greater selectivity of the SFE method compared to cold NSO, which ranged between 678 and 2102 𝜇mol/100 g oil.
press. Generally, extractions using cold press result in partial Compared to this study, the results are significantly higher
oil recovery [26]. In fact, the cold press gives low yields, for both CP oil and SFE oil. The SFE oil exhibited the highest
and usually the extraction process is time consuming [10] FRAP activity which indicated the advantages of the SFE
which may affect the concentration of the target compounds method.
from NSO quantity. The results for thymoquinone quantity Polyphenolic compounds are acknowledged to be antiox-
were similar to those of Solati et al. [13] who reported that idant active and there is a likelihood that these compounds
the NSO extracted by SFE had a high content of TQ with are the cause of abstracts activity [31]. In other studies, total
4.09 mg/mL. Furthermore, Ismail et al. [18] reported that phenolic contents of NSO extracted by solvent from Tunisian
NSO extracted with SFE was rich in thymoquinone with and Iranian Nigella sativa seeds were 245 and 309 mg/kg
the percentage of about four times higher compared to the expressed as gallic acid, respectively [14]. Viuda-Martos et
oil extracted by conventional solvent extraction method. TQ al. [32] determined the TPC in NSO and observed the
is a phytochemical compound of NSO, which is interesting high content of phenolic compounds (726.67 mg/L) in the
for study as it contributes to the oil’s general stability and commercial oil, and these results mirror those of CP oil
potential health benefits [27]. It has also been shown to be in this study, while these findings were much less than the
the reason for the biological antioxidant activity of NSO and oil extracted by SFE. This indicates that using SFE for the
also for the majority of the beneficial health effects related to extraction of the oil is preferred as it will yield a higher
the seeds and oils [13]. concentration of phenolic compounds.
The antiradical activity of NSO to scavenge DPPH was Differential scanning calorimetry DSC offers evidence of
determined based on their IC50 values, described as the the additional specific heat across a broad temperature range
quantum of the antioxidant needed to inhibit 50% of DPPH [33]. Any endothermic or exothermic event is noted as a peak
existing in the test material. Both the SFE and CP oils had in the chart, and its area is in proportion to the enthalpy
high ability to decrease the stable radical DPPH. The earlier achieved or lost, respectively. Figures 4(a) and 4(b) show
studies by [1, 13] reported higher IC50 (lowest antioxidant the DSC melting curves for NSO extracted by two different
activity) compared to this study’s IC50 of 2.26 mg/mL. These extraction methods. The melting curves of these seed oils
data confirm that SFE is a more appropriate method of show more endothermic peaks and shoulders. In general,
extraction for NSO. The SFE oil exhibited a high antioxidant both NSO samples showed similar DSC melting point and
power corresponding to that of CP oil. The ferric reducing profiles higher than 200∘ C temperature regions. This result
ability (FRAP assay) is very often employed to evaluate concurs with published data [14]. Thermogravimetric analy-
the antioxidant component in dietary polyphenols [28]. sis (TGA) and derivative thermograms analysis (DTG) were
Antioxidant activity is known to have a linear proportion conducted to determine the thermal stability of two samples
of the phenolic contents. Oktay et al. [29] mentioned the for NSO extracted by SFE and cold press. Thermogravimetric
likelihood of total phenolic contents and antioxidant activity analysis (TGA), also referred to as thermogravimetry (TG),
being very positively related between, a trend that seems to be is a technique where measuring the thermal behavior of mass
8 Evidence-Based Complementary and Alternative Medicine

6 2.0

1.5
4
1.0
Heat flow (W/g)

Heat flow (W/g)

2 0.5

0.0
0
−0.5

−1.0
−2
−1.5

−4 −2.0
50 100 150 200 250 300 350 100 150 200 250 300
Temperature (∘ C) Temperature (∘ C)
SFE CP
(a) (b)

Figure 4: Differential scanning calorimetry (DSC) thermal behavior of Nigella sativa oil: 529 (a) supercritical fluid and (b) cold press.

100 100

80 80
Weight loss (%)

Weight loss (%)

60 60

40 40

20 20

0 0

0 200 400 600 800 1000 0 200 400 600 800 1000
Temperature (∘ C) Temperature (∘ C)
TGA TGA
DTG DTG
(a) (b)

Figure 5: TGA and DTG curves of weight loss of Nigella sativa oil: (a) supercritical fluid 540 and (b) cold press.

as a function of heating is recorded. The TGA and the DTG 5. Conclusion

curves revealed an inverse linear correlation between the
weight and the temperature as the NSO lost weight with an In conclusion, the NSO extracted by using supercritical fluid
increase in the temperature. From TGA profile of both oils, had a high concentration of thymoquinone and total phenolic
there is no weight loss observed before 200∘ C. TGA and compounds. Moreover, the antioxidant activities were high
DTG curves’ behavior remained similar for the CP oil which with low IC50 value as established by DPPH and FRAP
showed similar thermal behavior with SFE oil. This might assays. The oil extracted by CP and SFE had no significant
be due to moisture evaporation and the presence of other differences in their thermal profiles with no weight loss
volatiles in oil samples. The onset temperature and peak value observed before 200∘ C and both oils have the same functional
of the SFE oil compared to CP oil are also similar. Thus, it is groups. The GC-MS showed significant differences in the
clear that the thermal stability of the N. sativa seed oils was two oil profiles which are directly affected by the extraction
not significantly affected by the type of extraction method. method employed. The SFE method can be considered as the
Evidence-Based Complementary and Alternative Medicine 9

optimum process for the extraction of NSO since the method extracts from Nigella sativa L. seeds,” Journal of the American Oil
enhances the quality of the yield in comparison with CP. Chemists’ Society, vol. 91, no. 2, pp. 295–300, 2014.
[14] S. Cheikh-Rouhou, S. Besbes, B. Hentati, C. Blecker, C.
Deroanne, and H. Attia, “Nigella sativa L.: chemical composi-
Competing Interests tion and physicochemical characteristics of lipid fraction,” Food
Chemistry, vol. 101, no. 2, pp. 673–681, 2007.
The authors declare no conflict of interests of any kind.
[15] M. B. Atta, “Some characteristics of nigella (Nigella sativa L.)
seed cultivated in Egypt and its lipid profile,” Food Chemistry,
Acknowledgments vol. 83, no. 1, pp. 63–68, 2003.
[16] A. Khoddami, H. M. Ghazali, A. Yassoralipour, Y. Ramakr-
This research was supported by Universiti Putra Malaysia ishnan, and A. Ganjloo, “Physicochemical characteristics of
grant (GP-IPS/2014/9438743). Nigella seed (Nigella sativa L.) oil as affected by different
extraction methods,” Journal of the American Oil Chemists’
Society, vol. 88, no. 4, pp. 533–540, 2011.
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